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S Sheriff

Publications and source records attributed to S Sheriff.

At least 19 recordsLinked to original sources
PubMed2006

Aging associated changes in amygdalar corticotropin-releasing hormone (CRH) and CRH-binding protein in Fischer 344 rats.

Behavioral adaptation in aging may become impaired from abnormal expression of amygdalar corticotropin-releasing hormone (CRH) and/or CRH-binding protein (CRH-BP). In this study, we serially sectioned the amygdala in 4-, 12-, and 24-month-old Fischer 344 rats following perfusion with 4% paraformaldehyde. We determined the amount of CRH and CRH-BP containing cells as well as the density of fibers expressing CRH or CRH-BP utilizing densitometric methods. Images were digitized using Zeiss Axiovision software and densitometrically analyzed using Scion Image. Both sides were analyzed in sections cut at 30 mum thickness. Cell counts of CRH-BP containing cells in the basolateral and lateral nucleus of the amygdala were lower in 24-month-old rats vs. 4-month-old rats, respectively (mean cells/section +/- SE): 31 +/- 6 vs. 72 +/- 10 (n = 3; P < 0.05 via ANOVA and Fisher's PLSD). There was a trend for cell counts of CRH containing cells in the central nucleus of the amygdala to be lower in 24-month-old rats vs. 4-month-old rats, respectively 28 +/- 7 vs. 47 +/- 9 (n = 3; P = 0.07 via ANOVA). Densitometric analysis of the number of CRH-BP positive fibers revealed no age differences in CeA; however, with regards to CRH-positive fibers, both 4- and 12-month rats had greater CeA CRH immunoreactivity relative to 24-month-old rats (Ps < 0.05 via ANOVA and Fisher's PLSD). These changes may contribute to impaired adaptations to stress, cognitive decline, and other pathophysiological processes during aging.

Age Factors↗
PubMed2005

Internalization of cloned pancreatic polypeptide receptors is accelerated by all types of Y4 agonists.

Internalization of cloned rat or human Y4 receptors expressed in Chinese hamster ovary (CHO) cells increased with concentration of all types of Y4 agonists, including human and rat pancreatic polypeptides, the Y1 receptor group co-agonists possessing C-terminal TRPRY.NH2 pentapeptide, and a C-terminally amidated dimeric nonapeptide related to neuropeptide Y, GR231118. These peptides also inhibited forskolin-stimulated adenylyl cyclase activity in Y4 receptor-expressing cells, and stimulated the binding of 35S-labeled GTP-gamma-S to pertussis toxin-sensitive G-proteins in particulates from these cells. Peptide VD-11 (differing from GR231118 only by C-terminal oxymethylation) acted as a competitive antagonist in all of the above processes. Agonist-induced stimulation of the Y4 receptor internalization persisted in the presence of allosteric inhibitors of hPP binding, N5-substituted amilorides, which also were relatively little active in G-protein stimulation and cyclase inhibition by Y4 agonists. Acceleration of Y4 receptor internalization by agonists apparently is related to relaxation of allosteric constraints to ligand attachment and sequestration of the receptor-ligand complex.

Adenylyl Cyclases↗
PubMed2003

In vitro regulation of corticotropin-releasing hormone.

Studies involving regulation of corticotropin-releasing hormone (CRH) in vitro have been used to validate findings obtained in vivo and more importantly have been used as model systems to better understand signalling mechanisms responsible for the expression of the CRH gene and peptide. Many in vitro studies examining CRH have utilized hypothalamic tissue while a few have focused on the amygdala. Clonal cell lines have also been utilized as models of central nervous system CRH neurons. Stimuli that have been implicated in regulating hypothalamic CRH regulation in vitro include protein kinase A (PKA) and protein kinase C (PKC) activators, glucocorticoids, biogenic amines, cytokines and the gaseous neurotransmitters. Amygdalar CRH levels in vitro are affected by some of the same stimuli that regulate hypothalamic CRH; however there is evidence supporting differential regulation of CRH in these two brain regions by some of the same stimuli. Only a few studies in aggregate have investigated signal transduction mechanisms and these studies have focused on PKA- and glucocorticoid-mediated changes in CRH expression. Thus, much more investigative work in better understanding CRH regulation in vitro is needed.

Amygdala↗
PubMed2003

Corticotropin-releasing hormone (CRH) expression and protein kinase A mediated CRH receptor signalling in an immortalized hypothalamic cell line.

