Biomedical subjects

A Sahu

Publications and source records attributed to A Sahu.

At least 55 records · Page 3Linked to original sources

PubMed1998

Effect of estrogen on hypothalamic transforming growth factor alpha and gonadotropin-releasing hormone gene expression in the female rhesus monkey.

In order to study whether hypothalamic transforming growth factor alpha (TGFalpha) gene expression in the monkey is estrogen-sensitive, long-term ovariectomized rhesus macaques were implanted subcutaneously with either estradiol-containing (n = 3) or blank (n = 3) Silastic capsules. Blood samples were collected every other day while the animals were lightly sedated with ketamine hydrochloride to monitor circulating LH and estradiol concentrations. Animals were killed with a lethal dose of pentobarbital sodium after a marked suppression of LH secretion was confirmed (81 days of estradiol treatment); the preoptic area (POA), mediobasal hypothalamus (MBH) and samples of cerebral cortex were dissected out, snap-frozen in liquid nitrogen and processed for the determination of TGFalpha messenger RNA (mRNA) by ribonuclease protection assay using a cRNA probe. The opportunity was also taken to study the action of estrogen on hypothalamic GnRH mRNA levels. Although circulating estradiol concentrations of 50-150 pg/ml achieved in the steroid-treated group produced a decrease in hypothalamic GnRH mRNA levels, which was significant in the MBH, TGFalpha mRNA levels in this hypothalamic region and in the POA were not influenced by estrogen treatment. These findings indicate that TGFalpha is probably not involved in mediating the inhibitory action of estradiol on GnRH neurons. Additionally, the relevance of our results to the understanding of the neurobiological mechanisms underlying the initiation of puberty in primates is discussed.

Animals↗

PubMed1998

Leptin decreases food intake induced by melanin-concentrating hormone (MCH), galanin (GAL) and neuropeptide Y (NPY) in the rat.

Recent evidence suggests that leptin reduces food intake (FI) by acting at the hypothalamic level. Leptin decreases hypothalamic neuropeptide Y (NPY), melanin-concentrating hormone (MCH) and galanin (GAL) gene expression in rats. The purpose of the present study was to test the hypothesis that leptin decreases FI by additionally modulating the action of NPY, MCH or GAL in the hypothalamus. Intracerebroventricular (i.c.v.) administration of NPY, MCH or GAL induced FI in satiated rats. A prior i.c.v. injection of leptin (4 microg) completely prevented the increase of FI either by MCH, GAL or NPY. These results suggest that modulation of post-synaptic actions of MCH, GAL and NPY is one of the mechanisms of leptin signaling in the hypothalamus.

Animals↗

PubMed1998

Absence of increased neuropeptide Y neuronal activity before and during the luteinizing hormone (LH) surge may underlie the attenuated preovulatory LH surge in middle-aged rats.

