advertisement
Browse Health
Internist, Nephrologist (kidney)
41 years of experience
Accepting new patients

Credentials

Education ?

Medical School
Middlesex Hospital Medical School, London (1971)
Foreign school

Awards & Distinctions ?

Awards  
One of America's Leading Experts on:
Glomerulonephritis (Kidney disease)
Associations
American Society of Hypertension

Affiliations ?

Dr. Wiggins is affiliated with 3 hospitals.

Hospital Affiliations

Score

Rankings

  • University of Michigan Hospitals & Health Centers
    1500 E Medical Center Dr, Ann Arbor, MI 48109
    •  
    Top 25%
  • Ann Arbor Veterans Affairs Medical Center
    2215 Fuller Rd, Ann Arbor, MI 48105
  • University of Michigan Health System
  • Publications & Research

    Dr. Wiggins has contributed to 178 publications.
    Title Contrast-enhanced Diagnostic Ultrasound Causes Renal Tissue Damage in a Porcine Model.
    Date January 2011
    Journal Journal of Ultrasound in Medicine : Official Journal of the American Institute of Ultrasound in Medicine
    Excerpt

    Glomerular capillary hemorrhage (GCH) has been reported and confirmed as a consequence of contrast-enhanced diagnostic ultrasound (CEDUS) imaging of rat kidney. This study assessed renal tissue injury in the larger porcine model.

    Title Nfkappab Promotes Inflammation, Coagulation, and Fibrosis in the Aging Glomerulus.
    Date April 2010
    Journal Journal of the American Society of Nephrology : Jasn
    Excerpt

    The peak prevalence of ESRD from glomerulosclerosis occurs at 70 to 79 years. To understand why old glomeruli are prone to failure, we analyzed the Fischer 344 rat model of aging under ad libitum-fed (rapid aging) and calorie-restricted (slowed aging) conditions. All glomerular cells contained genes whose expression changed "linearly" during adult life from 2 to 24 months: mesangial cells (e.g., MMP9), endothelial cells (e.g., ICAM and VCAM), parietal epithelial cells (e.g., ceruloplasmin), and podocytes (e.g., nephrin and prepronociceptin). Patterns of aging glomerular gene expression closely resembled atherosclerosis, including activation of endothelial cells, epithelial cells, and macrophages, as well as proinflammatory pathways related to cell adhesion, chemotaxis, blood coagulation, oxidoreductases, matrix metalloproteinases, and TGF-beta activation. We used a nonbiased data-mining approach to identify NFkappaB as the likely transcriptional regulator of these events. We confirmed NFkappaB activation by two independent methods: translocation of NFkappaB p50 to glomerular nuclei and ChIP assays demonstrating NFkappaB p50 binding to the kappaB motif of target genes in old versus young glomeruli. These data suggest that old glomeruli exhibit NFkappaB-associated up-regulation of a proinflammatory, procoagulable, and profibrotic phenotype compared with young glomeruli; these distinctions could explain their enhanced susceptibility to failure. Furthermore, these results provide a potential mechanistic explanation for the close relationship between ESRD and atherosclerotic organ failure as two parallel arms of age-associated NFkappaB-driven processes.

    Title In Vivo Gas Body Efficacy for Glomerular Capillary Hemorrhage Induced by Diagnostic Ultrasound in Rats.
    Date March 2010
    Journal Ieee Transactions on Bio-medical Engineering
    Excerpt

    Glomerular capillary hemorrhage (GCH) in rat kidney provided a model for assessing in vivo gas body efficacy in diagnostic or therapeutic applications of ultrasound. Two diagnostic ultrasound machines were utilized: one monitored the harmonic B-mode contrast enhancement of the left kidney and the other exposed the right kidney for GCH production. Definity contrast agent was infused at 1, 2, 5, or 10 microL/(kg x min) and infusion durations were 30, 60, 120, or 300 s. Exposure of the right kidney was at a peak rarefactional pressure amplitude of 2.3 MPa at 1.5 MHz. The circulating dose was estimated with a simple model of agent dilution and gas body loss. For 300 s infusion at 5 microL/(kg x min), the left kidney image brightness increased to a plateau with an estimated 6.4 +/- 1.3 microL/kg circulating dose with no GCH in histological sections. Exposure of the right kidney with a 1-s image interval reduced the estimated circulating dose to 1.3 +/- 0.3 microL/kg and induced 68.4% GCH. Dose and duration increases gave rapidly diminishing treatment effectiveness per gas body. The effective in vivo agent dose in rats can be reduced greatly due to high gas body destruction in the small animal, complicating predictions for similar conditions of human treatment.

    Title Antibodies to Protein Tyrosine Phosphatase Receptor Type O (ptpro) Increase Glomerular Albumin Permeability (p(alb)).
    Date August 2009
    Journal American Journal of Physiology. Renal Physiology
    Excerpt

    Glomerular capillary filtration barrier characteristics are determined in part by the slit-pore junctions of glomerular podocytes. Protein tyrosine phosphatase receptor-O (PTPro) is a transmembrane protein expressed on the apical surface of podocyte foot processes. Tyrosine phosphorylation of podocyte proteins including nephrin may control the filtration barrier. To determine whether PTPro activity is required to maintain glomerular macromolecular permeability, albumin permeability (P(alb)) was studied after incubation of glomeruli from normal animals with a series of monoclonal (mAb) and polyclonal antibodies. Reagents included mAbs to rabbit and rat PTPro and polyclonal rabbit immune IgG to rat PTPro. mAb 4C3, specific to the amino acid core of PTPro, decreased its phosphatase activity and increased P(alb) of rabbit glomeruli in a time- and concentration-dependent manner. In contrast, mAb P8E7 did not diminish phosphatase activity and did not alter P(alb). Preincubation of 4C3 with PTPro extracellular domain fusion protein blocked glomerular binding and abolished permeability activity. In parallel experiments, P(alb) of rat glomeruli was increased by two mAbs (1B4 and 1D1) or by polyclonal anti-rat PTPro. We conclude that PTPro interaction with specific antibodies acutely increases P(alb). The identity of the normal ligand for PTPro and of its substrate, as well as the mechanism by which phosphatase activity of this receptor affects the filtration barrier, remain to be determined.

    Title Glomerular Capillary Hemorrhage Induced in Rats by Diagnostic Ultrasound with Gas-body Contrast Agent Produces Intratubular Obstruction.
    Date June 2009
    Journal Ultrasound in Medicine & Biology
    Excerpt

    Glomerular capillary hemorrhage (GCH) induced by ultrasonic cavitation during diagnostic imaging represents a unique contrast agent-related nephron injury. Consequences of GCH during 1.5-MHz diagnostic ultrasound with contrast agent were examined by histologic methods in rats. Definity was infused at 10 microl/kg/min for 5 min at the start of 8 min of intermittent image-exposure, with 2.3 MPa in situ peak rarefactional pressure amplitude. Kidney samples were taken for histology at 5 min, 30 min, 4 h, 2 d, 1 week and 4 weeks post exposure. In addition, samples were taken at 4 h from groups treated with heparin or aminocaproic acid. GCH was found in 61% of glomeruli in the center of the scan plane 5 min after exposure, which declined (p < 0.05) to 36.3% after 4 h. The width of Bowman's space was significantly increased for glomeruli with GCH relative to glomeruli without GCH (p < 0.05), consistent with tubular obstruction. Antibody staining revealed fibrin clotting in Bowman's space in 4-h samples and this persisted in the 2-d samples. Heparin reduced and aminocaproic acid increased the GCH seen in 4-h samples. Tubular dilation was evident with injury to the epithelium after 2 d. After one week, areas of inflammatory cell infiltration were present. After four weeks, areas of interstitial fibrosis were revealed by Masson's trichrome stain. The consequences of GCH induced by diagnostic ultrasound with contrast agents include rupture of glomerular capillaries, procoagulant activity resulting in intratubular obstruction, and the potential for progression of the resulting tubular injury toward interstitial fibrosis.

    Title Urine Podocyte Mrnas Mark Progression of Renal Disease.
    Date May 2009
    Journal Journal of the American Society of Nephrology : Jasn
    Excerpt

    Because loss of podocytes associates with glomerulosclerosis, monitoring podocyte loss by measuring podocyte products in urine may be clinically useful. To determine whether a single episode of podocyte injury would cause persistent podocyte loss, we induced limited podocyte depletion using a diphtheria toxin receptor (hDTR) transgenic rat. We monitored podocyte loss by detecting nephrin and podocin mRNA in urine particulates with quantitative reverse transcriptase-PCR. Aquaporin 2 mRNA served as a kidney reference gene to account for variable kidney contribution to RNA amount and quality. We found that a single injection of diphtheria toxin resulted in an initial peak of proteinuria and podocyte mRNAs (podocin and nephrin) followed 8 d later by a second peak of proteinuria and podocyte mRNAs that were podocin positive but nephrin negative. Proteinuria that persisted for months correlated with podocin-positive, nephrin-negative mRNAs in urine. Animals with persistent podocyte mRNA in urine progressed to ESRD with global podocyte depletion and interstitial scarring. Podocytes in ectatic tubules expressed podocalyxin and podocin proteins but not nephrin, compatible with detached podocytes' having an altered phenotype. Parallel human studies showed that biopsy-proven glomerular injury associated with increased urinary podocin:aquaporin 2 and nephrin:aquaporin 2 molar ratios. We conclude that a single episode of podocyte injury can trigger glomerular destabilization, resulting in persistent podocyte loss and an altered phenotype of podocytes recovered from urine. Podocyte mRNAs in urine may be a useful clinical tool for the diagnosis and monitoring of glomerular diseases.

    Title Mutations in Plce1 Are a Major Cause of Isolated Diffuse Mesangial Sclerosis (idms).
    Date April 2008
    Journal Nephrology, Dialysis, Transplantation : Official Publication of the European Dialysis and Transplant Association - European Renal Association
    Excerpt

    BACKGROUND AND OBJECTIVES: Diffuse mesangial sclerosis (DMS) is a histologically distinct variant of nephrotic syndrome (NS) that is characterized by early onset and by progression to end-stage kidney disease (ESKD). Besides syndromic DMS, isolated (non-syndromic) DMS (IDMS) has been described. The etiology and pathogenesis of DMS is not understood. We recently identified by positional cloning recessive mutations in the gene PLCE1/NPHS3 as a novel cause of IDMS. We demonstrated a role of PLCE1 in glomerulogenesis. Mutations in two other genes WT1 and LAMB2 may also cause IDMS. We therefore determine in this study the relative frequency of mutations in PLCE1, WT1 or LAMB2 as the cause of IDMS in a worldwide cohort. METHODS: We identified 40 children from 35 families with IDMS from a worldwide cohort of 1368 children with NS. All the subjects were analyzed for mutations in all exons of PLCE1 by multiplex capillary heteroduplex analysis and direct sequencing, by direct sequencing of exons 8 and 9 of WT1, and all the exons of LAMB2. RESULTS: The median (range) age at onset of NS was 11 (1-72) months. We detected truncating mutations in PLCE1 in 10/35 (28.6%) families and WT1 mutations in 3/35 (8.5%) families. We found no mutations in LAMB2. CONCLUSIONS: PLCE1 mutation is the most common cause of IDMS in this cohort. We previously reported that one child with truncating mutation in PLCE1 responded to cyclosporine therapy. If this observation is confirmed in a larger study, mutations in PLCE1 may serve as a biomarker for selecting patients with IDMS who may benefit from treatment.

    Title Identification of Braf As a New Interactor of Plcepsilon1, the Protein Mutated in Nephrotic Syndrome Type 3.
    Date March 2008
    Journal American Journal of Physiology. Renal Physiology
    Excerpt

    Steroid-resistant nephrotic syndrome is a malfunction of the kidney glomerular filter that leads to proteinuria, hypoalbuminemia, edema, and renal failure. Recently, we identified recessive mutations in the phospholipase C epsilon 1 gene (PLCE1) as a new cause of early-onset nephrotic syndrome and demonstrated interaction of PLCepsilon1 with IQGAP1. To further elucidate the mechanism by which PLCE1 mutations cause nephrotic syndrome, we sought to identify new protein interaction partners of PLCepsilon1. We utilized information from the genetic interaction network of C. elegans. It relates the PLCE1 ortholog (plc-1) to the C. elegans ortholog (lin-45) of human BRAF (v-raf murine sarcoma viral oncogene homolog B1). We hypothesized that this may indicate a functional protein-protein interaction. Using GST pull down of HEK293T cell lysates in vitro and coimmunoprecipation of mouse kidney lysates in vivo, we show that BRAF interacts with PLCepsilon1. By immunohistochemistry in rat kidney, we demonstrate that both proteins are coexpressed and colocalize in developing and mature glomerular podocytes, reporting for the first time the expression of BRAF in the glomerular podocyte.

    Title Simulation of Diagnostic Ultrasound Image Pulse Sequences in Cavitation Bioeffects Research.
    Date November 2007
    Journal The Journal of the Acoustical Society of America
    Excerpt

    Research on cavitational bioeffects of diagnostic ultrasound (DUS) typically involves a diagnostic scanner as the exposure source. However, this can limit the ranges of exposure parameters for experimentation. Anesthetized hairless rats were mounted in a water bath and their right kidneys were exposed to ultrasound. Amplitude modulation with Gaussian envelopes simulated the image pulse sequences (IPSs) produced by diagnostic scanning. A 10 mulkgmin IV dose of Definity((R)) contrast agent was given during 1-5 min exposures. Glomerular capillary hemorrhage was assessed by histology. A stationary exposure approximated the bioeffects induced by DUS within the beam area. However, the use of five closely spaced exposures more faithfully reproduced the total effect produced within a DUS scan plane. Single pulses delivered at 1 s intervals induced the same effect as the simulated DUS. Use of 100 ms triangle-wave modulations for ramp-up or ramp-down of the IPS gave no effect or a large effect, respectively. Finally, an air-backed transducer simulating DUS without contrast agent showed a zero effect even operating at twice the present DUS guideline upper limit. Relatively simple single-element laboratory exposure systems can simulate diagnostic ultrasound exposure and allow exploration of parameter ranges beyond those available on present clinical systems.

    Title Doppler Mode Pulse Sequences Mitigate Glomerular Capillary Hemorrhage in Contrast-aided Diagnostic Ultrasound of Rat Kidney.
    Date November 2007
    Journal Ieee Transactions on Ultrasonics, Ferroelectrics, and Frequency Control
    Excerpt

    Glomerular capillary hemorrhage (GCH) induced in rat kidney by diagnostic ultrasound involving contrast agent destruction was characterized for different modes to explore possible mitigation strategies. Anesthetized hairless rats were scanned at 2.5 MHz in a water bath with contrast agent infused at 10 microl/kg/minute via tail vein. B mode flash echo imaging (FEI), color Doppler (CD) FEI and realtime Doppler imaging at 1 frame per second were tested, which had image pulse sequences of approximately 0.53 ms, 15.8 ms, and 83.5 ms duration, respectively. Bioeffects endpoints included grossly observed blood-filled tubules, histological evaluation of GCH, and detection of hematuria. B mode FEI for 1 minute induced GCH in 38.6+/-17.1% of glomeruli in histology from the scan plane for a peak rarefactional pressure amplitude (RPA) of 2.6 MPa. The threshold for GCH was approximately 1.5 MPa, confirmed by 10-minute exposure with agent infusion. Paradoxically, CD mode FEI delivered many more pulses but produced less GCH (P < 0.02), and real-time Doppler mode induced only 5.3 +/- 3.8% (P < 0.005). Hematuria results followed the GCH trends. These findings indicate a promising strategy, which is to use relatively slow ramp-up of pulse RPAs in agent-destroying image pulse sequences, for mitigating potential bioeffects in contrastaided diagnostic ultrasound.

    Title Nephron Injury Induced by Diagnostic Ultrasound Imaging at High Mechanical Index with Gas Body Contrast Agent.
    Date November 2007
    Journal Ultrasound in Medicine & Biology
    Excerpt

    The right kidney of anesthetized rats was imaged with intermittent diagnostic ultrasound (1.5 MHz; 1-s trigger interval) under exposure conditions simulating those encountered in human perfusion imaging. The rats were infused intravenously with 10 microL/kg/min Definity (Bristol-Myers Squibb Medical Imaging, Inc., N. Billerica, MA, USA) while being exposed to mechanical index (MI) values of up to 1.5 for 1 min. Suprathreshold MI values ruptured glomerular capillaries, resulting in blood filling Bowman's space and proximal convoluted tubules of many nephrons. The re-establishment of a pressure gradient after hemostasis caused the uninjured portions of the glomerular capillaries to resume the production of urinary filtrate, which washed some or all of the erythrocytes out of Bowman's space and cleared blood cells from some nephrons into urine within six hours. However, many of the injured nephrons remained plugged with tightly packed red cell casts 24 h after imaging and also showed degeneration of tubular epithelium, indicative of acute tubular necrosis. The additional damage caused by the extravasated blood amplified that caused by the original cavitating gas body. Human nephrons are virtually identical to those of the rat and so it is probable that similar glomerular capillary rupture followed by transient blockage and/or epithelial degeneration will occur after clinical exposures using similar high MI intermittent imaging with gas body contrast agents. The detection of blood in postimaging urine samples using standard hematuria tests would confirm whether or not clinical protocols need to be developed to avoid this potential for iatrogenic injury.

    Title The Spectrum of Podocytopathies: a Unifying View of Glomerular Diseases.
    Date July 2007
    Journal Kidney International
    Excerpt

    Glomerular diseases encompass a broad array of clinicopathologically defined syndromes which together account for 90% of end-stage kidney disease costing $20 billion per annum to treat in the United States alone. Recent insights have defined the central role of the podocyte as both the regulator of glomerular development as well as the determinant of progression to glomerulosclerosis. We can now place all glomerular diseases within this spectrum of podocytopathies with predictable outcomes based on podocyte biology impacted by temporal, genetic, and environmental cues. This simplified construct is particularly useful to rationalize clinical effort toward podocyte preservation and prevention of progression as well as to focus basic research effort on understanding podocyte biology and for clinical research toward development of practical monitoring strategies for podocyte injury, dysfunction, and loss.

    Title An in Vivo Rat Model Simulating Imaging of Human Kidney by Diagnostic Ultrasound with Gas-body Contrast Agent.
    Date June 2007
    Journal Ultrasound in Medicine & Biology
    Excerpt

    One kidney of anesthetized rats was imaged by diagnostic ultrasound with contrast agent under conditions simulating both the geometry and the attenuation encountered during human perfusion imaging. Contrary to earlier predictions, glomerular capillary rupture with blood loss into Bowman's space and proximal tubules occurred in our clinically relevant model system. Quantitative analysis of histologic sections showed that 37 +/- 5% of the glomeruli at the center of the scan plane had blood cells in Bowman's space after imaging for 1 min with 1.8 MPa (mechanical index equivalent, MIe = 1.5) with a 1 s image trigger interval during IV injection of 10 microl/kg/min of Definity contrast agent (as recommended by the manufacturer). This percentage decreased rapidly with decreasing peak rarefactional pressure amplitude to an apparent threshold of 0.73 MPa (MIe = 0.6). The percentage of glomeruli with hemorrhage decreased in proportion to dose when reduced below the recommended value, but leveled-off at doses above it. The percentage of glomerular hemorrhage increased with increasing numbers of image exposures, with an initial rate of 1.1% per image. The glomerular hemorrhage also depended on the frame trigger interval with no hemorrhage evident for continuous imaging but a maximal effect for trigger intervals greater than about 1 s. These results indicated that there is a potential for clinical diagnostic ultrasound with contrast agent to induce glomerular hemorrhage.

    Title Positional Cloning Uncovers Mutations in Plce1 Responsible for a Nephrotic Syndrome Variant That May Be Reversible.
    Date February 2007
    Journal Nature Genetics
    Excerpt

    Nephrotic syndrome, a malfunction of the kidney glomerular filter, leads to proteinuria, edema and, in steroid-resistant nephrotic syndrome, end-stage kidney disease. Using positional cloning, we identified mutations in the phospholipase C epsilon gene (PLCE1) as causing early-onset nephrotic syndrome with end-stage kidney disease. Kidney histology of affected individuals showed diffuse mesangial sclerosis (DMS). Using immunofluorescence, we found PLCepsilon1 expression in developing and mature glomerular podocytes and showed that DMS represents an arrest of normal glomerular development. We identified IQ motif-containing GTPase-activating protein 1 as a new interaction partner of PLCepsilon1. Two siblings with a missense mutation in an exon encoding the PLCepsilon1 catalytic domain showed histology characteristic of focal segmental glomerulosclerosis. Notably, two other affected individuals responded to therapy, making this the first report of a molecular cause of nephrotic syndrome that may resolve after therapy. These findings, together with the zebrafish model of human nephrotic syndrome generated by plce1 knockdown, open new inroads into pathophysiology and treatment mechanisms of nephrotic syndrome.

    Title Antioxidant Ceruloplasmin is Expressed by Glomerular Parietal Epithelial Cells and Secreted into Urine in Association with Glomerular Aging and High-calorie Diet.
    Date November 2006
    Journal Journal of the American Society of Nephrology : Jasn
    Excerpt

    Biologic aging is accelerated by high-calorie intake, increased free radical production, and oxidation of key biomolecules. Fischer 344 rats that are maintained on an ad libitum diet develop oxidant injury and age-associated glomerulosclerosis by 24 mo. Calorie restriction prevents both oxidant injury and glomerulosclerosis. Ceruloplasmin (Cp) is a copper-containing ferroxidase that functions as an antioxidant in part by oxidizing toxic ferrous iron to nontoxic ferric iron. Glomerular Cp mRNA and protein expression were measured in ad libitum-fed and calorie-restricted rats at ages 2, 6, 17, and 24 mo. In ad libitum-fed rats, Cp mRNA expression increased six-fold (P < 0.01) and protein expression increased five-fold (P = 0.01) between 2 and 24 mo of age. In calorie-restricted rats, Cp mRNA expression increased three-fold (P < 0.01) and protein expression increased 1.6-fold (NS) between 2 and 24 mo of age. Both the cell-associated alternately spliced variant and secreted variants of Cp were expressed. Immunofluorescent analysis showed that Cp was expressed by the parietal epithelial cells that line the inner aspect of Bowman's capsule in the glomerulus. Cp also was present in urine, particularly of old ad libitum-fed rats with high tissue Cp expression. Cp expression by Bowman's capsule epithelial cells therefore occurred in direct proportion to known levels of oxidant activity (older age and high-calorie diet) and is secreted into the urine. It is suggested that Cp expression at this site may be part of the repertoire of the glomerular parietal epithelial cell to protect the glomerular podocytes and the downstream nephron from toxic effects of filtered molecules, including ferrous iron.

    Title Recessive Missense Mutations in Lamb2 Expand the Clinical Spectrum of Lamb2-associated Disorders.
    Date October 2006
    Journal Kidney International
    Excerpt

    Congenital nephrotic syndrome is clinically and genetically heterogeneous. The majority of cases can be attributed to mutations in the genes NPHS1, NPHS2, and WT1. By homozygosity mapping in a consanguineous family with isolated congenital nephrotic syndrome, we identified a potential candidate region on chromosome 3p. The LAMB2 gene, which was recently reported as mutated in Pierson syndrome (microcoria-congenital nephrosis syndrome; OMIM #609049), was located in the linkage interval. Sequencing of all coding exons of LAMB2 revealed a novel homozygous missense mutation (R246Q) in both affected children. A different mutation at this codon (R246W), which is highly conserved through evolution, has recently been reported as causing Pierson syndrome. Subsequent LAMB2 mutational screening in six additional families with congenital nephrotic syndrome revealed compound heterozygosity for two novel missense mutations in one family with additional nonspecific ocular anomalies. These findings demonstrate that the spectrum of LAMB2-associated disorders is broader than previously anticipated and includes congenital nephrotic syndrome without eye anomalies or with minor ocular changes different from those observed in Pierson syndrome. This phenotypic variability likely reflects specific genotypes. We conclude that mutational analysis in LAMB2 should be considered in congenital nephrotic syndrome, if no mutations are found in NPHS1, NPHS2, or WT1.