Corticotropin-releasing hormone (CRH) is a 41 amino acid neuropeptide which plays an important role in the stress response in the hypothalamus. We describe the development of an immortalized hypothalamic cell line which expresses CRH. We hypothesized that this cell line would possess the relevant characteristics of parvocellular CRH-expressing neurones such as glucocorticoid receptor (GR) expression and vasopressin (VP) coexpression. For production of hypothalamic cells, embryonic day 19 rat pup hypothalami were dissected and dissociated into tissue culture dishes. They were immortalized by retrovirus-mediated transfer of the SV40 large T antigen gene at 3 days of culture and then screened for expression of CRH following dilution cloning. One cell line was chosen (IVB) which exhibited CRH-like immunoreactivity (CRH-LI) and expressed CRH, VP and CRH1 receptor RNA via the reverse transcriptase-polymerase chain reaction. In addition, the cell line expressed the neuronal marker, microtubule-associated protein-2. We verified that the CRH-LI from IVB cell lysates coeluted with CRH standard via reversed-phase high-performance liquid chromatography (HPLC). Furthermore, oxidation of the lysate converted its HPLC profile to that identical with oxidized CRH standard. In addition, IVB cells exhibited high affinity binding to CRH. Incubation of IVB cells with CRH lead to increases in cAMP levels and protein kinase A activity in a concentration-dependent manner. Incubation of IVB cells with CRH also resulted in increases in phospho-cyclic-AMP response element binding protein (CREB) immunostaining as detected by immunocytochemical analysis. Finally, CRH treatment of IVB cell lines has been linked to CREB-mediated gene expression as determined via the PathDetect CREB trans-reporting system. The characteristics of IVB cells, such as CRH and VP coexpression, GR expression and a biologically active CRH-R1-mediated signalling pathway, suggest that this neuronal cell line may serve as model of parvocellular CRH neurones.

Animals↗
PubMed2001

2-Aryl-2,2-difluoroacetamide FKBP12 ligands: synthesis and X-ray structural studies.

[structure: see text] 2-Aryl-2,2-difluoroacetamido-proline and pipecolate esters are high affinity FKBP12 ligands whose rotamase inhibitory activity is comparable to that seen for the corresponding ketoamides. X-ray structural studies suggest that the fluorine atoms participate in discrete interactions with the Phe36 phenyl ring and the Tyr26 hydroxyl group, with the latter resembling a moderate-to-weak hydrogen bond.

Acetamides↗
PubMed2001

Regulation of corticotropin-releasing hormone in vitro.

Studies examining regulation of corticotropin-releasing hormone (CRH) in vitro have been used to validate findings obtained in vivo and more importantly have been used as model systems to better understand signalling mechanisms responsible for the expression of the CRH gene and peptide. Most in vitro studies examining CRH have utilized hypothalamic tissue while a few have focused on the amygdala. Furthermore, clonal cell lines have also been utilized as models of central nervous system CRH neurons. Stimuli that have been implicated in regulating hypothalamic CRH in vitro include protein kinase A (PKA) and protein kinase C (PKC) activators, glucocorticoids, biogenic amines, cytokines and the gaseous neurotransmitters. CRH levels in the amygdala in vitro are affected by some of the same stimuli that regulate hypothalamic CRH; however there is evidence supporting differential regulation of CRH in these two brain regions by some of the same stimuli. Only a few studies in aggregate have investigated the signal transduction mechanisms responsible for CRH expression. These mechanistic studies have focused on PKA- and glucocorticoid-mediated changes in CRH expression. Clearly much more investigative work in better understanding CRH regulation in vitro is needed.

Animals↗
PubMed2001

Interaction of neuropeptide Y and corticotropin-releasing factor signaling pathways in AR-5 amygdalar cells.

Corticotropin-releasing factor (CRF) is a 41 amino acid neuropeptide which is involved in the stress response. CRF and neuropeptide Y (NPY) produce reciprocal effects on anxiety in the central nucleus of the amygdala. The molecular mechanisms of possible CRF-NPY interactions in regulating anxiety behavior is not known. In the central nervous system, the action of NPY leads to inhibition of cAMP production while CRF is known to stimulate levels of cAMP in the brain. Consequently, we hypothesized that NPY may antagonize anxiety-like behavior by counter-regulating CRF-stimulated cAMP accumulation and activation of the protein kinase A pathway. We have engineered an immortalized amygdalar cell line (AR-5 cells) which express via RT-PCR, the CRF(2alpha), Y(1) and Y(5) NPY receptor. In addition, in these cells CRF treatment results in significant concentration-dependent increases in cAMP production. Furthermore, incubation of 3 microM CRF with increasing concentrations of NPY was able to significantly inhibit the increases in cAMP compared to that observed with 3 microM CRF treatment alone. These findings suggest that CRF and NPY may counter-regulate each other in amygdalar neurons via reciprocal effects on the protein kinase A pathway.