A large body of evidence suggests that the neuroendocrine axis plays a major role in the reproductive aging of female rats. Since increased hypothalamic neuropeptide Y (NPY) neurosecretion is crucial in the preovulatory LH discharge in young rats, we tested the hypothesis that diminution in the preovulatory LH surge in middle-aged (MA) rats may be due to altered neurosecretory activity in NPYergic neurons. In Exp 1, we examined NPY levels in six microdissected hypothalamic nuclei, including median eminence (ME), arcuate nucleus (ARC), and medial preoptic area (MPOA), at 1000, 1200, 1400, 1600, 1800, 2000, or 2200 h on the day of proestrus in young (2.5- to 3-month old) and MA (7- to 9-month old) regularly cycling rats. At 1000 h, ME NPY levels in young rats were significantly lower than those in MA rats. In young rats, the ME NPY levels were significantly increased at 1400 h before the LH surge in the afternoon and thereafter decreased progressively during the interval of the LH surge. In MA rats, however, ME NPY levels decreased in the afternoon in association with an attenuated LH surge. In addition, in the ARC and MPOA, the other hypothalamic sites associated with induction of LH surge, NPY levels increased before and during the LH surge in young rats, no change in NPY levels in these nuclei was observed in association with the attenuated LH surge in MA rats. Also, NPY levels in the ARC and MPOA during the afternoon were significantly lower in MA compared with those in young animals. These results demonstrated the absence of an antecedent increase in NPY levels, specifically in the ME and ARC, during the afternoon of proestrus in MA animals. In a second experiment, we evaluated whether the absence of dynamic changes in NPY levels in the ME and ARC in MA rats was due to altered hypothalamic NPY gene expression. Regularly cycling young (2.5- to 3-month-old) and MA (8- to 10-month-old) rats were killed at 1000, 1200, 1400, 1600, 1800, 2000, or 2200 h on the day of proestrus. The medial basal hypothalamus was processed for prepro-NPY messenger RNA (mRNA) measurement by ribonuclease protection assay. In young rats, prepro-NPY mRNA levels were significantly increased at 1200 h and remained elevated throughout the afternoon. In contrast, in MA rats prepro-NPY mRNA levels remained unchanged before and during the attenuated LH surge. These results clearly indicate that the augmentation in NPY neuronal activity before and during the LH surge seen in young rats fails to manifest itself in middle-aged rats. As hypothalamic NPY participates in the induction of LHRH surge, our results suggest that reduced LHRH and LH surges in MA rats may be due to diminution in NPY secretion in these animals.

Aging↗

PubMed1998

Evidence suggesting that galanin (GAL), melanin-concentrating hormone (MCH), neurotensin (NT), proopiomelanocortin (POMC) and neuropeptide Y (NPY) are targets of leptin signaling in the hypothalamus.

Leptin (OB protein) reduces food intake by acting at the hypothalamic level. The purpose of the present study was to identify potential targets of leptin signaling in the hypothalamus in ad-lib fed rats. Central administration of leptin (5 microg) for 3 days decreased food intake and body weight gain in association with a decrease in hypothalamic galanin (GAL), melanin-concentrating hormone (MCH), proopiomelanocortin (POMC) and neuropeptide Y (NPY) gene expression and with an increase in neurotensin (NT) gene expression. In pair-fed rats, NPY gene expression was increased and there was no change in either MCH, GAL, POMC or NT gene expression. This study identifies GAL, MCH, POMC and NT as non-NPY targets of leptin signaling and suggests that leptin's action on food intake and body weight is most likely mediated by inhibiting excitatory (e.g. NPY, MCH, GAL, POMC) and stimulating inhibitory (e.g., NT) signals in the feeding circuitry.

Animals↗

PubMed1997

Evidence that hypothalamic neuropeptide Y gene expression and NPY levels in the paraventricular nucleus increase before the onset of hyperphagia in experimental diabetes.

Neuropeptide Y (NPY) is the most potent endogenous orexigenic signal. Several lines of evidence indicate that the site of NPY action in transducing feeding signal may reside in the paraventricular nucleus (PVN) and neighboring sites in the hypothalamus. To test the hypothesis that an increase in NPY activity in the ARC-PVN pathway precedes the onset of diabetic hyperphagia, we evaluated NPY levels in seven hypothalamic nuclei and NPY gene expression in the hypothalamus at 48, 72 or 96 h after streptozotocin (STZ) treatment in rat. In STZ-treated diabetic rats, NPY gene expression in the hypothalamus and NPY levels only in the PVN significantly elevated at 48 h, while hyperphagia occurred sometimes after 48 h post-injection. These results show that augmentation in NPY neuronal activity in the ARC-PVN axis precedes the onset of increased food intake produced by STZ-induced insulinopenia. These findings affirm the hypothesis that increased NPY neurosecretion in the PVN may underlie the diabetes-induced hyperphagia.

Animals↗

PubMed1997

Mechanism of complement inactivation by glycoprotein C of herpes simplex virus.

