Internists, Cardiologist (heart)
21 years of experience
Video profile
University Area
3841 Piper St
Ste T100
Anchorage, AK 99508
907-561-3211
Locations and availability (7)

Education ?

Medical School Score Rankings
University of Washington (1989)
  • Currently 4 of 4 apples
Top 25%

Awards & Distinctions ?

Awards  
Castle Connolly's Top Doctors™ (2013)
Patients' Choice Award (2010 - 2011, 2013)
Compassionate Doctor Recognition (2010 - 2011, 2013)
Top Ten Doctors (2012)
Tudor Municipal Campus
Cardiology
Associations
Heart Rhythm Society
American Board of Internal Medicine

Affiliations ?

Dr. Compton is affiliated with 6 hospitals.

Hospital Affilations

Score

Rankings

  • Alaska Regional Hospital
    Cardiology
    2801 Debarr Rd, Anchorage, AK 99508
    • Currently 3 of 4 crosses
    Top 50%
  • Valley Hospital
    Cardiology
    2500 S Woodworth Loop, Palmer, AK 99645
    • Currently 3 of 4 crosses
    Top 50%
  • Providence Alaska Medical Center
    Cardiology
    PO Box 196604, Anchorage, AK 99519
    • Currently 2 of 4 crosses
  • Providence Seward Wesley Care Center
    PO Box 365, Seward, AK 99664
  • Providence Extended Care Center
    4900 Eagle St, Anchorage, AK 99503
  • Alaska Heart Institute, LLC
  • Publications & Research

    Dr. Compton has contributed to 36 publications.
    Title Proteinase-activated Receptor-2 Activating Peptides: Distinct Canine Coronary Artery Receptor Systems.
    Date January 2008
    Journal American Journal of Physiology. Heart and Circulatory Physiology
    Excerpt

    In canine coronary artery preparations, the proteinase-activated receptor-2 (PAR(2)) activating peptides (PAR(2)-APs) SLIGRL-NH(2) and 2-furoyl-LIGRLO-NH(2) caused both an endothelium-dependent relaxation and an endothelium-independent contraction. Relaxation was caused at peptide concentrations 10-fold lower than those causing a contractile response. Although trans-cinnamoyl-LIGRLO-NH(2), like other PAR(2)-APs, caused relaxation, it was inactive as a contractile agonist and instead antagonized the contractile response to SLIGRL-NH(2). RT-PCR-based sequencing of canine PAR(2) revealed a cleavage/activation (indicated by underlines) sequence (SKGR/SLIGKTDSSLQITGKG) that is very similar to the human PAR(2) sequence (R/SLIGKV). As a synthetic peptide, the canine PAR-AP (SLIGKT-NH(2)) was a much less potent agonist than either SLIGRL-NH(2) or 2-furoyl-LIGRLO-NH(2), either in the coronary contractile assay or in a Madin-Darby canine kidney (MDCK) cell PAR(2) calcium signaling assay. In the MDCK signaling assay, the order of potencies was as follows: 2-furoyl-LIGRLO-NH(2) >> SLIGRL-NH(2) = trans-cinnamoyl-LIGRLO-NH(2) >> SLIGKT-NH(2), as expected for PAR(2) responses. In the coronary contractile assay, however, the order of potencies was very different: SLIGRL-NH(2) >> 2-furoyl-LIGRLO-NH(2) >> SLIGKT-NH(2), trans-cinnamoyl-LIGRLO-NH(2) = antagonist. Because of 1) the distinct agonist (relaxant) and antagonist (contractile) activity of trans-cinnamoyl-LIGRLO-NH(2) in the canine coronary contractile assays, 2) the different concentration ranges over which the peptides caused either relaxation or contraction in the same coronary preparation, and 3) the markedly distinct structure-activity profiles for the PAR-APs in the coronary contractile assay, compared with those for PAR(2)-mediated MDCK cell calcium signaling, we suggest that the canine coronary tissue possesses a receptor system for the PAR-APs that is distinct from PAR(2) itself.

    Title Inflammatory Mediators Modulate Thrombin and Cathepsin-g Signaling in Human Bronchial Fibroblasts by Inducing Expression of Proteinase-activated Receptor-4.
    Date April 2007
    Journal American Journal of Physiology. Lung Cellular and Molecular Physiology
    Excerpt

    Human lung fibroblasts express proteinase-activated receptor-1 (PAR1), PAR2 and PAR3, but not PAR4. Because PAR2 has inflammatory effects on human primary bronchial fibroblasts (HPBF), we asked 1) whether the inflammatory mediators TNF-alpha and LPS could modify HPBF PAR expression and 2) whether modified PAR expression altered HPBF responsiveness to PAR agonists in terms of calcium signaling and cell growth. TNF-alpha and LPS induced PAR4 mRNA expression (RT-PCR) at 6 h and 24 h, respectively. TNF-alpha and LPS also upregulated PAR2 mRNA expression with similar kinetics but had negligible effect on PAR1 and PAR3. Flow cytometry for PAR1, PAR2, and PAR3 also demonstrated selective PAR2 upregulation in response to TNF-alpha and LPS. Intracellular calcium signaling to SLIGKV-NH2 (a selective PAR2-activating peptide; PAR2-AP) and AYPGQV-NH2 (PAR4-AP) revealed that TNF-alpha and LPS induced maximal responses to these PAR agonists at 24 h and 48 h, respectively. Upregulation of PAR2 by TNF-alpha heightened HPBF responses to trypsin, while PAR4 induction enabled cathepsin-G-mediated calcium signaling. Cathepsin-G also disarmed PAR1 and PAR2 in HPBF, while tryptase disarmed PAR2. Induction of PAR4 also enabled thrombin to elicit a calcium signal through both PAR1 and PAR4, as determined by a desensitization assay. In cell growth assays the PAR4 agonists cathepsin-G and AYPGQV-NH2 reduced HPBF cell number only in TNF-alpha-treated HPBF. Moreover, the mitogenic effect of thrombin (a PAR1/PAR4 agonist) but not the PAR1-AP TFLLR-NH2, was ablated in TNF-alpha-treated HPBF. These findings point to an important mechanism, whereby cellular responses to thrombin and cathepsin-G can be modified during an inflammatory response.

    Title Multi-center Clinical Experience with a Lumenless, Catheter-delivered, Bipolar, Permanent Pacemaker Lead: Implant Safety and Electrical Performance.
    Date December 2006
    Journal Pacing and Clinical Electrophysiology : Pace
    Excerpt

    PURPOSE: Reduced lead diameter and reliability can be designed into transvenous permanent pacing leads through use of redundant insulation and removal of the stylet lumen. The model 3830 lead (Medtronic Inc., Minneapolis, MN, USA) is a bipolar, fixed-screw, steroid-eluting, lumenless, 4.1-Fr pacing lead. Implantation can be performed in a variety of right heart sites using a deflectable catheter (Model 10600, Medtronic). Lead performance and safety were studied. METHODS: Two prospective trials of 338 implanted subjects from 56 global sites were conducted. Electrical and safety data were obtained at implant, pre-discharge, and up to 18 months post-implant. Leads were implanted at traditional and alternate right heart sites. RESULTS: The study enrolled 338 subjects (204 males, 70.6 +/- 11.6 years) followed-up for a mean of 10.2 months (range, 0-21.6). Mean P-wave amplitudes ranged from 3.2 mV at 3 months to 2.9 mV at 18 months, while mean atrial pulse width thresholds at 2.5 V ranged from 0.07 ms at 3 months to 0.09 ms at 18 months. Mean R-wave amplitudes ranged from 11.3 mV to 11.1 mV and mean ventricular pulse width thresholds at 2.5 V ranged from 0.10 ms to 0.14 ms. There were 22 ventricular and 12 atrial lead complications within 3 months post-implant. Survival from lead-related complications improved to a clinically acceptable rate in the cohort of patients when revised implant techniques were employed. CONCLUSIONS: With the use of recommended implant techniques, the study results support the electrical efficacy and safety of a catheter-delivered, lumenless lead in traditional or alternate right atrium or right ventricle sites through 18 months post-implant.