Amygdala↗
PubMed2000

Structure-activity studies including a Psi(CH(2)-NH) scan of peptide YY (PYY) active site, PYY(22-36), for interaction with rat intestinal PYY receptors: development of analogues with potent in vivo activity in the intestine.

Peptide YY (PYY) is a gut hormone that inhibits secretion and promotes absorption and growth in the intestinal epithelium. We have performed structure-activity studies with the active site, N-alpha-Ac-PYY(22-36)-NH(2), for interaction with intestinal PYY receptors. Investigation of aromatic substitutions at position 27 resulted in analogues that exhibited potent in vitro antisecretory potencies with N-alpha-Ac-[Trp(27)]PYY(22-36)-NH(2) exhibiting even greater potency than intact PYY. In vivo studies in dogs revealed that this analogue also promoted intestinal absorption of water and electrolytes during continuous intravenous and intraluminal infusion. Investigations carried out to identify features that would enhance stability revealed that incorporation of Trp(30) increased affinity for PYY receptors. A "CH(2)-NH" scan revealed that incorporation of reduced bonds at position 28-29 or 35-36 imparted greater receptor affinity. In general, disubstituted analogues designed based on the results of single substitutions exhibited good receptor affinity with N-alpha-Ac-[Trp(27),CH(2)-NH(35-36)]PYY(22-36)-NH(2) having the greatest affinity (IC(50) = 0.28 nM). Conservative multiple substitutions with Nle-->Leu and Nva-->Val also imparted good affinity. An analogue designed to encompass most of the favored substitutions, N-alpha-Ac-[Nle(24,28),Trp(30),Nva(31), CH(2)-NH(35-36)]PYY(22-36)-NH(2), exhibited a proabsorptive effect in dogs comparable to, but longer lasting than, that of intact hormone. Selected analogues also exhibited good antisecretory potencies in rats with N-alpha-Ac-[Trp(30)]PYY(22-36)-NH(2) being even more potent than PYY. However, the potencies did not correlate well with the PYY receptor affinity or the proabsorptive potencies in dogs. These differences could be due to species effects and/or the involvement of multiple receptors and neuronal elements in controlling the in vivo activity of PYY compounds. PYY(22-36) analogues exhibited good affinity for neuronal Y2 receptors but poor affinity for Y1 receptors. Also, crucial analogues in this series hardly bound to Y4 and Y5 receptors. In summary, we have developed PYY(22-36) analogues which, via interacting with intestinal PYY receptors, promoted potent and long-lasting proabsorptive and antisecretory effects in in vivo models. These compounds or analogues based on them may have useful clinical application in treating malabsorptive disorders observed under a variety of conditions.

Animals↗
PubMed2000

[D-Trp(34)] neuropeptide Y is a potent and selective neuropeptide Y Y(5) receptor agonist with dramatic effects on food intake.

The neuropeptide Y (NPY) Y(5) receptor has been proposed to mediate several physiological effects of NPY, including the potent orexigenic activity of the peptide. However, the lack of selective NPY Y(5) receptor ligands limits the characterization of the physiological roles of this receptor. Screening of several analogs of NPY revealed that [D-Trp(34)]NPY is a potent and selective NPY Y(5) receptor agonist. Unlike the prototype selective NPY Y(5) receptor agonist [D-Trp(32)]NPY, [D-Trp(34)]NPY markedly increases food intake in rats, an effect that is blocked by the selective NPY Y(5) receptor antagonist CGP 71683A. These data demonstrate that [D-Trp(34)]NPY is a useful tool for studies aimed at determining the physiological roles of the NPY Y(5) receptor.

Animals↗
PubMed2000

Crystallization and preliminary X-ray analysis of CTLA-4 (CD152) membrane-external domain.

CTLA-4 (CD152) is involved in T-lymphocyte co-stimulatory pathways modulating both humoral and cellular immune response. The membrane-external domain has been prepared and crystallized. The unit-cell parameters are a = b = 43, c = 143 A with the symmetry of space group P3(1)21 or its enantiomer and the crystals diffract to 2. 7 A resolution at synchrotron beamlines.