Glycoprotein C (gC) of both herpes simplex virus type 1 (HSV-1) and HSV-2 interacts with complement C3b and protects the virus from complement-mediated neutralization. To study the mechanism by which gC modulates complement activation, we expressed both gC-1 and gC-2 in a baculovirus expression system. Baculovirus recombinants containing gC genes spanning the entire gC-1 sequence (gC-1-TMR) or only the extracellular domain(s) of gC-1, gC-2, or a deletion mutant of gC-1 lacking residues 33 through 123 were expressed in sf9 insect cells. Binding of the expressed proteins to human C3 and C3 fragments was assessed by direct and competition ELISA. All four expressed proteins bound to C3, C3b, and C3c but not to C3d, suggesting 1) that the binding sites for these proteins are located in the C3c region of C3; and 2) that gC, in contrast to other C3-binding proteins, interacts with native C3. We have also examined the interaction of native C3 with gC-1 expressed on the HSV-1-infected cells. Analogous to recombinant proteins, gC-1 expressed on the infected cells also bound to native C3. The ability of baculovirus-expressed gCs to inhibit the interaction of C3b with its ligands was also analyzed. We found that gC-1, but not gC-2, inhibited the binding of C5 and properdin to C3b and also inhibited the alternative pathway-mediated lysis of rabbit erythrocytes. Inhibition of alternative pathway-mediated lysis and properdin binding to C3b, but not of C5 binding to C3b, required the transmembrane segment of the gC-1. The specificity of gC interactions was examined by studying the interaction of gC with C3 from various species. In contrast to properdin, both gCs bound to cobra C3; this finding suggests that gC-1 and properdin bind to different sites on C3b. Further analyses suggested that gC-1 sterically hindered access of C5 and properdin to C3b.

Binding, Competitive↗

PubMed1996

Multiple forms of complement C3 in trout that differ in binding to complement activators.

In all other species analyzed to date, the functionally active form of complement component C3 exists as the product of a single gene. We have now identified and characterized three functional C3 proteins (C3-1, C3-3, and C3-4) in trout that are the products of at least two distinct C3 genes. All three proteins are composed of an alpha-and a beta-chain and contain a thioester bond in the alpha-chain. However, they differ in their electrophoretic mobility, glycosylation, reactivity with monospecific C3 antibodies, and relative ability to bind to various surfaces (zymosan, Escherichia coli, erythrocytes). A comparison of the partial amino acid sequences of the three proteins showed that the amino acid sequence identity/similarity of C3-3 to C3-4 is 87/91%, while that of C3-3 and C3-4 to C3-1 is 51.5/65.5% and 60/73% respectively. Thus, trout possess multiple forms of functional C3 that represent the products of several distinct genes and differ in their ability to bind covalently to various complement activators.

Amino Acid Sequence↗

PubMed1996

Inhibition of human complement by a C3-binding peptide isolated from a phage-displayed random peptide library.

We have screened a phage-displayed random peptide library for binding to C3b, the proteolytically activated form of complement component C3, and have identified a novel peptide that suppresses complement activation. This phage-displayed peptide bound to C3, C3b, and C3c, but not to C3d, indicating that it binds to the C3c region of C3. A synthetic 27-amino acid peptide corresponding to the phage-displayed peptide also bound to C3 and C3 fragments and inhibited both the classical and alternative pathways of complement activation. The inhibition of complement activation was reversible. Studies with overlapping peptides indicated that the functional activity was located in the cyclic 13-amino acid N-terminal region (ICVVQDWGHHRCT) of the parent peptide. Reduction and alkylation of this 13-residue synthetic peptide destroyed its inhibitory activity. Analysis of the mechanism of inhibition revealed that the peptide inhibited C3 cleavage in normal human serum as well as when the alternative pathway was reconstituted with purified complement components, and the observed inhibition was not due to sterically hindered access to the C3a/C3b cleavage site. Further, the peptide did not inhibit the cleavage of factor B, indicating that it did not affect the interaction of CA with factor B or the formation of C3b,Bb. The peptide also had no effect on the binding of properdin to C3, demonstrating that the observed inhibition of C3 cleavage in normal human serum was not due in part to its effect on the properdin-stabilized C3 convertase, C3b,Bb,P. These results indicate that the peptide we have identified interacts with C3 to inhibit its activation.

