Biomedical subjects

S Sheriff

Publications and source records attributed to S Sheriff.

78 records · Page 5Linked to original sources

PubMedDate not supplied

Characterization of neuropeptide Y binding sites in rat cardiac ventricular membranes.

Neuropeptide Y (NPY) binding sites in rat cardiac ventricular membranes have been characterized in detail. 125I-NPY bound to the membranes with high affinity. Binding was saturable, reversible and specific, and depended on time, pH and temperature. Analysis of the binding data obtained under optimal conditions, 2 hr, 18 degrees C and at pH 7.5, revealed the presence of low and high affinity binding sites. The high affinity binding sites had an apparent dissociation constant (Kd) of 0.38 nM and a binding capacity (Bmax) of 7.13 fmol/mg protein. The apparent Kd and Bmax for low affinity binding sites were 22.34 nM and 261.25 fmol/mg protein, respectively. Peptides unrelated to NPY did not compete with 125I-NPY for the binding sites even at 1 microM concentrations, whereas homologous peptides, peptide YY (PYY) and pancreatic polypeptide (PP), and NPY(13-36) inhibited 125I-NPY binding but with lower potency compared to NPY. 125I-NPY binding was sensitive to the nonhydrolyzable GTP analog, Gpp(NH)p, suggesting that the NPY receptor is coupled to the adenylate cyclase system. The ventricular membrane receptor characterized in this study may play an important role in mediating the physiological effects of NPY in the heart.

Animals↗

PubMedDate not supplied

N-alpha-biotinylated-neuropeptide Y analogs: syntheses, cardiovascular properties, and application to cardiac NPY receptor visualization.

Two monobiotinylated analogs of neuropeptide Y (NPY) were synthesized by coupling the N-hydroxysuccinimidyl esters of biotin and (6-biotinylamido)-hexanoic acid, respectively, to the free alpha-NH2 group of the side chain protected NPY peptide resin. Crude peptides obtained by HF cleavage were purified by RPLC and their integrities were confirmed by amino acid and mass spectral analysis. As with NPY, both biotinylated analogs inhibited 125I-NPY binding and adenylate cyclase activity of rat cardiac ventricular membranes in a dose-dependent manner. N-alpha-[(6-biotinylamido)-hexanoyl]-NPY exhibited potencies comparable to that of NPY whereas N-alpha-biotinyl-NPY was slightly less potent. In the in vivo experiments, however, both the biotinylated analogs exhibited responses comparable to NPY in increasing arterial blood pressure and decreasing heart rate in anesthetized rats. The responses of the biotinyl analogs were longer lasting than those of NPY. Histochemical studies revealed that N-alpha-[(6-biotinylamido)-hexanoyl]-NPY could label the NPY receptors in rat cardiac ventricular tissues. This labeling was specific since intact NPY inhibited the staining. These studies show that biotinyl-NPY analogs exhibit biological potencies comparable to intact NPY and can therefore be used to further probe the NPY-receptor interaction.

Adenylyl Cyclases↗

PubMedDate not supplied

Characterization of amylin binding sites in a human hepatoblastoma cell line.

Amylin binding sites in a human hepatoblastoma cell line (HepG2) have been characterized in detail. 125I-Amylin (rat) bound to HepG2 cells with high affinity. Binding was reversible and selective, and dependent on time and temperature. Scatchard analysis revealed the presence of high (Kd = 0.11 +/- 0.04 nM) and low (Kd = 1.3 +/- 0.4 microM) affinity binding sites for 125I-amylin in HepG2 cells. The dissociation experiments also showed that 125I-amylin dissociated from high- and low-affinity sites. The association data, however, indicated the presence of only one binding site. Rat amylin was more potent than human amylin and rat calcitonin gene-related peptide (CGRP) in displacing 125I-amylin bound to HepG2 cells. Nonhomologous peptides did not displace 125I-amylin. Rat amylin was, however, less potent than rat CGRP in displacing 125I[Tyr0]CGRP from HepG2 cells. Pretreatment of HepG2 cells with rat amylin (10 nM) reduced the specific binding of 125I-amylin by 75%, whereas rat CGRP (10 nM) pretreatment had no effect on amylin binding. Calcitonin gene-related peptide, as well as rat and human amylin, stimulated the adenylate cyclase activity of HepG2 cell membrane preparation in a dose-dependent manner, with an order of potency of CGRP > rat amylin > human amylin. A CGRP antagonist, CGRP(8-37), significantly attenuated the stimulatory effect of both amylin and CGRP on adenylate cyclase activity. These investigations show that distinct receptors of amylin and CGRP are present in HepG2 cells, and that amylin stimulates adenylate cyclase activity through CGRP receptors. This system could now be exploited for studying amylin receptors and amylin-mediated signal transduction.

