Internists, Pulmonologist (lungs)
22 years of experience

41 S Bedford Rd
Mount Kisco, NY 10549
914-393-4127
Locations and availability (1)

Education ?

Medical School Score
State University of New York Downstate (1988)
  • Currently 1 of 4 apples

Awards & Distinctions ?

Awards  
Patients' Choice Award (2011 - 2012)
Compassionate Doctor Recognition (2011 - 2012)
Associations
American Board of Internal Medicine
American Academy of Cosmetic Surgery
American College of Physicians

Affiliations ?

Dr. Fink is affiliated with 1 hospitals.

Hospital Affilations

Score

Rankings

  • Northern Westchester Hospital
    Pulmonary Disease
    380 E Main St, Mount Kisco, NY 10549
    • Currently 4 of 4 crosses
    Top 25%
  • Publications & Research

    Dr. Fink has contributed to 70 publications.
    Title Use of Intense Pulsed Light and a Retinyl-based Cream As a Potential Treatment for Cellulite: a Pilot Study.
    Date May 2008
    Journal Journal of Cosmetic Dermatology
    Excerpt

    BACKGROUND: There are few therapeutic treatments established for cellulite. OBJECTIVE: We studied the response of intense pulsed light (IPL) treatment with or without a compounded prescription retinyl-based cream on a small group of patients who had visible cellulite present on the buttocks and thigh regions. PATIENTS AND METHODS: Twenty patients were selected to complete a 12-week course of IPL treatment either with or without a retinyl-based cream. Assessment was based on visual evaluation, photographs, skin ultrasounds, and patient satisfaction. RESULTS: Fifteen (75%) completed the study, and nine (60%) had a self-improvement rating of >or= 50%. Seven (78%) of nine patients used IPL/cream. Of the remaining six (40%) completing the study, four (27%) had self-improvement ratings of 25-50% and two (13%; IPL only) were considered treatment failures with a rating of 10-25%. Both IPL/cream and IPL-only groups exhibited an improvement in the smoothness of the affected area even following weight gain. Skin ultrasounds confirmed an increase in the deposition of collagen. During an 8-month phone follow-up, 8 (67%) of 12 responding reported the same or improved results. CONCLUSION: IPL treatment with or without a retinyl-based cream can improve the appearance of peau d'orange cellulite, though the cream may augment cosmetic improvement. This approach is well tolerated, has minimal side effects, and is accompanied by a high degree of patient satisfaction.

    Title Characterization of Genomic Organization of the Adenosine A2a Receptor Gene by Molecular and Bioinformatics Analyses.
    Date June 2004
    Journal Brain Research
    Excerpt

    The adenosine A(2A) receptor (A(2A)R) is abundantly expressed in brain and emerging as an important therapeutic target for Parkinson's disease and potentially other neuropsychiatric disorders. To understand the molecular mechanisms of A(2A)R gene expression, we have characterized the genomic organization of the mouse and human A(2A)R genes by molecular and bioinformatic analyses. Three new exons (m1A, m1B and m1C) encoding the 5' untranslated regions (5'-UTRs) of mouse A(2A)R mRNA were identified by rapid amplification of 5' cDNA end (5' RACE), RT-PCR analysis and genome sequence analyses. Similar bioinformatics analysis also suggested six variants of the non-coding "exon 1" (h1A, h1B, h1C, h1D, h1E and h1F) in the human A(2A)R gene, which were confirmed by RT-PCR analysis, while three of the human exon 1 variants (h1D, h1E and h1F) were likewise verified by 5' oligonucleotide capping analysis suggesting multiple transcription start sites. Importantly, RT-PCR and quantitative PCR analysis demonstrated that the A(2A)R transcripts with different exon 1 variants displayed tissue-specific expression patterns. For instance, the mouse exon m1A mRNA was detected only in brain (specifically striatum) and the human exon h1D mRNA in lymphoreticular system. Furthermore, the determination of the three new transcription start sites of human A(2A)R gene by 5' oligonucleotide capping and bioinformatics analyses led to the identification of three corresponding promoter regions which contain several important cis elements, providing additional target for further molecular dissection of A(2A)R gene expression. Finally, our analysis indicates that A(2A)R mRNA and a novel transcript partially overlapping with the 3' exon h3, but in opposite orientation to the A(2A)R gene, could conceivably form duplexes to mutually regulate transcript expression. Thus, combined molecular and bioinformatics analyses revealed a new A(2A)R genomic structure, with conserved coding exons 2 and 3 and divergent, tissue-specific exon 1 variants encoding for 5'-UTR. This raises the possibility of generating multiple tissue-specific A(2A)R mRNA species by alternative promoters with varying regulatory susceptibility.

    Title Genetic and Pharmacological Inactivation of the Adenosine A2a Receptor Attenuates 3-nitropropionic Acid-induced Striatal Damage.
    Date February 2004
    Journal Journal of Neurochemistry
    Excerpt

    Adenosine A2A receptor (A2AR) antagonism attenuates 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced dopaminergic neurodegeneration and quinolinic acid-induced excitotoxicity in the neostriatum. As A2ARs are enriched in striatum, we investigated the effect of genetic and pharmacological A2A inactivation on striatal damage produced by the mitochondrial complex II inhibitor 3-nitropriopionic acid (3-NP). 3-NP was administered to A2AR knockout (KO) and wild-type (WT) littermate mice over 5 days. Bilateral striatal lesions were analyzed from serial brain tissue sections. Whereas all of the 3-NP-treated WT mice (C57BL/6 genetic background) had bilateral striatal lesions, only one of eight of the 3-NP-treated A2AR KO mice had detectable striatal lesions. Similar attenuation of 3-NP-induced striatal damage was observed in A2AR KO mice in a 129-Steel background. In addition, the effect of pharmacological antagonism on 3-NP-induced striatal neurotoxicity was tested by pre-treatment of C57Bl/6 mice with the A2AR antagonist 8-(3-chlorostyryl) caffeine (CSC). Although bilateral striatal lesions were observed in all mice treated either with 3-NP alone or 3-NP plus vehicle, there were no demonstrable striatal lesions in mice treated with CSC (5 mg/kg) plus 3-NP and in five of six mice treated with CSC (20 mg/kg) plus 3-NP. We conclude that both genetic and pharmacological inactivation of the A2AR attenuates striatal neurotoxicity produced by 3-NP. Since the clinical and neuropathological features of 3-NP-induced striatal damage resemble those observed in Huntington's disease, the results suggest that A2AR antagonism may be a potential therapeutic strategy in Huntington's disease patients.

    Title Sleep Regulation in Adenosine A2a Receptor-deficient Mice.
    Date February 2004
    Journal Neurology
    Title Renal Protection from Ischemia Mediated by A2a Adenosine Receptors on Bone Marrow-derived Cells.
    Date November 2003
    Journal The Journal of Clinical Investigation
    Excerpt

    Activation of A2A adenosine receptors (A2ARs) protects kidneys from ischemia-reperfusion injury (IRI). A2ARs are expressed on bone marrow-derived (BM-derived) cells and renal smooth muscle, epithelial, and endothelial cells. To measure the contribution of A2ARs on BM-derived cells in suppressing renal IRI, we examined the effects of a selective agonist of A2ARs, ATL146e, in chimeric mice in which BM was ablated by lethal radiation and reconstituted with donor BM cells derived from GFP, A2AR-KO, or WT mice to produce GFP-->WT, A2A-KO-->WT, or WT-->WT mouse chimera. We found little or no repopulation of renal vascular endothelial cells by donor BM with or without renal IRI. ATL146e had no effect on IRI in A2A-KO mice or A2A-KO-->WT chimera, but reduced the rise in plasma creatinine from IRI by 75% in WT mice and by 60% in WT-->WT chimera. ATL146e reduced the induction of IL-6, IL-1beta, IL-1ra, and TGF-alpha mRNA in WT-->WT mice but not in A2A-KO-->WT mice. Plasma creatinine was significantly greater in A2A-KO than in WT mice after IRI, suggesting some renal protection by endogenous adenosine. We conclude that protection from renal IRI by A2AR agonists or endogenous adenosine requires activation of receptors expressed on BM-derived cells.

    Title Adenosine A2a or A3 Receptors Are Required for Inhibition of Inflammation by Methotrexate and Its Analog Mx-68.
    Date February 2003
    Journal Arthritis and Rheumatism
    Excerpt

    OBJECTIVE: Low-dose weekly methotrexate therapy remains a mainstay in the treatment of inflammatory arthritis. Results of previous studies demonstrated that adenosine, acting at one or more of its receptors, mediates the antiinflammatory effects of methotrexate in animal models of both acute and chronic inflammation. We therefore sought to establish which receptor(s) is involved in the modulation of acute inflammation by methotrexate and its nonpolyglutamated analog MX-68 (N-[[4-[(2,4-diaminopteridin-6-yl)methyl]-3,4-dihydro-2H-1,4-benzothiazin-7-yl]-carbonyl]-L-homoglutamic acid). METHODS: We studied the effects of low-dose methotrexate (0.75 mg/kg intraperitoneally [IP] every week for 5 weeks), MX-68 (2 mg/kg IP 2 days and 1 hour before induction of inflammation), dexamethasone (1.5 mg/kg IP 1 hour before induction of inflammation), or vehicle control on acute inflammation in an air-pouch model in A(2A) and A(3) receptor knockout mice. RESULTS: Low-dose weekly methotrexate treatment increased the adenosine concentration in the exudates of all mice studied and reduced leukocyte and tumor necrosis factor alpha accumulation in the exudates of wild-type mice, but not in those of A(2A) or A(3) receptor knockout mice. Dexamethasone, an agent that suppresses inflammation by a different mechanism, was equally effective at suppressing leukocyte accumulation in A(2A) knockout, A(3) knockout, and wild-type mice, indicating that the lack of response was specific for methotrexate and MX-68. CONCLUSION: These findings confirm that adenosine, acting at A(2A) and A(3) receptors, is a potent regulator of inflammation. Moreover, these results provide strong evidence that adenosine, acting at either or both of these receptors, mediates the antiinflammatory effects of methotrexate and its analog MX-68.

    Title Adenosine Promotes Wound Healing and Mediates Angiogenesis in Response to Tissue Injury Via Occupancy of A(2a) Receptors.
    Date July 2002
    Journal The American Journal of Pathology
    Excerpt

    Recent evidence indicates that topical application of adenosine A(2A) receptor agonists, unlike growth factors, increases the rate at which wounds close in normal animals and promotes wound healing in diabetic animals as well as growth factors, yet neither the specific adenosine receptor involved nor the mechanism(s) by which adenosine receptor occupancy promotes wound healing have been fully established. To determine which adenosine receptor is involved and whether adenosine receptor-mediated stimulation of angiogenesis plays a role in promotion of wound closure we compared the effect of topical application of the adenosine receptor agonist CGS-21680 (2-p-[2-carboxyethyl]phenethyl-amino-5'-N-ethylcarboxamido-adenosine) on wound closure and angiogenesis in adenosine A(2A) receptor knockout mice and their wild-type littermates. There was no change in the rate of wound closure in the A(2A) receptor knockout mice compared to their wild-type littermates although granulation tissue formation was nonhomogeneous and there seemed to be greater inflammation at the base of the wound. Topical application of CGS-21680 increased the rate of wound closure and increased the number of microvessels in the wounds of wild-type mice but did not affect the rate of wound closure in A(2A) receptor knockout mice. Similarly, in a model of internal trauma and repair (murine air pouch model), endogenously produced adenosine released into areas of internal tissue injury stimulates angiogenesis because there was a marked reduction in blood vessels in the walls of healing air pouches of A(2A) receptor knockout mice compared to their wild-type controls. Inflammatory vascular leakage and leukocyte accumulation in the inflamed air pouch were similarly reduced in the A(2A) receptor knockout mice reflecting the reduced vascularity. Thus, targeting the adenosine A(2A) receptor is a novel approach to promoting wound healing and angiogenesis in normal individuals and those suffering from chronic wounds.