    Title Continuous Template Collection and Updating for Electrogram Morphology Discrimination in Implantable Cardioverter Defibrillators.
    Date September 2006
    Journal Pacing and Clinical Electrophysiology : Pace
    Excerpt

    INTRODUCTION: Electrogram morphology analysis improves discrimination of supraventricular tachycardias (SVTs) from ventricular tachycardias (VTs) in implantable cardioverter defibrillators (ICDs), but electrogram morphology may change with lead maturation, drugs, or disease progression. We report the clinical performance of an automatic algorithm that creates and updates templates from non-paced, slow rhythm and continuously checks the quality of the template used for arrhythmia discrimination. METHODS AND RESULTS: We studied this algorithm in 193 patients with single-chamber ICDs (Marquis VR, Medtronic Inc., Minneapolis, MN, USA). Of the 112 patients who completed 6-month follow-up, 99.1% of the patients had > or =1 automatic template created. Match scores between template and ongoing rhythm are computed using Haar Wavelets. Of the 435 automatic templates evaluated at follow-up, 423 (97.2%) had a median match score > or =70%. Intrinsic rhythm at 1 month had significantly higher match scores (P < 0.001) with automatic templates (90.3 +/- 7.0%) than with manual templates (85.7 +/- 10.9%) generated at pre-hospital discharge (PHD). The percentage of appropriately rejected SVTs was slightly higher with the automatic template (280/339 episodes) than with the manual template at PHD (272/339 episodes) while the Wavelet detection of VT was the same (218/220 episodes). CONCLUSIONS: In patients receiving ICDs, the automatic templates were successfully created during a 6-month follow-up period, and consistently matched the patients' intrinsic rhythm at the nominal match threshold. Both early (<1 month postimplant) and late (1- to 3-month follow-up period) changes in electrogram morphology were identified, confirming the need for automatic template updating.

    Title Reduction of Rv Pacing by Continuous Optimization of the Av Interval.
    Date September 2006
    Journal Pacing and Clinical Electrophysiology : Pace
    Excerpt

    BACKGROUND: In patients requiring permanent pacing, preservation of intrinsic ventricular activation is preferred whenever possible. The Search AV+ (SAV+) algorithm in Medtronic EnPulsetrade mark dual-chamber pacemakers can increase atrioventricular (AV) intervals to 320 ms in patients with intact or intermittent AV conduction. This prospective, multicenter study compared the percentage of ventricular pacing with and without AV interval extension. METHODS: Among 197 patients enrolled in the study, the percentage of ventricular-paced beats was evaluated via device diagnostics at the 1-month follow-up. Patient cohorts were defined by clinician assessment of conduction via a 1:1 AV conduction test at the 2-week follow-up. The observed percentage of ventricular pacing with SAV + ON and the predicted percentage of ventricular pacing with SAV + OFF were determined from the SAV + histogram data for the period between the 2-week and 1-month follow-up visits. RESULTS: Of 197 patients, 110 (55.8%) had intact 1:1 AV conduction, of which 109 had 1-month data. SAV + remained ON in 99/109 patients; 10 patients had intrinsic A-V conduction intervals beyond SAV + nominal and therefore SAV + disabled. The mean percentage of ventricular pacing in the 109 patients was SAV+ ON = 23.1% (median 3.7%) versus SAV + OFF = 97.2% (median 99.7%). In 87 patients without 1:1 AV conduction, SAV + was programmed OFF in 6, automatically disabled in 52, and remained ON in 29. In 8 of these patients, 80-100% reduction in ventricular pacing was observed with SAV + ON. CONCLUSION: The Search AV+ algorithm in the EnPulse pacemaker effectively promotes intrinsic ventricular activation and substantially reduces unnecessary ventricular pacing.

    Title Proteinase-activated Receptor2 Agonists Upregulate Granulocyte Colony-stimulating Factor, Il-8, and Vcam-1 Expression in Human Bronchial Fibroblasts.
    Date August 2006
    Journal American Journal of Respiratory Cell and Molecular Biology
    Excerpt

    Proteinase-activated receptors (PARs) are a novel family of G-protein-coupled receptors. PAR2 has been implicated in inflammatory airways disease. Although fibroblasts are pathologically important in the airways, the proinflammatory role of PAR2 in these cells remains unknown. We assessed PAR expression and functionality in human primary bronchial fibroblasts (HPBFs) before assessing PAR2-mediated HPBF proliferation, cytokine production, and adhesion molecule expression. RT-PCR and flow cytometry demonstrated that HPBFs express hPAR1, hPAR2, and hPAR3, but not hPAR4. Intracellular calcium signaling in HPBFs in response to PAR agonists showed that only hPAR1 and hPAR2 were functional receptors. We used the MTT assay to assess HPBF proliferation. Of the PAR2 agonist proteinases or selective PAR2-activating peptides (PAR2-APs) tested, none stimulated HPBF proliferation, whereas thrombin was a HPBF growth factor. mRNA for IL-8 and granulocyte colony-stimulating factor (G-CSF) was upregulated after addition of SLIGKV-NH2 when assessed by RT-PCR. No significant increase in G-CSF or IL-8 protein was detected. Trypsin stimulated IL-8 and G-CSF release from HPBF in a time- and dose-dependent manner. Leupeptin and soya trypsin inhibitor abrogated trypsin-stimulated cytokine release, indicating a requirement for trypsin's proteolytic activity. Trypsin and SLIGKV-NH2 stimulated an increase in VCAM-1 expression at 12 h after treatment, which declined thereafter. PAR2-driven upregulation of VCAM-1 cell surface expression and the release of IL-8 and G-CSF from bronchial fibroblasts may be important in promoting neutrophilic airways inflammation.