Adenylyl Cyclases↗

PubMedDate not supplied

Conformations of immunoglobulin hypervariable regions.

On the basis of comparative studies of known antibody structures and sequences it has been argued that there is a small repertoire of main-chain conformations for at least five of the six hypervariable regions of antibodies, and that the particular conformation adopted is determined by a few key conserved residues. These hypotheses are now supported by reasonably successful predictions of the structures of most hypervariable regions of various antibodies, as revealed by comparison with their subsequently determined structures.

Amino Acid Sequence↗

PubMedDate not supplied

Antagonism of NPY-induced feeding by pretreatment with cyclic AMP response element binding protein antisense oligonucleotide.

Although second messenger systems subserving neuropeptide Y (NPY)-mediated behaviors have been identified for a variety of receptors in several tissues, downstream signaling events are not well known. The nuclear binding protein, cyclic AMP response element binding protein (CREB) appears to be a transcription factor that is activated following injection of NPY into rat hypothalamus. To allow determination of the functional nature of CREB mediation of NPY-induced feeding, injection cannulae were implanted into the perifornical hypothalamus of 18 rats. Treatment of seven rats with CREB antisense oligonucleotide (15 ug) significantly antagonized NPY feeding for up to one week after treatment, while similar injections of CREB sense oligonucleotide (15 ug) had no significant effect on NPY-induced feeding. Two weeks after the antisense oligonucleotide treatment, feeding was once again elicited by the injection of NPY. Hypothalamic CREB protein was also reduced significantly two days after the CREB antisense oligonucleotide treatment. These results suggest that activation of CREB, probably through phosphorylation, may be a necessary event for the signal transduction of NPY stimulation into feeding behavior.

Animals↗

PubMedDate not supplied

Glucagon-like peptide-2 stimulates gut mucosal growth and immune response in burned rats.

Major burn trauma often leads to reduced gut barrier function, immunosuppression, and increased bacterial translocation. We hypothesized that treatments that maintain normal gut after burn trauma will also reduce immunosuppression and bacterial translocation. Recent studies suggest that treatment with glucagon-like peptide-2 (GLP-2), which is synthesized in the intestine and released after food intake, elicits mucosal hyperplasia in the small intestine of rodents and prevents parenteral nutrition-induced gut hypoplasia. Therefore, we determined whether GLP-2 would prevent loss of gut integrity after major burn trauma. Osmotic minipumps were implanted into the peritoneum of 22 adult, male, Sprague-Dawley rats to infuse saline (10 microl/hr; n = 14) or GLP-2 (1 microg/hr; n = 8). On the next day 8 saline-infused and 8 GLP-2-infused rats were subjected to a 25 sec duration 30% BSA open flame burn, with the remaining rats serving as sham-burn controls. Five days after burn, all rats were killed. Gut protein was assessed, and immunosuppression was estimated by the mitogenic response of cultured splenocytes to phytohemagglutinin, pokeweed, and concanavalin A. Bacterial translocation was determined by culturing the mesenteric lymph nodes. Although protein content was significantly decreased in the ileum of burned rats treated with saline, the burned rats treated with GLP-2 exhibited significant increases in protein levels in duodenum, jejunum. and ileum. Colon protein was not affected by GLP-2 infusion. Saline-treated burned rats also exhibited immunosuppression, as suggested by significantly decreased responses to each of the mitogens. Infusion of GLP-2 normalized the response by the burned rats to each of the mitogens. Lymph nodes taken from sham rats exhibited no colony forming units, whereas in both of the burn groups, 50% of the cultures were positive. However, more aggressive colonization may have occurred in the saline-infused burned rats as compared with the GLP-2-infused burned rats (81 +/- 63 vs 3 +/- 2 colony forming units). These results suggest that GLP-2 may stimulate gut mucosa and reduce immunosuppression in burned rats. However, there does not seem to be a statistically significant positive effect of GLP-2 on bacterial translocation. Thus, improving small intestine mucosa may increase immunity while being ineffective against bacterial translocation.

Analysis of Variance↗