    Title Synergistic Up-regulation of Vascular Endothelial Growth Factor Expression in Murine Macrophages by Adenosine A(2a) Receptor Agonists and Endotoxin.
    Date July 2002
    Journal The American Journal of Pathology
    Excerpt

    Under normoxic conditions, macrophages from C57BL mice produce low levels of vascular endothelial growth factor (VEGF). Hypoxia stimulates VEGF expression by approximately 500%; interferon-gamma (IFN-gamma) with endotoxin [lipopolysaccharide (LPS)] also stimulates VEGF expression by approximately 50 to 150% in an inducible nitric oxide synthase (iNOS)-dependent manner. Treatment of normoxic macrophages with 5'-N-ethyl-carboxamido-adenosine (NECA), a nonselective adenosine A(2) receptor agonist, or with 2-[p-(2-carboxyethyl)-phenylethyl amino]-5'-N-ethyl-carboxamido-adenosine (CGS21680), a specific adenosine A(2A) receptor agonist, modestly increases VEGF expression, whereas 2-chloro-N(6)-cyclopentyl adenosine (CCPA), an adenosine A(1) agonist, does not. Treatment with LPS (0 to 1000 ng/ml), or with IFN-gamma (0 to 300 U/ml), does not affect VEGF expression. In the presence of LPS (EC(50) < 10 ng/ml), but not of IFN-gamma, both NECA and CGS21680 synergistically up-regulate VEGF expression by as much as 10-fold. This VEGF is biologically active in vivo in the rat corneal bioassay of angiogenesis. Inhibitors of iNOS do not affect this synergistic induction of VEGF, and macrophages from iNOS-/- mice produce similar levels of VEGF as wild-type mice, indicating that NO does not play a role in this induction. Under hypoxic conditions, VEGF expression is slightly increased by adenosine receptor agonists but adenosine A(2) or A(1) receptor antagonists 3,7-dimethyl-1-propargyl xanthine (DMPX), ZM241385, and 8-cyclopentyl-1,3-dipropylxanthine (DCPCX) do not modulate VEGF expression. VEGF expression is also not reduced in hypoxic macrophages from A(3)-/- and A(2A)-/- mice. Thus, VEGF expression by hypoxic macrophages does not seem to depend on endogenously released or exogenous adenosine. VEGF expression is strongly up-regulated by LPS/NECA in macrophages from A(3)-/- but not A(2A)-/- mice, confirming the role of adenosine A(2A) receptors in this pathway. LPS with NECA strongly up-regulates VEGF expression by macrophages from C(3)H/HeN mice (with intact Tlr4 receptors), but not by macrophages from C(3)H/HeJ mice (with mutated, functionally inactive Tlr4 receptors), implicating signaling through the Tlr4 pathway in this synergistic up-regulation. Finally, Western blot analysis of adenosine A(2A) receptor expression indicated that the synergistic interaction of LPS with A(2A) receptor agonists does not involve up-regulation of A(2A) receptors by LPS. These results indicate that in murine macrophages there is a novel pathway regulating VEGF production, that involves the synergistic interaction of adenosine A(2A) receptor agonists through A(2A) receptors with LPS through the Tlr4 pathway, resulting in the strong up-regulation of VEGF expression by macrophages in a hypoxia- and NO-independent manner.

    Title The Role of the D(2) Dopamine Receptor (d(2)r) in A(2a) Adenosine Receptor (a(2a)r)-mediated Behavioral and Cellular Responses As Revealed by A(2a) and D(2) Receptor Knockout Mice.
    Date April 2001
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    The A(2A)R is largely coexpressed with D(2)Rs and enkephalin mRNA in the striatum where it modulates dopaminergic activity. Activation of the A(2A)R antagonizes D(2)R-mediated behavioral and neurochemical effects in the basal ganglia through a mechanism that may involve direct A(2A)R-D(2)R interaction. However, whether the D(2)R is required for the A(2A)R to exert its neural function is an open question. In this study, we examined the role of D(2)Rs in A(2A)R-induced behavioral and cellular responses, by using genetic knockout (KO) models (mice deficient in A(2A)Rs or D(2)Rs or both). Behavioral analysis shows that the A(2A)R agonist 2-4-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine reduced spontaneous as well as amphetamine-induced locomotion in both D(2) KO and wild-type mice. Conversely, the nonselective adenosine antagonist caffeine and the A(2A)R antagonist 8-(3-chlorostyryl)caffeine produced motor stimulation in mice lacking the D(2)R, although the stimulation was significantly attenuated. At the cellular level, A(2A)R inactivation counteracted the increase in enkephalin expression in striatopallidal neurons caused by D(2)R deficiency. Consistent with the D(2) KO phenotype, A(2A)R inactivation partially reversed both acute D(2)R antagonist (haloperidol)-induced catalepsy and chronic haloperidol-induced enkephalin mRNA expression. Together, these results demonstrate that A(2A)Rs elicit behavioral and cellular responses despite either the genetic deficiency or pharmacological blockade of D(2)Rs. Thus, A(2A)R-mediated neural functions are partially independent of D(2)Rs. Moreover, endogenous adenosine acting at striatal A(2A)Rs may be most accurately viewed as a facilitative modulator of striatal neuronal activity rather than simply as an inhibitory modulator of D(2)R neurotransmission.

    Title A Major Locus for Myoclonus-dystonia Maps to Chromosome 7q in Eight Families.
    Date December 2000
    Journal American Journal of Human Genetics
    Excerpt

    Myoclonus-dystonia (M-D) is an autosomal dominant disorder characterized by myoclonic and dystonic muscle contractions that are often responsive to alcohol. The dopamine D2 receptor gene (DRD2) on chromosome 11q has been implicated in one family with this syndrome, and linkage to a 28-cM region on 7q has been reported in another. We performed genetic studies, using eight additional families with M-D, to assess these two loci. No evidence for linkage was found for 11q markers. However, all eight of these families showed linkage to chromosome 7 markers, with a combined multipoint LOD score of 11.71. Recombination events in the families define the disease gene within a 14-cM interval flanked by D7S2212 and D7S821. These data provide evidence for a major locus for M-D on chromosome 7q21.

    Title Study of A(2a) Adenosine Receptor Gene Deficient Mice Reveals That Adenosine Analogue Cgs 21680 Possesses No A(2a) Receptor-unrelated Lymphotoxicity.
    Date November 2000
    Journal British Journal of Pharmacology
    Excerpt

    Cell surface A(2A) adenosine receptor (A(2A)R) mediated signalling affects a variety of important processes and adenosine analogues possess promising pharmacological properties. Demonstrating the receptor specificity of potentially lymphotoxic adenosine-based drugs facilitates their development for clinical applications. To distinguish between the receptor-dependent and -independent lymphotoxicity and apoptotic activity of adenosine and its analogues we used lymphocytes from A(2A)R-deficient mice. Comparison of A(2A)R-expressing (+/+) and A(2A)R-deficient (-/-) cells in cyclic AMP accumulation assays confirmed that the A(2A)R agonist CGS 21680 is indeed selective for A(2A) receptors in T-lymphocytes. Incubation of A(2A)R-expressing thymocytes with extracellular adenosine or CGS 21680 in vitro results in the death of about 7-15% of thymocytes. In contrast, no death was induced in parallel assays in cells from A(2A)R-deficient mice, providing genetic evidence that CGS 21680 does not display adenosine receptor-independent intracellular cytotoxicity. The A(2A) receptor-specific lymphotoxicity of CGS 21680 is also demonstrated in a long-term (6-day) in vitro model of thymocyte positive selection where addition of A(2A)R antagonist ZM 241,385 did block the effects of CGS 21680, allowing the survival of T cells. The use of cells from adenosine receptor-deficient animals is proposed as a part of the screening process for potential adenosine-based drugs for their receptor-independent cytotoxicity and lymphotoxicity.

    Title Porcine Xenografts in Parkinson's Disease and Huntington's Disease Patients: Preliminary Results.
    Date August 2000
    Journal Cell Transplantation
    Excerpt

    The observation that fetal neurons are able to survive and function when transplanted into the adult brain fostered the development of cellular therapy as a promising approach to achieve neuronal replacement for treatment of diseases of the adult central nervous system. This approach has been demonstrated to be efficacious in patients with Parkinson's disease after transplantation of human fetal neurons. The use of human fetal tissue is limited by ethical, infectious, regulatory, and practical concerns. Other mammalian fetal neural tissue could serve as an alternative cell source. Pigs are a reasonable source of fetal neuronal tissue because of their brain size, large litters, and the extensive experience in rearing them in captivity under controlled conditions. In Phase I studies porcine fetal neural cells grafted unilaterally into Parkinson's disease (PD) and Huntington's disease (HD) patients are being evaluated for safety and efficacy. Clinical improvement of 19% has been observed in the Unified Parkinson's Disease Rating Scale "off" state scores in 10 PD patients assessed 12 months after unilateral striatal transplantation of 12 million fetal porcine ventral mesencephalic (VM) cells. Several patients have improved more than 30%. In a single autopsied PD patient some porcine fetal VM cells were observed to survive 7 months after transplantation. Twelve HD patients have shown a favorable safety profile and no change in total functional capacity score 1 year after unilateral striatal placement of up to 24 million fetal porcine striatal cells. Xenotransplantation of fetal porcine neurons is a promising approach to delivery of healthy neurons to the CNS. The major challenges to the successful use of xenogeneic fetal neuronal cells in neurodegenerative diseases appear to be minimizing immune-mediated rejection, management of the risk of xenotic (cross-species) infections, and the accurate assessment of clinical outcome of diseases that are slowly progressive.

    Title Transplantation of Embryonic Porcine Mesencephalic Tissue in Patients with Pd.
    Date June 2000
    Journal Neurology
    Excerpt

    OBJECTIVE: To assess the safety and the effect on standardized clinical rating measures of transplanted embryonic porcine ventral mesencephalic (VM) tissue in advanced PD. METHODS: Twelve patients with idiopathic PD underwent unilateral implantation of embryonic porcine VM tissue; six received cyclosporine immunosuppression and six received tissue treated with a monoclonal antibody directed against major histocompatibility complex class I. Patients were followed for 12 months and assessed by clinical examination, MRI, and 18F-levodopa PET. Porcine endogenous retrovirus testing was conducted by PCR-based method on peripheral blood mononuclear cells. RESULTS: Cell implantation occurred without serious adverse events in all patients. Cultures were negative for bacterial and unknown viral contamination. No porcine endogenous retrovirus DNA sequences were found. MRI demonstrated cannula tracts within the putamen and caudate, with minimal or no edema and no mass effect at the transplant sites. In the medication-off state, total Unified Parkinson's Disease Rating Scale scores improved 19% (p = 0.01). Three patients improved over 30%. There were two patients with improved gait. 18F-levodopa PET failed to show changes on the transplanted side. CONCLUSIONS: Unilateral transplantation of porcine embryonic VM cells into PD patients was well tolerated with no evidence of transmission of porcine endogenous retrovirus. Changes in standardized clinical PD rating measures were variable, similar to the results of the first trials of unilateral human embryonic allografts that transplanted small amounts of tissue.

    Title Selective Attenuation of Psychostimulant-induced Behavioral Responses in Mice Lacking A(2a) Adenosine Receptors.
    Date June 2000
    Journal Neuroscience
    Excerpt

    A(2A) adenosine receptors are highly expressed in the striatum where they modulate dopaminergic activity. The role of A(2A) receptors in psychostimulant action is less well understood because of the lack of A(2A)-selective compounds with access to the central nervous system. To investigate the A(2A) adenosinergic regulation of psychostimulant responses, we examined the consequences of genetic deletion of A(2A) receptors on psychostimulant-induced behavioral responses. The extent of dopaminergic innervation and expression of dopamine receptors in the striatum were indistinguishable between A(2A) receptor knockout and wild-type mice. However, locomotor responses to amphetamine and cocaine were attenuated in A(2A) knockout mice. In contrast, D(1)-like receptor agonists SKF81297 and SKF38393 produced identical locomotor stimulation and grooming, respectively, in wild-type and A(2A) knockout mice. Similarly, the D(2)-like agonist quinpirole produced motor-depression and stereotypy that were indistinguishable between A(2A) knockout and wild-type mice. Furthermore, attenuated amphetamine- (but not SKF81297-) induced locomotion was observed in pure 129-Steel as well as hybrid 129-SteelxC57BL/6 mice, confirming A(2A) receptor deficiency (and not genetic background) as the cause of the blunted psychostimulant responses in A(2A) knockout mice.These results demonstrate that A(2A) receptor deficiency selectively attenuates psychostimulant-induced behavioral responses and support an important role for the A(2A) receptor in modulating psychostimulant effects.