    Title Expression of Proteinase-activated Receptor 2 on Human Primary Gastrointestinal Myofibroblasts and Stimulation of Prostaglandin Synthesis.
    Date February 2006
    Journal Canadian Journal of Physiology and Pharmacology
    Excerpt

    It is known that subepithelial myofibroblast-derived prostaglandin (PG)E2 can regulate intestinal epithelial cell functions, and that proteinase-activated receptor-2 (PAR2) is abundantly expressed in the gastrointestinal tract. Since PAR2 activation has previously been associated with stimulation of PGE2 synthesis, we hypothesized that PAR2 expressed on primary human gastrointestinal myofibroblasts regulates PGE2 synthesis via cyclooxygenase (COX)-1 and (or) COX-2, and associated PGE synthases. Primary human myofibroblasts were isolated from the resection tissue of the esophagus, small intestine, and colon. Expression of functional PAR2 was determined by RT-PCR and by calcium mobilization in Fura-2/AM-loaded cells. Trypsin and the selective PAR2-activating peptide (PAR2-AP) SLIGRL-NH2 stimulated PGE2 synthesis in a concentration-dependent manner, as measured by enzyme immunoassay. Selective COX inhibition showed PAR2-induced PGE2 synthesis to be COX-1 dependent in esophageal myofibroblasts and both COX-1 and COX-2 dependent in colonic cells, consistent with the distribution of COX-1 and COX-2 expression. Although both cytosolic and microsomal PGE synthases were expressed in cells from all tissues, microsomal PGE synthases were expressed at highest levels in the colonic myofibroblasts. Activation of PAR2 on gastrointestinal myofibroblasts stimulates PGE2 synthesis via different pathways in the colon than in the esophagus and small intestine.

    Title Characterization of a Novel Protein from Proatheris Superciliaris Venom: Proatherocytin, a 34-kda Platelet Receptor Par1 Agonist.
    Date February 2006
    Journal Toxicon : Official Journal of the International Society on Toxinology
    Excerpt

    Many toxins from viperid venoms have been characterised as powerful activators of platelets. Here, the venom from the East African Lowland viper, Proatheris superciliaris, was investigated for its effect on platelets and the coagulation system. Whole P. superciliaris venom stimulated platelet shape change and aggregation; however, the stimulation of platelet activation was unaffected by the structurally distinct Src family kinase inhibitors PP1 and PD0173952, suggesting that platelet activation was mediated by a G protein-coupled receptor. A platelet reactive 34-kDa protein was isolated from P. superciliaris venom which we have designated proatherocytin. This protein induced Src kinase-independent aggregation of both human and mouse platelets that was inhibited by the serine protease inhibitor AEBSF. Proatherocytin did not clot bovine or human fibrinogen, degrade fibrinogen or hydrolyse the serine protease substrate benzoyl-FVR-pNA. It activated human PAR1 on stably transfected rat kidney epithelial cells, whereas no activation of the trypsin receptor PAR2 was shown. Surprisingly, Edman degradation of proatherocytin revealed sequence identity with existing disintegrin-like domains of snake venom metalloproteinases. These results suggest that proatherocytin is a highly selective PAR1 agonist that also causes mouse platelet aggregation, probably through cleavage of PAR4.

    Title Optical Calcium Sensors: Development of a Generic Method for Their Introduction to the Cell Using Conjugated Cell Penetrating Peptides.
    Date September 2005
    Journal The Analyst
    Excerpt

    Probes Encapsulated By Biologically Localised Embedding (PEBBLEs) are optical sensors with nanometer dimensions fabricated by microemulsion polymerisation. The most beneficial characteristic of these sensors is the protection offered by the sensor matrix which decreases interaction between the fluorophore and the cell. These sensors have been introduced to the cell by a number of methods; however this paper discusses the development of a generic method to facilitate inclusion of this type of sensor in the cell by a simple incubation step. This was achieved by covalent linkage of a synthetic Cell Penetrating Peptide (CPP) based on the Human Immuno-deficiency Virus (HIV) -1 Tat, to the external sensor matrix. Calcium sensors were used to demonstrate this approach to incorporate the sensors within the cell. Characterisation revealed the calcium sensors were approximately 30 +/- 7 nm in diameter with a slightly negative zeta potential. The sensors demonstrated a linear range of 0-50 microM with negligible interference from a range of cellular ions and protein. Leaching of entrapped dyes from the calcium sensors was determined as 3% in a 24 h period, while photobleaching of the entrapped dye was minimal over a 40 min period. The sensors ability to cross the cell membrane using the covalently attached synthetic Tat peptide is demonstrated. Cellular inclusion of the sensors occurred within a 30 min incubation period.

    Title Expression and Characterization of the Intracellular Vanilloid Receptor (trpv1) in Bronchi from Patients with Chronic Cough.
    Date July 2005
    Journal Experimental Lung Research
    Excerpt

    TRPV1 is a modulator of noxious stimuli known to be important in the cough reflex. We have compared the expression of TRPV1 in normal human airways and those from patients with chronic cough and found that there is up regulation in airways smooth muscle in disease. This increased expression appears to be intracellular and we have therefore examined the role of intracellular rat and human TRPV1 activity was found using intracellular calcium signalling with human intracellular TRPV1 being located in a thapsigargin insensitive compartment. Increase in TRPV1 activity may have a role in the airway hypersensitivity seen in chronic cough.

    Title Remote Monitoring of Implantable Cardioverter Defibrillators: a Prospective Analysis.
    Date September 2004
    Journal Pacing and Clinical Electrophysiology : Pace
    Excerpt

    A prospective study evaluating the functionality and ease of use of the Medtronic CareLink Network, "CareLink," was conducted at ten investigational sites. This internet-based remote monitoring service allows clinicians to remotely manage their patients' implantable cardioverter defibrillators (ICDs) and chronic diseases. The network is comprised of a patient monitor, a secure server, and clinician and patient websites. Under clinician direction, patients interrogated their ICDs at home, and transmitted data to secure servers via a standard telephone line. Comprehensive device data and a 10-second presenting rhythm electrogram were captured by the monitor and available for access and review on the clinician website. The information could also be printed using a standard desktop computer with internet access. During this study, patients were asked to transmit device data twice, at least 7 days apart, as scheduled by the clinic. Monitor functionality was assessed, and ease of using the system components was evaluated via questionnaires completed by patients and clinicians following each data transmission and review. Fifty-nine patients (64 +/- 14 years, range 22-85 years) completed 119 transmissions with only 14 calls to the study support center. Clinician review of data transmissions revealed several clinically significant findings, including silent AF discovery, assessment of antiarrhythmic drug efficacy in a previously diagnosed AF patient, previously unobserved atrial undersensing, and ventricular tachycardia. ICD patients found the monitor easy to use. Clinicians were pleased with the performance of the network and the quality of the web-accessed data, and found it comparable to an in-office device interrogation. CareLink is a practical tool for routine device management and may allow timely identification of clinically important issues.

    Title Colitis Induced by Proteinase-activated Receptor-2 Agonists is Mediated by a Neurogenic Mechanism.
    Date August 2004
    Journal Canadian Journal of Physiology and Pharmacology
    Excerpt

    Proteinase-activated receptor-2 (PAR2) activation induces colonic inflammation by an unknown mechanism. We hypothesized that PAR2 agonists administered intracolonically in mice induce inflammation via a neurogenic mechanism. Pretreatment of mice with neurokinin-1 and calcitonin-gene-related peptide (CGRP) receptor antagonists or with capsaicin showed attenuated PAR2-agonist-induced colitis. Immunohistochemistry demonstrated a differential expression of a marker for the type-1 CGRP receptor during the time course of PAR2-agonist-induced colitis, further suggesting a role for CGRP. We conclude that PAR2-agonist-induced intestinal inflammation involves the release of neuropeptides, which by acting on their receptors cause inflammation. These results implicate PAR2 as an important mediator of intestinal neurogenic inflammation.