    Title Quantifying Leukocytes in First Catch Urine Provides New Insights into Our Understanding of Symptomatic and Asymptomatic Urethritis.
    Date August 2006
    Journal International Journal of Std & Aids
    Excerpt

    We quantitatively investigated inflammatory cells in the male urethra. Leukocytes in the first catch urine (FCU) from 87 men with and without urethritis were quantitated using haemocytometer counts and stained with an anti-CD45 pan-leukocyte antibody. An increased number of leukocytes in FCU specimens was associated with urethritis (P > 0.002), the presence of discharge and/or dysuria (P < 0.001), and detection of Chlamydia trachomatis (P < 0.001) and Neisseria gonorrhoeae (P < 0.001). In men with urethritis, higher leukocyte counts were also observed in the above groups (P = 0.07, 0.03 and P < 0.0001, respectively). As leukocyte number increased, the likelihood of detecting either pathogen increased. This study suggests that symptoms and signs are a surrogate marker for the degree of inflammation present, and that as urethral inflammation increases, the likelihood of detecting a sexually transmitted pathogen also increases. This would explain why men with asymptomatic urethritis are less likely to have a sexually transmitted infection detected than those with discharge and/or dysuria.

    Title Podocyte Depletion Causes Glomerulosclerosis: Diphtheria Toxin-induced Podocyte Depletion in Rats Expressing Human Diphtheria Toxin Receptor Transgene.
    Date March 2006
    Journal Journal of the American Society of Nephrology : Jasn
    Excerpt

    Glomerular injury and proteinuria in diabetes (types 1 and 2) and IgA nephropathy is related to the degree of podocyte depletion in humans. For determining the causal relationship between podocyte depletion and glomerulosclerosis, a transgenic rat strain in which the human diphtheria toxin receptor is specifically expressed in podocytes was developed. The rodent homologue does not act as a diphtheria toxin (DT) receptor, thereby making rodents resistant to DT. Injection of DT into transgenic rats but not wild-type rats resulted in dose-dependent podocyte depletion from glomeruli. Three stages of glomerular injury caused by podocyte depletion were identified: Stage 1, 0 to 20% depletion showed mesangial expansion, transient proteinuria and normal renal function; stage 2, 21 to 40% depletion showed mesangial expansion, capsular adhesions (synechiae), focal segmental glomerulosclerosis, mild persistent proteinuria, and normal renal function; and stage 3, >40% podocyte depletion showed segmental to global glomerulosclerosis with sustained high-grade proteinuria and reduced renal function. These pathophysiologic consequences of podocyte depletion parallel similar degrees of podocyte depletion, glomerulosclerosis, and proteinuria seen in diabetic glomerulosclerosis. This model system provides strong support for the concept that podocyte depletion could be a major mechanism driving glomerulosclerosis and progressive loss of renal function in human glomerular diseases.

    Title Podocyte Hypertrophy, "adaptation," and "decompensation" Associated with Glomerular Enlargement and Glomerulosclerosis in the Aging Rat: Prevention by Calorie Restriction.
    Date March 2006
    Journal Journal of the American Society of Nephrology : Jasn
    Excerpt

    Whether podocyte depletion could cause the glomerulosclerosis of aging in Fischer 344 rats at ages 2, 6, 17, and 24 mo was evaluated. Ad libitum-fed rats developed proteinuria and glomerulosclerosis by 24 mo, whereas calorie-restricted rats did not. No evidence of age-associated progressive linear loss of podocytes from glomeruli was found. Rather, ad libitum-fed rats developed glomerular enlargement over time. To accommodate the increased glomerular volume, podocytes principally underwent hypertrophy, whereas other glomerular cells underwent hyperplasia. Stages of hypertrophy through which podocytes pass en route to podocyte loss and glomerulosclerosis were identified: Stage 1, normal podocyte; stage 2, nonstressed podocyte hypertrophy; stage 3, "adaptive" podocyte hypertrophy manifest by changes in synthesis of structural components (e.g., desmin) but maintenance of normal function; stage 4, "decompensated" podocyte hypertrophy relative to total glomerular volume manifest by reduced production of key machinery necessary for normal podocyte function (e.g., Wilms' tumor 1 protein [WT1], transcription factor pod1, nephrin, glomerular epithelial protein 1, podocalyxin, vascular endothelial growth factor, and alpha5 type IV collagen) and associated with widened foot processes and decreased filter efficiency (proteinuria); and stage 5, podocyte numbers decrease in association with focal segmental glomerulosclerosis. In contrast, in calorie-restricted rats, glomerular enlargement was minor, significant podocyte hypertrophy did not occur, podocyte machinery was unchanged, there was no proteinuria, and glomerulosclerosis did not develop. Glomerular enlargement therefore was associated with podocyte hypertrophy rather than hyperplasia. Hypertrophy above a certain threshold was associated with podocyte stress and then failure, culminating in reduced podocyte numbers in sclerotic glomeruli. This process could be prevented by calorie restriction.

    Title Chronic Halothane Modification of Eeg-like Activity Recorded from Somatosensory Cortex and Deep Nuclei in Freely Behaving Rats.
    Date January 2005
    Journal Neurotoxicology
    Excerpt

    Chronic exposure to the anesthetic agent halothane has been implicated in morphological and biochemical alterations of central nervous system tissue. In the present experiments, analysis of electroencephalographic (EEG) recordings has been used to examine effects on brain electrical activity. EEGs were recorded from freely behaving rats with stereotaxically implanted permanent semimicroelectrodes. Recordings were taken from the somatosensory cortex (SC), nucleus parafasciculus thalami (PF), mesencephalic central gray (CG), and the ventromedial hypothalamus (VMH) before (control) and after 28 and 56 days of chronic intermittent halothane administration (0.5%, 3 hr/day, 5 days/week). On each recording day (0, 28 and 56), EEGs were obtained prior to halothane exposure and following exposure to 0.25%, 0.5% and 1.5% halothane. In halothane-naive rats (day 0), the EEG dominant frequency (DF) showed a dose-response pattern consisting of an initial increase with 0.25% (significant only for the PF) followed by suppression at 0.5% and a marked significant decrease in all regions at 1.5%. On day 28, the pre-drug DF recorded from three of four regions showed a slowing trend. Additionally, with 1.5% halothane, only the SC DF was significantly decreased. Following 56 days of intermittent exposure, the pre-drug EEG frequencies were significantly decreased in all regions as compared to naive values. Subsequent administration of 0.25% halothane produced a significant increase in all regional DFs which was also obtained with 0.5% and with 1.5% for the CG and VMH. The high DF values from the PF, CG and VMH at 0.5% and from the CG and VMH at 1.5% represent statistically significant increases over naive 1.5% values. Chronic halothane exposure is thus shown to progressively alter EEG activity and the EEG pattern of dose-responsiveness in four brain regions.

    Title Ethanol-induced in Vitro Invasion of Breast Cancer Cells: the Contribution of Mmp-2 by Fibroblasts.
    Date November 2004
    Journal International Journal of Cancer. Journal International Du Cancer
    Excerpt

    Ethanol is a tumor promoter and may promote metastasis of breast cancer. However, the underlying cellular/molecular mechanisms remain unknown. Overexpression and high activity of matrix metalloproteinase-2 (MMP-2) are frequently associated with metastatic breast cancers and serve as a prognostic indicator of clinical outcome. MMP-2 is predominantly expressed in stromal fibroblasts and plays a pivotal role in regulating the invasive behavior of breast tumor cells. We hypothesized that ethanol may enhance the invasion of breast tumor cells by modulating the activity of fibroblastic MMP-2. With in vitro models (HS68 and CCD1056SK human fibroblasts), we showed that ethanol at physiologically relevant concentrations (50-200 mg/dl) activated MMP-2; conversely, at a higher concentration (400 mg/dl), it inhibited the MMP-2 activity. Consistently, conditioned medium collected from ethanol (50-200 mg/dl)-exposed fibroblasts markedly enhanced the invasive potential of breast cancer cells and mammary epithelial cells overexpressing ErbB2/HER2 (BT474, SKBR-3 and HB2(ErbB2) cells) but had little effect on cells with low ErbB2 levels (BT20 and HB2 cells). In contrast, conditioned medium obtained from ethanol (400 mg/dl)-treated fibroblasts inhibited cell invasion. Selective inhibitors of MMP-2 (SB-3CT and OA-Hy) eliminated ethanol-stimulated invasion, indicating that the effect of ethanol was mediated by MMP-2. Ethanol activated conventional PKCs and JNKs in fibroblasts; inhibitors of PKC (Go6850 and Go6976) and JNKs (SP600125) significantly inhibited ethanol-mediated MMP-2 activation as well as cell invasion, indicating that PKCs and JNKs play a role in ethanol-induced MMP-2 activation and cell invasion in vitro. Thus, ethanol-promoted breast cancer cell invasion may be mediated by the modulation of fibroblastic MMP-2.

    Title Evaluation of a Thick and Thin Section Method for Estimation of Podocyte Number, Glomerular Volume, and Glomerular Volume Per Podocyte in Rat Kidney with Wilms' Tumor-1 Protein Used As a Podocyte Nuclear Marker.
    Date September 2004
    Journal Journal of the American Society of Nephrology : Jasn
    Excerpt

    Podocyte loss and glomerular hypertrophy are associated with development of glomerulosclerosis, suggesting that there may be a maximal area for each podocyte in terms of its capacity to support and maintain the glomerular filter. This study hypothesized that exceeding this maximal threshold will result in mesangial expansion and glomerulosclerosis. It may therefore be useful to measure podocyte number, glomerular volume, and glomerular volume per podocyte in clinical biopsy samples. An approach that uses thick and thin histologic sections cut from paraffin-embedded tissue to measure Wilms' tumor-1 protein-positive podocyte nuclear number and glomerular tuft area was studied. A rat model of aging has been used to track changes in glomerular podocyte number, glomerular volume per podocyte, and glomerular volume. Implications for clinical use of these variables are discussed.

    Title Feasibility of Applying Ultrasound Strain Imaging to Detect Renal Transplant Chronic Allograft Nephropathy.
    Date September 2004
    Journal Kidney International
    Excerpt

    Chronic renal transplantation fibrosis, often termed Chronic Allograft Nephropathy, may progress undetected. Since renal fibrosis may be accompanied by a change in measurable elastic tissue properties, ultrasound strain management may be useful in it detection. Ultrasound strain imaging was performed for two subjects with renal transplants; one with normal renal function and one with mild renal insufficiency and biopsy demonstrated fibrosis. Subjects underwent ultrasound examination with application of a controlled deformation using phase-sensitive, two-dimensional speckle tracking to evaluate internal tissue motion to measure tissue displacement and strain. Measurements over multiple beams for an equivalent deformational stress showed there was a threefold difference in renal cortical strain between the two subjects. These data suggest that ultrasound elasticity imaging may prove useful in measuring mechanical changes related to fibrosis with the transplant kidney.

    Title Podocyte-specific Expression of Cre Recombinase in Transgenic Mice.
    Date August 2003
    Journal Genesis (new York, N.y. : 2000)
    Excerpt

    We report a transgenic mouse line that expresses Cre recombinase exclusively in podocytes. Twenty- four transgenic founders were generated in which Cre recombinase was placed under the regulation of a 2.5-kb fragment of the human NPHS2 promoter. Previously, this fragment was shown to drive beta-galactosidase (beta-gal) expression exclusively in podocytes of transgenic mice. For analysis, founder mice were bred with ROSA26 mice, a reporter line that expresses beta-gal in cells that undergo Cre recombination. Eight of 24 founder lines were found to express beta-gal exclusively in the kidney. Histological analysis of the kidneys showed that beta-gal expression was confined to podocytes. Cre recombination occurred during the capillary loop stage in glomerular development. No evidence for Cre recombination was detected in any of 14 other tissues examined.

    Title Fyn Binds to and Phosphorylates the Kidney Slit Diaphragm Component Nephrin.
    Date July 2003
    Journal The Journal of Biological Chemistry
    Excerpt

    Recent investigations have focused on characterizing the molecular components of the podocyte intercellular junction, because several of these components, including Nephrin, are functionally necessary for development of normal podocyte structure and filter integrity. Accumulating evidence suggests that the Nephrin-associated protein complex is a signaling nexus. As such, Nephrin-dependent signaling might be mediated in part through Nephrin phosphorylation. Described are biochemical and mouse genetics experiments demonstrating that membrane-associated Nephrin is tyrosine-phosphorylated by the Src family kinase Fyn. Nephrin fractionated in detergent-resistant glomerular membrane fractions with Fyn and Yes. Fyn directly bound Nephrin via its SH3 domain, and Fyn directly phosphorylated Nephrin. Glomeruli in which Fyn, Yes, or Fyn and Yes were genetically deleted in mice were characterized to explore the relationship between these kinases and Nephrin. Fyn deletion resulted in coarsening of podocyte foot processes and marked attenuation of Nephrin phosphorylation in isolated glomerular detergent-resistant membrane fractions. Yes deletion had no identifiable effect on podocyte morphology but dramatically increased Nephrin phosphorylating activity. Similar to Fyn deletion, simultaneous deletion of Fyn and Yes reduced Nephrin phosphorylating activity. These results demonstrate that endogenous Fyn catalyzes Nephrin phosphorylation in podocyte detergent-resistant membrane fractions. Although Yes appears to effect the regulation of Nephrin phosphorylation, the mechanism by which this occurs requires investigation.

    Title Two Gene Fragments That Direct Podocyte-specific Expression in Transgenic Mice.
    Date November 2002
    Journal Journal of the American Society of Nephrology : Jasn
    Excerpt

    Transgenic manipulation of the glomerular visceral epithelial cell offers a powerful approach for studying the biology of this morphologically complex cell type. It has been previously demonstrated that an 8.3-kb and a 5.4-kb fragment of the murine Nphs1 (nephrin) promoter-enhancer drives lacZ expression in podocytes, brain, and pancreas of transgenic mice, recapitulating the expression pattern of the endogenous nephrin gene. In this present study, two truly podocyte-specific promoters were identified that drive transgene expression in podocytes without expression in extrarenal tissues in adult or embryonic mice. A 1.25-kb fragment driving a lacZ reporter gene (p1.25N-nlacF) was derived from murine Nphs1 promoter similar to a human NPHS1 promoter fragment previously reported. Transgenic mice were generated and beta-galactosidase (beta-gal) expression was analyzed. Four of twelve founder mice were found to express beta-gal in podocytes (33% penetrance). Expression in brain and pancreas was absent in all animals, suggesting that nephrin expression in these organs might be driven by distinct cis-regulatory elements that can be removed to obtain podocyte-specific expression. A 2.5-kb fragment derived from the human NPHS2 (podocin) gene was designed in a similar fashion to drive lacZ expression in transgenic mice (p2.5P-nlacF). Twelve of twlve NPHS2 mouse founder lines expressed beta-gal exclusively in podocytes (100% penetrance). Beta-gal activity was not observed extrinsic to the kidney in p1.25N-nlacF or p2.5P-nlacF mouse embryos at gestational time points between 8.5 d post coitus and birth. In conclusion, the 2.5-kb NPHS2 promoter fragment may be useful for podocyte-specific transgenic expression when extrarenal expression of a transgene is problematic.

    Title Lack of Correlation Between Facility-based Standardized Rates of Transplantation and Mortality.
    Date August 2002
    Journal American Journal of Kidney Diseases : the Official Journal of the National Kidney Foundation
    Excerpt

    The standardized mortality ratio (SMR) has been used to provide information about adjusted survival outcomes at dialysis facilities. There has been concern that high rates of transplantation could unjustly lead to unfavorable SMR profiles for individual dialysis units because healthier patients would be removed from dialysis therapy, leaving less healthy patients in the dialysis pool. We correlated 1999 overall adjusted SMR and 1999 standardized transplantation ratio (STR) weighted for mortality patient count and count of first transplantations of patients younger than 65 years. A total of 2,362 facilities were included in analyses. We found no correlation between rates of transplantation (by STR) and overall mortality profile (by SMR) based on Pearson's correlation coefficients (r), either unweighted, weighted by number of patients included in the 1999 mortality calculation (SMR), or weighted by number of patients included in the 1999 transplantation calculation (r = -0.016, r = -0.015, and r = -0.015, respectively; P > 0.40 for each). Sensitivity analyses using SMR and STR over 3- and 3.5-year periods (January 1997 to June 2000) also showed no correlation between SMR and STR, respectively. We conclude that reported standardized rates for transplantation do not correlate with those reported for mortality by dialysis facilities.

    Title Reconstructive Ultrasound Elasticity Imaging for Renal Transplant Diagnosis: Kidney Ex Vivo Results.
    Date September 2001
    Journal Ultrasonic Imaging
    Excerpt

    It may be possible to diagnose and monitor scarring, inflammation and edema in transplant kidney using reconstructive ultrasound elasticity imaging. Kidney elasticity is expected to change dramatically with scar, and to a lesser degree, with acute inflammation and edema. The hypothesis that changes in kidney elasticity can be imaged using a clinical ultrasound scanner was experimentally tested with an ex vivo canine kidney model, and results on a single pair of kidneys are reported in this paper. A cross-linking agent affected kidney elasticity both globally and locally. Elasticity changes were monitored with accurate estimates of internal displacement and strain followed by Young's modulus reconstruction. The results of this study strongly suggest that ultrasound elasticity imaging can detect elasticity changes in complex structures such as the kidney. Moreover, it has the potential to become an important clinical tool for renal transplant diagnosis.

    Title Up-regulation of Transcription of Smooth Muscle Myosin Alkali Light Chain by Ethanol in Human Breast Cancer Cells.
    Date July 2001
    Journal International Journal of Oncology
    Excerpt

    Both epidemiological and experimental studies indicate that ethanol is a tumor promoter and that chronic ethanol exposure enhances metastasis of breast cancer cells, and with an in vitro model (T47D human breast cancer cells), we have previously demonstrated that ethanol exposure stimulated the migration of breast cancer cells. In the present study, differential display reverse transcription polymerase chain reaction was used to identify ethanol-responsive genes in T47D cells. Three differentially displayed, ethanol-responsive gene fragments were identified, and their expression was confirmed by Northern blot hybridization. Sequence analysis revealed that one cDNA fragment represented the myosin alkali light chain (MLC 1sm) of human smooth muscle. The expression of MLC 1sm was found to be significantly higher in breast cancer cells than in normal mammary epithelial cells. With T47D cells, ethanol induced an additional duration- and concentration-dependent up-regulation of MLC 1sm. At 400 mg/dl, an ethanol-mediated increase was evident at 6 h (55% increase), peaked at 24 h (2.7-fold increase) following exposure, and diminished thereafter. At pharmacologically relevant concentrations (e.g., 100 mg/dl), ethanol produced a significant increase of MLC 1sm expression, and progressively higher ethanol concentrations resulted in more up-regulation. The half-life of MLC 1sm mRNA was not altered, however, the transcription rate of MLC 1sm was significantly increased by ethanol. MLC is a structural component of the cytoskeleton of eukaryotic cells, and it plays critical roles in the regulation of cell shaping, movement, and growth. Thus, ethanol-mediated up-regulation of MLC may be an underlying molecular mechanism for its tumor promoting effect.

    Title The Membrane-associated Guanylate Kinase Protein Magi-1 Binds Megalin and is Present in Glomerular Podocytes.
    Date July 2001
    Journal Journal of the American Society of Nephrology : Jasn
    Excerpt

    The transmembrane endocytic receptor glycoprotein 330/megalin (hereafter referred to as megalin) is localized to the apical membrane domain of epithelial cells, where it is involved in the uptake of proteins from extracellular sources. The cytoplasmic domain of megalin contains amino acid motifs that have the potential to bind to other proteins, which may influence its localization or function. The yeast two-hybrid system was used to search for proteins that bind to the cytoplasmic tail of megalin, and a protein fragment from a mouse embryonic cDNA library that contained a single PDZ domain was identified. This protein, which was named glycoprotein 330-associated protein (GASP), appears to be a truncated mouse counterpart of the human and rat proteins atrophin-1-interacting protein-1 and synaptic scaffolding molecule, respectively. The interaction of GASP with megalin is mediated by the PDZ domain of GASP binding to the DSDV motif found at the carboxyl-terminus of megalin. A mutant version of megalin that lacks the terminal valine is unable to bind to GASP, illustrating the PDZ domain-dependent interaction between these two proteins. A close homolog of GASP, i.e., membrane-associated guanylate kinase with inverted orientation-1 (MAGI-1), is more ubiquitous in its tissue distribution (including kidney) and is also able to specifically bind to megalin via its fifth PDZ domain. Immunofluorescence studies of adult kidney revealed that MAGI-1 is expressed in the glomerulus of the kidney, in a manner that parallels the expression of the podocyte-specific protein glomerular epithelial protein 1. Western analysis of endogenous MAGI-1 from glomerular preparations suggests that it is associated with the cytoskeleton and seems to be expressed in a different form, compared with cell line-derived endogenous MAGI-1. The association of megalin with MAGI-1 may allow the assembly of a multiprotein complex, in which megalin may serve a nonendocytic function in glomerular podocytes.

    Title Altered Podocyte Structure in Glepp1 (ptpro)-deficient Mice Associated with Hypertension and Low Glomerular Filtration Rate.
    Date January 2001
    Journal The Journal of Clinical Investigation
    Excerpt

    Glomerular epithelial protein 1 (GLEPP1) is a receptor tyrosine phosphatase present on the apical cell surface of the glomerular podocyte. The GLEPP1 gene (PTPRO:) was disrupted at an exon coding for the NH(2)-terminal region by gene targeting in embryonic stem cells. Heterozygote mating produced the expected genotypic ratio of 1:2:1, indicating that the Ptpro(-/-) genotype does not lead to embryonic or neonatal lethality. Kidney and glomerular structure was normal at the gross and light microscopic levels. Scanning and transmission electron microscopy showed that Ptpro(-/-) mice had an amoeboid rather than the typical octopoid structure seen in the wild-type mouse podocyte and that there were blunting and widening of the minor (foot) processes in association with altered distribution of the podocyte intermediate cytoskeletal protein vimentin. Reduced filtration surface area in association with these structural changes was confirmed by finding reduced glomerular nephrin content and reduced glomerular filtration rate in Ptpro(-/-) mice. There was no detectable increase in the urine albumin excretion of Ptpro(-/-) mice. After removal of one or more kidneys, Ptpro(-/-) mice had higher blood pressure than did their wild-type littermates. These data support the conclusion that the GLEPP1 (Ptpro) receptor plays a role in regulating the glomerular pressure/filtration rate relationship through an effect on podocyte structure and function.

    Title Hydrogen Peroxide Induces Rapid Digestion of Oligodendrocyte Chromatin into High Molecular Weight Fragments.
    Date November 2000
    Journal Neurochemistry International
    Excerpt

    High molecular weight (HMW) fragmentation of nuclear chromatin was studied in cultured rat oligodendrocytes (OL) exposed to hydrogen peroxide (H2O2). Intact genomic DNA was isolated by agarose embedding, and analyzed by field inversion gel electrophoresis, with and without S1 endonuclease digestion to detect and discriminate between single and double stranded fragmentations, respectively. The exposure of OL to H2O2 resulted in a very rapid degradation of chromosomal DNA into HMW fragments that reflect native chromatin structure. Hence, within 10 min after the addition of 1 mM H2O2, a discrete pool representing approximately 45% of the nuclear chromatin underwent single strand digestion into >400 kb fragments likely at AT-rich matrix attachment regions. Subsequent accumulation of single stand breaks at these regions led to bifilar scission. Ultimately, chromatin within this susceptible pool was cleaved at remaining matrix attachment regions into 50-200 kb fragments. Chromatin digestion could be elicited with H2O2 concentrations as low as 50 microM. After the removal of H2O2, most >400 kb fragments were religated within 2 h; however, digestion into 50-200 kb fragments was irreversible. The DNA digestion was not accompanied by the degradation of nuclear proteins, i.e., lamins A/C and poly (ADP-ribose) polymerase indicating that chromatin fragmentation is unlikely to be mediated by proteolysis. In conclusion, H2O2 at pathologically relevant concentrations induces a very rapid and extensive digestion of OL chromatin into HMW fragments. Because the chromatin fragmentation is only partly reversible, it may be a decisive factor in committing oxidatively stressed OL to degeneration and/or death.