    Title A(2a) Adenosine Receptor Deficiency Attenuates Brain Injury Induced by Transient Focal Ischemia in Mice.
    Date November 1999
    Journal The Journal of Neuroscience : the Official Journal of the Society for Neuroscience
    Excerpt

    Extracellular adenosine critically modulates ischemic brain injury, at least in part through activation of the A(1) adenosine receptor. However, the role played by the A(2A) receptor has been obscured by intrinsic limitations of A(2A) adenosinergic agents. To overcome these pharmacological limitations, we explored the consequences of deleting the A(2A) adenosine receptor on brain damage after transient focal ischemia. Cerebral morphology, as well as vascular and physiological measures (before, during, and after ischemia) did not differ between A(2A) receptor knock-out and wild-type littermates. The volume of cerebral infarction, as well as the associated neurological deficit induced by transient filament occlusion of the middle cerebral artery, were significantly attenuated in A(2A) receptor knock-out mice. This neuroprotective phenotype of A(2A) receptor-deficient mice was observed in different genetic backgrounds, confirming A(2A) receptor disruption as its cause. Together with complimentary pharmacological studies, these data suggest that A(2A) receptors play a prominent role in the development of ischemic injury within brain and demonstrate the potential for anatomical and functional neuroprotection against stroke by A(2A) receptor antagonists.

    Title Leukemia Inhibitory Factor and Ngf Regulate Signal Transducers and Activators of Transcription Activation in Sympathetic Ganglia: Convergence of Cytokine- and Neurotrophin-signaling Pathways.
    Date December 1998
    Journal Brain Research
    Excerpt

    We have used the response of the superior cervical ganglia (SCG) to axotomy to investigate interactions between neuropoietic cytokines and neurotrophins. Postganglionic sympathetic axotomy leads to a prolonged leukemia inhibitory factor (LIF)-dependent activation of signal transducers and activators of transcription (STAT) factors. To study regulation of LIF-dependent activation of STAT proteins and to mimic the loss of target-derived NGF resulting from postganglionic axotomy in vivo, SCG were explanted into media lacking NGF and activation of STAT proteins was assessed by electrophoretic mobility shift assay. Like postganglionic axotomy in vivo. STAT proteins were activated for up to 8 days after explantation of SCG in vitro. SCG cultured in the presence of NGF showed decreased STAT binding when compared to ganglia cultured in NGF-free media. This inhibition of STAT activation by NGF was only present in ganglia cultured for more than 5 days and was mimicked by brain-derived neurotrophic factor (BDNF). The serine kinase inhibitor H7 augmented the increase of STAT binding produced by explantation, suggesting the presence of a labile repressor of STAT activation in the SCG. These data indicated that the neuropoietic cytokine-signaling pathway interacts with neurotrophin and H7-sensitive-signaling pathways to regulate activation of STAT proteins in sympathetic neurons. Moreover, these data suggest that one of the mechanisms leading to prolonged activation of STAT proteins after postganglionic axotomy in vivo is loss of target-derived neurotrophins.

    Title Nfat Interactions with the Vasoactive Intestinal Peptide Cytokine Response Element.
    Date May 1998
    Journal Journal of Neuroscience Research
    Excerpt

    The vasoactive intestinal peptide cytokine response element (VIP CyRE) is responsible for mediating the transcriptional induction of the VIP gene to the neuropoietic cytokines leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF). In investigating the sequence and function of the CyRE, we found a region of DNA with homology to the distal NFAT site in the IL-2 promoter. In this paper we characterize this sequence and show that the VIP NFAT site recognizes T cell NFAT with similar affinity to the previously characterized IL-2 NFAT site. However, despite its location in the middle of the CyRE, we find no CNTF/LIF induced binding to it. Instead we show that in NBFL neuroblastoma cells, the calcium ionophore A23187 induces a protein to bind to the VIP NFAT site. This A23187-mediated induction of nuclear protein binding to an NFAT oligonucleotide is dependent on extracellular calcium but not dependent on de novo protein synthesis. Thus, this protein has the characteristics of an NFAT-like protein and is recognized by an NFAT3-specific antiserum suggesting that it is indeed an NFAT protein. The location of the NFAT site in the VIP CyRE suggests that this may be one mechanism through which different signaling pathways engage in cross talk to alter VIP gene transcription.

    Title Coordinate Regulation of Stat Signaling and C-fos Expression by the Tyrosine Phosphatase Shp-2.
    Date April 1998
    Journal The Journal of Biological Chemistry
    Excerpt

    The src homology 2 (SH2) domain-containing protein-tyrosine phosphatase SHP-2 has been implicated as an important positive regulator of several mitogenic signaling pathways. SHP-2 has more recently been shown to be tyrosine phosphorylated and recruited to the gp130 component of the ciliary neurotrophic factor (CNTF) receptor complex upon stimulation with CNTF. CNTF does not, however, have a proliferative effect on responsive cells, but rather enhances the survival and differentiation of sympathetic, motor, and sensory neurons. In this study, expression of an interfering mutant of SHP-2 in the neuroblastoma cell line NBFL increased CNTF induction of a vasoactive intestinal peptide (VIP) reporter gene, and in cultures of sympathetic neurons, it resulted in an up-regulation of endogenous VIP and substance P (SP) gene expression. Members of the CNTF family of cytokines transmit their signal by activating signaling pathways involving both STAT and Fos-Jun transcription factors. In CNTF-stimulated NBFL cells that constitutively express the SHP-2 interfering mutant, there was increased and prolonged formation of STAT/DNA complexes, but decreased AP-1 binding activity, that mirrored a down-regulation of c-fos expression both at the mRNA and protein level. Taken together, these data indicate that SHP-2 has dual and opposing roles in a signaling cascade triggered by the same ligand, as illustrated by its ability to differentially regulate the levels of activity of both STAT and AP-1 transcription factors.

    Title Transplantation in Parkinson's Disease.
    Date February 1998
    Journal Artificial Organs
    Excerpt

    The modification of nervous system function by cell replacement and the introduction of heterologous genes are being developed as possible therapeutic approaches in degenerative diseases of the nervous system. The use of cellular transplantation in the nervous system of patients with neurodegenerative diseases will be reviewed with an emphasis on Parkinson's disease.

    Title The Protein Tyrosine Phosphatase Shp-2 Negatively Regulates Ciliary Neurotrophic Factor Induction of Gene Expression.
    Date February 1998
    Journal Current Biology : Cb
    Excerpt

    Ciliary neurotrophic factor, along with other neuropoietic cytokines, signals through the shared receptor subunit gp130 [1-3], leading to the tyrosine phosphorylation of a number of substrates [4,5], including the transcription factors STAT1 and STAT3 and the protein tyrosine phosphatase SHP-2 [6,7] [8]. SHP-2 (also known as PTP1D, SHPTP2, Syp and PTP2C) is a positive regulatory molecule required for the activation of the mitogen-activated protein kinase pathway and the stimulation of gene expression in response to epidermal growth factor, insulin and platelet-derived growth factor stimulation [9-11]. We have previously shown that cytokines that signal via the gp130 receptor subunit activate transcription of the vasoactive intestinal peptide (VIP) gene through a 180 bp cytokine response element (CyRE) [12,13]. To characterize the role of SHP-2 in the regulation of gp130-stimulated gene expression, we examined the regulation of the VIP CyRE in two systems that prevented ligand-dependent SHP-2 phosphorylation. Inhibition of SHP-2, either by mutating the tyrosine residue in gp130 that mediates the SHP-2 interaction, or by expression of dominant-negative SHP-2, resulted in dramatic increases in gp130-dependent gene expression, through the VIP CyRE and more specifically through multimerized STAT-binding sites. These data suggest that SHP-2 has a negative role in gp130 signaling by modulating STAT-mediated transcriptional activation.

    Title Protein Kinase C Regulates Adenosine A2a Receptor Mrna Expression in Sh-sy5y Cells.
    Date January 1998
    Journal European Journal of Pharmacology
    Excerpt

    Protein kinase C regulates mRNAs encoding several G protein-linked receptors but its role in adenosine A2a receptor expression is not known. We tested the hypothesis that protein kinase C activated by tetradecanoyl phorbol acetate (TPA) regulates adenosine A2a receptor mRNA levels. SH-SY5Y human neuroblastoma cells express adenosine receptors which positively couple to adenylyl cyclase with a pharmacologic profile expected of the A2a subtype. Northern blotting demonstrated an adenosine A2a receptor mRNA species of similar molecular size in SH-SY5Y cells and in human brain. TPA increased adenosine A2a receptor mRNA in a dose- and time-dependent fashion. Transcription or translation inhibition prevented increases in adenosine A2a receptor mRNA. Bisindolylmaleimide blocked TPA effects. Adenosine A2a receptor mRNA stability was unchanged by TPA. This study identifies a human neuroblastoma cell line expressing functional adenosine A2a receptors. Protein kinase C activation appears to enhance transcription of the adenosine A2a receptor gene.

    Title A Sweat Gland-derived Differentiation Activity Acts Through Known Cytokine Signaling Pathways.
    Date December 1997
    Journal The Journal of Biological Chemistry
    Excerpt

    The sympathetic innervation of sweat glands undergoes a target-induced noradrenergic to cholinergic/peptidergic switch during development. Similar changes are induced in cultured sympathetic neurons by sweat gland cells or by one of the following cytokines: leukemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), or cardiotrophin-1 (CT-1). None of these is the sweat gland-derived differentiation activity. LIF, CNTF, and CT-1 act through the known receptors LIF receptor beta (LIFRbeta) and gp130 and well defined signaling pathways including receptor phosphorylation and STAT3 activation. Therefore, to determine whether the gland-derived differentiation activity was a member of the LIF/CNTF cytokine family, we tested whether it acted via these same receptors and signal cascades. Blockade of LIFRbeta inhibited the sweat gland differentiation activity in neuron/gland co-cultures, and extracts of gland-containing footpads stimulated tyrosine phosphorylation of LIFRbeta and gp130. An inhibitor (CGX) of molecules that bind the CNTFRalpha, which is required for CNTF signaling, did not affect the gland-derived differentiation activity. Soluble footpad extracts induced the same changes in NBFL neuroblastoma cells as LIF and CNTF, including increased vasoactive intestinal peptide mRNA, STAT3 dimerization, and DNA binding, and stimulation of transcription from the vasoactive intestinal peptide cytokine-responsive element. Thus, the sweat gland-derived differentiation activity uses the same signaling pathway as the neuropoietic cytokines, and is likely to be a family member.