    Title A New Oral Therapy for Long Qt Syndrome: Long-term Oral Potassium Improves Repolarization in Patients with Herg Mutations.
    Date January 2004
    Journal Journal of the American College of Cardiology
    Excerpt

    OBJECTIVES: We sought to determine whether oral potassium supplementation safely increases serum K(+) and results in sustained improvement of repolarization parameters in long QT syndrome type 2 (LQT2) subjects. BACKGROUND: Mutations in HERG (LQT2), the gene encoding the rapid delayed rectifier K(+) current I(Kr), account for a significant proportion of congenital long QT syndrome (LQTS). The magnitude of I(Kr) is paradoxically increased by an increase in extracellular K(+). We tested the hypothesis that long-term oral potassium supplementation results in a mild, sustainable increase in serum K(+) that improves repolarization abnormalities in subjects with LQT2. METHODS: After an initial evaluation consisting of electrocardiography, electrolytes, blood urea nitrogen, and creatinine, escalating doses of potassium chloride (KCl) and spironolactone were administered to eight subjects with six distinct HERG mutations. Medications were continued for four weeks, at which time, the final evaluation was undertaken. Beta-adrenergic blocking therapy was maintained. RESULTS: The subjects ranged in age from 11 to 52 years. The average daily KCl and spironolactone dose was 3.3 +/- 1.5 mEq/kg and 3.5 +/- 1.2 mg/kg, respectively, and this regimen resulted in an increase in serum K(+) from 4.0 +/- 0.3 to 5.2 +/- 0.3 mEq/l. There were no serious complications associated with therapy. The increase in serum K(+) resulted in a decrease in the corrected QT interval from 526 +/- 94 to 423 +/- 36 ms (mean +/- SD; lead V(2)). Both QT dispersion and T-wave morphology improved in most subjects. CONCLUSIONS: Long-term oral potassium administration increases serum K(+) in patients with LQT2. This can be achieved safely and results in improvement in repolarization. Further studies are warranted to determine whether this will reduce the incidence of life-threatening events in LQTS patients.

    Title Restricted Ability of Human Mast Cell Tryptase to Activate Proteinase-activated Receptor-2 in Rat Aorta.
    Date May 2003
    Journal Canadian Journal of Physiology and Pharmacology
    Excerpt

    We investigated the potential of human mast cell tryptase to induce relaxation of rat aorta. Trypsin and the selective PAR2-activating peptide (PAR2-AP) SLIGRL-NH2 stimulated robust relaxation of phenylephrine-precontracted rat aortic rings. However, human lung tryptase (1-100 nM) either in the presence or absence of heparin failed to induce any significant relaxation. Notwithstanding, incubation of the aorta with tryptase (100 nM), following the addition of a peptide corresponding to the cleavage/activation sequence of rat PAR2 (rPAR2), resulted in relaxation of precontracted tissue due to the proteolytic release of the PAR2-AP SLIGRL/ from the parent peptide. Thus, tryptase was enzymatically active in the bioassay system. Preincubation of aorta with neuraminidase to remove cell-surface sialic acid unmasked the ability of tryptase to induce relaxation of the aorta, but had no effect on relaxation induced by trypsin, SLIGRL-NH2, or acetylcholine (Ach). Like trypsin and SLIGRL-NH2, the tryptase-induced relaxation was inhibited by either removal of the endothelium or pretreatment of the tissue with NG-nitro-L-arginine methyl ester (L-NAME), suggesting an endothelium-derived nitric oxide mechanism. Interestingly, tryptase in the presence of heparin failed to induce relaxation of precontracted neuraminidase-treated rat aorta. We conclude that tryptase-induced relaxation of rat aorta, most likely via PAR2, is tightly regulated by heparin and cell-surface sialic acid.

    Title Glycosylation of Human Proteinase-activated Receptor-2 (hpar2): Role in Cell Surface Expression and Signalling.
    Date January 2003
    Journal The Biochemical Journal
    Excerpt

    We have analysed the role of N-linked glycosylation in regulating human proteinase-activated receptor-2 (hPAR(2)) expression and function. Epitope-tagged wild-type hPAR(2) (wt-hPAR(2)) or hPAR(2) that lacked glycosylation sequons (following site-directed mutagenesis) in either the N-terminus [hPAR(2)N30A (Asn(30)-->Ala)], extracellular loop 2 [ECL2; hPAR(2)N222Q (Asn(222)-->Gln) or hPAR(2)N222A (Asn(222)-->Ala)] or both (hPAR(2)N30A,N222A or hPAR(2)N30A,N222Q) were expressed in the Chinese-hamster ovary (CHO) fibroblast cell line, Pro5. Western blot analysis of wt-hPAR(2) showed mature wt-hPAR(2) to have a molecular mass of 55-100 kDa, and 33-48 kDa following N -glycosidase F deglycosylation. FACS analysis and immunocytochemistry of the wt-hPAR(2) and PAR(2) mutant cell lines revealed that removal of both glycosylation sequons decreases (50% of wt-hPAR(2)) cell surface expression. Western blot analysis indicated that both N-linked sites are glycosylated. In functional studies, hPAR(2)N30A displayed a selective and significant increase in sensitivity towards tryptase. Interestingly, hPAR(2)N222A displayed a loss in sensitivity towards all PAR(2) agonists tested. However, further analysis revealed receptor sensitivity to alanine mutations in this domain, as the more conservative substitution hPAR(2)N222Q displayed no change in response to PAR(2) agonists. hPAR(2)N30A,N222Q displayed increased sensitivity towards tryptase, but a loss in sensitivity towards trypsin and the synthetic peptide SLIGRL-NH(2), although this loss in sensitivity towards trypsin and SLIGRL-NH(2) was secondary to changes in cell-surface expression. Finally, expression of sialic-acid-deficient wt-hPAR(2) in the CHO Lec2 glycosylation-deficient mutant cell line, showed a 40 kDa loss in molecular mass, in addition to a marked and selective increase in sensitivity towards tryptase. We conclude that hPAR(2) N-linked glycosylation and sialylation regulates receptor expression and/or signalling.

    Title International Union of Pharmacology. Xxviii. Proteinase-activated Receptors.
    Date October 2002
    Journal Pharmacological Reviews
    Excerpt

    Proteinase-activated receptors (PARs) represent a unique subclass of G-protein-coupled receptors of which four family members have now been cloned from a number of species. The novel mechanism of receptor activation involves the proteolytic unmasking of a cryptic N-terminal receptor sequence that, remaining tethered, binds to and triggers receptor function. In addition, short (five to six amino acids) synthetic peptides, based on the proteolytically revealed motif, can activate PARs without the unmasking of the tethered ligand. This article summarizes the experiments leading to the pharmacological characterization and cloning of the four PAR family members and provides a rationale for their designation by the acronym "PAR". The ability to distinguish among the PARs pharmacologically 1) with selective proteinase activators, 2) with receptor-selective peptide agonists, and 3) with peptide and nonpeptide antagonists is discussed, as are the molecular mechanisms of receptor activation and desensitization/internalization. Finally, the potential physiological roles of the PARs, which are widely distributed in many organs in the settings of tissue injury, repair, and remodeling, including embryogenesis and oncogenesis are discussed, and the newly appreciated roles of proteinases as signaling molecules that can act as either functional agonists or antagonists are highlighted.