    Title Molecular Cloning, Expression, and Distribution of Glomerular Epithelial Protein 1 in Developing Mouse Kidney.
    Date May 2000
    Journal Kidney International
    Excerpt

    BACKGROUND: Glomerular epithelial protein 1 (GLEPP1) is a receptor-like membrane protein tyrosine phosphatase (RPTP) with a large ectodomain consisting of multiple fibronectin type III repeats, a single transmembrane segment, and a single cytoplasmic phosphatase active site sequence. In adult human and rabbit kidneys, GLEPP1 is found exclusively on apical membranes of podocytes and especially on surfaces of foot processes. Although neither ligand nor function for this protein is known, other RPTPs with similar topologies have been implicated in mediating adherence behavior of cells. METHODS: To evaluate potential roles of GLEPP1 further, we cloned the full-length mouse GLEPP1 cDNA and examined its expression patterns in developing kidney by Northern blot analysis, in situ hybridization, and immunofluorescence microscopy. RESULTS: Nucleotide sequencing showed that mouse GLEPP1 was approximately 80% identical to rabbit and human GLEPP1 and approximately 91% identical at the amino acid level. The membrane-spanning and phosphatase domains of mouse GLEPP1 shared> 99% homology with PTPphi, a murine macrophage cytoplasmic phosphatase. Northern analysis identified a single GLEPP1 transcript of approximately 5.5 kb in fetal kidney that became approximately threefold more abundant in adults. In situ hybridization of newborn mouse kidney revealed GLEPP1 mRNA in visceral epithelial cells (developing podocytes) of comma- and S-shaped nephric figures, and expression increased in capillary loop and maturing stage glomeruli. Beginning on embryonic day 14, GLEPP1 protein was first observed on cuboidal podocytes of capillary loop stage glomeruli, but nascent podocytes of earlier comma- and S-shaped nephric figures were negative. At later stages of glomerular maturation, where foot process elongation and interdigitation occurs, GLEPP1 immunolabeling intensified on podocytes and then persisted at high levels in fully developed glomeruli. CONCLUSION: Our findings are consistent with a role for GLEPP1 in mediating and maintaining podocyte differentiation specifically.

    Title Differentiation Dependent Activation of the Myelin Genes in Purified Oligodendrocytes is Highly Resistant to Hypoglycemia.
    Date January 2000
    Journal Metabolic Brain Disease
    Excerpt

    We have previously demonstrated that the developmental upregulation of myelin-specific genes in mixed glial cultures is strongly attenuated by hypoglycemia. The present study was designed to evaluate the effect of hypoglycemia on differentiation-dependent upregulation of myelin genes in purified oligodendrocyte cultures. The expression of major myelin protein genes, i.e., proteolipid protein (PLP), basic protein (BP) and myelin associated glycoprotein (MAG) were monitored by Northern blot analysis. In control cultures maintained at 6 mg/ml of glucose, the expression of all the genes upregulated rapidly, and plateaued at approximately day 4. A similar pattern of differentiation-dependent upregulation was observed for the gene encoding a lipogenic enzyme, i.e., malic enzyme (ME). In contrast to mixed glial cultures, however, this developmental gene upregulation was not significantly affected by severe hypoglycemia (approximately 0.02 mg/ml). The results indicate that the effect of glucose deprivation on oligodendrocyte genes observed in mixed glial cultures is mediated by other cells. The upregulation of the genes in differentiating oligodendrocytes was accompanied by the production of myelin-related membrane that was isolated by density gradient fractionation. In contrast to the effect on gene expression, this anabolic activity was highly dependent on glucose, as seen from a profound suppression by severe hypoglycemia.

    Title Antisense Oligodeoxynucleotides Targeted to Mag Mrna Profoundly Alter Bp and Plp Mrna Expression in Differentiating Oligodendrocytes: a Caution.
    Date January 2000
    Journal Metabolic Brain Disease
    Excerpt

    The applicability of antisense technology to suppress the expression of myelin associated glycoprotein (MAG) in cultured oligodendrocytes was evaluated. Differentiating oligodendrocyte precursor cells obtained by the shake-off method were exposed to nine unmodified antisense oligodeoxynucleotides (ODNs) targeted to the first seven exons of MAG mRNA. After four days, steady-state levels of MAG, proteolipid protein (PLP) and basic protein (BP) mRNAs were determined by Northern blot analysis. Only ODN annealing to 599-618 nt of the MAG mRNA (the junction of exon 5 and 6) resulted in a significant, 75% decrease in the MAG mRNA level. Unexpectedly, six other anti-MAG ODNs which had no significant effect on the MAG message, greatly increased the level of BP mRNA. The highest upregulation of approximately 12 fold was observed with ODN annealing to 139-168 nt (junction of exon 3 and 4). On the other hand, the 997-1016 ODN decreased the levels of BP and PLP messages by 70-80%. The 599-618 ODN also decreased the PLP mRNA by 85%. The results demonstrate that antisense ODNs targeted to one gene may profoundly alter the expression of other genes, and hence, complicate functional analysis of the targeted protein.

    Title Delayed Oligodendrocyte Degeneration Induced by Brief Exposure to Hydrogen Peroxide.
    Date May 1999
    Journal Journal of Neuroscience Research
    Excerpt

    An in vitro model system of cultured oligodendrocytes was used to determine the susceptibility of these cells to oxidative stress induced by 15 min exposure to millimolar concentrations of hydrogen peroxide (H2O2). Following the exposure, the cells were incubated in normal growth medium, and analyzed at different time points. Although no cell loss was observed during the exposure period, there was a progressive depletion of adherent cells during the postexposure period as seen from either the number of recoverable nuclei, or from total RNA content of the cultures. Both the rate and the extent of cell deletion was directly dependent on H2O2 concentration. Cell death was preceded by structural alterations in the nuclear envelope resulting in "fragile" nuclei which disintegrated during isolation. Northern blot analysis showed that the expression of myelin-specific genes was rapidly downregulated in H2O2-treated cells. On the other hand, the expression of antiapoptotic gene, bcl-2 featured massive but transient upregulation. Oligodendrocyte degeneration also featured genomic DNA degradation into high molecular weight fragments, which are likely to represent cleaved chromosomal loops. The results demonstrate vulnerability of oligodendrocytes to oxidative stress that induces rapid degeneration and ultimately leads to delayed cell death. This feature is highly relevant to oligodendrocyte damage and depletion following ischemic, traumatic, or inflammatory insults to the central nervous system (CNS).

    Title Structural Characterization of Myelin-associated Glycoprotein Gene Core Promoter.
    Date March 1998
    Journal Journal of Neuroscience Research
    Excerpt

    Myelin-associated glycoprotein (MAG) is emerging as an important molecule involved in the plasticity and regeneration of the central nervous system. In this study, the structure of MAG gene promoter was characterized in cultured rat oligodendrocyte lineage cells. Heterogeneous transcription initiation with five major and eight minor start sites scattered within 72 bp was shown by primer extension analysis. This TATA-less core promoter contains no prominent initiator (Inr) elements associated with the transcription initiation sites, and hence, appears to utilize novel positioning mechanisms. Genomic footprinting analysis revealed several putative protein-binding regions overlapping the initiation sites and containing a multitude of CG-rich sequences. However, no conspicuous alterations in the protein-binding pattern were evident between O2A progenitors in which the gene is inactive, and mature oligodendrocytes with fully upregulated gene. The core promoter DNA features a differentiation-dependent demethylation as shown by genomic sequencing analysis. Three of eight cytosines are totally demethylated in oligodendrocyte chromosomes, indicating that these unmodified bases may be critical for full activation of the promoter. The core promoter is located within an internucleosomal linker, and the upstream regulatory region appears to be organized into an array of nucleosomes with hypersensitive linkers.

    Title Renal Biopsy Collagen I Mrna Predicts Scarring in Rabbit Anti-gbm Disease: Comparison with Conventional Measures.
    Date January 1998
    Journal Kidney International
    Excerpt

    Progressive loss of normal structure associated with scarring is the hallmark of chronic diseases of most organs. To test the hypothesis that measurement of interstitial collagen mRNA levels would be a useful index to predict future scarring, we developed an assay to quantitate alpha 1(I) procollagen mRNA factored for GAPDH mRNA using RT-PCR (the "CI:G ratio"). We first defined conditions under which the assay could be used for analysis of renal biopsy samples. The CI:G ratio was then used to determine whether mRNA measurements performed at an early stage of inflammation (day 7) in a model of anti-GBM disease in the rabbit would predict outcome at day 30 as measured by interstitial and glomerular scarring and renal cortical hydroxyproline accumulation. The predictive value of this assay was compared to functional (serum creatinine and urine protein:creatinine ratio) and histologic (glomerular and interstitial scoring) parameters also measured at day 7. We found that the CI:G ratio alone provided a sensitive and discriminating assay over a wide range of renal injury that predicted various parameters of scarring with an average coefficient of determination (r2) of 0.69. This predictive power was higher than that found for conventional measures, which tended to have good discriminatory capacity over limited ranges of renal injury. The CI:G ratio provided significant additional predictive power over and above that available from combinations of conventional functional or histologic parameters. We conclude that measurement of the CI:G ratio in biopsy samples deserves further assessment as a potentially useful quantitative predictor of outcome that could lead to improved clinical decision-making.

    Title Assignment of the Human Podocalyxin-like Protein (podxl) Gene to 7q32-q33.
    Date November 1997
    Journal Genomics
    Title Molecular Cloning and Characterization of Human Podocalyxin-like Protein. Orthologous Relationship to Rabbit Pclp1 and Rat Podocalyxin.
    Date July 1997
    Journal The Journal of Biological Chemistry
    Excerpt

    Human renal cortex and heart cDNA libraries were screened for a human homolog of rabbit PCLP1 using the rabbit PCLP1 cDNA as a probe. Clones spanning 5869 base pairs with an open reading frame coding for a 528-amino acid peptide were obtained. The putative peptide contains a potential signal peptide and a single membrane-spanning region. The extracellular domain contains multiple potential sites for N- and O-linked glycosylation and 4 cysteines for potential disulfide bonding similar to rabbit PCLP1. On Northern blot a major transcript is seen at 5.9 kilobases. Antibodies to this protein show a doublet at 160/165 kDa on Western blots of human glomerular extract and a pattern of intense glomerular staining and vascular endothelial staining on immunofluorescence of human kidney sections. Comparison of the rabbit and human peptide sequences shows a high degree of identity in the transmembrane and intracellular domains (96%) with a lower degree of identity in the extracellular domain (36%). An antibody to the intracellular domain reacted across species (human, rabbit, and rat) and recognized both rabbit PCLP1 and rat podocalyxin. An interspecies Southern blot probed with a cDNA coding for the intracellular domain showed strong hybridization to all vertebrates tested in a pattern suggesting a single copy gene. We conclude that this cDNA and putative peptide represent the human homolog of rabbit PCLP1 and rat podocalyxin.

    Title Selenium is Required for Normal Upregulation of Myelin Genes in Differentiating Oligodendrocytes.
    Date June 1997
    Journal Journal of Neuroscience Research
    Excerpt

    The purpose of this study was to characterize the selenium requirement for the normal differentiation of oligodendrocyte lineage cells. In primary mixed glial cultures prepared from newborn rat brains, the overall growth of cultures, as seen from the total RNA yield, was not significantly affected by selenium. However, 30 nM selenium was required for the normal upregulation the proteolipid protein, basic protein, and myelin-associated glycoprotein gene expression assessed by Northern blot analysis. Selenium deprivation during initial, rapid phase of the gene upregulation irreversibly suppressed the genes, indicating the existence of a critical period in oligodendrocyte differentiation. In purified oligodendrocyte cultures prepared by mechanical dislodging of progenitor (O-2A) cells from mixed glial cultures, total cell number and total RNA yield were virtually unaffected by selenium deprivation; however, the developmental upregulation of the myelin genes was profoundly attenuated. Immunocytochemical analysis confirmed the suppressive effect of selenium deficiency on the differentiation of oligodendrocyte lineage cells, as seen from a significant decrease in the population of GalC+ and O4+ cells. Because the number of GC+ cells was more reduced than the number of O4+ cells, the results indicate that selenium deficiency may specifically inhibit the progression from immature to mature oligodendrocytes.

    Title Molecular Cloning, Expression, and Characterization of Podocalyxin-like Protein 1 from Rabbit As a Transmembrane Protein of Glomerular Podocytes and Vascular Endothelium.
    Date January 1996
    Journal The Journal of Biological Chemistry
    Excerpt

    Podocytes are responsible in part for maintaining the size and charge filtration characteristics of the glomerular filter. The major sialoprotein of the podocyte foot process glycocalyx is a 140-kDa sialoprotein named podocalyxin. Monoclonal antibodies raised against isolated rabbit glomeruli that recognized a podocalyxin-like protein base upon size, Alcian blue staining, wheat germ agglutinin binding, and distribution in renal cortex were used to expression clone cDNAs from a rabbit glomerular library. On Northern blot the cDNAs hybridized to a 5.5-kilobase pair transcript predominantly present in glomerulus. The overlapping cDNAs spanned 5,313 base pairs that contained an open reading frame of 1,653 base pairs and were not homologous with a previously described sequence. The deduced 551-amino acid protein contained a putative 21-residue N-terminal signal peptide and a 26-amino acid transmembrane region. The mature protein has a calculated molecular mass of 55 kDa, an extracellular domain that contains putative sites for N- and O-linked glycosylation, and a potential glycosaminoglycan attachment sites. The intracellular domain contains potential sites for phosphorylation. Processing of the full-length coding region in COS-7 cells resulted in a 140-kDa band, suggesting that the 55-kDa core protein undergoes extensive post-translational modification. The relationship between the cloned molecule and the monoclonal antibodies used for cloning was confirmed by making a fusion protein that inhibited binding of the monoclonal antibodies to renal cortical tissue sections and then raising polyclonal antibodies against the PCLP1 fusion protein that also recognized glomerular podocytes and endothelial cells in tissue sections in a similar distribution to the monoclonal antibodies. We conclude that we have cloned and sequenced a novel transmembrane core glycoprotein from rabbit glomerulus, which has many of the characteristics of podocalyxin. We have named this protein podocalyxin-like protein 1.

    Title Elasticity Imaging for Early Detection of Renal Pathology.
    Date January 1996
    Journal Ultrasound in Medicine & Biology
    Excerpt

    Early detection of renal pathology may be possible with elasticity imaging. This hypothesis was experimentally tested by quantitatively imaging internal mechanical strain due to surface deformations in an in vitro animal model of nephritis. Preliminary data support the hypothesis that kidney elasticity changes with renal damage and concomitant scarring before problems are detectable by traditional diagnostic techniques such as laboratory measurements of renal function.

    Title Fibronectin Mrna in the Developing Glomerular Crescent in Rabbit Antiglomerular Basement Membrane Disease.
    Date December 1995
    Journal Journal of the American Society of Nephrology : Jasn
    Excerpt

    Fibronectin is a multifunctional matrix protein important in wound healing that is markedly increased in glomerular crescents. A previous report established two phases of fibronectin metabolism in crescent formation in an anti-glomerular basement membrane model of crescentic nephritis in the rabbit. Phase I was associated with increased glomerular fibronectin content from plasma. Phase II was associated with increased fibronectin mRNA in glomeruli. To examine the hypothesis that fibronectin is synthesized in the developing crescent, rabbit fibronectin cDNA was cloned, sense and antisense riboprobes were prepared and their specificity under the conditions to be used was validated and in situ hybridization studies were performed in the model. The results showed that the cells in the developing glomerular crescent express an intense fibronectin mRNA signal at Day 7 and that this signal persisted in cells of the crescent at Day 14. This result shows that fibronectin synthesis does indeed take place in cells of the developing crescent in this model and supports the hypothesis that fibronectin may be an important agent regulating crescent formation and fibrosis.

    Title Molecular Cloning of Cdnas Encoding Human Glepp1, a Membrane Protein Tyrosine Phosphatase: Characterization of the Glepp1 Protein Distribution in Human Kidney and Assignment of the Glepp1 Gene to Human Chromosome 12p12-p13.
    Date October 1995
    Journal Genomics
    Excerpt

    Human glomerular epithelial protein 1 (GLEPP1), a receptor-like membrane protein tyrosine phosphatase (PTPase), was cloned and sequenced from a human renal cortical cDNA library. The human nucleotide and derived amino acid sequences were, respectively, 90 and 97% identical to those of rabbit. Human GLEPP1 is predicted to contain 1188 amino acids. The predicted mature protein is 1159 amino acids long and contains a large extracellular domain, a single transmembrane domain, and a single intracellular PTPase domain. Monoclonal and polyclonal antibodies raised against a human GLEPP1 fusion protein recognized a protein with distribution restricted to the glomerulus in human kidney and with an apparent molecular weight of approximately 200 kDa. The GLEPP1 gene was assigned to human chromosome 12p12-p13 by fluorescence in situ hybridization.

    Title Glioma-stimulated Chemoattraction of Endothelial Cells and Fibroblasts in Vitro: a Model for the Study of Glioma-induced Angiogenesis.
    Date April 1995
    Journal Metabolic Brain Disease
    Excerpt

    Induction of angiogenesis is essential for the continued growth of solid tumors, and one critical component of tumor-induced angiogenesis involves the stimulation of microvascular cells to migrate into the growing mass. We have developed a convenient model system utilizing dual co-culture chambers to study cellular chemotaxis induced by glioma cells in vitro. In this system, rat C6 glioma cells induced migration of fibroblasts and brain capillary endothelial cells. The migratory response was directly related to the number of C6 cells serving as stimulus in the lower chamber. Similar migratory responses were induced by C6 cell conditioned medium in a concentration dependent fashion. Medium conditioned by cultured human anaplastic astrocytoma cells was also found to contain potent chemotactic factor(s). This system may ultimately be employed in the identification of particular glioma cell population(s) and secreted factor(s) responsible for the chemoattraction of microvascular cells.

    Title Differentiation-specific Demethylation of Myelin Associated Glycoprotein Gene in Cultured Oligodendrocytes.
    Date March 1995
    Journal Journal of Neuroscience Research
    Excerpt

    The methylation status of a 4.4-kb 5' end of the myelin-associated glycoprotein (MAG) gene was assessed in cells with different levels of transcriptional activity of the gene, i.e., liver, brain, O-2A oligodendrocyte precursors cells, mature oligodendrocytes, and glioma C6 cells. Purified DNA was digested with methylation-sensitive restriction enzymes, and the cuts were mapped by the indirect end-labeling technique. The restriction sites within the 4.4-kb fragment revealed a highly heterogenous methylation pattern among cells and tissues, and liver DNA was the most heavily methylated. Most of the restriction sites were partly demethylated in the nervous system cells. Notably, two adjacent Hha1 sites at +94 and +96 were fully methylated in liver, but partially demethylated in the brain, OL, and O2A. Two Hpa2 site located at -1836 and at -39 were progressively demethylated in oligodendrocyte lineage cells, indicating specific hypomethylation associated with the oligodendrocytic differentiation. Most of the restriction sites were weakly methylated in the DNA from neoplastic C6 cells, although the Hha1 sites were fully methylated. No clear-cut correlation between the extent of CpG dinucleotide methylation and the chromatin conformation was found. For example, out of four heavily methylated sites only two comapped with MNase hypersensitive sites. Also, the -1836 Hpa2 site whose demethylation is concomitant with oligodendrocytic differentiation seems to be localized within precisely positioned nucleosomal arrays of the MAG gene chromatin. The results indicate that the MAG gene undergoes progressive demethylation concomitant with the oligodendrocyte differentiation/maturation. However, certain CpG dinucleotides remain heavily methylated even in the fully active gene in mature oligodendrocytes, indicating that they may be essential in maintaining proper chromatin structure.

    Title Evaluation of Quantitative Magnetic Resonance Imaging As a Noninvasive Technique for Measuring Renal Scarring in a Rabbit Model of Antiglomerular Basement Membrane Disease.
    Date October 1994
    Journal Journal of the American Society of Nephrology : Jasn
    Excerpt

    Renal function tests are an insensitive means of detecting progressive scarring of the kidney such as occurs in chronic allograft rejection and lupus nephritis. Alternative approaches for the measurement of small progressive changes in renal structure on a repetitive basis are needed. Quantitative magnetic resonance imaging (proton T1 relaxation time) was assessed for this purpose. A rabbit model of antiglomerular basement membrane disease that develops glomerular and interstitial inflammation followed by scarring of the renal cortex was used. Longitudinal studies of cortical T1 showed a marked prolongation of T1 during the initial inflammation and edema associated with glomerular crescent formation (Day 7). The T1 remained prolonged up to Day 120 in animals with significant fibrosis and crescent formation when the wet/dry weight ratio (a measure of edema) had returned to baseline. T1 was a more sensitive index of renal injury than was serum creatinine for all of the end points measured (cortical hydroxyproline per dry weight, percent crescent formation, or histologic fibrosis score). It was concluded from these studies that measurement of renal cortical T1 is quite a sensitive index of renal injury, probably more sensitive than the measurement of serum creatinine, but that this method does not discriminate between edema and scarring.

    Title Glepp1, a Renal Glomerular Epithelial Cell (podocyte) Membrane Protein-tyrosine Phosphatase. Identification, Molecular Cloning, and Characterization in Rabbit.
    Date September 1994
    Journal The Journal of Biological Chemistry
    Excerpt

    Podocytes are specialized epithelial cells with delicate interdigitating foot processes which cover the exterior basement membrane surface of the glomerular capillary. They are in part responsible for the extraordinary charge and size filtration characteristics of the glomerulus. To better understand disease processes affecting the glomerular filter, we searched for proteins with relative specificity to the podocyte using a monoclonal antibody strategy. The first such protein characterized (designated glomerular epithelial protein 1 (GLEPP1)) is a membrane protein-tyrosine phosphatase (PTPase) with a large extracellular domain containing eight fibronectin type III-like repeats, a hydrophobic transmembrane segment, and a single PTPase domain. The GLEPP1 PTPase domain shows homology with two other single domain transmembrane PTPases (PTP beta and Drosophila central nervous system PTP10D). This homology includes 2 cysteines in the PTPase domain not present in intracellular or tandem domain membrane PTPases. GLEPP1 PTPase protein is distributed to the podocyte foot processes themselves. RNase protection assay shows that GLEPP1 mRNA is also present in brain. By analogy with the CD45 PTPase of T cells, we expect that this receptor might play a role in maintaining foot process structure and/or function by regulating tyrosine phosphorylation of podocyte proteins.