    Title Apomorphine Priming Alters the Response of Striatal Outflow Pathways to D2 Agonist Stimulation in 6-hydroxydopamine-lesioned Rats.
    Date August 1997
    Journal Neuroscience
    Excerpt

    Chronic treatment with dopaminergic agonists is associated with response fluctuations to L-dihydroxyphenylalanine in Parkinson's disease and enhanced motor activity to D1 and D2 dopamine agonists in rats with 6-hydroxydopamine lesions of the nigrostriatal pathway. In dopamine-depleted rodents this phenomenon has been referred to as "priming" or reverse tolerance. The neurochemical changes that underlie "priming" of dopaminergic agonist responses are poorly understood. Some aspects of priming of D1 agonist-mediated rotation in the 6-hydroxydopamine-lesioned rat have been characterized, but priming of D2-agonist-dependent motor responses has been less thoroughly studied. In this study, examination of rotational behaviour and induction of Fos-like immunoreactivity were used to investigate changes in the striatal outflow systems in response to treatment with the D2 agonist quinpirole in 6-hydroxydopamine-lesioned rats that had been primed with apomorphine. Administration of apomorphine (0.5 mg/kg; three injections at three to six day intervals) permitted an otherwise inactive dose of quinpirole (0.25 mg/kg) to produce robust contralateral rotation and to induce the expression of Fos in striatal neurons belonging to the striato-nigro-entopeduncular ("direct") pathway. The increase in contralateral rotation and ipsilateral striatal Fos expression following administration of quinpirole to apomorphine-primed rats was mediated by a D2-like receptor and did not appear to be due to a change in sensitivity of D2 receptors. Apomorphine priming also enhanced the ability of quinpirole to induce Fos expression in the globus pallidus, a target of the striatopallidal ("indirect") pathway. Western blot analysis confirmed that treatment with quinpirole induced the expression of c-Fos protein with no change in the expression of 35-37,000 mol. wt Fos-related antigens in apomorphine-primed rats treated with water or quinpirole. Induction of Fos expression in the striatum generally results from blockade of D2 receptors and the striato-nigro-entopeduncular pathway preferentially expresses D1 receptors. Thus, the quinpirole-dependent induction of striatal Fos in apomorphine-primed 6-hydroxydopamine-lesioned rats represents a qualitative alteration in striatal outflow. These studies demonstrate that pretreatment of 6-hydroxydopamine-lesioned rats with apomorphine increases the activity of the "direct" and "indirect" striatal outflow pathways in response to D2 receptor stimulation. These changes have the net result of enhancing thalamocortical activity and likely underlie the enhanced contralateral rotation produced by quinpirole in apomorphine-primed rats. Changes in striatal outflow, particularly in the striato-nigro-entopeduncular pathway, may contribute to alterations in D2-dependent motor responses observed after chronic dopaminergic stimulation in the dopamine-depleted striatum.

    Title Enhanced Creb Phosphorylation and Changes in C-fos and Fra Expression in Striatum Accompany Amphetamine Sensitization.
    Date June 1997
    Journal Brain Research
    Excerpt

    Expression in striatum of c-Fos, a 35 kDa Fos-related antigen (FRA) and the phosphorylated form of cyclic AMP response element binding protein (phosphoCREB) was assessed using Western blots in rats that developed behavioral sensitization following repeated amphetamine administration. Treatment with d-amphetamine (5 mg/kg) for 5 consecutive days produced behavioral sensitization. Similar to previous observations using chronic cocaine administration, amphetamine sensitized animals had decreased c-Fos and increased FRA proteins in striatum. Supershift analysis with antisera to c-Fos and FRA proteins demonstrated that 4-Fos and the 35 kDa FRA are components of the striatal AP-1 binding complex from sensitized rats. Thus, amphetamine sensitization is accompanied by alterations in the composition of the AP-1 DNA binding complex. An increased amount of phosphoCREB protein was also present in the striatum of amphetamine sensitized rats. These results suggest that alterations in Fos, FRA and CREB transcription factors are common neuronal responses to chronic psychostimulant administration and may contribute to regulation of genes important to the neuroplastic changes underlying psychostimulant sensitization.

    Title Integration of Jak-stat and Ap-1 Signaling Pathways at the Vasoactive Intestinal Peptide Cytokine Response Element Regulates Ciliary Neurotrophic Factor-dependent Transcription.
    Date May 1997
    Journal The Journal of Biological Chemistry
    Excerpt

    Ciliary neurotrophic factor (CNTF)-dependent induction of expression of the neuropeptide vasoactive intestinal peptide (VIP) gene is mediated by a 180-base pair cytokine response element (CyRE) in the VIP promoter. To elucidate the molecular mechanisms mediating the transcriptional activation by CNTF, intracellular signaling to the CyRE has been studied in a neuroblastoma cell line. It has been shown previously that CNTF induces Stat proteins to bind to a site within the CyRE. CNTF also induces a second protein to bind to a C/EBP-like site within the CyRE. In this report, we show that this inducible CyRE binding protein is composed of the AP-1 proteins c-Fos, JunB, and JunD. These proteins bind to a non-canonical AP-1 site located near the previously characterized C/EBP site. The serine/threonine kinase inhibitor H7 prevents CNTF-dependent induction of AP-1 binding and CyRE-mediated transcription, suggesting that an H7-sensitive kinase is important to mediating CNTF effects on VIP transcription. The integration at the VIP CyRE of the Jak-Stat and AP-1 signaling pathways with other pre-existing proteins provides a cellular mechanism for cell- and cytokine-specific signaling.

    Title Synergistic Interaction Between an Adenosine Antagonist and a D1 Dopamine Agonist on Rotational Behavior and Striatal C-fos Induction in 6-hydroxydopamine-lesioned Rats.
    Date April 1997
    Journal Brain Research
    Excerpt

    The interaction between adenosine and D1 dopamine systems in regulating motor behavior and striatal c-Fos expression was examined in rats with unilateral 6-hydroxydopamine (6-OHDA) lesions. These results were compared to the synergistic interaction between D1 and D2 dopamine systems in 6-OHDA rats. Coadministration of the adenosine antagonist 3,7-dimethyl-1-propargylxanthine (DMPX: 10 mg/kg) and the D1 dopamine agonist SKF38393 (0.5 mg/kg) to 6-OHDA-lesioned rats produced significant contralateral rotation and c-Fos expression in the ipsilateral striatum compared to 6-OHDA rats treated with either drug alone. However, the regional pattern of striatal c-Fos activation following treatment of 6-OHDA rats with SKF38393 and DMPX was different from the dorsolateral pattern of striatal c-Fos induction observed after coadministration of D1 and D2 dopamine agonists (SKF38393: 0.5 mg/kg + quinpirole: 0.05 mg/kg). These data are consistent with a functional interaction between D1 dopamine and adenosine systems in the striatum, but suggest that activation of different subsets of striatal neurons underlie the behavioral synergy observed following combined adenosine antagonist-D1 dopamine agonist and combined D1 dopamine agonist-D2 dopamine agonist treatment.

    Title Region-specific Central Nervous System Expression and Axotomy-induced Regulation in Sympathetic Neurons of a Vip-beta-galactosidase Fusion Gene in Transgenic Mice.
    Date April 1997
    Journal Brain Research. Molecular Brain Research
    Excerpt

    To assess the activity of cis-acting elements that direct human vasoactive intestinal peptide (VIP) expression in vivo, two independent transgenic mouse lines were created using a transgene comprised of 1.9 kb of 5'-flanking sequence of the human VIP gene joined to the Escherichia coli beta-galactosidase reporter gene. Transgene expression in brain was assessed using beta-galactosidase histochemistry and compared to the distribution of endogenous VIP expression. Transgene expression was observed in most central and peripheral nervous system sites in which endogenous VIP is expressed. We investigated whether the VIP-beta-galactosidase transgene was regulated in sympathetic neurons in experimental paradigms in which VIP regulation is dependent on the release of leukemia inhibitory factor (LIF). After dissociation in vitro and postganglionic axotomy in vivo there were parallel increases in endogenous VIP and transgene expression in superior cervical ganglia. These results indicate that the 1.9 kb region of 5'-flanking sequence of the human VIP gene includes genomic elements important for cell-specific expression and LIF-dependent regulation in neurons.

    Title Effects of Selective Adenosine A1 and A2a Agonists on Amphetamine-induced Locomotion and C-fos in Striatum and Nucleus Accumbens.
    Date December 1996
    Journal Brain Research
    Excerpt

    Low to moderate doses of amphetamine produce locomotion which is dependent on release of dopamine in the anteromedial striatum and nucleus accumbens. The effects of selective adenosine A1 and A2a receptor agonists on locomotion and c-Fos induction following a moderate dose of amphetamine was assessed in rats. Pretreatment with the adenosine A1 receptor agonist N6-cyclohexyladenosine (CHA) or the adenosine A2a receptor agonist 2-[(2-aminoethylamino)carbonylethylphenylethylamino]-5'-N- ethylcarboxamidoadenosine (APEC) inhibited locomotion following an injection of amphetamine (1.5 mg/kg). This dose of amphetamine induced Fos-like immunoreactivity in an antero-dorsomedial distribution in the caudate-putamen and uniformly in the core and shell of the nucleus accumbens. Pretreatment with the adenosine A2a receptor agonist APEC, but not the adenosine A1 receptor agonist CHA, attenuated c-Fos induction in caudate-putamen and nucleus accumbens by amphetamine. These findings indicate that amphetamine-induced behavior is subject to modulation by adenosine receptors through mechanisms which are both related to and independent of c-Fos induction.

    Title Quantitative Analysis of the Expression of a Vip Transgene.
    Date October 1996
    Journal Brain Research. Molecular Brain Research
    Excerpt

    We have analyzed the expression of a transgene bearing 2 kilobases of the 5' flanking region of the human vasoactive intestinal polypeptide (VIP) gene coupled to beta-galactosidase. Expression was assayed by beta-galactosidase histochemistry and by mRNA quantitation using polymerase chain reaction (PCR)-mediated amplification; we compared beta-galactosidase activity against both transgene and endogenous VIP mRNA levels. We found that the human 5' flanking sequence in this construct is able to direct tissue-specific expression of beta-galactosidase similar to the pattern for endogenous VIP. However, the transgene is also expressed in smooth muscle and Schwann cells, where VIP mRNA is rare. In various tissues where the transgene and endogenous gene are both active, the ratio between their message levels differs dramatically--transgene mRNA is more abundant where VIP is relatively scarce, but is much less abundant than the endogenous message at sites where VIP mRNA is most concentrated. These results suggest that sequence elements that may restrict VIP transcription or cause tissue-specific VIP mRNA accumulation are missing from the transgene. In the testis there is a high level of transgene message but no significant beta-galactosidase activity; this discrepancy is caused by transcription from a cryptic promoter within the beta-galactosidase sequence.

    Title Stat Proteins Are Activated by Ciliary Neurotrophic Factor in Cells of Central Nervous System Origin.
    Date September 1996
    Journal Journal of Neuroscience Research
    Excerpt

    Neuropoietic cytokines, including ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF), have survival effects on cells of the peripheral and central nervous systems (PNS and CNS). CNTF and LIF also produce differentiation in some cells of the PNS. We have shown previously that CNTF activates the signal transducers and activators of transcription (STAT) family of transcription factors, and that this signaling pathway may be one of several employed by CNTF to regulate the expression of the vasoactive intestinal peptide (VIP) gene in cells of the PNS (Symes et al.: Proc Natl Acad Sci USA 90:572-576, 1993; Symes et al.: Mol Endocrinol 8:1750-1763, 1994). To investigate the mechanisms of action of CNTF in the CNS, we have analyzed the activation of STAT proteins in a septal-derived cell line, SN48, and in primary CNS cultures. CNTF treatment of SN48 cells produces a sustained activation of Stat3. CNTF treatment of SN48 cells also activated transcription mediated by the VIP cytokine responsive element (CyRE) which contains a STAT binding site. Mutation of the STAT site in the CyRE attenuated transcriptional activation by CNTF, indicating the importance of STAT proteins to CNTF-dependent transcriptional activation of SN48 cells. In cultures of embryonic rat septum and other brain areas, in addition to Stat3, CNTF also activates Stat1. As in cells of the PNS and non-neuronal cells, the Janus kinase (Jak)-STAT pathway is activated in CNS cells by cytokines. The SN48 cell line may be valuable in further characterization of regulation of the Jak-STAT pathway by neuropoietic cytokines.

    Title Lif-mediated Activation of Stat Proteins After Neuronal Injury in Vivo.
    Date April 1996
    Journal Neuroreport
    Excerpt

    Leukemia inhibitory factor (LIF) is a member of a family of neuropoietic cytokines which influence neuronal survival, differentiation and response to injury. In cell lines LIF activates the Jak/Tyk tyrosine kinases and the STAT family of transcription factors. We have investigated whether the Jak-STAT intracellular signaling pathway is activated by LIF after neuronal injury in vivo. Axotomy of postganglionic sympathetic nerves resulted in sustained activation of members of the STAT transcription factor family. This activation is dependent on LIF as axotomy failed to activate STAT proteins in LIF-deficient mice. These data indicate that LIF-dependent activation of STAT proteins is one of the signal transduction pathways activated after neuronal injury.