    Title Glycosylation and the Activation of Proteinase-activated Receptor 2 (par(2)) by Human Mast Cell Tryptase.
    Date December 2001
    Journal British Journal of Pharmacology
    Excerpt

    1. Human mast cell tryptase appears to display considerable variation in activating proteinase-activated receptor 2 (PAR(2)). We found tryptase to be an inefficient activator of wild-type rat-PAR(2) (wt-rPAR(2)) and therefore decided to explore the factors that may influence tryptase activation of PAR(2). 2. Using a 20 mer peptide (P20) corresponding to the cleavage/activation sequence of wt-rPAR(2), tryptase was as efficient as trypsin in releasing the receptor-activating sequence (SLIGRL.). However, in the presence of either human-PAR(2) or wt-r PAR(2) expressing cells, tryptase could only activate PAR(2) by releasing SLIGRL from the P20 peptide, suggesting that PAR(2) expressed on the cells was protected from tryptase activation. 3. Three approaches were employed to test the hypothesis that PAR(2) receptor glycosylation restricts tryptase activation. (a) pretreatment of wt-rPAR(2) expressing cells or human embryonic kidney cells (HEK293) with vibrio cholerae neuraminidase to remove oligosaccharide sialic acid, unmasked tryptase-mediated PAR(2) activation. (b) Inhibiting receptor glycosylation in HEK293 cells with tunicamycin enabled tryptase-mediated PAR(2) activation. (c) Wt-rPAR(2) devoid of the N-terminal glycosylation sequon (PAR(2)T25(-)), but not rPAR(2) devoid of the glycosylation sequon located on extracellular loop-2 (PAR(2)T224A), was selectively and substantially (>30 fold) more sensitive to tryptase compared with the wt-rPAR(2). 4. Immunocytochemistry using antisera that specifically recognized the N-terminal precleavage sequence of PAR(2) demonstrated that tryptase released the precleavage domain from PAR(2)T25(-) but not from wt-rPAR(2). 5. Heparin : tryptase molar ratios of greater than 2 : 1 abrogated tryptase activation of PAR(2)T25(-). 6. Our results indicate that glycosylation of PAR(2) and heparin-inhibition of PAR(2) activation by tryptase could provide novel mechanisms for regulating receptor activation by tryptase and possibly other proteases.

    Title Tryptase and Agonists of Par-2 Induce the Proliferation of Human Airway Smooth Muscle Cells.
    Date December 2001
    Journal Journal of Applied Physiology (bethesda, Md. : 1985)
    Excerpt

    Airway remodeling with smooth muscle cell (SMC) hyperplasia is a feature of chronic asthma. We investigated the potential for tryptase, the major secretory product of human mast cells, to act as a growth factor for human airway SMCs. Because this serine protease can activate proteinase-activated receptor-2 (PAR-2), we also examined the actions of SLIGKV, a peptide agonist of PAR-2. Incubation with lung tryptase provoked a twofold increase in [(3)H]thymidine incorporation; a similar increase in cell numbers was found when we used the MTS assay. The effect was catalytic site dependent, being abolished by the protease inhibitors leupeptin and benzamidine and by heat inactivation of the enzyme. Tryptase-induced DNA synthesis was inhibited by preincubation of the cells with pertussis toxin, calphostin C, or genistein. Transduction mechanisms are thus likely to involve a pertussis toxin-sensitive G protein, protein kinase C, and tyrosine kinase. SLIGKV elicited a response on SMCs similar to that of tryptase. Tryptase could provide an important stimulus for SMC proliferation in asthmatic airways, by acting on PAR-2.

    Title Intra-atrial Conduction Block Along the Mitral Valve Annulus During Accessory Pathway Ablation: Evidence for a Left Atrial "isthmus".
    Date December 2001
    Journal Journal of Cardiovascular Electrophysiology
    Excerpt

    INTRODUCTION: We observed a change in the atrial activation sequence during radiofrequency (RF) energy application in patients undergoing left accessory pathway (AP) ablation. This occurred without damage to the AP and in the absence of a second AP or alternative arrhythmia mechanism. We hypothesized that block in a left atrial "isthmus" of tissue between the mitral annulus and a left inferior pulmonary vein was responsible for these findings. METHODS AND RESULTS: Electrophysiologic studies of 159 patients who underwent RF ablation of a left free-wall AP from 1995 to 1999 were reviewed. All studies with intra-atrial conduction block resulting from RF energy delivery were identified. Fluoroscopic catheter positions were reviewed. Intra-atrial conduction block was observed following RF delivery in 11 cases (6.9%). This was evidenced by a sudden change in retrograde left atrial activation sequence despite persistent and unaffected pathway conduction. In six patients, reversal of eccentric atrial excitation during orthodromic reciprocating tachycardia falsely suggested the presence of a second (septal) AP. A multipolar coronary sinus catheter in two patients directly demonstrated conduction block along the mitral annulus during tachycardia. CONCLUSION: An isthmus of conductive tissue is present in the low lateral left atrium of some individuals. Awareness of this structure may avoid misinterpretation of the electrogram during left AP ablation and may be useful in future therapies of atypical atrial flutter and fibrillation.

    Title Proteinase-activated Receptor-2 and Hyperalgesia: A Novel Pain Pathway.
    Date July 2001
    Journal Nature Medicine
    Excerpt

    Using a combined pharmacological and gene-deletion approach, we have delineated a novel mechanism of neurokinin-1 (NK-1) receptor-dependent hyperalgesia induced by proteinase-activated receptor-2 (PAR2), a G-protein-coupled receptor expressed on nociceptive primary afferent neurons. Injections into the paw of sub-inflammatory doses of PAR2 agonists in rats and mice induced a prolonged thermal and mechanical hyperalgesia and elevated spinal Fos protein expression. This hyperalgesia was markedly diminished or absent in mice lacking the NK-1 receptor, preprotachykinin-A or PAR2 genes, or in rats treated with a centrally acting cyclooxygenase inhibitor or treated by spinal cord injection of NK-1 antagonists. Here we identify a previously unrecognized nociceptive pathway with important therapeutic implications, and our results point to a direct role for proteinases and their receptors in pain transmission.

    Title Mass, Charge, and Subcellular Localization of a Unique Secretory Product Identified by the Basophil-specific Antibody Bb1.
    Date June 2001
    Journal The Journal of Allergy and Clinical Immunology
    Excerpt

    BACKGROUND: BB1 is a basophil-specific mAb (Lab Invest 1999;79:27-38). The identity of the corresponding antigen has not been determined, but it gives a granular appearance on staining and is secreted on activation of basophils. OBJECTIVE: We sought to further characterize the basophilspecific antigen identified by BB1. METHODS: Intracellular localization was determined by flow cytometry and by immunogold labeling and electron microscopy. Physical chemical properties were investigated by gel filtration chromatography and preparative isoelectric focusing. RESULTS: In flow cytometry, permeabilization of cells increased immunofluorescence 100-fold, confirming the predominantly intracellular localization of the antigen. It was further localized to the secretory granules by immunoelectron microscopy. Double labeling with a CD63-specific antibody demonstrated selective binding of BB1 to the granule matrix. Gel filtration chromatography indicated that the antigen is secreted as a complex of approximately 5 x 10(6) d, which was well resolved from the 210-kd supramolecular complex containing tryptase. The antigen was degraded by pronase. Isoelectric focusing indicated a highly basic protein with an isoelectric point of 9.6. CONCLUSION: With its granule localization, release on cell activation, and unique properties, the antigen identified by BB1 could be a novel mediator of allergic disease. We propose the name basogranulin for this novel basophil-specific protein.