    Title Transcriptional Regulation of Myelin Associated Glycoprotein Gene Expression by Cyclic Amp.
    Date August 1994
    Journal Journal of Neuroscience Research
    Excerpt

    The treatment of rat glioma C6 cells with 10 microM isoproterenol (Ipt) for 4 days upregulated the expression of the myelin-associated glycoprotein (MAG) gene by approximately 55-fold over the control value. The constant presence of Ipt in the medium was required for the maximal upregulation, as time-restricted exposures to the drug produced only partial, or no upregulation of the gene. No difference in the MAG mRNA stability could be detected in Ipt-treated vs untreated cells indicating that the drug upregulates the MAG gene at transcriptional level. Serum (FCS) strongly attenuated the response of the MAG gene to Ipt. The stimulatory effect of Ipt was profoundly reduced by spermine and H-89, indicating that protein kinase A-dependent protein phosphorylation is involved in the MAG gene activation. Within 30 min after Ipt administration, the c-fos gene was upregulated by 10-fold, and thereafter, its message level decreased and stabilized at approximately 3-fold over control. In contrast, the c-jun gene was downregulated to approximately 20% of control within 30 min after Ipt administration. Subsequently, its message level rose and fell once again within 12 h to approximately half of control, and returned to control level within 72 h.(ABSTRACT TRUNCATED AT 250 WORDS)

    Title Spatiotemporal Expression of Scip and Myelin Genes in Rat Brain During Early Postnatal Development.
    Date May 1994
    Journal Developmental Neuroscience
    Excerpt

    The expression of the SCIP and myelin genes (PLP, BP and MAG) was quantitated by Northern blot analysis during early postnatal rat brain development. The SCIP gene was already profoundly upregulated on day 1 postpartum. The SCIP message level in the forebrain was 4- and 17-fold higher than the message level in the cerebellum and brainstem, respectively. By day 20, the SCIP gene expression declined in the forebrain to approximately 5% of the day-1 level, and became undetectable in the brainstem and cerebellum. The myelin gene expression sharply upregulated at approximately day 7-8, and in general, was highest in the brainstem, and lowest in the forebrain. The results demonstrate that the expression of the SCIP gene in the oligodendrocytic lineage cells is relatively low, whereas other cellular elements feature very active expression of the gene during the early postnatal development. Additionally, the variation in the relative ratios of the myelin messages among the brain regions indicates that the levels of expression of the myelin genes are not tightly synchronized.

    Title Glucocorticoid-induced Upregulation of Proteolipid Protein and Myelin-associated Glycoprotein Genes in C6 Cells.
    Date May 1994
    Journal Journal of Neuroscience Research
    Excerpt

    The effect of dexamethasone on the expression of proteolipid protein (PLP) and myelin-associated glycoprotein (MAG) genes was investigated in rat C6 glioma cells. The steady state level of the respective mRNAs was quantitated by Northern blot analysis. The treatment of cells with dexamethasone transiently upregulated the expression of both genes with peak mRNA levels of approximately 10-fold over control levels occurring at day 3 for the PLP gene and at day 5 for the MAG gene. The effect was directly related to the drug concentration in the range from 10(-9) to 10(-5) M. Combined exposure of the cells to dexamethasone and retinoic acid featured an additive effect on PLP gene expression, whereas MAG gene expression was depressed below detectability level. The dissimilarity in the response of the genes to dexamethasone and retinoic acid supports the contention that the genes are controlled by different mechanisms. Furthermore, the results indicate that the effects of dexamethasone and retinoic acid on the myelin genes are mediated by different regulatory pathways.

    Title Myelin Gene Activation: a Glucose Sensitive Critical Period in Development.
    Date January 1994
    Journal Journal of Neuroscience Research
    Excerpt

    Glucose deprivation was employed to model caloric undernutrition in newborn rat mixed glial cultures. Six day-old cultures were placed in serum-free media containing glucose concentrations from 0.55 mg/ml to 10 mg/ml. The expression of the myelin PLP, BP, and MAG genes was determined by Northern blot analysis. The activation of the myelin genes began at approximately 6 days in vitro (DIV), and a period of rapid upregulation followed through 14 DIV. The gene activity was directly related to the glucose concentration. The increase in glucose concentration from 0.55 to 1.5 mg/ml (which spans the physiological range) resulted in 2-3 fold increases in expression of the myelin genes, whereas further increases in glucose (2-10 mg/ml) produced only slight additional elevation in the gene activity. We used high glucose (5-6 mg/ml) as control, or low glucose (0.55 mg/ml) as deprived, to delineate possible critical periods of oligodendrocytic differentiation. Cultures were deprived for 4-day intervals, staggered from 6 to 22 DIV. Deprivation from 6 to 10 DIV produced an 80-90% suppression of the myelin gene upregulation at 22 DIV; deprivation from 10 to 14 DIV produced 60-70% suppression; whereas deprivation from 14 to 18 DIV was fully recoverable by 22 DIV. These results show that mixed glial cultures model the developmental pattern of myelin gene expression, as well as their vulnerability. Furthermore, the period of rapid upregulation of the myelin genes appears to be a critical period in development, during which glucose deprivation irreversibly alters oligodendrocyte differentiation.

    Title Cocaine Cytotoxicity in Serum-free Environment: C6 Glioma Cell Culture.
    Date September 1993
    Journal Neurotoxicology
    Excerpt

    Rat glioma C6 cells were employed to determine the vulnerability of the CNS-derived cells to cocaine. The cells were cultured either in the presence of serum or in serum-free (defined) medium to model glial development in the normal brain. In serum-containing medium, cocaine in amounts up to 100 micrograms/ml neither retarded cell proliferation nor altered cell morphology. In the absence of serum, the culture growth was profoundly retarded and cell death was observed with amounts as small as 0.1 micrograms/ml. Even brief (24 hrs) exposure to low cocaine concentrations in serum-free medium irreversibly decreased the cell number. However, an initial 24 hr exposure to 0.1-2.5 micrograms/ml cocaine prolonged survival of cells subsequently exposed to a lethal concentration (100 micrograms/ml). Benzoylecgonine in amounts up to 100 micrograms/ml had no effect on cell proliferation, with or without serum. These data show that cocaine in the absence of serum is highly cytotoxic, which indicates a possibility that the blood-brain-barrier-protected CNS cells may be particularly vulnerable to the drug when it enters the nervous system.

    Title Myelin Isolation: Comparison of Sedimentation and Flotation Techniques.
    Date June 1993
    Journal Neurochemistry International
    Excerpt

    Brains from young (20 day old) and adult rats were used to compare myelin yields obtained by sedimentation and flotation techniques. The flotation method consistently gave approx 70% higher yields of myelin than the sedimentation method. Both myelin preparations have virtually identical protein composition as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Electrophoretic analysis revealed substantial concentrations of myelin proteins in the non-myelin particulate fraction obtained by the sedimentation but not by the flotation method. The study indicates that the paradigm of the sedimentation method results in a significant loss of myelin during isolation, and that this loss can be avoided or minimized by employing the flotation method.

    Title How Much Undernourishment is Required to Retard Brain Myelin Development.
    Date June 1993
    Journal Neurochemistry International
    Excerpt

    This study employs a large population of developing rats designed to range continuously from above a normal, average weight to severely undernourished. The purpose of the study is to determine if brain myelin development is vulnerable to mild growth retardation from chronic hunger, or if brain myelin development is altered only after a certain tolerable amount of growth retardation is exceeded. The brains were examined at a landmark age, weaning, since myelination is one of the most vulnerable features of brain development and its rate of synthesis is highest at this age. Brain size was logarithmically related to body weight, and brain growth retardation increased as the severity of food deprivation increased. There was an additional reduction in the concentration of brain myelin. In contrast to brain weight, the reduction in myelin concentration was linearly related to body size over the full range from well nourished to undernourished. From a population perspective, these data indicate growth retardation from undernourishment in any amount slows brain growth and additionally lowers the concentration of brain myelin; however, individuals, in both well nourished and undernourished groups, vary widely. Implications and cautions of extrapolation to human populations are discussed.

    Title Abnormal Upregulation of Myelin Genes Underlies the Critical Period of Myelination in Undernourished Developing Rat Brain.
    Date June 1993
    Journal Brain Research
    Excerpt

    Since myelin gene expression is suppressed during active myelination of the undernourished brain, this study was designed to determine the effects of undernourishment on the upregulation of myelin genes and the relationship between upregulation and the 'critical period' associated with permanent hypomyelination of the brain. Long-Evans rat dams were given either ad libitum or restricted access to rat chow to produce two populations of developing offsprings. The food deprivation schedule was designed to produce a degree of growth retardation comparable to our earlier studies of hypomyelination in undernourished brain. The expression of myelin genes, at various developmental ages, was determined in the forebrains from undernourished and normal, well fed controls by Northern analysis. In well nourished forebrain, proteolipid protein (PLP), myelin associated glycoprotein (MAG), and basic protein (BP) messages began to increase polynomially after day 8 post partum, leading to a rapid accumulation of message during the following several days. In undernourished forebrain, PLP, MAG, and BP messages did not show any increase until day 10, and then increased at a diminished rate as compared to well nourished forebrain. Additionally, the two PLP messages (1.6 kb and 3.2 kb) showed different vulnerabilities to protein-calorie undernourishment, which explains the abnormal ratio of the 3.2 and 1.6 kb forms we previously found in undernourished brain. This study shows a pattern of temporal specificity when the myelin PLP, MAG, and BP genes are synchronously upregulated in the normal forebrain to a high rate of transcription between day 7 to 9, which is several days before the onset of rapid myelination of the brain.(ABSTRACT TRUNCATED AT 250 WORDS)

    Title Differential Upregulation of Plp and Mag Genes in C6 Glioma Cells by N2a Neuroblastoma Conditioned Medium.
    Date December 1992
    Journal Metabolic Brain Disease
    Excerpt

    The effect of factors released from N2A neuroblastoma cells on the expression of myelin protein genes in glioma C6 cells, i.e., proteolipid protein (PLP) and myelin-associated glycoprotein (MAG), was studied. Both cells lines were propagated in serum-free DMEM-F10 (1:1) medium. The addition of 50% N2A conditioned medium (N2ACM) stimulated the proliferation of C6 cells by approximately 4.5 fold as compared to control cells. The N2ACM-treated cells formed aggregates indicating increased cell-cell affinity. The exposure of C6 cells to N2ACM transiently stimulated the expression of both the MAG-specific and the PLP-specific messages up to eight and four fold over the control values, respectively. The maximal upregulation of the PLP gene occurred two days after N2ACM administration and preceded that of the MAG gene by two days. The effect of N2ACM was dose-dependent in the range of 12.5 to 50%. The secretion of N2A paracrine factors that stimulated the myelin gene expression was also time-dependent. The optimal conditioning time for the release of the PLP gene-stimulating activity was one day, while the maximal MAG gene-stimulating activity was found in the medium conditioned for 3 days. This cellular system may provide a convenient model for studies on trophic neuronal-glial interaction. Furthermore, the results indicate a difference in the regulatory mechanisms between the PLP and the MAG genes.

    Title Ascorbic Acid Upregulates Myelin Gene Expression in C6 Glioma Cells.
    Date December 1992
    Journal Metabolic Brain Disease
    Excerpt

    The effect of ascorbic acid (AA) on rat glioma C6 cells was studied. At physiological AA concentrations of 0.1 and 1 mM, no morphological and no proliferative alterations in the C6 cultures were detectable. Although the total RNA content per cell was not affected by the AA-treatment, AA upregulated the expression of myelin-specific genes, i.e. proteolipid protein (PLP) and myelin-associated glycoprotein (MAG) genes as assessed by northern blot analysis. The steady-state level of the specific mRNAs increased transiently in the AA-treated cells. Three days after AA administration the message level reached a maximum of 10- and 2-fold over control for the PLP and MAG genes, respectively. The upregulation of the genes was directly related to AA concentration. The present data indicate a possible involvement of AA in the regulation of myelin gene activity in the CNS.

    Title Cyclic Amp-induced Upregulation of Proteolipid Protein and Myelin Associated Glycoprotein Gene Expression in C6 Cells.
    Date August 1992
    Journal Journal of Neuroscience Research
    Excerpt

    A model culture system of C6 rat glioma cells was used to test the involvement of cAMP in the regulation of the myelin PLP and MAG genes. The treatment of cells with isoproterenol (10(-5) to 10(-8) M) upregulated the expression of the PLP and MAG genes in a concentration-dependent manner. The mRNA for PLP reached a maximum (sevenfold higher than in control cells) after about 12-24 hr, then declined to approximately fourfold over the control level. The response of MAG gene was delayed by at least 36 hr, and the level of MAG mRNA reached a maximum of approximately 48-fold over the control level on the fourth day in culture. The co-administration of propranolol blocked the effect of isoproterenol, whereas 10(-5) M forskolin simulated the effect of isoproterenol, indicating a role of cAMP in the signal transduction cascades leading to upregulation of the myelin genes. However, the dissimilarity in the timing and the extent of upregulation of the PLP and MAG genes by cAMP-stimulating agents indicate the existence of different intracellular mechanisms for the activation of these two genes. Cycloheximide blocked the stimulatory effect of isoproterenol on both the PLP and MAG genes, indicating that the effect of cAMP on the myelin genes is mediated by protein product(s) of other cAMP-response gene(s).

    Title In Vitro and in Vivo Interleukin-8 Production in Human Renal Cortical Epithelia.
    Date July 1992
    Journal Kidney International
    Excerpt

    The signals resulting in leukocytes infiltrating the tubulointerstitial compartment during renal inflammatory disease are not well understood. A recently described cytokine, interleukin-8 (IL-8), has been demonstrated to be chemotactic for lymphocytes and neutrophils at picomolar and nanomolar concentrations, respectively. Cytokeratin positive, renal cortical epithelial cells (RCEC) with tubular attributes were cultured from kidney tissue from six human subjects. We report that these human renal cortical epithelial cells in primary cell culture respond to either IL-1 beta, TNF or LPS in both a time- and dose-dependent manner by expressing IL-8 mRNA and secreting antigenic IL-8 peptide. In addition, RCEC were found to be strongly positive for cell-associated antigenic IL-8 peptide by immunostaining after 24 hour incubation with IL-1 beta, TNF and LPS. To ascertain whether IL-8 was present in renal disease associated with infiltrating leukocytes, we performed immunohistochemistry on renal biopsy specimens from patients with acute allograft rejection. Both proximal and distal tubular epithelial cells were found to be strongly positive for cell-associated antigenic IL-8. These findings suggest that the human renal tubule epithelial cell may actively participate in acute inflammatory processes in the kidney, including allograft rejection, by effecting and directing leukocyte chemotaxis via the production of IL-8.

    Title Retinoic Acid-regulated Expression of Proteolipid Protein and Myelin-associated Glycoprotein Genes in C6 Glioma Cells.
    Date June 1992
    Journal Journal of Neuroscience Research
    Excerpt

    The effect of retinoic acid (RA) on the expression of myelin-specific genes, i.e., proteolipid protein (PLP) and myelin-associated glycoprotein (MAG) in rat glioma C6 cells, was analyzed by Northern blot hybridization. RA-treatment increased the steady-state level of the PLP-specific messages within one day after RA administration and the upregulation reached a maximum on the third day. Concomitantly, the expression of MAG-specific messages in the RA-treated C6 cells dropped below the detectability limit. The expression of the PLP gene was directly related to the RA concentration increasing to approximately 44-fold over the control (untreated cells) level at 10(-6) M RA. The stimulatory effect was vitiated by cycloheximide indicating the involvement of intermediate genes in the PLP gene activation. The total cellular RNA content and the level of cyclophilin mRNA was not changed by the RA-treatment. The present data indicate that RA can be a potent modulator of the myelin-specific gene expression. Furthermore, the reciprocal response of PLP versus MAG genes to RA demonstrates that these two genes utilize different regulatory mechanisms.

    Title Down Regulation of Myelin-specific Mrnas in the Mechanism of Hypomyelination in the Undernourished Developing Brain.
    Date May 1992
    Journal Brain Research. Developmental Brain Research
    Excerpt

    The expression of myelin-specific protein genes, i.e. myelin proteolipid (PLP), basic (BP), and myelin associated glycoproteins (MAG) was studied in normal and severely undernourished 20-day-old rats. The undernutrition paradigm resulted in reductions of approximately 50, 25 and 65% in body weight, brain weight and brain myelin yield, respectively. The amount of total brain RNA was not significantly altered, although the amount of cyclophilin (CYC) mRNA was increased. In contrast, the steady-state levels of myelin specific mRNAs were significantly decreased by approximately 40, 20 and 40% for PLP, BP and MAG, respectively. In addition, polyadenylation of the PLP transcript was altered, producing an abnormal ratio of the 1.6 kb to the 3.2 kb PLP mRNAs. The results indicate that down-regulation of myelin-specific gene expression is involved in the mechanisms of hypomyelination in hunger disease, although the individual genes are differently altered. Furthermore, undernutrition may have additional effects on the posttranscriptional processing of the transcripts as indicated by the abnormal size distribution of PLP messages.

    Title Pharmacokinetics of Cocaine in Pregnancy and Effects on Fetal Maturation.
    Date April 1992
    Journal Clinical Pharmacokinetics
    Title Effect of Culture Conditions on Plp and Mag Gene Expression in Rat Glioma C6 Cells.
    Date March 1992
    Journal Metabolic Brain Disease
    Excerpt

    The effects of culture conditions on the expression of myelin-specific genes, i.e. proteolipid protein (PLP) and myelin-associated glycoprotein (MAG) in rat glioma C6 cells was studied. Early passage (40-46) cells had higher steady-state level of PLP- and MAG-specific mRNA than late (100) passage cells when grown in defined (serum-free) medium. The PLP gene expression was increased whereas the MAG gene expression was reduced in the presence of 10% fetal calf serum in either passage. The level of both PLP- and MAG-specific messages was also directly related to the cell density indicating cell contact-induced stimulation of the gene expression. Furthermore, the cells apparently secrete factors into the medium, which upregulate the gene expression in autocrine fashion. The results also indicate a dissimilarity of regulatory mechanisms involved in the expression of the PLP and MAG genes.

    Title Turbidity As a Measure of Particulate Subcellular Fraction Yield.
    Date December 1991
    Journal Journal of Biochemical and Biophysical Methods
    Excerpt

    Turbidity measurement, as an estimate of the concentration of a particulate subcellular membrane fraction, is an effective alternative to protein assay. As exemplified by purified myelin membrane the technique is fast, accurate, and consumes no sample, giving it certain advantages over protein assay in some applications.

    Title Moderate Protection of Renal Function and Reduction of Fibrosis by Colchicine in a Model of Anti-gbm Disease in the Rabbit.
    Date October 1991
    Journal Journal of the American Society of Nephrology : Jasn
    Excerpt

    A rabbit model of renal glomerulosclerosis induced by anti-glomerular basement membrane antibody was used to determine whether colchicine would protect renal function and reduce fibrosis. Initial studies established the time course of renal function changes and fibrosis. Colchicine at a dose of 0.02 to 0.04 mg/kg per day injected ip was begun at day 4 when injury had been initiated, and the experiment was ended at day 21 when fibrotic changes were established. Colchicine significantly reduced the rise in serum creatinine (serum creatinine = 2.7 +/- 0.3 mg% in vehicle-treated animals versus 1.8 +/- 0.1 mg% in colchicine-treated animals) and interstitial fibrosis (fibrosis score = 2.6 +/- 0.2 in vehicle-treated versus 1.5 +/- 0.2 in colchicine-treated animals). Colchicine treatment did not significantly affect weight, anti-guinea pig immunoglobulin level, % fibrocellular crescents formed, hydroxyproline per gram (dry weight) in tissue, or urine protein: creatine ratio. Regression analysis was performed to examine the interrelationships between variables for all animals and the effect of colchicine on pairs of variables. No clear-cut site of colchicine action could be identified. These data show that colchicine, in doses that could be used in humans, protected renal function by about 25% and reduced interstitial fibrosis in a model of severe crescentic nephritis.

    Title Cocaine Interaction with Sulpiride, Methysergide, Naloxone and Desipramine: Neurophysiological Effects of Mesolimbic and Neostriatal Neuronal Activity.
    Date September 1991
    Journal Nida Research Monograph
    Title Generation of High Efficiency, Single-stranded Dna Hybridization Probes by Pcr.
    Date September 1991
    Journal Biotechniques
    Title Differential Regulation of Myelin Gene Expression in Sv40 T Antigen-transfected Rat Glioma C6 Cells.
    Date August 1991
    Journal Metabolic Brain Disease
    Excerpt

    Rat glioma C6 cells were stably transfected with a pSV3-neo plasmid containing SV40 T antigen gene, and geniticin-resistant transfectants (designated C6T cells) were cloned. The C6T cells grew as well-defined foci of cells showing squamous or irregular morphology. The doubling time for transfected cells was reduced by approximately 40% as compared to control C6 cells. The transfection with T-antigen also affected the expression of genes coding for structural myelin proteins and for myelin-associated enzymes. The steady-state level of proteolipid protein (PLP)-specific mRNA in C6T cells was 44% lower than in parental C6 cells. On the other hand, the transfection upregulated the expression of myelin-associated glycoprotein (MAG) by 153%. The activity of 2':3' cyclic AMP phosphodiesterase (CNP) was increased by approximately 80% in the C6T cells as compared to untransfected, control cells. The activity of calcium-activated neutral proteinase (CANP) was also significantly elevated in the transfectants by approximately 50% and 220% for millimolar and micromolar form respectively. The results indicate that T antigen affects the expression of myelin genes, although, individual genes appear to be differently regulated implying the existence of several independent regulatory mechanisms.

    Title Consequences of Glomerular Injury. Glomerular Crescent Formation.
    Date July 1991
    Journal Seminars in Nephrology
    Title Tissue Factor Expression in Endothelial Cell/monocyte Cocultures Stimulated by Lipopolysaccharide And/or Aggregated Igg. Mechanisms of Cell:cell Communication.
    Date March 1991
    Journal Journal of Immunology (baltimore, Md. : 1950)
    Excerpt

    A human umbilical vein endothelial cell (EC)/monocyte (MC) coculture system was used to dissect cell:cell interactions associated with production of procoagulant activity (PCA) in response to two common stimuli of intravascular coagulation in vivo (LPS and immune complexes). We found that the presence of MC at a ratio of 1 MC:10 EC increased the sensitivity of EC to LPS by 4 logs and the maximal response approximately 20-fold. Aggregated IgG alone did not stimulate the system, but in the presence of small amounts of LPS (1 to 10 ng/ml) aggregated IgG was a powerful stimulus. More than 90% of the PCA was tissue factor as shown by clotting studies and mRNA analysis. PCA was not produced by either cell alone under the conditions of study, but was produced in large amounts when the EC and MC were cocultured. The supernatant from the coculture stimulated virgin EC, but not MC, to synthesize tissue factor. The major factor in the supernatant was IL-1 beta as shown by measuring IL-1 beta, IL-1 alpha, and TNF-alpha in supernatants and by blocking the production of PCA by preincubation of supernatants with anti-cytokine antibodies. Small amounts of TNF-alpha were present in the supernatant but anti-TNF-alpha did not inhibit PCA production. Studies using recombinant cytokines established that IL-1 beta was the most potent of the cytokines tested, that cytokines potentiated each other, and that the results could be explained in quantitative terms by the amounts of IL-1 beta measured. These data emphasize that cell:cell interactions are likely to modulate procoagulant events in vivo in the presence of both LPS and immune complexes, and that IL-1 beta may be an important cytokine in these events.

    Title Analysis of Alpha 1 (i) Procollagen Alpha 1 (iv) Collagen, and Beta-actin Mrna in Glomerulus and Cortex of Rabbits with Experimental Anti-glomerular Basement Membrane Disease. Evidence for Early Extraglomerular Collagen Biosynthesis.
    Date January 1991
    Journal Laboratory Investigation; a Journal of Technical Methods and Pathology
    Excerpt

    Renal cortical and glomerular mRNA for alpha 1 (I) and alpha 1 (IV) collagen were measured by filter hybridization during experimental anti-glomerular basement membrane disease in the rabbit. The abundance of alpha 1 (IV) mRNA was 5 times greater in total RNA isolated from glomeruli as compared with whole renal cortex from normal rabbits. In contrast, there was no difference in the relative amounts of alpha 1 (I) procollagen mRNA in these two fractions. Four days after the administration of anti-glomerular basement membrane antisera, a time histologically characterized by glomerular inflammatory cell infiltration without crescent formation, beta-actin mRNA were increased 17-fold in glomeruli and 4-fold in whole renal cortex. Renal cortical mRNA for alpha 1 (I) and alpha 1 (IV) were increased 7-fold (p = 0.07) and 9-fold (p less than 0.05), respectively compared with normal rabbit kidney cortex. In contrast, there was no significant difference in the abundance of these mRNA in glomeruli at day 4. By day 7, cortical alpha 1 (I) and alpha 1 (IVP mRNA had increased 17- and 10-fold, respectively, and these transcripts had increased 13- and 7-fold in glomeruli. Cortical alpha 1 (I) mRNA remained elevated for 35 days. These data show that large changes in collagen mRNA levels occur early in this model of crescentic nephritis in the rabbit, and that extraglomerular collagen mRNA accumulates very rapidly when glomerular inflammation occurs. Extraglomerular collagen synthesis associated with intraglomerular inflammation may help to explain the common association of interstitial fibrosis with glomerulonephritis, particularly in the periglomerular area.