    Title Adenosine Antagonists Potentiate D2 Dopamine-dependent Activation of Fos in the Striatopallidal Pathway.
    Date March 1996
    Journal Neuroscience
    Excerpt

    Adenosine antagonists potentiate dopamine-mediated behaviours. A2a adenosine and D2 dopamine receptors are abundantly co-expressed within the striatopallidal subset of striatal neurons, suggesting that this is the site of interaction between A2a and D2 receptors. We show that the D2-dependent induction of the immediate early gene c-Fos occurs in striatopallidal neurons 3 h following injection of reserpine (10 mg/kg). We used this paradigm to test whether adenosine antagonists modulate D2-dependent activation of striatopallidal neurons. The non-selective A1-A2 adenosine antagonists theophylline (25 mg/kg) or 3,7-dimethyl-1-propargylxanthine (25 mg/kg) potentiated the effect of a submaximal dose of the D2 dopamine agonist quinpirole (0.05 mg/kg) to prevent the induction of striatal c-Fos following reserpine. Co-administration of the A2a receptor antagonist 8-(3-chlorostyryl) caffeine (5 mg/kg) with quinpirole (0.05 mg/kg) also attenuated striatal c-Fos induction following reserpine to a greater extent than 0.05 mg/kg quinpirole alone. When administered prior to reserpine, theophylline (25 mg/kg) or 3,7-dimethyl-1-propargylxanthine (25 mg/kg) partially attenuate the induction of striatal c-fos. These results demonstrate that systemic administration of adenosine antagonists enhance D2 dopamine receptor-dependent regulation of c-Fos in the striatopallidal pathway. These results support a functional interaction between A2a adenosine and D2 dopamine receptors in striatopallidal neurons.

    Title Characterization and Expression of the Human A2a Adenosine Receptor Gene.
    Date January 1996
    Journal Journal of Neurochemistry
    Excerpt

    The actions of the neurotransmitter adenosine are mediated by a family of high-affinity, G protein-coupled receptors. We have characterized the gene for the human A2a subtype of adenosine receptor (hA2aR) and determined levels of A2aR mRNA in human brain regions and nonneural tissues. Human genomic Southern blot analysis demonstrates the presence of a single gene encoding the hA2aR located on chromosome 22. Two overlapping cosmids containing the hA2aR gene were isolated from a chromosome 22 library and characterized. Southern blot and sequence analyses demonstrate that the hA2aR gene spans approximately 9-10 kb with a single intron interrupting the coding sequence between the regions encoding transmembrane domains III and IV. The sequence of the hA2aR gene diverged from the reported cDNA structure in the 5' untranslated region. This divergence appears to result from an artifact in the construction of the original cDNA library. By northern blot analysis, high expression of the hA2aR gene was identified in the caudate nucleus with low levels of expression in other brain regions. High expression was also seen in immune tissues; lesser A2aR expression was detected in heart and lung. The gene for the A2a subtype of receptor for the neurotransmitter adenosine falls in the class of intron containing G protein-coupled receptor genes. Expression in the basal ganglia is consistent with a role for the hA2aR in motor control. Activation of the A2aR may also regulate immune responses and cardiopulmonary function.

    Title Cellular Localization of Dopamine D2 Receptor Messenger Rna in the Rat Trigeminal Ganglion.
    Date December 1995
    Journal Anesthesia and Analgesia
    Excerpt

    The actions of dopamine are mediated by specific, high-affinity, G protein-coupled receptors. Multiple subtypes of dopamine receptors have been characterized, including the D2 subtype (D2R). Cells within the dorsal root and petrosal ganglia of the rat express D2R messenger RNA (mRNA) consistent with D2R expression by primary sensory neurons. We hypothesized that neurons of the trigeminal ganglion express D2R mRNA. Total cellular RNA from rat trigeminal ganglia was analyzed on Northern blots under high stringency conditions. Hybridization of trigeminal ganglion RNA resulted in a signal which comigrated with striatal, pituitary, and hypothalamic D2R mRNA. To determine the distribution of D2R expressing cells in the trigeminal ganglion, cryostat sections were analyzed by in situ hybridization followed by emulsion autoradiography. We identified a population of clustered cells labeled with dense grain concentrations over their cytoplasms. These findings demonstrate the expression of D2 dopamine receptor mRNA in discrete subpopulations of neurons in the rat trigeminal ganglion. Our observations suggest that drugs active at dopamine receptors of the D2 subtype are potential modulators of sensory activity of neurons whose cell bodies reside in the trigeminal ganglion. D2 dopamine receptors may thus have a role in clinical pain syndromes involving the head and neck.

    Title Differences in Nuclear Signaling by Leukemia Inhibitory Factor and Interferon-gamma: the Role of Stat Proteins in Regulating Vasoactive Intestinal Peptide Gene Expression.
    Date December 1995
    Journal Journal of Neurochemistry
    Excerpt

    To investigate the importance of STAT protein activation in leukemia inhibitory factor (LIF)-mediated induction of neuropeptide gene transcription, we compared signaling to the 180-bp cytokine response element (CyRE) in the vasoactive intestinal peptide (VIP) promoter by interferon-gamma (IFN-gamma) and LIF. We show that LIF and IFN-gamma activate STAT proteins but only LIF activates VIP gene transcription. Thus STAT activation is not sufficient for VIP transcriptional activation. In a CyRE reporter plasmid, in which the STAT site has been deleted, LIF, but not IFN-gamma, activates transcription, indicating that sequences within the CyRE distinct from the STAT site are important to LIF-mediated transcriptional activation. The CyRE does not mediate transcriptional activation to LIF in a non-VIPergic cell line, suggesting that cell-specific factors exist which are permissive for cytokine-dependent regulation of gene expression. Human and mouse sequences are highly conserved in the region of the CyRE, consistent with the functional importance of multiple regions of the CyRE. These findings show that regions within the CyRE distinct from the STAT site are important to the LIF-dependent regulation of VIP gene expression and enable the CyRE to respond in a cell-specific and cytokine-specific manner.

    Title C/ebp-related Sites in Addition to a Stat Site Are Necessary for Ciliary Neurotrophic Factor-leukemia Inhibitory Factor-dependent Transcriptional Activation by the Vasoactive Intestinal Peptide Cytokine Response Element.
    Date May 1995
    Journal The Journal of Biological Chemistry
    Excerpt

    The neuropoietic cytokines ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF) regulate VIP gene expression through a cytokine response element (CyRE) which interacts with members of the STAT transcription factor family. The CyRE STAT site is, however, insufficient to mediate full transcriptional activation by CNTF/LIF, suggesting that other sequences and nuclear proteins are also important. As C/EBP proteins participate in the transcriptional effects of the related cytokine, interleukin-6, we investigated the role of possible C/EBP-binding sites in the response of the VIP CyRE to CNTF/LIF. Using DNase I footprinting, transactivation studies, DNA mobility shift assays, and mutational analysis, three sites within the VIP CyRE were identified as C/EBP-related binding sites and shown to be important to CNTF/LIF-mediated transcriptional activation. The CyRE C/EBP-related sites interact with nuclear proteins from the human neuroblastoma cell line, NBFL, including a novel, protein synthesis-dependent, nuclear protein complex, induced by CNTF treatment. These nuclear proteins are not, however, recognized by antisera to known C/EBP proteins. Therefore, other nuclear proteins regulated by independent pathways act in concert with the JAK-STAT pathway to mediate CNTF/LIF regulation of VIP gene expression through the CyRE.

    Title Stat Proteins Participate in the Regulation of the Vasoactive Intestinal Peptide Gene by the Ciliary Neurotrophic Factor Family of Cytokines.
    Date May 1995
    Journal Molecular Endocrinology (baltimore, Md.)
    Excerpt

    Ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF) are members of a family of neuropoietic cytokines that have a broad range of actions on many different neuronal populations. In cultured sympathetic neurons, CNTF and LIF induce transcription of the VIP and other neuropeptide genes as part of a program of differentiation. To gain insight into the nuclear events involved in cytokine-mediated activation of the neuropeptide genes involved in neuronal differentiation, we have investigated the mechanisms of transcriptional activation of the vasoactive intestinal peptide (VIP) gene by the CNTF family of cytokines. In the neuroblastoma cell line NBFL, CNTF, LIF, and a related cytokine, oncostatin-M, activate VIP gene transcription through a 180-base pair cytokine response element (CyRE). Deletion analysis of the VIP CyRE showed that multiple regions within the 180 base-pairs are important for cytokine-mediated transcriptional activation of the VIP gene. To one of these regions within the CyRE, cytokine treatment induces binding of a protein complex composed of members of the signal transducers and activators of transcription (STAT) transcription factor family. Mutation of this STAT-binding site attenuates cytokine-mediated transcriptional activation. LIF treatment of primary sympathetic neurons also induced binding of a STAT-containing protein complex to the VIP CyRE. Thus, activation of STAT transcription factors contributes to the induction of the VIp gene by the CNTF family of cytokines and may be involved in cytokine-mediated differentiation of sympathetic neurons.

    Title Leukemia Inhibitory Factor and Ciliary Neurotrophic Factor Increase Activated Ras in a Neuroblastoma Cell Line and in Sympathetic Neuron Cultures.
    Date October 1994
    Journal Journal of Neurochemistry
    Excerpt

    The cytokines leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) have been implicated in determination of neuronal phenotype as well as promotion of neuronal survival. However, the intracellular mechanisms by which their signals are transduced remain poorly understood. We have previously studied the regulation of vasoactive intestinal polypeptide gene expression by LIF and CNTF in the NBFL neuroblastoma cell line. Because these cytokines induce tyrosine phosphorylation that may lead to Ras activation, we explored a possible role for Ras in LIF- and CNTF-induced signal transduction. In NBFL cells LIF increases activated Ras in a rapid, transient, and concentration-dependent manner. CNTF and a related cytokine, oncostatin M, produce similar increases. CNTF and LIF also increase activated Ras in neuron-enriched dissociated cultures of sympathetic ganglia. Moreover, these cytokines rapidly and transiently induce specific tyrosine-phosphorylated proteins, p165 and p195. The protein kinase inhibitors K252a and staurosporine block LIF-induced increases in tyrosine phosphorylation, activated Ras, and vasoactive intestinal polypeptide mRNA in NBFL cells. These data support a possible role for Ras in the cell differentiation effects of LIF and CNTF.

    Title Hereditary Variations in Monoamine Oxidase As a Risk Factor for Parkinson's Disease.
    Date August 1994
    Journal Movement Disorders : Official Journal of the Movement Disorder Society
    Excerpt

    Parkinson's disease (PD) is a common neurodegenerative disorder caused by loss of dopaminergic neurons in the brainstem. Recent studies suggest that several genes may have a role in determining individual susceptibility to this disease, and the degradative enzyme monoamine oxidase (MAO) has been implicated in the disease process. Wide differences in activity levels for both forms of this enzyme (MAO-A and MAO-B) exist in the human population, and levels of both are genetically determined. Here we have compared the frequency of haplotypes at the MAOA and MAOB loci on the X chromosome in 91 male patients with PD and 129 male controls. Alleles were marked using two restriction fragment length polymorphisms (RFLPs), a (GT)n repeat in the MAOA locus, and a (GT)n repeat in the MAOB locus. One particular haplotype marked by the RFLP's at MAOA was three times more frequent in patients with PD as compared with controls, and the overall distribution of these alleles was significantly different (p = 0.03) between these two groups. Another MAOA haplotype was about threefold more common in controls than in patients with PD (p = 0.005). No associations were observed between individual MAOB alleles and the disease state, but the frequency distribution for all alleles was significantly different in the two populations (p = 0.046). These findings support the idea that the MAO genes may be among the hereditary factors that influence susceptibility of individuals to PD.