    Title Amiodarone is Safe and Highly Effective Therapy for Supraventricular Tachycardia in Infants.
    Date March 2001
    Journal American Heart Journal
    Excerpt

    BACKGROUND: The clinical effectiveness of amiodarone must be weighed against the likelihood of adverse effects. Adverse effects are less common in children than in adults, yet there have been no large studies assessing the efficacy and safety of amiodarone in the first 9 months of life. We sought to assess the safety and efficacy of amiodarone as primary therapy for supraventricular tachycardia in infancy. METHODS: We evaluated the clinical course of 50 consecutive infants and neonates (1.0+/-1.5 months, 35 male) treated with amiodarone for supraventricular tachyarrhythmias between July 1994 and July 1999. At presentation, congenital heart disease, congestive heart failure, or ventricular dysfunction were present in 24%, 36%, and 44% of the infants, respectively. Infants received a 7- to 10-day load of amiodarone at either 10 or 20 mg/kg/d. If this failed to control the arrhythmia, oral propranolol (2 mg/kg/d) was added. Patients were followed up for 16.0+/-13.0 months, and antiarrhythmic drugs were discontinued as tolerated. RESULTS: Rhythm control was achieved in all patients. Of the 34 patients who have reached 1 year of age, 23 (68%) have remained free of arrhythmia, despite discontinuation of propranolol and amiodarone. Growth and development remained normal for age. Higher loading doses of amiodarone were associated with an increase in the corrected QT interval, but no proarrhythmia was seen. There were no side effects necessitating drug withdrawal. CONCLUSIONS: Amiodarone is an effective and safe therapy for tachycardia control in infancy.

    Title A Polymorphic Protease-activated Receptor 2 (par2) Displaying Reduced Sensitivity to Trypsin and Differential Responses to Par Agonists.
    Date February 2001
    Journal The Journal of Biological Chemistry
    Excerpt

    Protease-activated receptor 2 (PAR2) is a trypsin-activated member of a family of G-protein-coupled PARs. We have identified a polymorphic form of human PAR2 (PAR(2)F240S) characterized by a phenylalanine to serine mutation at residue 240 within extracellular loop 2, with allelic frequencies of 0.916 (Phe(240)) and 0.084 (Ser(240)) for the wild-type and mutant alleles, respectively. Elevations in intracellular calcium were measured in permanently transfected cell lines expressing the receptors. PAR(2)F240S displayed a significant reduction in sensitivity toward trypsin ( approximately 3.7-fold) and the PAR2-activating peptides, SLIGKV-NH(2) ( approximately 2.5-fold) and SLIGRL-NH(2) ( approximately 2.8-fold), but an increased sensitivity toward the selective PAR2 agonist, trans-cinnamoyl-LIGRLO-NH(2) ( approximately 4-fold). Increased sensitivity was also observed toward the selective PAR-1 agonist, TFLLR-NH(2) ( approximately 7-fold), but not to other PAR-1 agonists tested. Furthermore, we found that TLIGRL-NH(2) and a PAR4-derived peptide, trans-cinnamoyl-YPGKF-NH(2), were selective PAR(2)F240S agonists. By introducing the F240S mutation into rat PAR2, we observed shifts in agonist potencies that mirrored the human PAR(2)F240S, suggesting that Phe(240) is involved in determining agonist specificity of PAR2. Finally, differences in receptor signaling were paralleled in a cell growth assay. We suggest that the distinct pharmacological profile induced by this polymorphism will have important implications for the design of PAR-targeted agonists/antagonists and may contribute to, or be predictive of, an inflammatory disease.

    Title Spectrum of St-t-wave Patterns and Repolarization Parameters in Congenital Long-qt Syndrome: Ecg Findings Identify Genotypes.
    Date January 2001
    Journal Circulation
    Excerpt

    BACKGROUND: Congenital long-QT syndrome (LQTS) is caused by mutations of genes encoding the slow component of the delayed rectifier current (LQT1, LQT5), the rapid component of the delayed rectifier current (LQT2, LQT6), or the Na(+) current (LQT3), resulting in ST-T-wave abnormalities on the ECG. This study evaluated the spectrum of ST-T-wave patterns and repolarization parameters by genotype and determined whether genotype could be identified by ECG. METHODS AND RESULTS: ECGs of 284 gene carriers were studied to determine ST-T-wave patterns, and repolarization parameters were quantified. Genotypes were identified by individual ECG versus family-grouped ECG analysis in separate studies using ECGs of 146 gene carriers from 29 families and 233 members of 127 families undergoing molecular genotyping, respectively. Ten typical ST-T patterns (4 LQT1, 4 LQT2, and 2 LQT3) were present in 88% of LQT1 and LQT2 carriers and in 65% of LQT3 carriers. Repolarization parameters also differed by genotype. A combination of quantified repolarization parameters identified genotype with sensitivity/specificity of 85%/70% for LQT1, 83%/94% for LQT2, and 47%/63% for LQT3. Typical patterns in family-grouped ECGs best identified the genotype, being correct in 56 of 56 (21 LQT1, 33 LQT2, and 2 LQT3) families with mutation results. CONCLUSIONS: Typical ST-T-wave patterns are present in the majority of genotyped LQTS patients and can be used to identify LQT1, LQT2, and possibly LQT3 genotypes. Family-grouped ECG analysis improves genotype identification accuracy. This approach can simplify genetic screening by targeting the gene for initial study. The multiple ST-T patterns in each genotype raise questions regarding the pathophysiology and regulation of repolarization in LQTS.

    Title Human Mast Cell Tryptase Stimulates the Release of an Il-8-dependent Neutrophil Chemotactic Activity from Human Umbilical Vein Endothelial Cells (huvec).
    Date August 2000
    Journal Clinical and Experimental Immunology
    Excerpt

    Tryptase, the major product of human mast cell activation, is a potent stimulus of vascular leakage and neutrophil accumulation in vivo in animal studies, but the mechanisms of action remain unclear. Using HUVEC cultures we have sought to investigate the potential of tryptase to alter monolayer permeability or induce the release of neutrophil chemotactic activity. Tryptase (1-100 mU/ml) failed to alter the permeability of endothelial cell monolayers as assessed by albumin flux over 1 h. However, supernatants from endothelial cells treated with tryptase (1-50 mU/ml) for a 24-h period induced neutrophil migration across Transwell filters, with maximal migration observed at 10 mU/ml tryptase. Pretreatment of tryptase with the protease inhibitor leupeptin abolished the chemotactic activity, indicating a dependence on the catalytic site. Moreover, this effect was abolished by addition of an IL-8 neutralizing antibody, suggesting that IL-8 release makes an important contribution to the chemotactic activity. The interaction of mast cell tryptase with endothelial cells could be important in stimulating the ingress of neutrophils following mast cell activation in inflammatory disease.