    Title Development Under the Influence of Cocaine. Ii. Comparison of the Effects of Maternal Cocaine and Associated Undernutrition on Brain Myelin Development in the Offspring.
    Date September 1990
    Journal Metabolic Brain Disease
    Excerpt

    Pregnant and lactating Long-Evans rats were treated daily with oral cocaine at a dosage rate of 60 mg/kg/day, which is the highest dosage tolerated during chronic treatment. Brain myelin concentrations were compared in the offspring during early myelination (day 15) and peak myelination (day 20). Body and brain weights in the offspring of cocaine-treated and pair-fed dams were transiently (but insignificantly) retarded, and by day 20 brain weights of the normal controls, cocaine-exposed, and pair-fed pups were the same (1.36 +/- 0.01-1.35 +/- 0.01 g). Similarly, specific myelin yields in the offspring of pair-fed dams were retarded by no more than 1-2% of the myelin concentrations obtained in normal controls, which is consistent with studies showing that such minimal growth retardation would not be expected to produce hypomyelination. In contrast, myelin concentrations from the brains of cocaine-exposed pups were reduced about 10%. In addition, cross-fostering indicated that the fetal period of cocaine exposure presents a greater risk to postnatal myelination than exposure during the suckling period, which is the reverse of the vulnerability resultant from undernourishment. Thus, the hypomyelination observed in the offspring of cocaine-treated dams indicates a toxic effect of cocaine acting directly on the developing offspring.

    Title Development Under the Influence of Cocaine. I. A Comparison of the Effects of Daily Cocaine Treatment and Resultant Undernutrition on Pregnancy and Early Growth in a Large Population of Rats.
    Date September 1990
    Journal Metabolic Brain Disease
    Excerpt

    After administering cocaine to pregnant and lactating dams in oral dosages ranging from 5 to 90 mg/kg/day, we observed a slight increase in fatalities starting at 60 mg/kg/day, followed by a sharp rise fatalities at higher dosages. Therefore, a dosage near 60 mg/kg/day by the oral route appears to mark a useful threshold between highly lethal dosages and an acceptable, sublethal dosage for chronic studies of pregnant rats. At 60 mg/kg/day, there was a marginal trend toward less weight gain in the cocaine-treated dams during pregnancy, followed by a much more pronounced lag in weight recovery following parturition. Neither prematurity nor any reduction in litter sizes and birth weights was consistently observed at dosages below 90 mg/kg/day; however, poor maternal care was evident when the dams received dosages of 80-90 mg/kg/day, producing a very high death rate in the neonatal offspring. Internal bleeding (intracranial and subcutaneous) was also observed in three neonates whose dams received cocaine at rates of 60-90 mg/kg/day, confirming a similar clinical observation and indicating a direct action of cocaine toxicity on the fetus. Although fetal growth and development were not significantly altered by administering the pregnant dams 60 mg/kg/day, there was a higher death rate in the offspring during the initial 24 hr after delivery. Weight gain appeared transiently retarded early in the suckling period, although similar growth retardation was observed in the offspring of pair-fed controls. These results indicate that the dam's undernourishment contributes much to the early growth retardation resultant from cocaine administration. The most striking effect of cocaine on the offspring, after considering undernourishment, appears to be an increased mortality in the neonatal period (60 mg/kg/day and higher), apparently resulting from poor maternal care. The greatest risk of all was the death of the dam at any point beginning with the second administration of cocaine at 60 mg/kg/day and increasing precipitously at any higher dosage.

    Title Microiontophoresis of Cocaine, Desipramine, Sulpiride, Methysergide, and Naloxone in Habenula and Parafasciculus.
    Date July 1990
    Journal Experimental Neurology
    Excerpt

    The effects of microiontophoretically applied cocaine, desipramine (DES), sulpiride (SUL), methysergide (METH), and naloxone (NAL) on the responses of physiologically identified single neurons in the habenula (Hab) and parafasciculus thalami nucleus (PF) were examined in rats. Three cell types were identified in both nuclei on the basis of the responses obtained following noxious stimulation that were classified as "nociceptive-on," "nociceptive-off," and "nonnociceptive" cells. Administration of cocaine generally resulted in a decrease in the firing rate of nociceptive-on and nonnociceptive neurons in both Hab and PF. In contrast, cocaine generally induced an excitation in the baseline firing of the nociceptive-off cells. Cocaine application concomitant with noxious stimulation prevented the evoked responses of the nociceptive-on and the nociceptive-off cells. DES, when applied alone, was found to induce excitation in neuronal discharge of all three cell types in both sites. Combined application of cocaine with DES resulted in no observable change in discharge frequency for the nociceptive-on and nonnociceptive cells, while inducing an additive excitatory effect on the nociceptive-off cells. SUL, in contrast, induced no observable effect on baseline firing when given alone, yet consistently antagonized cocaine-induced effects on all three cell types. Finally, METH and NAL induced no effects on baseline firing or cocaine-induced modifications in neuronal discharge frequency.

    Title Tissue Factor Production by Cultured Rat Mesangial Cells. Stimulation by Tnf Alpha and Lipopolysaccharide.
    Date June 1990
    Journal Kidney International
    Excerpt

    Fibrin formation plays an important role in glomerular injury. We therefore examined the procoagulant signal produced by cultured rat mesangial cells. Actively growing mesangial cells produced procoagulant activity (PCA) that was present in intact cells (surface-associated), was inhibitable by cyclohexamide and which, by clotting assay, had the characteristics of tissue factor. This PCA decreased with incubation of cells in serum-deprived medium. Incubation with bacterial lipopolysaccharide (LPS) and tumor necrosis factor (TNF alpha) induced increased detectable tissue factor by mesangial cells within two hours which was maximal by four hours. We conclude that quiescent mesangial cells produce a small amount of tissue factor-like procoagulant activity, and that this PCA can be stimulated by incubation with TNF alpha, LPS or when cells are actively growing in high serum medium. Therefore mesangial cells have the capability of contributing to fibrin formation during inflammatory glomerular injury or sepsis.

    Title Effects of Microiontophoretic Application of Cocaine, Alone and with Receptor Antagonists, Upon the Neurons of the Medial Prefrontal Cortex, Nucleus Accumbens and Caudate Nucleus of Rats.
    Date June 1990
    Journal Neuropharmacology
    Excerpt

    The spontaneous extracellular electrical activity of 102 neurons, within the caudate nucleus (CN), medial prefrontal cortex (MPC), nucleus accumbens (NAc) and a control site, the lateral thalamic nucleus (LT), was studied. Cocaine depressed spontaneous activity in the majority of the cells studied from all regions except the lateral thalamus. Desipramine, which has been used clinically for the treatment of withdrawal of cocaine, also depressed neuronal activity in the caudate nucleus. In addition, of the three receptor antagonists tested, sulpiride, methysergide and naloxone, only the dopamine antagonist (sulpiride) affected cocaine-induced neuronal responses. This study further emphasizes the emerging importance of midbrain dopaminergic systems in the pharmacological effects of this important drug of abuse.

    Title Tumor Necrosis Factor Production and Accumulation of Inflammatory Cells in the Corpus Luteum of Pseudopregnancy and Pregnancy in Rabbits.
    Date June 1990
    Journal Biology of Reproduction
    Excerpt

    The potential involvement of macrophages, T lymphocytes, and the cytokine tumor necrosis factor (TNF) in regression of the corpus luteum was investigated at different stages of pseudopregnancy and pregnancy by use of immunocytochemical methods and a TNF bioassay. Few macrophages (11 +/- 6 per high power field of 8-microns frozen sections of corpus luteum, Day 10 of pseudopregnancy) were observed until the very end of pseudopregnancy, when the number of macrophages increased greatly (176 +/- 42 per high power field, Day 19 of pseudopregnancy). Pregnancy, of 32 days duration, delayed large-scale macrophage accumulation until 3 days after parturition (154 +/- 30 per high power field). Low TNF activity (approximately 1.0 U/mg protein) was detected in incubations of luteal tissue at all stages; in response to lipopolysaccharide, TNF values in medium increased 10- to 30-fold at times of luteal regression and macrophage accumulation (1 day postpartum and Day 19 of pseudopregnancy). Class II-positive T lymphocytes were observed in luteal tissue, but unlike macrophages, the number of lymphocytes did not increase at the time of regression of the corpus luteum. These data are consistent with the hypothesis that involution of the corpus luteum is promoted through the interactions of inflammatory cells and action of TNF, although the action of TNF has not been determined in this luteal tissue. Through unknown mechanisms, pregnancy postpones the accumulation of macrophages in the corpus luteum, in association with the prolongation of luteal function until the time of parturition.

    Title Pharmacokinetics of Cocaine: Basic Studies of Route, Dosage, Pregnancy and Lactation.
    Date April 1990
    Journal Neurotoxicology
    Excerpt

    As a preface to the pharmacokinetic analysis of cocaine in pregnant and lactating rats (using oral administration of drug), young Long-Evans rats were used to compare the relative concentrations of cocaine in blood, brain, and liver after administering cocaine by iv or oral routes. Cocaine and its metabolites were determined using 3H-cocaine as a tracer, followed by homogenization and solvent extraction of tissues, and quantitative analysis by HPTLC and LSC. From 30 min postinjection to several hrs later, the concentration of cocaine was higher in brain (3-4 fold) and liver (3-5 fold) than in blood, using the iv route. Using the oral route, the concentration in brain was 2-3 fold higher than in blood, and in liver, 10-20 fold higher. The metabolites of cocaine were largely excluded from entry into brain tissue, whereas the accumulation of metabolites in liver was typically an order of magnitude higher, or more, than in blood (iv or oral route). The ratio of cocaine to metabolites increased in all three tissues, as the dosage increased, indicating that more and more of an administered dose actually reaches the tissues as cocaine as the dosage level increases. During the period from 30 to 90 min following the administration of cocaine to pregnant dams, cocaine appeared in fetal brain at a rate of 50-90% of the concentration in the dam's brain (presumably because of the lower lipid content in fetal brain compared to adult), but still at a rate of 109-151% of the concentration in the dam's blood. Cocaine is sufficiently stable in milk to assume that any cocaine entering breast milk from the blood stream will be available to the suckling infant, and after administering radioactive cocaine to lactating dams, the milk/blood ratio for cocaine averaged 7.8. These data indicate that both the fetus and suckling infant are at considerable risk from cocaine use by the mother.

    Title Peroxidative Aggregation of Myelin Membrane Proteins.
    Date June 1989
    Journal Metabolic Brain Disease
    Excerpt

    The exposure of CNS myelin to reactive oxygen species (ROS) generated by a Cu2+-H2O2 system results in the aggregation of membrane proteins. Integral and peripheral membrane proteins are equally vulnerable and the denaturation is not mediated by the SH groups. The aggregated proteins retain their original antigenicity as determined by immunoblot technique. The aggregation of proteins is not limited to myelin and can be elicited in the preparation of other cerebral membranes. The effect of ROS on membrane proteins can also be demonstrated in cerebral slices incubated in the presence of the ROS-generating system. Furthermore, the peroxidation inactivates membrane-bound enzymes as exemplified by myelin cyclic nucleotide phosphatase (CNP). Competitive inhibition studies with various scavengers and quenchers of ROS implicate singlet oxygen as a major mediator in the Cu2+-H2O2 oxidizing system responsible for the peroxidative aggregation of membrane proteins.

    Title Restraint Stress During Late Pregnancy in Rats Elicits Early Hypermyelination in the Offspring.
    Date June 1989
    Journal Metabolic Brain Disease
    Excerpt

    Female rats were subjected to a regular, daily schedule of 2 hr of restraint stress during the final 6 days of pregnancy. During the first 2 postnatal weeks, adrenal weights were greater than normal in the offspring of the stressed dams. The concentration of brain myelin was higher than control at 14 and 21 days of age but similar to normal by day 40. Early hypermyelination may be partly responsible for early motor development, as previously observed in prenatally stressed rats.

    Title Effects of Aging and Alcohol on the Biochemical Composition of Histologically Normal Human Brain.
    Date February 1989
    Journal Metabolic Brain Disease
    Excerpt

    Human brains were removed at autopsy and examined grossly and histologically for any abnormality or evidence of disease. Sixty-two brains appearing normal by these criteria were examined further. First, a detailed record of alcohol consumption was obtained. Second, frozen punches of gray and white matter were used to determine the compositional change associated with age and drinking patterns. Increased age was associated with an increase in the water content, particularly in the white matter, a decline in RNA content in gray matter, a decline in total protein in white matter, and a decline in both myelin and the myelin-like subfraction. The loss of myelin membrane in white matter corresponded to a similar increase in water content, although there was an additional loss of some nonmyelin protein. There was no significant shift in the density between the myelin and the myelin-like membranes, and the protein composition of myelin was not significantly altered by age. A history of heavy alcohol consumption was associated with a relative increase in total protein in white matter even though heavy drinking accelerated the age-related loss of myelin. Presumably, alcohol produced a lag in the rate at which nonmyelin proteins are lost or accelerated the accumulation of abnormal protein. Alcohol consumption did not influence the myelin composition or the ratio of myelin and myelin-like membranes. The interval between patient death and autopsy was shown to have little or no effect on the samples used in this study. These data show that normal aging, uncomplicated by other disease processes, can have a significant effect on the composition of brain tissue, particularly the white matter, and that heavy alcohol consumption accelerates degenerative change, even in tissue appearing normal by histology.

    Title Development of Axonal-oligodendroglial Relationships and Junctions During Myelination of the Optic Nerve.
    Date February 1989
    Journal International Journal of Developmental Neuroscience : the Official Journal of the International Society for Developmental Neuroscience
    Excerpt

    The early stages of myelination were examined in optic nerves of rats aged 12-15 days. The initial association between oligodendroglial processes and bare axons involves no junctional specialization, as the axoglial extracellular space remains unaltered. Following ensheathment by a collar of glial cytoplasm, at least one full rotation of mesaxon was evident before compact myelin formed. Furthermore, myelin was generally evident before a second rotation was completed. In longitudinal sections, an axoglial junction was always observed beginning on the first paranodal loop, continuing through to the last (or outermost) loop. Thus, the formation of myelin and elaboration of a junctional complex in the paranodal region follow a promyelination phase and appear to be synchronous (and possibly related) events. Although the paranodal plasmalemma and axolemma are in close apposition, there is a material in the extracellular space that precipitates phosphotungstic acid, a characteristic that appears to be featured in a number of different types of cell junctions.

    Title Tocopherol in Brain Metabolism and Disease: a Review.
    Date February 1989
    Journal Metabolic Brain Disease
    Title The Appended Curve Technique for Deconvolutional Analysis--method and Validation.
    Date December 1988
    Journal European Journal of Nuclear Medicine
    Excerpt

    Deconvolutional analysis (DCA) is useful in correction of organ time activity curves (response function) for variations in blood activity (input function). Despite enthusiastic reports of applications of DCA in renal and cardiac scintigraphy, routine use has awaited an easily implemented algorithm which is insensitive to statistical noise. The matrix method suffers from the propagation of errors in early data points through the entire curve. Curve fitting or constraint methods require prior knowledge of the expected form of the results. DCA by Fourier transforms (FT) is less influenced by single data points but often suffers from high frequency artifacts which result from the abrupt termination of data acquisition at a nonzero value. To reduce this artifact, we extend the input (i) and response curves to three to five times the initial period of data acquisition (P) by appending a smooth low frequency curve with a gradual taper to zero. Satisfactory results have been obtained using a half cosine curve of length 2-3P. The FTs of the input and response I and R, are computed and R/I determined. The inverse FT is performed and the curve segment corresponding to the initial period of acquisition (P) is retained. We have validated this technique in a dog model by comparing the mean renal transit times of 131I-iodohippuran by direct renal artery injection to that calculated by deconvolution of an intravenous injection. The correlation was excellent (r = 0.97, P less than 0.005). The extension of the data curves by appending a low frequency "tail" before DCA reduces the data termination artifact. This method is rapid, simple, and easily implemented on a microcomputer.(ABSTRACT TRUNCATED AT 250 WORDS)

    Title Fetal Ethanol Exposure: a Morphometric Analysis of Myelination in the Optic Nerve.
    Date October 1988
    Journal International Journal of Developmental Neuroscience : the Official Journal of the International Society for Developmental Neuroscience
    Excerpt

    Pregnant Long-Evans rats were fed a liquid diet containing ethanol during gestation. Controls consisted of both pair-fed dams and dams fed ad libitum with an equivalent, iso-caloric diet lacking ethanol. Subsequent effects of ethanol measured in the offspring include a significant lag in the rate at which non-myelinated axons are lost in association with the initial overproduction of neurons. Additionally, there was a slight lag in the rate of acquisition of myelinated axons; and altogether there was a large increase in the ratio of non-myelinated to myelinated axons. Frequency spectra of myelinated and non-myelinated axons by size were normal, and the relationship between axon size and myelin lamellae was also normal. Measured against the dynamic, normal background of rapid cell-loss and the progressive development of myelin, morphometric demonstration and evaluation of the comparatively small divergences associated with fetal alcohol exposure are difficult: nevertheless, these results are consistent with and help account for the marginal hypomyelination previously observed by quantitative neurochemistry.

    Title Analysis of Renal Fibrosis in a Rabbit Model of Crescentic Nephritis.
    Date October 1988
    Journal The Journal of Clinical Investigation
    Excerpt

    The pathogenesis of renal fibrosis in crescentic nephritis is incompletely understood. To improve our understanding of this process, crescentic nephritis was induced in New Zealand White rabbits by administration of guinea pig antiglomerular basement membrane IgG after sensitization with guinea pig IgG, and their kidneys were analyzed for the development of fibrosis. Collagen synthesis in renal cortical tissue was significantly elevated by day 3, peaked at days 7-15, and returned towards baseline by day 21. Collagen content of both glomeruli and cortex were increased starting on days 14-16, and remained constant in cortex thereafter. Light microscopic analysis was much less sensitive, revealing fibrosis only after day 21. Immunofluorescence revealed that type IV collagen was distributed primarily in the glomerulus, while types I and III were increased in the glomerulus and interstitium. Thus, in this model of crescentic nephritis, fibrosis, as assessed biochemically, developed early at time points when morphologic analysis failed to detect such a development. Hence early therapeutic intervention, before morphologic evidence of fibrosis is evident, may be more successful in arresting the progression of this disease before it reaches irreversible terminal stages.

    Title Dose-related Differential Accumulation of Morphine in Specific Regions of Rat Brain Determined by Mass Fragmentography.
    Date May 1988
    Journal The International Journal of Neuroscience
    Excerpt

    Regional morphine accumulation was examined in 8 brain areas (cerebral cortex, hippocampus, striatum, midbrain, hypothalamus, thalamus, medulla oblongata and cerebellum) following either a single dose or incremental doses administered by intraperitoneal injection. Morphine levels were determined by gas chromatography-mass spectrometry with chemical ionization detection. Prior to morphine assay, the vasculature was cleared of blood by saline perfusion to eliminate distortion of tissue-morphine by blood-morphine. Results indicate a dose-dependent, differential accumulation of morphine in different brain regions following incremental morphine administration. Three distinct uptake profiles were obtained, with the cerebellum, hippocampus, medulla and cortex showing roughly linear accumulation the midbrain, striatum and thalamus showing nonlinear accumulation, and the hypothalamus showing significantly higher absolute morphine levels than any other brain region.

    Title Tumor Necrosis Factor-a (tnf-a) Production and Localization of Macrophages and T Lymphocytes in the Rabbit Corpus Luteum.
    Date April 1988
    Journal Endocrinology
    Excerpt

    Utilizing immunocytochemistry numerous macrophages were localized in regressing corpora lutea. In contrast, few macrophages were observed in young corpora lutea. Regressing corpora lutea readily produced TNF-a in vitro in response to lipopolysaccharide, whereas young corpora lutea produced significantly less TNF-a. T lymphocytes were identified in young corpora lutea preceding the appearance of macrophages. These observations suggest that cells of the immune system and cytokines could be important participants in physiological regression of the corpus luteum.

    Title Procoagulant Activity in Kidneys of Normal and Bacterial Lipopolysaccharide-treated Rabbits.
    Date October 1987
    Journal Kidney International
    Excerpt

    Fibrin formation in the kidney is frequently associated with clinically-significant renal dysfunction. We therefore measured and characterized the procoagulant activity (PCA) which is present in normal kidneys and in kidneys of rabbits with the Shwartzman phenomenon induced by two injections of bacterial lipopolysaccharide (LPS; E. coli LPS 055:B5,25 micrograms/kg and 50 micrograms/kg administered 24 hrs apart with rabbits sacrificed 12 hrs after the second injection). PCA was measured in sonicated tissue by one-stage coagulation assay. In normal kidneys the amounts of PCA in the inner medulla, outer medulla and inner cortex were 18.2 +/- 3.2, 44.1 +/- 3.8 and 78.5 +/- 5.7 percent, respectively, of that in the outer cortex (N = 31). Glomeruli (purified by the iron oxide magnetic method to greater than 95 percent homogeneity) contained 21.6 +/- 8.8 arbitrary units/micrograms protein compared with tubular fragments which contained 13.9 +/- 2.6 U/micrograms protein (N = 9). In LPS-treated rabbits PCA (in units/micrograms) increased in outer cortex from 33.7 +/- 3.9 (control) to 73.4 +/- 10.4 (LPS, P less than 0.01), in inner cortex from 26.7 +/- 2.9 (control) to 83.3 +/- 17 (LPS, P less than 0.02), in outer medulla from 12.9 +/- 2.4 (control) to 54.5 +/- 16.5 (LPS, P less than 0.05), and in inner medulla from 12.2 +/- 2.4 (control) to 32.1 +/- 4.9 (LPS, P less than 0.01). Glomerular PCA increased from 21.6 +/- 8.8 (control) to 88.8 +/- 20.7 (LPS) units/micrograms (P = 0.01), while tubular fragment preparation PCA increased from 13.9 +/- 2.6 (control) to 44.6 +/- 12.7 (LPS) U/micrograms (P = 0.02) (N = 9 per group).(ABSTRACT TRUNCATED AT 250 WORDS)

    Title Uromucoid (tamm-horsfall Glycoprotein) Forms Different Polymeric Arrangements on a Filter Surface Under Different Physicochemical Conditions.
    Date June 1987
    Journal Clinica Chimica Acta; International Journal of Clinical Chemistry
    Excerpt

    Normal human urine cannot be forced through a 0.2 micron filter. To investigate the reason for this phenomenon, uromucoid (Tamm-Horsfall protein) was purified from human urine and its capacity to block a 0.2 micron Millipore filter was measured under different conditions. In the presence of cations (H+, Na+, Ca2+) uromucoid blocked the filter. The blocking varied with cation concentration. Scanning electron microscopy of the filter surface revealed different arrangements of polymerized uromucoid coating the filter surface depending on ionic conditions. In the presence of 100 mmol/l NaCl or 1 mmol/l CaCl2 uromucoid polymers were present in a fibrous arrangement. In the presence of both NaCl and CaCl2 a dense mat of uromucoid polymers was present together with clumps of aggregated polymer. In the absence of ions uromucoid formed a homogeneous coat on the filter surface (as demonstrated by scanning electron microscopy, Western blotting and 125I-uromucoid binding studies) but did not block the filter. Similar fibrous and highly aggregated arrangements of uromucoid polymer were seen in hyaline casts from urine. These data are consistent with the concept that the uromucoid glycoprotein can exist in several different polymeric forms under different ionic conditions.