    Title Coordinate Regulation of Choline Acetyltransferase, Tyrosine Hydroxylase, and Neuropeptide Mrnas by Ciliary Neurotrophic Factor and Leukemia Inhibitory Factor in Cultured Sympathetic Neurons.
    Date August 1994
    Journal Journal of Neurochemistry
    Excerpt

    The neurotransmitter phenotype switch that occurs in cultures of rat superior cervical ganglion neurons after treatment with leukemia inhibitory factor or ciliary neurotrophic factor is a useful model permitting investigation of the mechanisms of cytokine-mediated differentiation. Recently the actions of leukemia inhibitory factor and ciliary neurotrophic factor have been linked through their interactions with related receptor complexes. Here we compare the effects of these two cytokines on gene expression in sympathetic neuronal cultures and begin to investigate their mechanisms. We report that, as has been shown for leukemia inhibitory factor, ciliary neurotrophic factor regulates peptides and classical transmitters in these cultures at the mRNA level. In addition, we find that the induction of substance P mRNA by these cytokines is rapid, dependent on protein synthesis, and occurs in 40-50% of superior cervical ganglion neurons in dissociated culture.

    Title Mapping of a Human A2a Adenosine Receptor (adora2) to Chromosome 22.
    Date August 1994
    Journal Genomics
    Title Differential Localization of A2a Adenosine Receptor Mrna with D1 and D2 Dopamine Receptor Mrna in Striatal Output Pathways Following a Selective Lesion of Striatonigral Neurons.
    Date March 1994
    Journal Brain Research
    Excerpt

    We have used the suicide transport agent volkensin to produce selective lesions of striatonigral neurons. By in situ hybridization histochemistry unilateral volkensin injections in the substantia nigra decreased the number of D1 receptor mRNA-expressing neurons in the ipsilateral striatum but did not change the number of D2 receptor and A2a adenosine receptor mRNA-expressing neurons. These findings confirm that striatonigral neurons express D1 receptors and suggest that D2-A2a receptor expressing neurons are predominantly localized to other neuronal populations within the striatum.

    Title Multiple Regions Within the Cytoplasmic Domains of the Leukemia Inhibitory Factor Receptor and Gp130 Cooperate in Signal Transduction in Hepatic and Neuronal Cells.
    Date January 1994
    Journal Molecular and Cellular Biology
    Excerpt

    The receptor for leukemia inhibitory factor (LIFR), in combination with the signal-transducing subunit for interleukin-6-type cytokine receptors, gp130, and LIF, activates transcription of acute-phase plasma protein genes in human and rat hepatoma cells and the vasoactive intestinal peptide gene in a human neuroblastoma cell line. To identify the regions within the cytoplasmic domain of LIFR that initiate signal transduction independently of gp130, we constructed a chimeric receptor by linking the extracellular domain of the granulocyte colony-stimulating factor receptor (G-CSFR) to the transmembrane and cytoplasmic domain of human LIFR. The function of the chimeric receptor protein in transcriptional activation was assessed by G-CSF-mediated stimulation of cotransfected cytokine-responsive reporter gene constructs in hepatoma and neuroblastoma cells. By using the full-length cytoplasmic domain and mutants with progressive carboxy-terminal deletions, internal deletions, or point mutations, we identified the first 150 amino acid residues of LIFR as the minimal region necessary for signaling. The signaling reaction appears to involve a cooperativity between the first 70-amino-acid region containing the two sequence motifs conserved among hematopoietin receptors (box 1 and box 2) and a critical sequence between residues 141 and 150 (box 3). Analogous analyses of the cytoplasmic domains of G-CSFR and gp130 indicated similar arrangements of functional domains in these receptor subunits and the requirement of a box 3-related motif for signaling.

    Title Molecular Cloning and Functional Expression of a Sheep A3 Adenosine Receptor with Widespread Tissue Distribution.
    Date October 1993
    Journal Molecular Pharmacology
    Excerpt

    Using the polymerase chain reaction, an A3 adenosine receptor has been cloned from the hypophysial par tuberalis of sheep. The clone encodes a 317-amino acid protein that is 72% identical to the rat A3 adenosine receptor. In contrast to rat, where abundant A3 mRNA transcript is found primarily in testis, the sheep transcript is most abundant in lung, spleen, and pineal gland and is present in moderate levels in brain, kidney, and testis. The agonist N6-amino[125I]iodobenzyladenosine binds with high affinity (Kd congruent to 6 nm) and specificity to recombinant A3 adenosine receptors expressed transiently in COS-1 cells or stably in CHO K1 cells. The potency order of agonists is N6-aminoiodobenzyladenosine > N-ethylcarboxamidoadenosine > or = (R)-phenylisopropyladenosine >> cyclopentyladenosine. Little or no binding of purine nucleotides was detected. The potency order of antagonists is 3-(3-iodo-4-aminobenzyl)-8-(4-oxyacetate)phenyl-1- propylxanthine (I-ABOPX) (Ki = 3 nM) > 1,3-dipropyl-8-(4-acrylate)phenylxanthine (BW-A1433) > 1,3-dipropyl-8-sulfophenylxanthine = xanthine amine cogener >> 8-cyclopentyl-1,3-dipropylxanthine. Enprofylline does not bind. These data indicate that, in contrast to A1 adenosine receptors, A3 adenosine receptors preferentially bind ligands with aryl rings in the N6-position of adenine and in the C8-position of xanthine. Among antagonists, the A3 adenosine receptor preferentially binds 8-phenylxanthines with acidic versus basic para-substituents (I-ABOPX > BW-A1433 > 1,3-dipropyl-8-sulfophenylxanthine = xanthine amine cogener). Agonists reduce forskolin-stimulated cAMP accumulation in Chinese hamster ovary cells stably transfected with recombinant sheep A3 adenosine receptors; the reduction is blocked by BW-A1433 but not by 8-cyclopentyl-1,3-dipropylxanthine. These data suggest that (i) A3 adenosine receptors display unusual structural diversity for species homologs, (ii) in contrast to rat, sheep A3 adenosine receptors have a broad tissue distribution, and (iii) some xanthines with acidic side chains bind with high affinity to A3 adenosine receptors.

    Title Calcium Regulation of Vasoactive Intestinal Polypeptide Mrna Abundance in Sh-sy5y Human Neuroblastoma Cells.
    Date August 1993
    Journal Journal of Neurochemistry
    Excerpt

    Second messenger regulation of gene expression provides a mechanism by which neurons can transduce environmental stimuli into long-term changes in the expression of molecules involved in neuronal signaling. We have investigated calcium-dependent induction of vasoactive intestinal polypeptide (VIP) mRNA and compared it with induction of VIP mRNA by cyclic AMP. Depolarization with 60 mM KCl or exposure to the calcium ionophore A23187 increases VIP mRNA levels in SH-SY5Y cells. The increase in VIP mRNA content in response to Ca2+ mobilization is slow, independent of adenylate cyclase activation, and requires de novo protein synthesis. The increase in VIP mRNA content in response to elevation of cyclic AMP levels by forskolin/isobutylmethylxanthine is independent of Ca2+ influx and does not require new protein synthesis. The mRNA for the transcription factors ATF-3, c-fos, c-jun, junB, and zif/268 is induced by A23187. Of these, ATF-3 showed the greatest relative induction by A23187 compared with induction by forskolin/isobutylmethylxanthine.

    Title Ciliary Neurotrophic Factor Coordinately Activates Transcription of Neuropeptide Genes in a Neuroblastoma Cell Line.
    Date February 1993
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    Differentiation factors have been identified that influence the phenotype of sympathetic neurons by altering expression of classical neurotransmitters and neuropeptides. Investigation of the molecular mechanisms through which such factors act would be facilitated by the availability of a neuronal cell line that responds to these factors in a fashion similar to sympathetic neurons. We have identified a human neuroblastoma cell line, NBFL, that responds to the differentiation factor ciliary neurotrophic factor (CNTF) by coordinately inducing multiple neuropeptide genes as do sympathetic neurons. Treatment of NBFL cells with CNTF increases vasoactive intestinal polypeptide (VIP), somatostatin, and calcitonin gene-related peptide (CGRP) mRNAs but does not change other neurotransmitter properties. The induction of VIP mRNA by CNTF in NBFL cells is dose dependent, rapid, sustained, and independent of new protein synthesis. Genomic 5' flanking sequences located within a 1.59-kilobase region of the human VIP gene and distinct from the previously defined cAMP-responsive element subserve transcriptional activation by CNTF. Further examination of NBFL cells should permit the elucidation of the molecular mechanisms by which CNTF and other differentiation factors coordinately activate neuropeptide gene transcription to influence neuronal differentiation. Similar mechanisms may mediate the effect of CNTF on neuronal survival.

    Title Oncostatin M Regulates Vip Expression in a Human Neuroblastoma Cell Line.
    Date December 1992
    Journal Neuroreport
    Excerpt

    Oncostatin-M (OM), a recently described glycoprotein cytokine, is structurally and functionally related to cholinergic differentiation factor/leukemia inhibitory factor (CDF/LIF) and ciliary neurotrophic factor (CNTF). To determine whether OM, like CDF/LIF and CNTF, possesses trophic or differentiative functions for neurons we examined the effects of recombinant human OM on ciliary neuron survival and neurotransmitter expression in sympathetic neurons. Like CDF/LIF, but in contrast to CNTF, OM had no effect on ciliary neuronal survival at any concentration tested. OM produced small but reproducible increases in choline acetyl transferase (ChAT) activity and vasoactive intestinal peptide (VIP) levels in rat sympathetic neuron cultures, but this effect was significantly less than that of CNTF or CDF/LIF. To determine if human OM would elicit a more robust response from human cells, we utilized a human neuroblastoma cell line, NBFL, that responds to CNTF and CDF/LIF by altering vasoactive intestinal peptide (VIP) levels. OM specifically elevated VIP and c-fos mRNA levels in NBFL cells and was as potent as CDF/LIF in this assay. Our data provides evidence that OM acts on neurons and identifies a neural cell line responsive to OM, CNTF, CDF/LIF.

    Title Molecular Cloning of the Rat A2 Adenosine Receptor: Selective Co-expression with D2 Dopamine Receptors in Rat Striatum.
    Date December 1992
    Journal Brain Research. Molecular Brain Research
    Excerpt

    A cDNA fragment homologous to other G protein-coupled receptors was isolated from rat brain using the PCR method and demonstrated to be abundantly expressed in striatum. Using this fragment as a probe, a 2.1 kb full-length cDNA was isolated from a rat striatal cDNA library. This cDNA encodes a protein of 410 amino acids and is highly homologous to previously isolated adenosine receptor cDNAs. Expression of this cDNA in COS cells revealed high affinity (Kd = 38.6 nM) and saturable binding of the A2 adenosine receptor-selective ligand [3H]CGS 21680. Agonist displacement profile of [3H]CGS 21680 binding was consistent with an adenosine receptor of the A2 subtype (NECA greater than (R)-PIA greater than CPA greater than (S)-PIA). In situ hybridization demonstrated that rat A2 adenosine receptor mRNA was co-expressed in the same striatal neurons as D2 dopamine receptor mRNA, and never co-expressed with striatal D1 dopamine receptor mRNA. Several lines of evidence have previously suggested that dopamine-induced changes in motor behavior can be modulated by adenosine analogs acting at the A2 subtype of adenosine receptor in the forebrain. The co-expression of D2 dopamine and A2 adenosine receptors in a subset of striatal cells provides an anatomical basis for dopaminergic-adenosinergic interactions on motor behavior.

    Title Dopamine D1 Receptor Stimulation of Cyclic Amp Accumulation in Cos-1 Cells.
    Date March 1992
    Journal European Journal of Pharmacology
    Excerpt

    Dopamine is shown to stimulate cAMP accumulation in COS-1 cells via endogenously expressed dopamine D1 receptors. A dissociation of dopamine and beta-adrenoceptor responses is demonstrated by the use of selective antagonists and different desensitization patterns following exposure of the cells to dopamine or the beta-adrenoceptor agonist, isoproterenol. The dopamine response in COS-1 cells exhibits a pharmacological profile similar to that found in dopamine D1 tissues such as rat striatum and fish retina. The presence of DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, Mr 32,000) immunoreactivity in COS-1 cells is shown by Western blotting and is consistent with the endogenous expression of a dopamine D1 receptor in these cells. It is concluded that a dopamine D1 receptor is expressed in COS-1 cells and the implications of this are discussed.