Abatacept↗
PubMed2000

Maintaining gut integrity during parenteral nutrition of tumor-bearing rats: effects of glucagon-like peptide 2.

Maintaining tumor-bearing rats on total parenteral nutrition (TPN) for eight days significantly reduced mass, protein, and DNA in small intestine and colon. Coinfusion of glucagon-like peptide 2 (GLP-2) significantly increased each of these variables in the duodenum, jejunum, and ileum, but not in the colon. Histological analysis of tissue revealed normal mucosa thickness and villus height in the small intestine of GLP-2-treated rats, whereas non-treated rats maintained on TPN exhibited villus shortening and thinning of the mucosa. Compared with TPN alone, no significant effects of GLP-2 were noted on tumor growth, liver weight, or heart weight. Coinfusion of GLP-2 with TPN had no significant effect on TPN-associated immunosuppression, as measured by mitogen-induced proliferation of cultured splenocytes. Although translocation of bacteria to the mesenteric lymph nodes appeared to be reduced in GLP-2-treated rats, the difference between groups was not statistically significant. These results suggest that hormonal alterations may be more important than an absence of luminal nutrition in TPN-associated mucosa changes in tumor-bearing rats. Additionally, maintenance of gut integrity during TPN does not appear to be a sufficient condition for the avoidance of the negative sequelae associated with this route of supplemental nutrition.

Animals↗
PubMed2000

CL1-GFP: an androgen independent metastatic tumor model for prostate cancer.

PURPOSE: The mechanisms responsible for tumor progression to androgen independence in prostate cancer (CaP) remain unknown. To characterize these changes and provide a basis for rational therapeutic strategies for advanced CaP, an in vivo model from a highly aggressive androgen independent CaP cell line with distinct cellular and molecular properties was developed. MATERIALS AND METHODS: An aggressive androgen-independent cell line designated CL1 was derived from a slow-growing, and androgen-dependent, parental LNCaP cell line through in-vitro androgen-deprivation and selection. CL1 was stably transfected with a green fluorescence protein gene (CL1-GFP) and orthotopically injected into SCID mice. The pathologic behavior, histology, and molecular determinants of CL1 tumor and metastases were determined and characterized by standard light and fluorescent microscopy, and quantitative RT-PCR analysis. RESULTS: CL1 is an anaplastic prostate cancer cell line which demonstrates extensive local invasion and metastases to various organs that can be visualized via GFP expression. When compared with parental LNCaP cells, RT-PCR analysis of the tumor revealed an over-expression of EGFR, b-FGF, VEGF, TGF-beta, IL-8, IL-6, and bcl-2 and a down regulated expression of the p53, E-cadherin and PTEN. In contrast to LNCaP cells, CL1 tumors express lower levels of androgen receptor and barely detectable PSA mRNA. CONCLUSIONS: CL1-GFP represents an aggressive androgen-independent CaP tumor model derived through androgen deprivation whose pathologic development and molecular properties in animals resembles the clinical characteristics of hormone refractory prostate cancer (HRPC). Metastatic sites of CL1-GFP can be visualized with fluorescence microscopy offering a unique therapeutic model for the evaluation of drug sensitivity and other therapeutic modalities.

Androgens↗
PubMed1999

Regulation of corticotropin-releasing factor messenger RNA by nicotine in an immortalized amygdalar cell line.

Preliminary data suggest that amygdalar corticotropin-releasing factor (CRF) is regulated by nicotinic agonists. We sought to confirm and extend these observations by determining the effects of various concentrations of nicotine on CRF messenger RNA expression in the AR-5 immortalized amygdalar cell line. Nicotine produced concentration- and time-dependent increases in CRF mRNA. This cell line thus confirms that nicotinic agonists stimulate amygdalar CRF and appears to be a useful model for studying molecular factors important in this interaction.

Amygdala↗
PubMed1999

Coordinate and divergent regulation of corticotropin-releasing factor (CRF) and CRF-binding protein expression in an immortalized amygdalar neuronal cell line.