    Title Amount of Antibody is Critical for Immune Complex Displacement by Charge Competition from Both Rabbit Glomeruli and Anionic Beads.
    Date January 1987
    Journal Clinical and Experimental Immunology
    Excerpt

    The purpose of this study was to determine the feasibility of displacing cationized bovine serum albumin (CBSA) immune complexes from glomeruli by charge competition. An in vitro model identified protamine as an effective agent for displacing 125I-CBSA from anionic beads (dextran sulfate-coated Sepharose 4B). Anti-CBSA serum prevented displacement of 125I-CBSA from anionic beads in a dose-dependent fashion. When 125I-CBSA was injected intravenously into rabbits 98% of 125I-CBSA disappeared from blood within 5 min, at which time CBSA was visualized by immunofluorescence in glomerular capillary walls but not in liver, muscle, skin, spleen or lung. By 24 h 90% of 125I-CBSA had disappeared from glomeruli. In contrast, injection of anti-CBSA antibody caused persistence of 125I-CBSA in kidney (particularly along glomerular capillary walls) for more than 7 days (detected by counting 125I in kidney, by radionuclide imaging and by immunofluorescence). Protamine administration (50 mg intravenously daily for 6 days) caused significant reduction of 125I-CBSA trapped in kidney only if the amount of anti-CBSA injected was small. Protamine did not significantly displace 125I-CBSA from glomeruli if the anti-CBSA dose was larger. Therefore both in vivo and in vitro displacement of 125I-CBSA by protamine depended upon the amount of antibody. We conclude that although charge dependent displacement of immune complexes from glomeruli is probably feasible using protamine this approach would only work in the presence of small amounts of antibody.

    Title Myelination: a Critical Stage in Development.
    Date January 1987
    Journal Neurotoxicology
    Title A Map of Urine Proteins Based on One-dimensional Sds-polyacrylamide Gel Electrophoresis and Western Blotting Using One Microliter of Unconcentrated Urine.
    Date September 1986
    Journal Clinica Chimica Acta; International Journal of Clinical Chemistry
    Excerpt

    A sensitive one-dimensional SDS-polyacrylamide gel electrophoretic system was devised whereby the proteins in 1 microliter of unconcentrated urine could be visualized by silver staining over the range 9,000-900,000 molecular weight. Identification of urine proteins was confirmed by Western blotting using peroxidase labelled antibodies. A map of the major proteins visualized in urine from individuals with renal disease was constructed. We conclude that the information available from the simple analysis of proteins according to their size is limited to general conclusions regarding whether proteinuria is likely to be of tubular or glomerular or mixed origin. More specific identification of individual proteins is not feasible because simple protein staining is not sufficiently reliable to identify individual proteins. The reasons for this conclusion are as follows: many proteins in urine migrate with similar apparent molecular weights, some proteins are not visualized by silver staining, and albumin polymeric complexes and fragments can be present at almost any molecular weight. However, one-dimensional SDS-polyacrylamide gel electrophoresis together with Western blotting does provide reliable information which might be clinically and experimentally useful.

    Title Regulation of Macrophage Tumor Necrosis Factor Production by Prostaglandin E2.
    Date July 1986
    Journal Biochemical and Biophysical Research Communications
    Excerpt

    We have studied the role of prostaglandin E2 on the modulation of tumor necrosis factor by immunologically elicited and lipopolysaccharide treated murine macrophages. Indomethacin, a potent inhibitor of prostaglandin E2 production, caused a dose dependent augmentation of lipopolysaccharide induced tumor necrosis factor production (2-3 fold at 10(-7) molar). Tumor necrosis factor was released into the extracellular environment and no activity was found to be associated with membrane or cytosolic fractions. Prostaglandin E2 added to the lipopolysaccharide treated cultures suppressed tumor necrosis factor in a dose dependent manner. In these studies, 10(-7) molar PGE2 reduced tumor necrosis factor production to basal levels. These data suggest that PGE2 may be a potent autoregulatory factor that dramatically influences tumor necrosis factor production.

    Title A Different Cleavage Site for High Molecular Weight Kininogen in Vivo Following Intravenous Injection of Dextran Sulfate in the Rabbit.
    Date June 1986
    Journal Circulation Research
    Excerpt

    Purified radiolabeled rabbit Hageman factor, prekallikrein, and high molecular weight kininogen were used to examine Hageman factor system molecular dynamics after the intravenous injection of heparin-like dextran sulfate polymer in the rabbit. Hageman factor system proteins rapidly disappeared from the circulation following dextran sulfate injection, as measured by radial immunodiffusion, by kaolin-releasable kinin formation, and by measuring circulating levels of radiolabeled Hageman factor, prekallikrein, and high molecular weight kininogen. 125I-Hageman factor was distributed mainly to lung, liver, and spleen following dextran sulfate injection. Proteolysis of circulating 125I-Hageman factor occurred at a site within a disulfide loop into fragments of 50,000 and 30,000 molecular weight. Proteolysis of 125I-prekallikrein also occurred with visualization of a 50,000 molecular weight fragment. Although extensive proteolysis of 131I-high molecular weight kininogen was observed, the cleavage fragments were not the same as those generated during contact activation in vitro. The major fragment of high molecular weight kininogen observed in vivo was at 80,000 molecular weight, in contrast to the 65,000 molecular weight fragment generated by kallikrein in vitro. These results indicate that high molecular weight kininogen can undergo proteolysis in vivo into fragments not known to be associated with kinin release.

    Title Procoagulant Activity in Normal Human Urine Associated with Subcellular Particles.
    Date June 1986
    Journal Kidney International
    Excerpt

    Procoagulant activity (PCA) in normal human urine was found to be sedimented by centrifugation at X 100,000g. Therefore, studies were done to identify the structures associated with the procoagulant activity. Transmission electron microscopy of the X 100,000g pellet revealed numerous membrane-bound vesicles as well as fibrous material. Filtration of normal urine through a 0.2-micron filter removed more than 90% of the procoagulant activity. Scanning electron microscopy of the filter surface revealed 0.1 to 1.1 micron particles and fibrous material. By centrifugation at pH 3 and 5 the fibrous material and particles were separated. The procoagulant activity remained with the particles in each case. The fibrous material was shown to be Tamm-Horsfall protein by SDS-PAGE and Western blotting using anti-Tamm-Horsfall protein serum. Purified Tamm-Horsfall protein itself was not procoagulant. Therefore, PCA in normal human urine is associated with particles 0.1 to 1.1 micron in diameter which appear to be lipid membranes in various arrangements.

    Title A Morphometric Analysis of Pyramidal Tract Structures During Postnatal Undernourishment and Recovery.
    Date June 1986
    Journal Brain Research
    Excerpt

    Rats were postnatally undernourished during the suckling period (up to 20 days) and the brainstems of the perfused rats were dissected and prepared for electronmicroscopy at 21, 35 and 63 days of age. The effects on myelin were relatively mild and consisted primarily of a slight reduction in the relative numbers of myelinated fibers, most likely caused by a lag in the rate of loss of non-myelinated fibers, and fewer lamellae in myelinated axons of less than 2.5 micron circumference. Organelles were examined in the interfasicular oligodendroglia and in paragigantocellular reticular neurons immediately dorsal to the pyramidal tract. The numbers of mitochondrial particles in neuronal perikarya were significantly increased by postnatal undernourishment, although the numbers of other organelles appeared normal. Increased numbers of mitochondria persisted in nutritionally rehabilitated rats. Mitochondrial particles in oligodendroglia were not altered.

    Title Glomerular Injury and Proteinuria in Rats After Intrarenal Injection of Cobra Venom Factor. Evidence for the Role of Neutrophil-derived Oxygen Free Radicals.
    Date April 1986
    Journal The American Journal of Pathology
    Excerpt

    The purpose of these studies was to determine how intravascular complement activation could lead to glomerular injury. Cobra venom factor (CVF) infused into the renal artery of rats resulted in increased excretion of protein in urine, which was maximal over the first 24 hours (51.2 +/- 6.0 mg/24 hours in CVF versus 14.1 +/- 0.9 mg/24 hours in saline-treated animals; P less than 0.001). Depletion of circulating neutrophils with anti-neutrophil serum significantly reduced the CVF-induced proteinuria in the first 24 hours (neutrophil depleted rats 22.7 +/- 2.8 mg/24 hours versus 63.4 +/- 9.9 mg/24 hours in neutrophil intact rats; P less than 0.005). Morphologic abnormalities (which were quantitated morphometrically) included accumulation of neutrophils in glomerular capillary loops, blebbing of endothelial cells, and epithelial cell foot process fusion. The increased protein excretion was reduced by 70% by simultaneous administration of catalase (23 +/- 4.3 mg/24 hours in CVF plus catalase versus 52.1 +/- 10 mg/24 hours in CVF alone; P less than 0.05). Catalase reduced glomerular endothelial cell blebbing and epithelial cell foot process fusion but not neutrophil accumulation in glomeruli as assessed by morphometry. In similar experiments superoxide dismutase, dimethyl sulfoxide, and deferoxamine did not prevent CVF-induced proteinuria. These studies, therefore, suggest that intravascular activation of complement in the rat causes glomerular injury and proteinuria which is dependent on neutrophils and upon the generation of hydrogen peroxide and/or its metabolites.

    Title Prolonged Daily Inhalation of Halothane Modifies the Dose-response Pattern to Acute Administration of Halothane. An Electrophysiological Study.
    Date February 1986
    Journal Neuropharmacology
    Excerpt

    Sensory-evoked field potentials were obtained from freely moving rats implanted sterotaxically with permanent electrodes in the parafasciculus thalami (PF), mesencephalic central gray (CG), ventromedial hypothalamus (VMH) and somatosensory cortex (SCX). Animals were exposed to chronic, subanesthetic inhalation of halothane (0.5%, 3 hr/day, 5 days/week) for 56 days. The averaged acoustic evoked responses (AAER) were recorded on day 0, as well as at 28 and 56 days after a 48-hr halothane-free period ("control") and after acute doses of halothane (0.25, 0.5 and 1.5%). In general, the averaged sensory-evoked responses from each structure were affected at day 0 of the experiment in dose-response manner, and suppression of the responses was the main effect of halothane. Chronic exposure to subanesthetic inhalation of halothane produced marked alteration of the "control" recording from 3 CNS structures; mainly from the mesencephalic central gray, the parafasciculus thalami and the somatosensory cortex and the direction (increase or decrease) of the averaged acoustic evoked responses in all the four CNS sites studied. The total responsiveness was modified as well, i.e. the recordings obtained from the mesencephalic central gray and somatosensory cortex exhibited hypersensitivity while the recordings obtained from the parafasciculus thalami and ventromedial hypothalamus exhibited tolerance. It is concluded that prolonged and intermittent inhalation of halothane can alter the electrophysiological properties of the four structures investigated.

    Title Effect of Reactive Oxygen Species on Myelin Membrane Proteins.
    Date October 1985
    Journal Journal of Neurochemistry
    Excerpt

    Fresh myelin, isolated from brainstems of adult rats, was incubated in the presence of Cu2+ and H2O2. Electrophoretic analysis of the reisolated myelin membrane revealed a gradual loss of the protein moiety from the characteristic pattern and an increase in aggregated material appearing at the origin of the gel. The aggregation of proteins was time-dependent and was concomitant with the accumulation of lipid peroxidation products reactive with thiobarbituric acid. Furthermore, during the course of incubation, there was a gradual decrease in the amount of recovered light myelin and a quantitatively similar increase in heavier myelin subfractions. The aggregation of proteins seems not to be directly related to the buoyant densities of myelin fragments. The peroxidative damage to the myelin proteins may be an important contributor to pathochemistry of myelin sheath, in particular, and in general it implies the susceptibility of the protein moiety of cell membranes to oxygen-induced deterioration.

    Title Hageman Factor in Experimental Nephrotoxic Nephritis in the Rabbit.
    Date October 1985
    Journal Laboratory Investigation; a Journal of Technical Methods and Pathology
    Excerpt

    This study was performed to investigate whether the Hageman factor (HF) system might contribute to glomerular damage in vivo. HF was purified from rabbit plasma. The proteolytic activation pattern of 80,000-dalton rabbit HF was the same as that previously reported for human HF. Anti-HF IgG, raised in a goat, was monospecific as judged by immunodiffusion analysis and inhibited HF activity in rabbit plasma. A telescoped model of nephrotoxic nephritis in the rabbit was developed using guinea pig antirabbit glomerular basement membrane IgG injected into rabbits preimmunized against guinea pig IgG. In this model protein excretion was increased by days 3 to 4 in association with glomerular influx of acute inflammatory cells. By days 5 and 6 fibrin was present within glomerular capillaries, beneath endothelial cells, in Bowman's space, and in proximal tubules. By fluorescent microscopic analysis rabbit IgG and C3 had accumulated along the glomerular capillary wall; however, no HF was detectable in glomerular capillary wall over the initial 10 days of glomerular injury. Positive fluorescence for HF was seen within Bowman's space and in tubules along with albumin and plasmin- and fibrin-related antigens. Although the circulating antigenic HF concentration did not change during the glomerular injury, the rate of turnover of 125I-HF did increase. However, when this was factored for turnover of 131I-albumin in a paired study, the relative turnover of 131I-albumin was found to be faster than that of 125I-HF. Proteolysis of 125I-HF in plasma consistent with HF activation was noted in only one of these rabbits in spite of a decrease in antigenic C3 level to 54% of baseline. The 125I-HF appearing in urine of nephritic rabbits had undergone proteolysis from the native 80,000-dalton parent molecule to form fragments of 50,000 and 30,000 daltons, compatible with HF activation. Urine from nephritic rabbits also contained procoagulant activity that was HF dependent. These results are compatible with the concept that HF passively crosses the damaged glomerular filter where it may become activated in Bowman's space or in fluid draining damaged glomeruli in this model of nephrotoxic nephritis in the rabbit.

    Title Fragmentation and Polymeric Complexes of Albumin in Human Urine.
    Date September 1985
    Journal Clinica Chimica Acta; International Journal of Clinical Chemistry
    Excerpt

    Analysis of urine proteins of some individuals with proteinuria by SDS-PAGE and silver staining revealed protein bands in urine which did not appear to be present in plasma. The bands migrated with apparent molecular weights of 260 000, 180 000, 110 000, 45 000, 40 000, 30 000, 24 000, 18 000 and 11 000. These bands were shown to be albumin polymer and fragments by using a polyclonal antibody to (a) immunoprecipitate radiolabelled urine proteins, and (b) identify bands blotted from SDS-PAGE gels onto nitrocellulose paper. The specificity of the polyclonal anti-albumin antibody was confirmed by using two mouse monoclonal antibodies raised against human albumin which, between them, recognized the same protein bands on nitrocellulose paper as did the polyclonal antibody. The results of these studies of albumin in human urine confirm that albumin exists as polymer and also show that albumin fragmentation occurs in urine. Fragmentation occurs by proteolysis of the albumin molecule both at sites within and outside disulfide loops. The predominant cleavage site appears to be approximately two-fifths of the distance from one end of the albumin molecule to produce disulfide-linked fragments of about 45 000 and 30 000 molecular weight.

    Title Procoagulant Activity in Glomeruli and Urine of Rabbits with Nephrotoxic Nephritis.
    Date September 1985
    Journal Laboratory Investigation; a Journal of Technical Methods and Pathology
    Excerpt

    A telescoped model of nephrotoxic nephritis in the rabbit, using guinea pig antiglomerular basement membrane IgG in rabbits preimmunized with guinea pig IgG, reproducibly induced crescentic nephritis. Procoagulant activity (PCA) was measured in sieve-isolated glomeruli that had been either sonicated or cultured for 48 hours. In both sonicated and cultured glomeruli PCA peaked on days 5 and 6. The time course for appearance of PCA corresponded precisely with the appearance of proteinaceous material containing fibrin in Bowman's space as measured by a light microscopic histologic scoring system and confirmed by immunofluorescence and electron microscopy. Glomerular PCA returned to baseline by days 9 and 10 in spite of progression of glomerular injury. PCA also appeared in urine. Urine PCA peaked on day 8 and persisted through day 12 when glomerular PCA had returned to baseline. Glomerular and urine PCA were characterized using human coagulation factor-deficient plasmas and antithromboplastin IgG. Both glomerular PCA and urine PCA were inhibited by antithromboplastin IgG, showing that thromboplastin (tissue factor) contributed to PCA. The PCA in glomerular sonicates was dependent on factor X, but independent of factor VII or Hageman factor, suggesting that factor VII was present. Following glomerular culture for 48 hours the PCA had changed and in some cases was dependent on Hageman factor, factor IX, and factor VII for full PCA expression. Urine PCA was uniformly Hageman factor dependent and sometimes independent of factors VII and X. No active thrombin was present. The forms of glomerular and urine PCA were, therefore, complex. They seemed to be primarily driven by thromboplastin but also appeared to require the presence of the intrinsic coagulation pathway for full expression of PCA.

    Title Acute Hypotension Due to Platelet Serotonin-induced Chemoreflexes After Intravenous Injection of Dextran Sulfate in the Rabbit.
    Date September 1985
    Journal Circulation Research
    Excerpt

    The hypotension and bradycardia observed after intravenous injection of dextran sulfate in rabbits was prevented by prior depletion of circulating platelets, but was not prevented by depletion of the third component of complement or Hageman factor. Dextran sulfate injection caused immediate thrombocytopenia with temporary localization of platelets within lungs. Morphological analysis revealed platelet aggregates in lung capillaries. The platelets had changed shape and were in the process of degranulating. Serotonin and histamine levels in blood increased approximately 5-fold and 7-fold, respectively, after dextran sulfate injection. The cardiovascular events following dextran sulfate injection were mimicked by intravenous serotonin but not by intravenous histamine injection, although a combination of serotonin and histamine reproduced the pattern of blood pressure changes better than did either agent alone. Quantification of platelets trapped in lung revealed that the potential release of serotonin from trapped platelets could account for the rise in plasma serotonin concentration and the hemodynamic changes observed. Both the dextran sulfate-induced cardiovascular effects and serotonin-induced hypotension were markedly diminished by cutting vagus and depressor nerves, and were virtually abolished by carotid ligation in addition to nerve section. These results support the concept that platelet activation within rabbit lungs may cause hypotension via serotonin-induced chemoreflexes.

    Title Role of Oxygen Radicals in Phorbol Myristate Acetate-induced Glomerular Injury.
    Date July 1985
    Journal Kidney International
    Excerpt

    Phorbol myristate acetate (PMA) is known to be a potent activator of neutrophils and macrophages resulting in the generation of large amounts of oxygen-free radicals by these cells. When injected into the left renal artery of 250 to 300 g male Sprague-Dawley rats, PMA caused significant proteinuria compared to control rats which received normal saline (35.4 +/- 4 mg/24 hr in PMA treated vs. 14.1 +/- 0.9 mg/24 hr in saline control, P less than 0.02). The proteinuria was associated with evidence of glomerular injury. These PMA-induced alterations were not prevented by complement depletion but were prevented by prior depletion of neutrophils. The coinstillation of catalase prevented the development of the proteinuria (catalase + PMA 12.7 +/- 2.3 mg/24 hr vs. PMA alone 38.2 +/- 5.7 mg/24 hr, P less than 0.001) suggesting that H2O2 and/or its metabolites derived from neutrophils were important in the PMA-induced proteinuria. In contrast, superoxide dismutase (SOD) had no effect. We conclude that, following the intra-arterial injection of PMA, neutrophil-derived hydrogen peroxide and/or its metabolic products are capable of causing acute proteinuria in association with morphological alterations in glomeruli of rats.

    Title Intracellular Translocation of Myelin Proteolipid Protein.
    Date May 1985
    Journal Journal of Neurochemistry
    Excerpt

    Brainstem slices prepared from 22-day-old rats were employed to study the intracellular translocation of radioactively labeled myelin proteolipid protein (PLP). Double-isotope and short pulse-chase procedures allowed us to demonstrate the flux of PLP through nine different subcellular membrane fractions that were isolated on the basis of their particle size and buoyant density. Tagged PLP was rapidly depleted from microsomes, showed transient passage through a number of presumably intermediate membranous pools, and accumulated in myelin. On the basis of the kinetics of PLP labeling and isotope ratios, the membranes can be arranged as they participate in the intracellular translocation of PLP and consistently show a pattern indicating possible precursor-product relationships.

    Title The Corpus Callosum During Postnatal Undernourishment and Recovery: a Morphometric Analysis of Myelin and Axon Relationships.
    Date April 1985
    Journal Brain Research
    Excerpt

    This study was designed to compare morphometric relationships between myelin lamellae and axons in undernourished and well nourished developing rats, and in rats nutritionally rehabilitated for two weeks. Although sampling techniques employed in this study were not specifically designed to compare numbers of myelinated fibers in test and control populations, we did observe a trend indicating a reduction in the numbers of myelinated fibers. The mean numbers of myelin lamellae, from an average of all myelinated axons, were not different in control and test population. However, regression analysis of axon sizes by numbers of myelin lamellae revealed significant differences from the normal in 21-day-old undernourished rats. For callosal axons of any size, there were too few myelin lamellae in the undernourished rats. A partial recovery was observed in relatively small fibers by 35 days of age, but no recovery was observed in larger sized fibers. Comparison of the frequency distribution of axon circumferences of myelinated fibers revealed an increase in average axonal caliber. Computation shows that although mean numbers of lamellae were not altered by undernourishment, the axons themselves are increased in size by about 10%. This unexpected result indicates that the relationship normally governing the numbers of myelin lamellae is altered by postnatal nutritional deprivation, and that the relatively larger axon calibers do not produce in the ensheathing oligodendroglia any compensatory increase in the layers of myelin.

    Title Polymeric Complexes and Fragments of Albumin in Normal Human Plasma.
    Date December 1984
    Journal Clinica Chimica Acta; International Journal of Clinical Chemistry
    Excerpt

    Nitrocellulose blots of normal human plasma proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were examined for polymeric complexes and fragments of albumin using an immunoperoxidase-labelled mouse monoclonal anti-human albumin antibody. Under reducing conditions, no polymeric complexes were seen. Under non-reducing conditions, polymeric complexes were detected at the following molecular weights: 210 000, 168 000, 147 000, 132 000, and 110 000. These probably represent both homo- and heteropolymers of albumin. Fresh plasma samples were also analyzed by S-200 chromatography with the same results indicating that detection of polymeric complexes was not an artifact of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis technique. In quantitative terms, polymeric complexes constituted 0.3-2.8% of the total albumin present. Fragments of albumin were also seen in normal human plasma with molecular weights of 45 000, 28 000 and 19 000. These fragments probably represent breakdown products of albumin in normal blood, and they constituted less than 2% of the total albumin present.

    Title Undernutrition and the Development of Brain Neurotransmitter Systems.
    Date December 1984
    Journal Life Sciences
    Excerpt

    While there are homeostatic mechanisms to protect the brain against wide fluctuations in the availability of essential nutrients, food deprivation is known to influence brain neurochemistry. Given the growing problem of infant undernutrition and the fact that the developing nervous system appears to be especially vulnerable to this type of insult, numerous studies have been conducted to define the relationship between nutritional factors and cellular growth and maturation in the brain. The data suggest that the development of both neural and nonneural elements are significantly affected by undernutrition. This includes processes and substances important for neurotransmission such as transmitter synthesis, degradation and receptor sites. Because many neuropsychiatric conditions can be traced to dysfunctions in synaptic neurochemistry, it is possible that some of the central nervous system abnormalities which result from childhood undernutrition may be a consequence of a modification in synaptic biochemistry. The present report reviews data relating to this issue with the aim of assessing its relevance to developmental neurobiology.