    Title A Review of 32 Cases of Tardive Dystonia.
    Date August 1991
    Journal The American Journal of Psychiatry
    Excerpt

    OBJECTIVE: Tardive dystonia, historically combined with tardive dyskinesia, is now viewed as probably having a different pathophysiology, course, outcome, and treatment response than tardive dyskinesia. In addition, patients with tardive dystonia are reported to be younger, and most are men. This study evaluates characteristics of 32 patients with tardive dystonia and compares results to other reports. METHOD: Twenty-four patients had been referred for research purposes and were videotaped, while eight had been followed clinically. Two of the authors reviewed all available videotapes and clinical reports to assess the course of symptoms over time. For global ratings and ratings of affected body parts, two scales were used: the Abnormal Involuntary Movement Scale (AIMS) for tardive dyskinesia and a similar scale for tardive dystonia. The method of case finding does not provide incidence or prevalence data for tardive dystonia. RESULTS: Fifty-nine percent of the patients experienced onset of tardive dystonia symptoms within 6 years of antipsychotic drug exposure; women had a shorter exposure time. No patient had complete remission of tardive dystonia symptoms, and 22 were moderately or severely impaired when their movements were most prominent. CONCLUSIONS: While epidemiological studies of tardive dystonia have yet to be performed, these results support the observations of others that most patients with tardive dystonia are men, have a short history of exposure to antipsychotic drugs, and may initially present with blepharospasm. Tardive dystonia rarely remits completely, can cause notable disability, and may partially respond to anticholinergic agents.

    Title Cyclic Amp- and Phorbol Ester-induced Transcriptional Activation Are Mediated by the Same Enhancer Element in the Human Vasoactive Intestinal Peptide Gene.
    Date March 1991
    Journal The Journal of Biological Chemistry
    Excerpt

    Transcription of the human vasoactive intestinal peptide (VIP) gene is regulated by both cyclic AMP and phorbol esters. A 17-nucleotide enhancer element within the human VIP gene mediates transcriptional activation by both phorbol esters and forskolin. Mutations of this element decrease responses to both agents, suggesting that the trans-acting proteins that mediate both modes of transcriptional regulation have similar DNA-binding characteristics. The response of the VIP enhancer element to forskolin, but not to 12-O-tetradecanoylphorbol-13-acetate, was attenuated by treatment with a recombinant inhibitor of the cAMP-dependent protein kinase, suggesting that the cAMP-dependent protein kinase and protein kinase C second messenger pathways that converge on this single enhancer element are distinct. The transcriptional activator cAMP-responsive element-binding (CREB) proteins and the c-fos.c-Jun complex interact with the VIP enhancer. The dual second messenger responses of the VIP gene may result from the interaction of this second messenger enhancer with different transcriptional activator proteins.

    Title Induction of a Cyclic Amp-responsive Gene in Living Cells Requires the Nuclear Factor Creb.
    Date March 1991
    Journal Molecular and Cellular Biology
    Excerpt

    We constructed cell lines containing various enhancer elements cloned upstream from a marker gene. By microinjection of specific antibodies directed against transcriptional activator proteins into these cell lines, we have developed a functional assay for factors which regulate the activity of target promoters. Here we show that microinjection of a highly specific antibody to the cyclic AMP enhancer element-binding (CREB) protein diminishes gene expression in response to cyclic AMP in living fibroblasts.

    Title Day-night Variation in Prepro Vasoactive Intestinal Peptide/peptide Histidine Isoleucine Mrna Within the Rat Suprachiasmatic Nucleus.
    Date February 1990
    Journal Brain Research. Molecular Brain Research
    Excerpt

    Neurons within the suprachiasmatic nuclei of the hypothalamus (SCN) appear to function as a circadian clock that controls the timing of many physiological systems. The SCN contain several chemically distinct neuronal subpopulations, including a large group of interneurons within the ventrolateral SCN that exhibit co-localizable immunoreactivity for both vasoactive intestinal peptide (VIP) and peptide histidine isoleucine (PHI). The purpose of the present study was to determine whether VIP/PHI neurons within the rat SCN exhibit rhythmicity in the cellular levels of the messenger RNA encoding the precursor from which both VIP and PHI are derived. Using both quantitative in situ and solution hybridization prepro-VIP/PHI mRNA levels early in the dark phase were demonstrated to be significantly higher than those 5 h after the onset of the daily light period. Since no statistically reliable (P greater than 0.05) day-night variation was observed in the levels of prepro-VIP/PHI mRNA within cortex, these data suggest that the rhythmicity in prepro-VIP/PHI mRNA is an intrinsic property of VIP/PHI-containing SCN neurons, or rhythmically driven by local synaptic events within the SCN.

    Title Distinguishable Promoter Elements Are Involved in Transcriptional Activation by E1a and Cyclic Amp.
    Date December 1989
    Journal Molecular and Cellular Biology
    Excerpt

    The sequence motif CGTCA is critical for binding of a group of cellular transcription factors (ATF, CREB, E4F, and EivF) and for activation of certain E1a-inducible and cyclic AMP (cAMP)-inducible promoters. We have tested different promoter elements containing the CGTCA motif (referred to here as ATF-binding sites) for the ability to function as E1a or cAMP response elements. The adenovirus E4 promoter and the cellular vasoactive intestinal peptide (VIP) promoter responded differently to E1a and cAMP, demonstrating that the activating potential of ATF-binding sites within these promoters is not equivalent. While particular ATF-binding sites were sufficient for the activity of both the E4 (E1a inducibility) and VIP (cAMP inducibility) enhancers, these two enhancers had contrasting effects on E1a- and cAMP-inducible transcription. Thus, the relationship between E1a- and cAMP-inducible transcription is not simply explained by the action of these two inducers through the same promoter elements.

    Title Thyroid Hormone Regulates Vasoactive Intestinal Peptide (vip) Mrna Levels in the Rat Anterior Pituitary Gland.
    Date November 1989
    Journal Endocrinology
    Excerpt

    Vasoactive intestinal peptide (VIP) is a secretagogue for pituitary prolactin, but the importance of this peptide in the normal control of prolactin secretion is unclear. Recent studies suggest VIP synthesis within the rat anterior pituitary. We have shown (Endocrinology 124:1077) that the content of rat pituitary VIP increases in hypothyroidism. To confirm in situ pituitary synthesis of VIP and determine whether thyroid hormone effects on pituitary VIP relate to changes in VIP mRNA, Northern and in situ hybridization analyses of VIP mRNA in rat pituitaries were performed. Northern hybridization demonstrated an RNA species from rat pituitary consistent with rat VIP mRNA. Hypothyroidism increased the content of pituitary VIP mRNA, and replacement with 1-thyroxine prevented this increase. In situ hybridization showed multiple, widely-distributed hybridizing cells in pituitaries from hypothyroid animals. A distinct population of VIP-producing pituitary cells exists which may serve to modulate prolactin secretion in a paracrine or autocrine fashion.

    Title The Catalytic Subunit of Camp-dependent Protein Kinase Induces Expression of Genes Containing Camp-responsive Enhancer Elements.
    Date November 1988
    Journal Nature
    Excerpt

    Transcriptional regulation of eukaryotic genes by cyclic AMP requires a cAMP-dependent protein kinase (A kinase). Two hypotheses have been proposed to explain how the holoenzyme of the A kinase induces transcription. The regulatory subunits of the A kinase, which bind cAMP and DNA, and have amino-acid homology with the Escherichia coli catabolite activator protein could directly stimulate gene expression. Alternatively, phosphorylation by the catalytic subunits could induce transcription by activating proteins involved in gene transcription. To distinguish between these models, we microinjected purified preparations of the catalytic and regulatory subunits of A kinase into tissue culture cells and monitored expression of a stably integrated fusion gene containing a cAMP-responsive human promoter fused to a bacterial reporter gene, or of the endogenous c-fos gene. The catalytic subunit stimulated expression of these genes, whereas the regulatory subunit did not. These results indicate that the catalytic subunit of A kinase is sufficient to induce expression of two cAMP-responsive genes, without increasing levels of cAMP.

    Title The Cgtca Sequence Motif is Essential for Biological Activity of the Vasoactive Intestinal Peptide Gene Camp-regulated Enhancer.
    Date October 1988
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    cAMP-regulated transcription of the human vasoactive intestinal peptide gene is dependent upon a 17-base-pair DNA element located 70 base pairs upstream from the transcriptional initiation site. This element is similar to sequences in other genes known to be regulated by cAMP and to sequences in several viral enhancers. We have demonstrated that the vasoactive intestinal peptide regulatory element is an enhancer that depends upon the integrity of two CGTCA sequence motifs for biological activity. Mutations in either of the CGTCA motifs diminish the ability of the element to respond to cAMP. Enhancers containing the CGTCA motif from the somatostatin and adenovirus genes compete for binding of nuclear proteins from C6 glioma and PC12 cells to the vasoactive intestinal peptide enhancer, suggesting that CGTCA-containing enhancers interact with similar transacting factors.

    Title Identification of a Region in the Human Vasoactive Intestinal Polypeptide Gene Responsible for Regulation by Cyclic Amp.
    Date July 1987
    Journal The Journal of Biological Chemistry
    Excerpt

    Transcription of the vasoactive intestinal polypeptide (VIP) gene is regulated by cAMP. To identify the nucleotide sequences in the human VIP gene responsible for this regulation, we constructed chimeric genes containing different portions of the 5'-flanking region of the human VIP gene fused to the structural sequence encoding the bacterial reporter enzyme chloramphenicol acetyltransferase (CAT). The transcriptional activities of the fusion genes introduced into the rat pheochromocytoma cell line PC12 were assayed by measuring CAT activity in the cell lysates. Forskolin, an adenylate cyclase-activating agent, stimulated the expression of VIP-CAT fusion genes. Deletional analysis demonstrated that a region between -86 and -70 nucleotides upstream from the transcriptional origin of the human VIP gene was responsible for stimulation by forskolin. This region was able to confer cAMP-responsiveness to a gene that is not normally regulated by cAMP. Two copies of a 5 base pair motif, 5'-CGTCA-3', are required for activity of the VIP cAMP regulatory region. This motif is also present in the cAMP regulatory region of several other eukaryotic genes.

    Title Biosynthesis of Pancreatic Islet Hormones.
    Date March 1987
    Journal Hepatology (baltimore, Md.)
    Excerpt

    We have outlined the various strategies used to characterize the precursors of three pancreatic islet hormones--somatostatin, pancreatic polypeptide and VIP. In each case, isolation of the cDNA clones was facilitated by the use of gastrointestinal tissues that were extremely rich in specific mRNA. Characterization of the structures of the precursors is clearly only the first step in understanding the regulation of pancreatic hormone biosynthesis. It is likely that the availability of the cDNA clones will allow us to define the actual mechanisms underlying hormone production within the pancreas.

    Title Mesolimbic and Mesocortical Dopaminergic Neurons Are Necessary for Normal Exploratory Behavior in Rats.
    Date June 1983
    Journal Neuroscience Letters
    Excerpt

    After ablation of mesolimbic and mesocortical dopaminergic neurons produced by 6-hydroxydopamine (6-OHDA) injections in the anterolateral hypothalamus, rats investigate a novel object less. Systemic administration of the dopamine (DA) agonist apomorphine increases investigatory movements in these DA-denervated rats when the object is novel, but not when the object is familiar. Pimozide pretreatment prevents the increased investigation produced by apomorphine in DA-denervated rats. These findings suggest that mesolimbic and mesocortical DA neurons are necessary for normal exploratory behavior in the rat.