    Title Sesquiflutter.
    Date November 1999
    Journal Journal of Cardiovascular Electrophysiology
    Title Mast Cell Tryptase As a Mediator of Hyperresponsiveness in Human Isolated Bronchi.
    Date July 1999
    Journal Clinical and Experimental Allergy : Journal of the British Society for Allergy and Clinical Immunology
    Excerpt

    BACKGROUND: Although the role of mediators and cytokines produced by mast cells is well established in asthmatic bronchial inflammation, the contribution of mast cell-derived proteases to the development of hyperresponsiveness remains unclear. There have been reports indicating that tryptase alters the mechanical activity of animal airway smooth muscle or spontaneously sensitized human isolated airways. OBJECTIVE: The aim of this study was to analyse the effect of purified mast cell tryptase on non-sensitized human isolated bronchi. METHODS: Both central and peripheral bronchi, dissected from lung specimens obtained at thoracotomy, were studied in terms of both mechanical activity i.e. isometric contraction in response to a variety of agonists and distribution of inflammatory cells i.e. immunohistochemistry. RESULTS: In both proximal and distal bronchi, the reactivity to histamine was significantly increased by a previous incubation in the presence of 1 microg/mL of tryptase (increase in maximal force, DeltaFmax was 12.1 +/- 3.8%, and 8.8 +/- 3.1%, respectively). This effect of tryptase on histamine-induced contraction was completely abrogated in the presence of the protease inhibitor benzamidine (100 micromol/L). Histological examination of specimens exposed to tryptase demonstrated an increase in mast cell number within the subepithelial tissue whereas mast cell numbers in the epithelial layer concomittently decreased. CONCLUSION: These results indicate that human mast cell tryptase alters the contractile response of non-sensitized human isolated bronchi and that this alteration is accompanied by a change in the mast cell distribution within the airway wall.

    Title Interaction of Human Mast Cell Tryptase with Endothelial Cells to Stimulate Inflammatory Cell Recruitment.
    Date June 1999
    Journal International Archives of Allergy and Immunology
    Title Rantes, Macrophage-inhibitory Protein 1alpha, and the Eosinophil Product Major Basic Protein Are Released into Upper Respiratory Secretions During Virus-induced Asthma Exacerbations in Children.
    Date March 1999
    Journal The Journal of Infectious Diseases
    Excerpt

    The presence of cytokines and the toxic eosinophil granule product major basic protein (MBP) was investigated in nasal aspirates from children with naturally occurring virus-induced asthma exacerbations and compared with levels in nasal aspirates taken from the same children when asymptomatic. Increased levels of MBP accompanied by increased levels of the chemokines RANTES and macrophage-inhibitory protein 1alpha were observed in nasal aspirates from children during the virus-induced exacerbations. Granulocyte-macrophage colony-stimulating factor was mostly undetectable in samples obtained during both symptomatic and asymptomatic periods. Interleukin-5 levels were low, but tended to increase in samples from symptomatic children. These data confirm that the eosinophil product MBP and the eosinophil chemoattractant chemokines RANTES and macrophage-inhibitory protein 1alpha are increased in upper respiratory viral infections associated with asthma exacerbations and suggest an important role for these chemokines in regulating eosinophil influx and activation. These chemokines may represent targets for therapeutic intervention in virus-induced asthma exacerbations.

    Title Development and Characterization of a Monoclonal Antibody Specific for Human Basophils and the Identification of a Unique Secretory Product of Basophil Activation.
    Date February 1999
    Journal Laboratory Investigation; a Journal of Technical Methods and Pathology
    Excerpt

    Despite increasing evidence that basophils can infiltrate into inflamed tissues during allergic reactions, determination of the extent of infiltration and elucidation of their role in allergic disease has been frustrated by the lack of reliable means for detecting this cell type in tissues. In the present study, we report on a new monoclonal antibody specific for basophils and on the initial characterization of the antigen it recognizes. Basophils were isolated from peripheral blood by Percoll density gradient centrifugation and a positive-selection immunomagnetic procedure and injected into mice to produce monoclonal antibodies. A hybridoma clone, designated BB1, secreted antibody of the IgG2a isotype; this antibody bound selectively to basophils on immunocytochemistry but did not react with any other cell type or tissue structure, although it did stain a proportion of cells from the basophilic cell line KU812F. In sections of mixed populations of peripheral blood cells, similar numbers of cells stained with Alcian blue dye and BB1 over a wide range of basophil purity. BB1 antibody was effective in identifying basophils in sections of mixed cells or in tissues after fixation with ethanol, Carnoy's solution, or formalin. Staining of basophils with BB1 gave a granular appearance, although flow cytometry indicated that some antigen was also present on the surface of the cell. Activation of these cells with anti-IgE antibody or with the calcium ionophore A23187 provoked release of the antigen in parallel with that of histamine. BB1 antibody did not, by itself, stimulate histamine release. The molecular mass of the antigen was determined on Hedrick-Smith gels to be 124+/-11 kd. This new monoclonal antibody will be a valuable experimental tool in future studies, allowing the reliable detection of basophils in tissues of patients with allergic and chronic inflammatory disease; in addition, the antigen it identifies has potential as a unique marker of basophil activation.

    Title The Role of Mast Cell Tryptase in Regulating Endothelial Cell Proliferation, Cytokine Release, and Adhesion Molecule Expression: Tryptase Induces Expression of Mrna for Il-1 Beta and Il-8 and Stimulates the Selective Release of Il-8 from Human Umbilical Vein Endothelial Cells.
    Date August 1998
    Journal Journal of Immunology (baltimore, Md. : 1950)
    Excerpt

    Mast cells are found frequently in close proximity to blood vessels, and endothelial cells are likely to be exposed to high concentrations of their granule mediators. We have investigated the proinflammatory actions of the major mast cell product tryptase on HUVEC. Addition of purified tryptase was found to stimulate thymidine incorporation, but induced little alteration in cell numbers, suggesting it is not a growth factor for HUVEC. Expression of ICAM-1, VCAM-1, and E-selectin was not altered following incubation with tryptase, but the potent granulocyte chemoattractant IL-8 was released in a dose-dependent fashion in response to physiologically relevant concentrations, with maximal levels in supernatants after 24 h. The actions of tryptase on HUVEC were inhibited by heat inactivation of the enzyme, or by preincubating with the protease inhibitors leupeptin or benzamidine, suggesting a requirement for an intact catalytic site. Reverse-transcription PCR analysis indicated up-regulation of mRNA for IL-8 as well as for IL-1 beta in response to tryptase or TNF-alpha. However, tryptase was a more selective stimulus than TNF-alpha and did not induce increased expression of mRNA for granulocyte-macrophage CSF or stimulate the release of this cytokine. Leukocyte accumulation in response to tryptase may be mediated in part through the selective secretion of IL-8 from endothelial cells.