    Title Differential Growth Recovery Within the Brains of Postnatally Undernourished Rats.
    Date November 1984
    Journal Brain Research
    Title Brain Myelination in the Offspring of Ethanol-treated Rats: in Utero Versus Lactational Exposure by Crossfostering Offspring of Control, Pairfed and Ethanol Treated Dams.
    Date November 1984
    Journal Brain Research
    Excerpt

    Pregnant Long-Evans rats were received on day 5 of gestation and divided into 4 treatment groups: (1) 27% calories provided as ethanol in a liquid diet; (2) pairfed i.e., isocaloric liquid diet restricted to match group (1); (3) liquid diet provided ad libitum; and (4) laboratory chow and water provided ad libitum. Litters were culled to 8 pups at birth and crossfostered across dams in all 4 groups to provide offspring falling into 16 different experimental groups, including some exposed to ethanol in utero only and some exposed only during lactation. At birth, blood alcohol levels of dams, culled pups and alcohol levels in the stomach contents of culled pups were measured. All pups were weaned and fed laboratory chow and water ad libitum from 21 days onward. At ages 16, 21, 30 and 52 days, pups were sacrificed, and organ/body weight ratios and brain myelin concentrations were determined. Ethanol treated dams had longer gestational periods. The offspring of ethanol treated dams which were crossfostered to pairfed and well nourished dams during lactation had delayed eye opening, persistent lag in body growth and slightly lower brain myelin concentrations. Offspring of dams which were either pairfed or well nourished during gestation, but crossfostered during lactation to ethanol treated dams, had abnormal organ weights, abnormal brain weights and severely depressed brain myelin concentrations persisting through 52 days of age. Thus, lactational ethanol effects on brain myelin were more severe than gestational effects; body growth was affected more severely by gestational exposure, and gestational effects were generally less severe with adequate nutrition.

    Title Myelination of the Rat Optic Nerve During Postnatal Undernourishment and Recovery: a Morphometric Analysis.
    Date November 1984
    Journal Brain Research
    Excerpt

    Newborn rats were undernourished from the second postnatal day through 20 days of age and weaned to a diet of laboratory chow ad libitum. Optic nerve development was examined by various light and electron microscopic techniques at 14, 21, 35 and 63 days of age. The degree of undernourishment achieved resulted in body growth lag comparable to results obtained in our previous studies. Although cellularity (cells per photomicrograph area) of the oligodendroglia was unaffected, there was an apparent significant relative reduction in the total number of myelinated fibers by 21 days of age, as determined by light microscopic sampling. Ratios of unmyelinated-to-myelinated fibers were thus estimated by electron microscopy, and results indicated an early increase in the ratio (14 days). Either as the result of catch up in a developmental lag, or as a result of possible restorative effects of rehabilitation, these differences were significant by 35 days of age. The relationship between axon circumference and numbers of myelin lamellae was determined by regression analysis, which revealed a significant reduction in numbers of lamellae over axons of all sizes at 14 days. By 21 days, only fibers in the size range of 1-2 micron of circumference showed a difference, and by 35 days there were no significant differences. These results all indicate that there is a significant myelin reduction in optic nerve of the undernourished rat.

    Title Evidence for the Role of Oxygen Radicals in Acute Nephrotoxic Nephritis.
    Date November 1984
    Journal Laboratory Investigation; a Journal of Technical Methods and Pathology
    Excerpt

    Acute glomerular injury in the rat has been induced by the intrarenal, intraarterial infusion of sheep antibody to glomerular basement membrane (antiglomerular basement membrane). The antiglomerular basement membrane antibody has been verified to be of the variety that is complement and neutrophil dependent for the induction of acute proteinuria, which peaks during the first 24 hours. Following injection of the antibody, acute, intense, glomerular injury resulted, with the denuding of glomerular vascular basement membrane associated with extensive damage or destruction of glomerular endothelial cells and fusion of epithelial cell foot processes. Treatment of animals with catalase produced, in a dose-dependent manner, as much as 75% protection against glomerular injury, as assessed by reduction in the proteinuria. Treatment of animals with superoxide dismutase caused a small reduction in the degree of glomerular injury, again assessed by a reduction in proteinuria. However, this protective effect of superoxide dismutase was not found to be statistically significant. The hydroxyl radical scavenger, dimethyl sulfoxide, which has been shown to protect against endothelial cell injury following systemic activation of complement, was not protective in the anti-GBM model. Morphologically, glomeruli from catalase-protected rats showed numerous neutrophils but little or no evidence of injury of either glomerular endothelial or epithelial cells. These data suggest that acute glomerular injury produced by antiglomerular basement membrane is related to H2O2 production from activated neutrophils.

    Title The Synthesis of Myelin and Brain Subcellular Membrane Proteins in the Offspring of Rats Fed Ethanol During Pregnancy.
    Date August 1984
    Journal Brain Research
    Excerpt

    Pregnant Long-Evans rats received either: (1) liquid diet containing 5.15% ethanol; (2) liquid diet pair fed to (1) for total calories; or (3) liquid diet ad libitum. These special diets were administered from the 5th through the 18th days of gestation. Dams received standard laboratory chow and water ad libitum before and after the test interval. Additional dams received standard chow and water throughout the study. Birth weights of offspring in the ethanol group were lower than for offspring of the pair-fed or control groups, and their subsequent growth lagged behind the other groups. Neonate deaths in the ethanol group outnumbered other deaths. Eye opening was delayed, and brain weights appeared low from 16 to 30 days postnatal age, The onset of myelin synthesis was delayed by several days; however, by 30 days of age, the rate of myelin synthesis and net accumulation was comparable to the offspring of pair-fed controls. Thus, the effect of ethanol on brain myelination in the offspring of subject females appears as a delay in myelin initiation and cannot be fully explained by caloric undernourishment. An unexpected observation involved offspring of females fed standard chow throughout the study. The brain myelin concentration in this group was lower than for any of the other groups, which may relate to the higher fat content of the liquids diets and/or the comparatively slow weight gain of pregnant rats on standard chow.

    Title In Vitro Activation of the Contact (hageman Factor) System of Plasma by Heparin and Chondroitin Sulfate E.
    Date July 1984
    Journal Blood
    Excerpt

    A large number of negatively charged macromolecules, including DNA, glycosaminoglycans, and proteoglycans, were tested as possible activators of the contact (Hageman factor) system in vitro. Activation was assessed by conversion of prekallikrein to kallikrein, as determined by amidolytic assay and by cleavage of 125I-Hageman factor into 52,000- and 28,000-dalton fragments. Of particular interest to these studies, heparin proteoglycan and glycosaminoglycan from rat peritoneal mast cells, and squid chondroitin sulfate E, which is representative of the glycosaminoglycan from cultured mouse bone marrow derived mast cells, induced the reciprocal activation between Hageman factor and prekallikrein. In addition, naturally occurring heparin glycosaminoglycans from pig mucosa, bovine lung, and rat mast cells also induced activation. In contrast, native connective tissue matrix glycosaminoglycans and proteoglycans from several sources were inactive, although when one such chondroitin sulfate was further sulfated in vitro, it gained activity. When the negative charge of the activating agents was blocked by the addition of hexadimethrine bromide, the cleavage of 125I-Hageman factor in the presence of prekallikrein was prevented. The active negatively charged macromolecules induced cleavage of 125I-high molecular weight kininogen in normal plasma but not in Hageman factor-deficient or prekallikrein-deficient plasmas. Reconstitution of prekallikrein-deficient plasma with purified prekallikrein restored the kininogen cleavage upon addition of the active proteoglycans. These results suggest that both heparin from connective tissue mast cells and highly sulfated chondroitin sulfate E from cultured mouse bone marrow derived mast cells (which are considered synonomous with mucosal mast cells) could activate the contact system of plasma subsequent to an activation secretion response.

    Title Brain Hypomyelination During Postnatal Undernourishment: a Comparison of Proteolipid Protein Synthesis Versus Assembly into Membrane.
    Date June 1984
    Journal Experimental Neurology
    Excerpt

    A double isotope methodology which permits the separate analysis of (i) the synthesis of myelin proteolipid protein and (ii) its subsequent assembly into myelin membrane was applied to the case of postnatal undernourishment of rats. Results showed that brain proteolipid protein synthesis was greatly depressed, indicating that undernourishment limited the production of myelin components. This is a different result from that obtained in the mouse Quaking mutant, in which case component formation is normal whereas their assembly into myelin membrane is blocked. The results have possible implications regarding cellular development of oligodendroglia of undernourished rats.

    Title The Relationship Between Nutritional Adequacy and Brain Myelin Accumulation: a Comparison of Varying Degrees of Well Fed and Undernourished Rats.
    Date March 1984
    Journal Brain Research
    Excerpt

    Rats undernourished through 17 days of age to produce mild weight lag (less than 20%) had either the same amount, or slightly more myelin than well fed controls. Schedules of undernourishment producing approximately 30, 40 and 50% body weight lags produced corresponding rank-ordered myelin deficits of 25, 55 and 60%, respectively. Brain growth was relatively spared in all cases, never exceeding a deficit of 10%. Absolute myelin deficits did not recover following nutritional rehabilitation, although myelin continued to increase in both normal and all test populations.

    Title Distribution of Radioactivity Among Total Myelin Protein Amino Acids Following Administration of Labeled Glycine, Leucine, or Methionine.
    Date January 1984
    Journal Journal of Neuroscience Research
    Excerpt

    This paper analyzes the distribution of radioactivity in the different amino acids of brain myelin protein for up to 6-7 weeks after an intracranial pulse administration of radioactive leucine, glycine, or methionine. Results show that there is no significant accumulation or reutilization of protein radioactivity in any form other than the one administered.

    Title Postnatal Increase in the Metabolism of Leucine and Valine.
    Date January 1984
    Journal Biology of the Neonate
    Excerpt

    The catabolism of tritium-labeled leucine, valine, and glycine was determined by measuring the appearance of tritium-labeled water at various postnatal and adult ages in rats. Results for leucine and valine show a marked increase in the formation of labeled water during the 3rd postnatal week. The partial exclusion of leucine and valine from brain, as a result of the blood-brain barrier, particularly enhances observation of labeled water formation in that the product (water) and precursor (amino acid) are largely separated (supporting experiments demonstrate the prior existence of the blood-brain barrier for leucine). Results for glycine indicate its extensive metabolic degradation at all postnatal ages. These data indicate that the metabolic rate of intraperitoneally administered, radioactively labeled leucine and valine changes appreciably during early postnatal development. The early postnatal manifestation of human disorders of branched-chain amino acid metabolism is consistent with the chronology of development in the rat.

    Title Inhibition of Serotonin-mediated Cardiovascular Reflexes by Cimetidine in the Rabbit.
    Date November 1983
    Journal General Pharmacology
    Title Kinin Release from High Molecular Weight Kininogen by the Action of Hageman Factor in the Absence of Kallikrein.
    Date August 1983
    Journal The Journal of Biological Chemistry
    Excerpt

    Proteolysis of 125I-high molecular weight (Mr) kininogen occurred in kaolin-activated plasma which was deficient in prekallikrein, but not in plasma which lacked both prekallikrein and Hageman factor (HF) activity. The implication of this observation is that HF itself might be capable of releasing kinin from high Mr kininogen. This concept was further supported by the following studies. In a purified protein system rabbit (Mr = 80,000) two-chain activated HF (alpha-HFa) incubated with human 125I-high Mr kininogen caused rapid proteolysis of the kininogen in the presence, but not in the absence, of kaolin. This was in contrast to the effect of kallikrein on proteolysis of high Mr kininogen which was inhibited 10-fold by the presence of kaolin. The possibilities that the alpha-HFa preparation was contaminated by rabbit kallikrein or that the human high Mr kininogen preparation was contaminated by human prekallikrein were excluded by using specific antibodies (IgG) against these proteins. The proteolytic fragments of 125I-high Mr kininogen generated by both alpha-HFa and kallikrein were indistinguishable by sodium dodecyl sulfate-polyacrylamide gel analysis. Bioassayable kinin was released from high Mr kininogen following incubation with alpha-HFa in the presence of kaolin. The amount of kinin released in proportion to the extent of proteolysis was the same for both kallikrein and alpha-HFa. The data show that activated Hageman factor may cause release of kinin by proteolytic cleavage of high Mr kininogen. This phenomenon occurs not only in a purified system of proteins but also in kaolin-activated plasma.

    Title Relative Halothane Accumulation in Brain Subcellular Membranes in Vitro.
    Date June 1983
    Journal Neurochemical Research
    Excerpt

    The accumulation of halothane in brain homogenates was compared with halothane accumulation in brain during inhalation at anesthetic and subanesthetic levels. Anesthesia is achieved at a tissue concentration well below the halothane solubility in brain tissue. Analysis of halothane in the particulate solids of brain homogenate and in purified subcellular membranes indicates that a membrane constituent (presumably the lipids) acts as an ideal solvent in which halothane is fully miscible. Therefore, membranes offer a local microenvironment in which halothane accumulation deviates from Henry's law. Specifically, we observe that even slight increases of halothane in a saline medium result in a relatively large increase in the concentration of halothane in subcellular membranes suspended in the medium, eventually leading to solvation of the membrane in halothane. This observation offers a ready explanation for the high degree of positive correlation between MAC and lipid solubility and the small difference between anesthetic and lethal concentrations of halothane during inhalation. The rate of halothane increase in myelin exceeded the rate in other brain subcellular membranes, indicating that a major site of halothane localization is within this subcellular membrane.

    Title Catecholamines in Rat Brain Following Postnatal Undernutrition and Nutritional Rehabilitation.
    Date April 1983
    Journal Journal of Neuroscience Research
    Excerpt

    Norepinephrine and dopamine were examined in 19 discrete brain areas from the telencephalon, diencephalon, and mesencephalon of nutritionally rehabilitated adult rats following postnatal undernutrition from birth through 21 days of age. Following rehabilitation, catecholamine levels were not significantly different from control values in any of the areas examined. Catecholamine concentrations in young, undernourished rats are generally elevated (either from stress or from nutritional insufficiency). Data presented here show that whichever case is true, the early effect of undernourishment is transient and that normal values are restored by nutritional rehabilitation.

    Title Agarose Drop Method for Loading Thin Polyacrylamide Gels.
    Date March 1983
    Journal Analytical Biochemistry
    Title The Effect of Postnatal Caffeine Administration on Brain Myelination.
    Date January 1983
    Journal Brain Research
    Title Propagation of Photic Evoked Responses Recorded from the Retina, Optic Chiasm, Lateral Geniculate Body, and Visual Cortex of the Nutritionally Rehabilitated Rat Visual System.
    Date December 1982
    Journal Experimental Neurology
    Title Brain Maturation Following Administration of Phenobarbital, Phenytoin, and Sodium Valproate to Developing Rats or to Their Dams: Effects on Synthesis of Brain Myelin and Other Subcellular Membrane Proteins.
    Date December 1982
    Journal Journal of Neurochemistry
    Excerpt

    The anticonvulsant drugs phenobarbital, phenytoin, sodium valproate, and phenytoin-sodium valproate in combination were administered daily to (a) pregnant rats starting on the 5th day after conception, and continued through 17 days postpartum, or (b) to developing rats between 3 and 17 days of age. Each drug was prepared in water and administered at either a therapeutic dose (TD), three times therapeutic dose (3TD), or 9TD. Drug administration had no discernible effect on litter size or sex ratio in the offspring; however, phenobarbital administration to dams caused small but significant reductions in birth weights. Body weights of developing rats treated with anticonvulsant drugs either via dams of directly by intraperitoneal injection lagged behind controls. At 20-24 days of age the brain weights of the offspring of phenobarbital (9TD)-exposed dams lagged control weights by 5% whereas brain weights in the offspring of the other treated groups were indistinguishable from controls. In contrast, administration of phenobarbital directly to developing rats caused no significant brain weight deficits whereas significant deficits were observed with phenytoin (9TD), sodium valproate (9TD), and phenytoin-sodium valproate (9TD) in combination. AT 20-24 days of age the relative incorporation of radioactive leucine into purified myelin and crude nuclear proteins of drug-treated rats or the offspring of drug-treated dams was reduced by 10-20% in all cases. Dose-related differences were not observed however, and the effects of phenytoin and sodium valproate in combination approximated those of phenytoin administered alone.

    Title Accumulation and Disappearance of Enflurane from Rat Brain.
    Date December 1982
    Journal Neurochemical Research
    Title Myelin Development and Nutritional Insufficiency.
    Date October 1982
    Journal Brain Research
    Excerpt

    Postnatal undernourishment does not greatly retard the generation of rat brain cells, although there is a slight reduction in total cell numbers and brain size. Possibly the maturation of cells is more severely affected. The ratio of myelinated to non-myelinated fibers is greatly reduced in the corpus callosum and pyramidal tract, and presumably in other areas as well. There is only a slight reduction in the numbers of myelin lamellae for axons of a given size. The recovery of brain myelin and the incorporation of radioactive precursors into purified myelin proteins and lipids are all greatly reduced, leading to a comparatively severe reduction in the brain myelin concentration. The myelin composition is only slightly altered, possibly as a result of delay in its normal chemical maturation. The actual vulnerable period that produces a lasting myelin deficit is the early period that includes oligodendroglia cell proliferation, whereas undernutrition restricted to a later period that includes the actual peak of myelin does not cause a lasting reduction in the brain myelin concentration. The belief that stunting the postnatal proliferation of oligodendroglia largely accounts for the myelin effect has not been substantiated by direct analysis of cell numbers. Consequently, the observed hypomyelination likely results from a failure of oligodendroglia to mature and to initiate myelin formation. The myelin deficit appears largely uniform throughout the brain. Indirect evidence in human studies indicate that comparable effects appear in undernourished infants.

    Title Chronic Subanesthetic Halothane Exposure Causes Selective Alterations in Neurotransmitter Systems in Discrete Brain Regions.
    Date August 1982
    Journal Experimental Neurology
    Title Synthesis of Myelin, Particulate, and Soluble Protein Subfractions of Rat Sciatic Nerve During the Early Stage of Wallerian Degeneration: a Comparison of Metabolic Studies Using Double and Single Isotope Methods and Recovery.
    Date June 1982
    Journal Neurochemical Research
    Excerpt

    The recovery, electrophoretic composition and synthesis of the myelin, particulate protein and soluble protein subfractions of rat sciatic nerve were compared in normal, sham-operated, and degenerating rat sciatic nerve at one, three and five days after neurotomy. Both single and double isotope methods were used to measure changes in synthesis in vitro and double isotope methods were used in vivo. The wet weights of nerves undergoing Wallerian degeneration for 5 days increased by 40 percent compared to normal and sham-operated nerves. The recovery, specific radioactivity, and synthesis of the myelin was reduced. The effect on myelin protein synthesis was similar in vitro and in vivo. The myelin loss was relatively constant in amount (30-40 microgram) regardless of differences in nerve sizes of young and old rats, consequently the percentage of myelin loss was inversely proportional to nerve size. The recovery of particulate protein increased, its rate of synthesis remained unchanged, and accordingly the specific radioactivity was decreased. The recovery, specific radioactivity, and the rate of synthesis of the soluble protein fraction were all elevated. The protein composition of the three fractions, as analyzed qualitatively by polyacrylamide disc gel electrophoresis, remained essentially unchanged through five days of degeneration. With regard to comparisons of the single and double isotope methods, results shows that the latter are more ideally suited to measuring changes in synthesis during the non-steady state conditions that are characteristics of rapid degeneration.

    Title Cerebral Energy Metabolism During the Onset and Recovery from Halothane Anesthesia.
    Date June 1982
    Journal Neurochemical Research
    Excerpt

    Halothane (1%) was administered to twenty-two gram female Swiss-Albino mice which were sacrificed at times of 15 seconds, 45 seconds, 79 seconds and 5 minutes. Additional animals were exposed for 5 minutes and sacrificed 10 minutes after removal from halothane (recovery). Selected energy metabolites were measured in 100-500 nanogram samples from the inferior colliculus and the ascending reticular activating system. Results from this study showed an increase in glucose levels at 79 seconds, when the animals first lost their righting response. The glucose increase was similar in the inferior colliculus and reticular formation. ATP and phosphocreatine were increased at 45 seconds, and during the sleep period in the ascending reticular activating system, and returned to normal during the recovery period. In the inferior colliculus. ATP was similarly increased from 45 seconds throughout the time course, whereas phosphocreatine was elevated at 79 seconds, and during recovery only. These data suggest a decrease in utilization of energy metabolites during halothane anesthesia, both in cells of the inferior colliculus and ascending reticular activating system.

    Title Does Chronic Halothane Exposure Alter Brain Electrical Activity? Sensory Evoked Potentials Recorded from Cortex, Diencephalon, and Mesencephalon in Freely Behaving Rats.
    Date March 1982
    Journal Substance and Alcohol Actions/misuse
    Title Ethanol-induced Modification of Sensory Evoked Potentials Recorded from the Caudate Nucleus, Substantia Nigra, Hypothalamus, and Pineal.
    Date December 1981
    Journal Neuropharmacology
    Title Analysis of Distribution of Rat Sciatic Nerve Protein Among Soluble, Insoluble, and Myelin Subfraction.
    Date November 1981
    Journal Neurochemical Research
    Title Halothane Actions in the Rabbit Hippocampus: Correlative Neurophysiologic and Neurochemical Effects.
    Date October 1981
    Journal Experimental Neurology
    Title Chemotactic Activity Generated from the Fifth Component of Complement by Plasma Kallikrein of the Rabbit.
    Date September 1981
    Journal The Journal of Experimental Medicine
    Excerpt

    Rabbit plasma kallikrein incubated with rabbit C5 resulted in the generation of chemotactic and secretagogue activity for rabbit neutrophils. This effect on C5 appeared to be due to kallikrein itself and not to a contaminating enzyme, because it could be inhibited by anti-kallikrein IgG or by soybean trypsin inhibitor to the same extent the kinin generation by the same kallikrein preparation was inhibited by these agents. The chemotactic response was consistent with the generation of a C5a-like peptide from C5 because the effect could be partially inhibited by carboxypeptidase N and was related to the generation of a small (approximately 14,000 mol wt) fragment of C5. No direct chemotactic response was detectable for kallikrein, activated Hageman factor, high-molecular weight kininogen, or intact C5. Incubation of Kallikrein, high-molecular weight kininogen, and Hageman factor together, so that activation of all three proteins occurred, did not results in the generation of detectable chemotactic activity.

    Title The Relative Numbers of Oligodendroglia in Different Brain Regions of Normal and Postnatally Undernourished Rats.
    Date September 1981
    Journal Brain Research Bulletin
    Excerpt

    The relative numbers of oligodendroglia were compared in representative brain regions of 21 day old undernourished and control rats. As a result of postnatal undernutrition which produced half normal body weights and a 10-15 percent reduction in brain weight, the relative numbers of oligodendroglia were slightly increased in photomicrographs of corticospinal tract (a motor tract), medial lemniscus (a sensory tract), red nucleus (a motor nucleus) and somatosensory cortex. Relative numbers were reduced in the corpus callosum, and the thickness of the corpus callosum was significantly reduced. Cell sizes of oligodendroglia were essentially normal throughout the brain, although some reductions of 5 to 6 percent were observed. Areas of brain structures in cross section were essentially unchanged. We have previously hypothesized that nutritionally induced brain hypomyelination results from a reduction in the specific numbers of oligodendroglia and consequently a lasting reduction in the brain myelin concentration. The present results are inconsistent with this hypothesis, as both the density of oligodendroglia and sizes of brain regions are essentially normal. We know from prior work using the same model of nutritional deprivation that myelin synthesis is greatly reduced. Consequently an important depressant effect of undernourishment on oligodendroglia in the developing brain involves either the communication between axons and oligodendroglia leading to myelin induction or the synthetic capacity to make myelin.