    Title Genetic Control of the Number of Dopamine Neurons in the Brain: Relationship to Behavior and Responses to Psychoactive Drugs.
    Date March 1983
    Journal Research Publications - Association for Research in Nervous and Mental Disease
    Title Genetic Control of Dopamine Receptors in Mouse Caudate Nucleus: Relationship on Cataleptic Response to Neuroleptic Drugs.
    Date February 1983
    Journal Neuroscience Letters
    Title Genetic Variations in Midbrain Dopamine Cell Number: Parallel with Differences in Responses to Dopaminergic Agonists and in Naturalistic Behaviors Mediated by Central Dopaminergic Systems.
    Date December 1981
    Journal Brain Research
    Excerpt

    Mice of the inbred strain BALB/cJ have more midbrain dopaminergic cell bodies and greater activity of the catecholamine-synthesizing enzyme, tyrosine hydroxylase (TH), in the nigrostriatal and mesolimbic dopaminergic systems than mice of the CBA/J strain. This difference in cell number and TH activity in the midbrain dopaminergic systems are paralleled by differences in drug responses and behaviors which are dependent on the release of dopamine in midbrain dopaminergic system. BALB/cJ mice showed greater locomotion and stereotypy than CBA/J mice after D-amphetamine (2-20 mg/kg, i.p.). There was no difference in the amount of amphetamine accumulated in brain at the peak of drug response or in the duration of drug effect, suggesting that the differences in behavioral effect were not due to strain differences in pharmacokinetic distribution of the drug. In contrast to the greater stereotypy to D-amphetamine, BALB/cJ mice showed less stereotypy after apomorphine (2-10 mg/kg, i.p.) than CBA/J mice. BALB/cJ mice also showed more exploration than CBA/J mice, measured as locomotion and rearing in a novel open field and investigation of a novel object. Genetically determined differences in the number of midbrain dopaminergic cell bodies and in the relative density of innervation of DA terminals in target fields and in TH activity in the nigrostriatal and mesolimbic dopaminergic systems are paralleled by difference in behavioral responses mediated by release of dopamine. The number of cells of a particular neurochemical class may dictate the magnitude of behaviors, drug-induced or spontaneous, mediated by those neurons.

    Title Relationships Between Selective Denervation of Dopamine Terminal Fields in the Anterior Forebrain and Behavioral Responses to Amphetamine and Apomorphine.
    Date December 1980
    Journal Brain Research
    Excerpt

    To investigate the relationship between denervation of dopamine (DA) terminal fields in the anterior forebrain and the behavioral responses to amphetamine (1.5 mg/kg) and apomorphine (1 mg/kg), we injected 6-hydroxydopamine (6-OHDA) bilaterally into the anterolateral hypothalamus (ALH) or into specific mesolimbic and anterior striatal terminal fields after pretreatment with desmethylimipramine to protect noradrenergic axons and terminals from 6-OHDA toxicity. After drug testing was completed, the extent of denervation was determined by fluorescent histochemical analysis. When nearly all of the mesolimbicocortical and anteroventral striatal DA terminal fields were denervated by bilateral ALH 6-OHDA, the locomotor response to amphetamine was abolished, and the locomotor and stereotyped sniffing responses to apomorphine were increased. When fewer DA terminal fields were denervated, different results were obtained: the locomotor response to amphetamine decreased or did not change; stereotyped sniffing elicited by apomorphine did not increase or sniffing was replaced by stereotyped licking and biting. The results suggest a mass action relationship between DA terminal fields in the anterior forebrain and the locomotor response to amphetamine. The relationship between the same DA lesions and responses to apomorphine appears to be more complex.

    Title Mesolimbicocortical Dopamine Terminal Fields Are Necessary for Normal Locomotor and Investigatory Exploration in Rats.
    Date December 1980
    Journal Brain Research
    Excerpt

    Rats explore a novel open field or novel object less after denervation of mesolimbicocortical dopaminergic terminal fields produced by bilateral 6-hydroxydopamine (6-OHDA) microinjections into the anterolateral hypothalamus after pretreatment with desmethylimipramine (DMI). These behavioral deficits were correlated with complete or nearly complete loss of fluorescent dopaminergic (DA) terminals in the nucleus accumbens, olfactory tubercle, dorsal bed nucleus of the stria terminalis, lateral septal nucleus and the deep layers of the frontal and piriform cortices. There were also fewer A10, medial A9, and A8 DA fluorescent cells after the 6-OHDA-DMI injections; this suggests retrograde degeneration of the cells of origin of the mesolimbicocortical DA system. When the DMI pretreatment was omitted, identical bilateral 6-OHDA microinjections also produced severe loss of norepinephrine (NE) fibers in the neocortex, hippocampus, lateral hypothalamus and ventral bed nucleus of the stria terminalis. The addition of this noradrenergic damage did not change the exploratory deficits observed after mesolimbicocortical DA denervation alone. Systemic administration of the DA agonist apomorphine, but not the adrenergic agonist clonidine, to the 6-OHDA-DMI rats repaired the deficits in exploration of a novel open field or novel object. The increased locomotion in a novel open field and investigation of a novel object produced by apomorphine in 6-OHDA-DMI rats were blocked by the DA antagonist, pimozide. This is evidence that apomorphine restored exploratory responses by stimulating dopaminergic receptors. The exploratory responses produced by apomorphine were also blocked by testing rats in a familiar open field or with a familiar novel object. This is evidence that apomorphine facilitates exploratory responding to novel stimuli by 6-OHDA-DMI rats, but that the same dose of apomorphine does not increase activity when 6-OHDA-DMI rats are confronted by familiar stimuli. We conclude: (1) that mesolimbicocortical dopaminergic terminals are necessary for normal exploratory behavior in rats; and (2) that DA released by these terminals may facilitate optimal sensorimotor integration in these terminal fields during spontaneous exploratory behavior.

    Title Abnormal Pattern of Amphetamine Locomotion After 6-ohda Lesion of Anteromedial Caudate.
    Date December 1979
    Journal Pharmacology, Biochemistry, and Behavior
    Excerpt

    6-Hydroxydopamine (6-OHDA) injections into the anteromedial caudate nucleus (AMCN) produced severe loss of dopamine (DA) fibers in this region of the caudate. After a low dose of d'amphetamine (1.5 mg/kg), AMCN 6-OHDA rats made fewer traverses of the length of the activity cage than control rats. In contrast, AMCN 6-OHDA rats interrupted a photocell bema that passed across the middle of the long axis of the activity cage as often as control rats. 6-OHDA injections into the nucleus accumbens (NAc) produced severe loss of DA fibers in NAc without significantly damaging the adjacent anteromedial caudate or olfactory tubercle. After d-amphetamine (1.5 mg/kg), NAc 6-OHDA rats interrupted the photocell beam and traversed the length of the activity cage as frequently as control rats. We conclude that the DA innervation to the anteromedial caudate, but not to the nucleus accumbens, is necessary for that part of the normal locomotor response to a low dose of d'amphetamine that is required for the performance of long traverses of an activity cage.

    Title Decreased Locomotor and Investigatory Exploration After Denervation of Catecholamine Terminal Fields in the Forebrain of Rats.
    Date August 1979
    Journal Journal of Comparative and Physiological Psychology
    Excerpt

    Exploratory behaviors were examined after bilateral microinjections of 6-hydroxydopamine into two hypothalamic sites that produced different patterns of denervation of forebrain catecholamine terminal fields. After anterolateral injections rats locomoted and reared less in a novel open field, responded abnormally to changes in the degree of novelty of the open field, and investigated a novel object less. These are deficits in exploratory behavior because they were not secondary to the inhibition of open-field behavior by hyperemotionality, by general motor disability, or by the failure to detect novel spaces or objects. Such anterolateral injections produced loss of catecholamine fibers, determined histochemically, in neocortical, hippocampal, anterolateral hypothalamic, mesolimbic, mesocortical, and anteromedioventral striatal terminal fields and loss of dopaminergic perikarya in the A10 and anteromedial A9 cell groups. No deficits in exploratory behaviors occurred, however, after bilateral anteromedial 6-hydroxydopamine injections that denervated neocortical, hippocampal, and anteromedial hypothalamic catecholamine terminal fields. A critical forebrain catecholaminergic innervation for exploratory responses to novel stimuli may be within areas that were denervated by anterolateral but not by anteromedial hypothalamic 6-nydroxydopamine injections. These areas are mesolimbic, mesocortical, anteromedioventral, and anterolateral hypothalamic terminal fields.

    Title L-dopa Repairs Deficits in Locomotor and Investigatory Exploration Produced by Denervation of Catecholamine Terminal Fields in the Forebrain of Rats.
    Date August 1979
    Journal Journal of Comparative and Physiological Psychology
    Excerpt

    In a previous study rats were shown to have decreased locomotor and investigatory exploration after bilateral microinjections of 6-hydroxydopamine into the anterolateral hypothalamus. These deficits correlate with the loss of catecholamine terminals in neocortical, limbic, and anteromedioventral striatal brain sites. To test whether this correlation was causal, central catecholamines were increased by the intraperitoneal administration of L-3,4-dihydroxyphenylalanine (L-dopa), 10--40 mg/kg) after inhibition of extracerebral L-amino acid decarboxylase. Such treatment repaired the deficits in locomotor exploration and investigation in 6-hydroxydopamine rats. Pretreatment with the catecholamine antagonist chlorpromazine (1--2 mg/kg) blocked the increase in locomotor exploration and investigation produced by L-dopa in 6-hydroxydopamine rats. The results suggest, but do not prove, that L-dopa produced these behavioral effects by increasing central catecholamines at the denervated catecholamine receptor sites in the forebrain. These data and the data from the previous study are complementary evidence for the hypothesis that forebrain catecholamine synaptic action is necessary for normal exploratory behavior.

    Title Different Behavioral Responses to L-dopa After Anterolateral or Posterolateral Hypothalamic Injections of 6-hydroxydopamine.
    Date December 1977
    Journal Brain Research
    Excerpt

    After bilateral microinjections of 6-hydroxydopamine (6-OHDA) into the anterolateral (AL) or posterolateral (PL) hypothalamus L-DOPA (1,3,4-dihydroxy-phenylalanine) produced running and rearing in AL6-OHDA rats and oral stereotypies in PL 6-OHDA rats. Since the same dose of L-DOPA had no behavioral effect in vehicle injected rats, the responses to L-DOPA of both AL and PL 6-OHDA rats are examples of behavioral supersensitivity. The different forms of behavioral supersensitivity correlated with different patterns of catecholamine (CA) denervation determined by fluorescent microscopy. The major regions of CA denervation in AL 6-OHDA rats were neocortex, hippocampus, limbic forebrain, anteromedial striatum and anterolateral hypothalamus. PL 6-OHDA had these same areas denervated and, in addition, had severe denervation of the entire striatum, parts of the amygdala and thalamus, and of the posterolateral hypothalamus. We conclude that the supersensitive behavioral response to a fixed dose of L-DOPA is determined by the pattern and/or extent of CA denervation.

    Title Method for Identifying Ligands That Bind to Cloned G(s)- or G(i)-coupled Receptors.
    Date
    Journal Molecular and Cellular Neurosciences
    Excerpt

    We describe a fast, simple assay for testing ligands for binding to an unknown receptor after transfection of that receptor cDNA into tissue culture cells. The assay is based on a mouse L cell line (LVIP2.OZc) that contains a cyclic AMP responsive reporter construct and is performed in 96-well plates. If the appropriate agonist binds to a receptor clone coupled to G(s) proteins, activation of adenylyl cyclase produces cAMP which in turn induces the enzyme beta-galactosidase in LVIP2.0Zc cells. beta-Galactosidase activity is detected by staining cells with a chromogenic substrate. After cell lysis, incubation with o-nitrophenyl beta-d-galactopyranoside (ONPG) results in a yellow color. Color development can be observed with the naked eye or read with a plate reader at 405 nm. Agonists that bind to G(i)-coupled receptors can be identified by inhibition of forskolin-induced expression of beta-galactosidase.


    Similar doctors nearby

    Dr. Ernest Terranova

    Internal Medicine
    32 years experience
    Mount Kisco, NY

    Dr. Jim Koo

    Internal Medicine
    29 years experience
    Mount Kisco, NY

    Dr. Ronen Marmur

    Internal Medicine
    12 years experience
    Mount Kisco, NY

    Dr. Jeffrey Gross

    Internal Medicine
    18 years experience
    Mount Kisco, NY

    Dr. David Duncan

    Internal Medicine
    22 years experience
    Mount Kisco, NY

    Dr. Dayna Yardeni

    Internal Medicine
    5 years experience
    Mount Kisco, NY
    Search All Similar Doctors