    Title Accurate Quantitative Analysis of Cellular Proteins Using a Computerised Scanning-aided Dot Blot Technique.
    Date December 1996
    Journal British Journal of Biomedical Science
    Excerpt

    The aim of this study was to use a dot blot technique (DB) aided by a computerised scanning programme (CSDBT) to assess expression of cellular proteins in various tumour cell lines. Two groups of proteins, i.e. major histocompatibility complex (MHC) Class I and cell adhesion molecules (e.g. ICAM-1) were investigated. The constitutive expression of Class I antigens was measured and found to vary for different tumour lines. Exposure of SKV14 (SV40 transformed epithelial cells) and 5637 lines (bladder) to interferons (IFN) alpha or gamma resulted in upregulation of Class I antigens. IFN gamma, but not IFN alpha, induced ICAM-1 on all the lines studied. Thus, the stimulatory indices (SI) for Class I antigens in the case of SKV14, J82 (bladder) and 5637 lines after IFN gamma and IFN alpha stimulation were 2.02, 1.77, 4.68 and 1.65, 1.36, 2.08 respectively. The corresponding values for ICAM-1 were 1.65, 1.87, 3.43 and 1.75, 1.0, 0.96. In all the cases, the P values (using a paired t-test), except ICAM-1 for IFN alpha-stimulated J82 cells, were below 0.05. Following exposure of cells to combinations of drugs and IFN alpha, the percentage inhibition for Class I and ICAM-1 in the case of SKV14 and 5367 lines in the presence of cycloheximide (10 micrograms/mL) were 82%, 85% and 57%, 45% respectively. The corresponding values for indomethacin (10 micrograms/mL) were -23%, -52% and -20%, -24%. In all cases, the P values (using a paired t-test) were below 0.05. To our knowledge this is the first report describing a computerised scanning programme for assessing the presence of cellular proteins. The results were consistent with those obtained by radiobinding and immunocytochemistry. This simple and sensitive approach highlighted two important issues: (a) that it can be used to detect minor changes, under various conditions, of cellular proteins whether cytoplasmic or otherwise; and (b) that the accuracy of the measurements was enhanced by the use of computer scanning.

    Title Genetically Defined Therapy of Inherited Long-qt Syndrome. Correction of Abnormal Repolarization by Potassium.
    Date October 1996
    Journal Circulation
    Excerpt

    BACKGROUND: Many members of families with inherited long-QT (LQT) syndrome have mutations in HERG, a gene encoding a cardiac potassium channel that is modulated by extracellular potassium. We hypothesized that an increase in serum potassium would normalize repolarization in these patients. METHODS AND RESULTS: We studied seven subjects with chromosome 7-linked LQT syndrome and five normal control subjects. Repolarization was measured by ECG and body surface potential mapping during sinus rhythm, exercise, and atrial pacing, before and after serum potassium increase. Potassium administration improved repolarization in the LQT syndrome. At baseline, LQT subjects differed from control subjects: resting corrected QT interval (QTc, 627 +/- 90 versus 425 +/- 25 ms, P = .0007), QTc dispersion (133 +/- 62 versus 36 +/- 9 ms, P = .009), QT/RR slope (0.35 +/- 0.08 versus 0.24 +/- 0.07, P = .04), and global root-mean-square QT interval (RMS-QTc; 525 +/- 68 versus 393 +/- 22, P = .002). All LQT subjects had biphasic or notched T waves. After administration of potassium, the LQT group had a 24% reduction in resting QTc interval (from 617 +/- 92 to 469 +/- 23 ms, P = .004) compared with a 4% reduction among control subjects (from 425 +/- 25 to 410 +/- 45 ms, P > .05). The reduction was significantly greater in LQT subjects (P = .018). QT dispersion became normal in LQT subjects and did not change in control subjects. The slope of the relation between QT interval and cycle length (QT/RR slope) decreased toward normal. T-wave morphology improved in six of seven LQT subjects. The LQT group had a greater reduction in RMS-QTc than control subjects (P = .04). CONCLUSIONS: An increase in serum potassium corrects abnormalities of repolarization duration, T-wave morphology, QT/ RR slope, and QT dispersion in patients with chromosome 7-linked LQT.

    Title Accurate and Rapid Assessment of Mhc Antigen Upregulation Following Cytokine Stimulation.
    Date December 1993
    Journal Journal of Immunological Methods
    Excerpt

    Three human tumour cell lines, A431 (cervical), Scaber (bladder) and Fen (bladder), were studied using immunohistochemical staining (IC), radiobinding (RB), immunoprecipitation (IP), enzyme-linked immunosorbent assay (ELISA) and dot-blot (DB) techniques in order to assess major histocompatibility antigen (MHC) induction in response to interferon-gamma (IFN-gamma). Induction of class II antigens by IFN-gamma was observed on all three cell lines using all techniques. Monoclonal antibody (Mab) staining showed that both Scaber and A431 lines were positive for intact class I (Mab W6/32), class I free heavy chain (Mab HC10) and beta 2 microglobulin (beta 2-m) (Mab BBM.1), while Fen cells were positive only with HC10. The IP technique demonstrated the presence of a 45 kDa band on precipitation of the Fen lysate with HC10 Mab, whereas no such band was observed when W6/32 was used. The DB technique confirmed the negative reaction with W6/32 and BBM.1 Mabs, while HC10 showed positive staining which was upregulated by IFN-gamma. Transfection of the Fen cells with the beta 2-m gene resulted in the surface expression of fully assembled class I molecules. The DB technique showed upregulation of class I antigens following IFN-gamma stimulation, while RB detected no significant increase in cell surface expression (t test; p = 0.104). The binding values for transfected Fen cells before and after IFN-gamma stimulation were 2000 +/- 48 and 2161 +/- 156 cpm respectively. These results demonstrate that the DB technique facilitates an accurate assessment of cytokine induced antigens, corrected against a background of total cellular protein synthesis. The ease of execution, simplicity, non-radioactive nature and economy make it the method of choice for routine screening prior to the selection of suitable patients for cytokine therapy.

    Title Trichinosis with Ventilatory Failure and Persistent Myocarditis.
    Date July 1993
    Journal Clinical Infectious Diseases : an Official Publication of the Infectious Diseases Society of America
    Excerpt

    Life-threatening infections with Trichinella spiralis are rare in countries that have adopted laws requiring cooking of raw garbage fed to pigs. Thus such infections may pose a diagnostic dilemma for clinicians unfamiliar with their presentation. We report a case of imported trichinosis in a Mexican national who developed respiratory failure, myocarditis, and sinus arrest. The patient recovered uneventfully after the administration of benzimidazole and corticosteroid drugs, although a pacemaker was required to maintain normal cardiac rhythm. Symptomatic myocarditis is a rare complication of trichinosis that is often associated with increased morbidity and mortality. This report illustrates and reviews important features of the epidemiology, clinical presentation, and management of trichinosis.

    Title Mechanism of Dye Response and Interference in the Bradford Protein Assay.
    Date March 1986
    Journal Analytical Biochemistry
    Excerpt

    Bradford Coomassie brilliant blue G-250 protein-binding dye exists in three forms: cationic, neutral, and anionic. Although the anion is not freely present at the dye reagent pH, it is this form that complexes with protein. Dye binding requires a macromolecular form with certain reactive functional groups. Interactions are chiefly with arginine rather than primary amino groups; the other basic (His, Lys) and aromatic residues (Try, Tyr, and Phe) give slight responses. The binding behavior is attributed to Van der Waals forces and hydrophobic interactions. Assay interference by bases, detergents, and other compounds are explained in terms of their effects upon the equilibria between the three dye forms.


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