    Title A Possible Effect of the Methylxanthines Caffeine, Theophylline and Aminophylline on Postnatal Myelination of the Rat Brain.
    Date September 1981
    Journal Brain Research
    Excerpt

    A double isotope methodology was used to assess the effect of methylxanthine administration on membrane protein synthesis in developing rat brain. Rat pups were given either aminophylline, theophylline, or caffeine in a dosage of 40 mg/kg or 80 mg/kg daily from the second postnatal day through 20 days of age. Results show depressed myelin protein synthesis at 21.24 days by theophylline (80 mg/kg) and caffeine (40 and 80 mg/kg). Synthesis was essentially normal at 27-28 days of age, indicating a possible delay in development followed by a 'catch-up' phenomenon.

    Title Adrenergic Neuroplasticity is Maintained in the Nutritional Rehabilitated Adult Rat.
    Date August 1981
    Journal Experientia
    Title Halothane Accumulation in Rat Brain and Liver.
    Date June 1981
    Journal Neurochemical Research
    Excerpt

    Halothane concentrations (microgram/g wet weight) was measured in rat brain and liver following exposure to various concentrations of halothane in air. Because of the difficulty of determining the amount of a volatile compound in brain, we analyzed tissue fixed by two different methods. The apparent concentration of halothane in brain was higher following direct decapitation into liquid nitrogen, than after decapitation, removal of fresh tissue, and then freezing. However, the relative effects of altering the inspired concentration were essentially the same in each case. Thus, absolute quantitative accuracy remains a point for discussion; however, we can reach several conclusions regarding the relative accumulation of halothane in brain tissue following various conditions of exposure. Resultant tissue concentrations of halothane were not linearly related to ambient concentrations. Above an inspired concentrations of 1.0%, an increase to 1.5% inspired concentration caused little further increase in the halothane concentration in brain, although the liver concentration increased in proportion to the dose increase. Below an inspired concentration of 0.5%, tissue concentrations were less expected, probably as a result of metabolic degradation occurring at a rate that becomes more noticeable at lower inspired concentrations. Body size was shown to be an important variable affecting the time required for each tissue to reach equilibrium at a given inspired concentration. These data indicate that tissue concentrations at low exposure levels may be less than proportional at dose and that concentrations in small laboratory animals may be expected to exceed values in humans under equivalent conditions of exposure.

    Title A Morphometric Comparison of Central and Peripheral Hypomyelination Induced by Postnatal Undernourishment of Rats.
    Date June 1981
    Journal The Journal of Nutrition
    Excerpt

    Developing Long-Evans rats were undernourished to produce a body-weight deficit of 39% at the age of weaning. Well-nourished litter mates were used as controls. Morphometric analyses were made of pyramidal tracts and posterior tibial nerves of each animal. In measuring pyramidal tract, we observed that axonal circumferences of myelinated fibers were smaller in the undernourished rats and that the number of myelin lamellae per axon appeared reduced by a small amount. The most striking observation in the undernourished rats compared to the controls was that the proportion of myelinated fibers was decreased by 40% at 20 days of age. Axon circumferences of nonmyelinated axons were not measured. Similar decreases were observed in myelinated axon circumference and myelin lamellae of posterior tibial nerves of undernourished rats. Because of sampling problems, we did not attempt to compare the proportion of myelinated and nonmyelinated fibers in posterior tibial nerves of undernourished and well-nourished rats. These morphometric data, particularly the reduction of myelinated fibers, are consistent with biochemical studies of brain hypomyelination in undernourished rats. The data indicate that the mechanism of hypomyelination in nutritionally deprived rats involves a failure of the "trigger" by which myelin-forming cells begin to sheath axons in myelin.

    Title The Diagnosis of Primary Hyperaldosteronism.
    Date April 1981
    Journal Lancet
    Excerpt

    An aldosterone-suppression test based on a simple method of extracellular-fluid volume expansion over three days reliably discriminated between patients with aldosterone-producing adenomas, idiopathic adrenal hyperplasia, and essential benign hypertension. In patients with primary hyperaldosteronism adrenal-vein plasma aldosterone/cortisol concentration ratios successfully lateralised all 21 adenomas. In patients with an adenoma the contralateral adrenal gland was always suppressed, as indicated by a ratio which was less than that seen in the lower inferior vena cava, whereas in patients with hyperplasia the adrenal-vein aldosterone/cortisol concentration ratio from each adrenal was always greater than that seen in the lower inferior vena cava. Thus adrenal-vein sampling not only lateralises solitary adenomas but also discriminates between patients with an adenoma or hyperplasia. However, in view of the diagnostic reliability of the suppression test, it is suggested that adrenal-vein sampling is unnecessary in hyperaldosteronism due to adrenal hyperplasia.

    Title Immune-complex-mediated Biologic Effects.
    Date March 1981
    Journal The New England Journal of Medicine
    Title Brain Cyclic Nucleotide Responses to Anesthesia with Halothane Delivered in Air or Purified Oxygen.
    Date March 1981
    Journal Journal of Neurochemistry
    Excerpt

    Inhalation of either 0.5% or 1.0% halothane in air caused a slight decrease in the cAMP concentration in rat cerebral cortex and cerebellum. During recovery, concentrations returned to normal in 3 h, or less. In contrast, cGMP decreased sixfold in cerebellum, but increased twofold in cortex. Recovery time for cerebellum was several hours. When oxygen was used as the carrier gas for halothane delivery, cAMP in the cortex doubled, in striking contrast to the case with halothane in air. Oxygen alone had no apparent effect. The cGMP effect of halothane delivered in oxygen appeared the same as for halothane in air. Thus, the cAMP effects of brain halothane are related to the enrichment of oxygen.

    Title A Halothane-related Effect on Rat Brain Myelination: a Comparison of Chronic Prenatal or Postnatal Exposure.
    Date March 1981
    Journal Journal of Neurochemistry
    Excerpt

    Rats were exposed to 0.5% halothane in air for 8 h per day during the intervals (1) 5 days postconception to birth, (2) birth to 5 days postnatal age, or (3) birth to 10 days postnatal age. Controls were exposed to an equivalent flow of air. Prenatal exposure had no significant effect on body or brain weight and no subsequent effect on the relative synthesis of brain subcellular membranes. Five days of postnatal exposure caused a 10% reduction in body and brain weight and a 10% relative reduction in the synthesis of brain myelin. The effect persisted throughout the period rapid postnatal brain myelination. Ten days of postnatal exposure produced equivalent, more severe effects on body and brain weights and a more severe effect on myelin synthesis. Postnatal exposure had no apparent effect on the relative synthesis of non-myelin particulate proteins.

    Title Brain Cyclic Nucleotide and Energy Metabolite Responses to Subanesthetic and Anesthetic Concentrations of Halothane.
    Date December 1980
    Journal Experientia
    Excerpt

    Adult rats were exposed to 0.5, 1.0 and 1.5% halothane, delivered in air, for 1 h. Whole brain 3',5'-cyclic adenosine monophosphate (cAMP) of halothane-exposed rats showed only a slight increase relative to control values. 3',5'-Cyclic guanosine monophosphate (cGMP) was increased significantly in halothane-exposed rats, and the response was directly related to the halothane concentrations. Adenosine triphosphate (ATP) and phosphocreatine (PC) remained unchanged relative to control values. Correspondence of these values to apparent discrepancies in the literature is discussed.

    Title Pattern of Myelin Breakdown During Sciatic Nerve Wallerian Degeneration: Reversal of the Order of Assembly.
    Date December 1980
    Journal The Journal of Cell Biology
    Excerpt

    Myelin sheaths of rapidly growing rats were sequentially labeled with the 3H and 14C isotopes of leucine as precursors of protein synthesis. The two injections were separated by time intervals ranging from 2 to 12 d. Wallerian degeneration was initiated by sciatic nerve neurotomy at 2 or 10 d after the second injection of radioactivity. After 5 d of degeneration, myelin was purified and the ratio of isotopes was determined in the delipidated protein. Regardless of the order in which the two isotopes were administered, the relative recovery of radioactivity resultant from the second injection was greatly reduced in degenerating nerves compared with sham-operated controls. Radioactivity incorporated from the first injection was also reduced, but to a lesser extent. Consequently, the isotope ratio corresponding to the first/second injection was greater in degenerating nerves than in controls, and the ratio increased in proportion to the time interval separating the two injections. The magnitude of the effect of degeneration was only slightly greater when degeneration was initiated 2 d after the second injection than when initiated 10 d after the last injection. Consequently, myelin disintegration rather than diminished incorporation of radioactivity accounts for the losses of radioactivity. Furthermore, the pattern of myelin degeneration preferentially involves the last myelin to be formed.

    Title The Electroretinogram of Adult Rats Following a Period of Postnatal Undernutrition and Prolonged Nutritional Rehabilitation.
    Date September 1980
    Journal Life Sciences
    Title Accumulation and Persistence of Halothane in Adult and Fetal Rat Brain As a Result of Subanesthetic Exposure.
    Date August 1980
    Journal Journal of Neurochemistry
    Title A Comparison of Starvation Models in Studies of Brain Myelination.
    Date July 1980
    Journal Neurochemical Research
    Title The Effect of Postnatal Undernourishment on Epileptiform Kindling of Dorsal Hippocampus.
    Date May 1980
    Journal Experientia
    Excerpt

    Rats were undernourished postnatally from birth through 20 days of age. They were subsequently tested for susceptibility to motor seizures kindled in hippocampus in adulthood. Compared to littermate control animals the postnatally undernourished rats were more susceptible to the kindling treatment. We conclude that early postnatal undernourishment has a permanent effect on susceptibility of the hippocampus to electrically-induced seizures.

    Title Phosphorylation and Fucosylation of Myelin Protein in Vitro by Sciatic Nerve from Developing Rats.
    Date April 1980
    Journal Journal of Neurochemistry
    Title Activation of Rabbit Hageman Factor by Homogenates of Cultured Rabbit Endothelial Cells.
    Date February 1980
    Journal The Journal of Clinical Investigation
    Excerpt

    Rabbit Hageman factor was proteolytically cleaved and activated by a homogenate prepared from cultured rabbit endothelial cells. Cleavage of radiolabeled Hageman factor was monitored by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Endothelial cell-mediated cleavage of Hageman factor was demonstrated both in a purified system and in plasma, was time and concentration dependent, and was associated with formation of the characteristic 28,000 M(r) form of active Hageman factor. The rate of cleavage of Hageman factor was not affected by Triton X-100 (Rohm and Haas, Co., Philadelphia, Pa.), hexadimethrine bromide (Polybrene, Aldrich Chemical Co., Inc., Milwaukee, Wis.), hirudin, soybean trypsin inhibitor, or antisera to plasminogen or prekallikrein. However, cleavage was enhanced by kaolin, and was inhibited by diisopropyl-fluorophosphate. The enzyme responsible for cleavage of Hageman factor was localized to the 100,000-g-sedimentable, subcellular fraction of the endothelial cell homogenate and was relatively specific, because neither radiolabeled rabbit Factor XI nor rabbit prekallikrein were themselves proteolytically cleaved by the endothelial cell homogenate. However, when these molecules were incubated with the homogenate in the presence of Hageman factor, both Factor XI and prekallikrein were cleaved, demonstrating that Hageman factor had been activated by the endothelial cell homogenate. Furthermore, the kallikrein generated by endothelial cell homogenate-activated Hageman factor was capable of liberating kinin from high molecular weight kininogen as measured by bioassay. Cultured rabbit endothelial cells, therefore, possess the capacity to activate Hageman factor by proteolysis. This may be one mechanism for Hageman factor activation in vivo.

    Title The Autoactivation of Rabbit Hageman Factor.
    Date January 1980
    Journal The Journal of Experimental Medicine
    Title Rabbit Blood Coagulation Factor Xi. Purification and Properties.
    Date December 1979
    Journal Thrombosis Research
    Title Rabbit Blood Coagulation Factor Xi. Mechanism of Activation of Rabbit Hageman Factor (factor Xii).
    Date December 1979
    Journal Thrombosis Research
    Title Decreased Myelin Synthesis in Developing Rats Following Repeated Pre- and Perinatal Exposure to Subanesthetic Amounts of Halothane.
    Date September 1979
    Journal Journal of Neurochemistry
    Title Incorporation of Leucine Metabolites into Brain and Sciatic Nerve Myelin.
    Date July 1979
    Journal Journal of Neurochemistry
    Title Relative Synthesis of Myelin in Different Brain Regions of Postnatally Undernourished Rats.
    Date April 1979
    Journal Brain Research
    Excerpt

    We used a double isotope procedure and starved and normal littermate rats to compare relative protein synthesis in the cerebellar nuclear, myelin, synaptosomal, mitochondrial, and microsomal subfractions of postnatally starved animals. The remaining brain tissue was dissected into 6 additional regions (cerebral cortex, medulla oblongata, midbrain, hippocampus, striatum, and hypothalamus) and these were frozen for similar subcellular fractionation and analysis at a later date. The microsomal fraction derived from frozen tissues was discarded. The results show that early postnatal starvation specifically depresses myelin synthesis to about the same extent in all major brain regions at 18 and 21 days of age.

    Title Myelin of the Peripheral Nerve of the Dystrophic Mouse.
    Date December 1978
    Journal Journal of Neurochemistry
    Title Early Postnatal Starvation Causes Lasting Brain Hypomyelination.
    Date September 1978
    Journal Journal of Neurochemistry
    Title Effect of Arterial Pressure and Inheritance on the Sodium Excretory Capacity of Normal Young Men.
    Date August 1978
    Journal Clinical Science and Molecular Medicine
    Excerpt

    1. In normal young adult sons of normotensive parents the rate of renal sodium excretion is highly correlated with mean arterial pressure after a large intravenous isotonic fluid load. The correlation appeared to strengthen with time and was improved when the rate of sodium excretion was corrected for variations in the rate of glomerular filtration. 2. There was no such correlation in normal, age-matched sons of hypertensive parents. 3. In eight of the 20 normotensive sons of hypertensive parents studied, the rate of renal sodium excretion per unit of mean arterial pressure was significantly higher than in the sons of normotensive parents. 4. Because the sons of hypertensive parents are much more likely to become hypertensive than those of normotensive parents, we suggest that an abnormality of renal sodium handling precedes the development of demonstrable hypertension.

    Title Role of High-molecular-weight Kininogen in Surface-binding and Activation of Coagulation Factor Xi and Prekallikrein.
    Date January 1978
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    In the contact phase of activation of the kinin-forming, intrinsic clotting, and fibrinolytic systems, high-molecular-weight kininogen acts as a cofactor for the activation of Factor XI, prekallikrein, and Hageman factor. One mechanism by which high-molecular-weight kininogen acts as a cofactor has been studied by using 125I-labeled Factor XI and prekallikrein in kaolin-activated normal human plasma and plasmas deficient in high-molecular-weight kininogen and Hageman factor. High-molecular-weight kininogen was found to be essential for normal binding and cleavage of both Factor XI and prekallikrein on the kaolin surface. Hageman factor was essential for cleavage but not for binding of Factor XI and prekallikrein to kaolin. In normal plasma 80% of the activated Factor XI remained surface-bound, whereas 80% of the kallikrein was not surface-bound. These findings are consistent with the hypothesis that, in the initial phase of contact activation, high-molecular-weight kininogen links both Factor XI and prekallikrein to the exposed surface where they are activated by surface-bound activated Hageman factor. Once activated, the Factor XI molecules remain localized at the site of activation, in contrast to the kallikrein molecules which are found largely in the surrounding plasma.

    Title Vasovagal Hypotension and Vasopressin Release.
    Date August 1977
    Journal Clinical Endocrinology
    Excerpt

    Mild vasovagal hypotension occurred in two normal male volunteers during insertion of indwelling venous cannulae. In spite of a rapid intravenous fluid load (3.6-4.6 litres in 90 min) both subjects passed little urine for 100 min. When the experiment was repeated without vasovagal hypotension, a rapid large diuresis followed the fluid load. The prolonged oliguria after vasovagal hypotension was the result of vasopressin release. This was demonstrated by measuring vasopressin in the blood and urine and by observing the renal response to a water load. The observation in man that a mild transient hypotensive episode may reduce urine flow for 100 min has clinical significance.

    Title Myelin Synthesis During Postnatal Nutritional Deprivation and Subsequent Rehabilitation.
    Date August 1976
    Journal Brain Research
    Excerpt

    Newborn Long-Evans rats were undernourished by maternal deprivation so that by 20 days of age their body and brain weights were about 45 and 80%, respectively, of the values obtained for control (well-nourished) values. Proteins from myelin of undernourished and control rats were separated by polyacrylamide gel electrophoresis in buffers containing sodium dodecyl sulfate. At 15 and 20 days of age the proportion of basic and proteolipid protein was reduced in the starved animals relative to controls, indicative of a delay in maturation. However, by 30 days of age the composition of myelin from starved and control animals appeared similar. At all ages the yield of myelin from brains of starved rats was less than 25% of that obtained from control animals. A series of isotope labeling experiments, using a double label design, was carried out to compare relative rates of incorporation of radioactive amino acids into individual proteins of various brain subcellular fractions. In 20-day-old rats the incorporation of [3H] OR [14C] leucine or glycine into myelin proteins, relative to incorporation into proteins of other subcellular fractions, is preferentially depressed (about 60%) in starved animals. Synthesis of all the myelin proteins was depressed, supporting the hypothesis that the high molecular weight proteins isolated with myelin are true myelin constituents. Similar experiments were conducted using [3H]-and [14C] acetate, choline, or glycerol as precursors of lipids. Incorporation of isotope into lipids of myelin, relative to lipids of other subcellular fractions, was also depressed by about 60% in starved animals. In several experiments we studied synthesis during rehabilitation (ad libitum feeding) following 20 days of postnatal starvation. After 6 days of rehabilitation, incorporation of radioactive precursors into myelin, relative to other subcellular fractions, was still depressed. This result was true for both proteins and lipids, and was interpreted as evicence against the initiation of a process leading to a net recovery of myelin (i.e., an irreversible deficit of myelin synthesis is induced by this regime of nutritional deprivation).

    Title Polyacrylamide Gel Electrophoresis of Myelin Proteins: a Caution.
    Date January 1976
    Journal Analytical Biochemistry
    Title Appearance of Myelin Proteins in Rat Sciatic Nerve During Development.
    Date November 1975
    Journal Brain Research
    Excerpt

    Rats between 5 and 45 days of age were sacrificed and their sciatic nerves dissected. Myelin was prepared from these sciatic nerves by a procedure involving purification on discontinuous sucrose gradients. The proteins of whole sciatic nerves at different ages and the proteins derived from the myelin isolated from these sciatic nerves were examined by discontinuous polyacrylamide gel electrophoresis in buffers containing sodium dodecyl sulfate. Over half of the proteins of sciatic nerve myelin migrated in a single band on the gel (P0). There were only minor changes in the protein distribution of sciatic nerve mylein during development. In contrast, the polyacrylamide gel patterns of whole sciatic nerve homogenate changed markedly during development between 5 and 15 days of age. The amount of P0 protein as a proportion of the total sciatic nerve protein increased from 3% at 5 days of age to 13% at 15 days of age after which it remained constant. Several other proteins which were also characteristic of the isolated myelin increased in relative importance during this time period. Parallel experiments dealing with a metabolic parameter of myelinogenesis, incorporation of intraperitoneally injected [35S]sulfate into sulfatide, were conducted. The maximum synthesis of sulfatide occurred between 6 and 16 days of age, coincident with the marked accumulation of myelin proteins in sciatic nerve.

    Title Synthesis of Myelin Proteins During Starvation.
    Date January 1975
    Journal Brain Research
    Title Nh2-terminal Analysis of Wolfgram and Folch-lees Proteolipid Proteins.
    Date July 1974
    Journal Journal of Neurochemistry
    Title Characterization of Wolfgram Proteolipid Protein of Bovine White Matter and Fractionation of Molecular Weight Heterogeneity.
    Date May 1974
    Journal Journal of Neurochemistry
    Title Haptoglobins and Acid Phosphatases of Galago.
    Date February 1971
    Journal Folia Primatologica; International Journal of Primatology
    Title Plasma-calcitonin in Man.
    Date April 1969
    Journal Lancet
    Title 32 Molecular Cloning and Expression of Glomerular Epithelial Protein 1 (glepp1) in Developing Mouse Kidney
    Date
    Journal The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society
    Title Prion Stability and Infectivity in the Environment.
    Date
    Journal Neurochemical Research
    Excerpt

    The biology of normal prion protein and the property of infectivity observed in abnormal folding conformations remain thinly characterized. However, enough is known to understand that prion proteins stretch traditional views of proteins in biological systems. Numerous investigators are resolving details of the novel mechanism of infectivity, which appears to feature a protein-only, homologous replication of misfolded isoforms. Many other features of prion biology are equally extraordinary. This review focuses on the status of infectious prions in various natural and man-made environments. The picture that emerges is that prion proteins are durable under extreme conditions of environmental exposure that are uncommon in biological phenomena, and this durability offers the potential for environmental reservoirs of persistent infectivity lasting for years. A recurrent theme in prion research is a propensity for these proteins to bind to mineral and metal surfaces, and several investigators have provided evidence that the normal cellular functions of prion protein may include metalloprotein interactions. This structural propensity for binding to mineral and metal ions offers the hypothesis that prion polypeptides are intrinsically predisposed to non-physiological folding conformations that would account for their environmental durability and persistent infectivity. Similarly, the avidity of binding and potency of prion infectivity from environmental sources also offers a recent hypothesis that prion polypeptides bound to soil minerals are actually more infectious than studies with purified polypeptides would predict. Since certain of the prion diseases have a history of epidemics in economically important animal species and have the potential to transmit to humans, urgency is attached to understanding the environmental transmission of prion diseases and the development of protocols for their containment and inactivation.

    Title Frequency Dependence of Kidney Injury Induced by Contrast-aided Diagnostic Ultrasound in Rats.
    Date
    Journal Ultrasound in Medicine & Biology
    Excerpt

    This study was performed to examine the frequency dependence of glomerular capillary hemorrhage (GCH) induced by contrast-aided diagnostic ultrasound (DUS) in rats. Diagnostic ultrasound scanners were used for exposure at 3.2, 5.0 and 7.4 MHz, and previously published data at 1.5 and 2.5 MHz was also included. A laboratory exposure system was used to simulate DUS exposure at 1.0, 1.5, 2.25, 3.5, 5.0 and 7.5 MHz, with higher peak rarefactional pressure amplitudes (PRPAs) than were available from our DUS systems. The right kidneys of rats mounted in a water bath were exposed to intermittent image pulse sequences at 1 s intervals during infusion of diluted ultrasound contrast agent. The percentage of GCH was zero for low PRPAs, and then rapidly increased with increasing PRPAs above an apparent threshold, p(t). The values of p(t) were approximately proportional to the ultrasound frequency, f, such that p(t) /f was approximately 0.5 MPa/MHz for DUS and 0.6 MPa/MHz for laboratory system exposures. The increasing thresholds with increasing frequency limited the GCH effect for contrast-aided DUS, and no GCH was seen for DUS at 5.0 or 7.4 MHz for the highest available PRPAs.

    Similar doctors nearby

    Dr. Jason Knight

    Internal Medicine
    5 years experience
    Ann Arbor, MI

    Dr. Robert Hyzy

    Internal Medicine
    30 years experience
    Ann Arbor, MI

    Dr. Jessica Slocum

    Internal Medicine
    8 years experience
    Ann Arbor, MI

    Dr. D'Anna Saul

    Internal Medicine
    6 years experience
    Ann Arbor, MI

    Dr. Thuy Ledesai

    Internal Medicine
    21 years experience
    Ann Arbor, MI

    Dr. John Del Valle

    Internal Medicine
    32 years experience
    Ann Arbor, MI
    Search All Similar Doctors