CRF is a 41-amino acid neuropeptide best known for its hypophysiotropic actions. CRF is widely distributed in the central nervous system in areas beyond the hypothalamus. CRF-binding protein (CRF-BP) regulates the bioavailability of CRF, and knowledge of the regulation of CRF-BP synthesis is an integral component of understanding the actions of CRF. To better study the regulation of CRF and CRF-BP, primary amygdalar cultures were immortalized by transfection with the SV 40 large T antigen. A clonal line that expresses CRF immunoreactivity and messenger RNA was selected. The production of CRF peptide and message by this line is regulated in a manner indistinguishable from primary cultures. We also observed that the immortalized cells express CRF-BP immunoreactivity and messenger RNA. The expression of both CRF and CRF-BP is positively regulated by forskolin and interleukin-6. Unlike CRF, the expression of CRF-BP message and peptide was increased by phorbol 12-myristate 13-acetate or dexamethasone. These results demonstrate that the synthesis of CRF and CRF-BP in this clonal cell line may be regulated in parallel by some agents but not by others. These data also suggest that dexamethasone may decrease the biological availability of CRF in the amygdala by increasing the expression of CRF-BP, rather than by decreasing CRF expression.

Amygdala↗
PubMed1998

NPY upregulates genes containing cyclic AMP response element in human neuroblastoma cell lines bearing Y1 and Y2 receptors: involvement of CREB.

Four NPY receptor subtypes have been cloned, and shown to be coupled to both Ca2+ and cAMP. However, very little is known about the downstream elements mediating NPY actions. It has recently been demonstrated in our laboratory that intrahypothalamic (i.h.t.) administration of NPY induces hypothalamic CaM kinase activity, cyclic AMP response element binding protein (CREB) phosphorylation and cyclic AMP response element (CRE) binding activity in rat hypothalamic nuclear proteins. In the present study, we have investigated whether these changes in CRE binding transcriptional factors activated by NPY results in gene regulation using a human neuroblastoma cell line (SK-N-BE2). This cell line which expresses the Y2 subtype of NPY receptors was transfected with a fusion gene containing 1.305 kb of human CRF 5' flanking region with a perfect palindromic CRE site linked to firefly luciferase gene. NPY treatment increased CaM kinase II activity, CREB phosphorylation and CRE binding in these cells. In transfected cells, luciferase activity was also increased by NPY (1.8-4-fold) within 4 h of treatment. Moreover, forskolin (7-30-fold), which stimulates cAMP production, and thapsigargin (6-8-fold), which mobilizes intracellular calcium, also increased luciferase activity within 4 h of treatment. PMA (phorbol-12-myristate-13-acetate), an activator of protein kinase-C, induced luciferase activity by 1.8-fold. NPY augmented forskolin-stimulated luciferase activity from 11- to 15-fold, but had no significant effect on thapsigargin-induced luciferase activity. These findings suggest that activation of protein kinase A (PKA) or CaM kinase leads to the induction of fusion gene. NPY treatment upregulated fusion gene expression through Ca2+ pathway in SK-N-BE2 cell line. Pretreatment with CREB antisense, but not the sense oligodeoxynucleotides, inhibited forskolin-, thapsigargin- and NPY-stimulated luciferase activity. However, CREB sense or antisense oligodeoxynucleotide treatment had no effect on PMA-stimulated luciferase activity. Furthermore, NPY induced CRE binding activity and the expression of CRE containing Y1 receptor gene in SK-N-MC cell line. These findings suggest that NPY can upregulate CRE containing reporter gene including Y1 receptor gene and NPY-induced reporter gene regulation in SK-N-BE2 cells is mediated by intracellular Ca2+ and CREB protein.

Animals↗
PubMed1998

NPY messenger RNA is increased in medial hypothalamus of anorectic tumor-bearing rats.

Previous investigations suggest that neuropeptide Y (NPY) feeding mechanisms and corticotropin releasing factor (CRF) are altered in anorectic tumor-bearing (TB) rats. To better determine the relationship of NPY and CRF synthesis to cancer anorexia we measured mRNA for these peptides in medial and lateral hypothalamus of TB and control rats. NPY and CRF mRNA were reliably detected by Northern blot analysis only in medial hypothalamus, where NPY message was elevated significantly in TB rats. CRF mRNA tended to be reduced in both pair-fed (PF) and TB rats, but did not reach statistical significance. Concentrations of NPY or CRF were not altered significantly in either the lateral or medial hypothalamus of TB or PF rats. These results suggest that the transcription of NPY is elevated in PF rats and is increased further in anorectic TB rats. The lack of significant increases in levels of peptides may be related to dilution, due to measuring a relatively large block of hypothalamic tissue. Alternatively, translation of the signal for NPY production may be inhibited, or degradation of peptide levels may be increased.

Animals↗