Amino Acid Sequence↗

PubMed1996

Investigation of mechanism-based inhibitors of complement targeting the activated thioester of human C3.

An intramolecular thioester bond in complement protein C3 is vital for covalent attachment of C3b (the proteolytically activated form of C3) to biological surfaces and for activation of the complement system. Proteolytic removal of C3a from C3 activates the thioester in the C3b fragment. Activated C3b primarily forms ester bonds with hydroxyl groups of carbohydrates on complement activating surfaces, but it has also been shown to react with the hydroxyl group of tyrosine and with specific Ser and Thr residues on IgG and on complement protein C4b. To examine the reactivity of the thioester, several families of hydroxylated compounds were examined. Reactivity of a series of substituted phenols varied over two orders of magnitude and demonstrated a linear correlation between reactivity and the Hammett substituent constants. Hydroxylated drugs including members of the L-DOPA/epinephrine family and hydroxamic acids also were examined. Compounds were identified that were 20,000 times more reactive than carbohydrates. These compounds were found to inhibit both the classical and alternative pathways of complement activation. Although the specificity of the thioester for its natural biological targets appears to be determined by many structural features, the data presented here demonstrate that increasing the nucleophilic character of the target hydroxyl group can increase the potency of a synthetic inhibitor many orders of magnitude.

Complement C3↗

PubMed1996

Differential increase in neuropeptide Y-like levels and myenteric neuronal staining in diabetic rat intestine.

Neuropeptide Y is a regulatory peptide found in adrenergic and non-adrenergic neurons. Diabetes, which may cause autonomic neuropathy, induces an increase in hypothalamic neuropeptide Y (NPY) levels; thereby we measured the effects of chronic diabetes on neuropeptide Y in the intestine. Rats were injected with streptozotocin (65 mg/kg) and maintained for up to 20 weeks. Another group of rats was injected with 6-hydroxydopamine (50 mg/kg) x 2 to induce sympathectomy. Ileum and colon were harvested and both whole and microdissected intestine were (1) stained with antibodies to neuropeptide Y, vasoactive intestine polypeptide, and neurofilaments or (2) extracted for neuropeptide Y radioimmunoassay. Neuropeptide Y levels were similar under all conditions in the colon, but there was a trend toward an increase in the diabetic whole ileum. NPY levels were significantly increased in the dissected myenteric plexus ileal layer in diabetics. We noted an increase in the number of neuropeptide Y and vasoactive intestine polypeptide immunoreactive myenteric neurons in diabetics and after 6-hydroxydopamine-induced sympathectomy. Diabetes, and to a lesser extent sympathectomy, induced an increase in ileal neuropeptide Y levels and neuropeptide Y-staining myenteric but not submucosal neurons. Altered tissue levels of neuropeptide Y may account for certain of the gastrointestinal disturbances commonly seen in diabetes.

Animals↗

PubMed1996

Disinhibition from opioid influence augments hypothalamic neuropeptide Y (NPY) gene expression and pituitary luteinizing hormone release: effects of NPY messenger ribonucleic acid antisense oligodeoxynucleotides.

It is well known that hypothalamic endogenous opioid peptides (EOP) exert an inhibitory influence on LH release, and that a restraint on this inhibitory tone triggers preovulatory LHRH and LH hypersecretion. Recent evidence suggests that hypothalamic neuropeptide Y (NPY) is an important component of the neural circuitry that participates in induction of the LH surge. We have reported previously that blockade of EOP influence by the opioid receptor antagonist, naloxone (NAL), stimulated NPY accumulation in the median eminence (ME) in association with increased LH release in estradiol-17 beta-primed ovariectomized rats. To evaluate whether a restraint on the EOP system will result in an increase in NPY synthesis, the effects of NAL infusion on preproNPY messenger RNA (mRNA) levels in the medial basal hypothalamus were studied by solution hybridization/RNase protection assay and by in situ hybridization. NAL (2 mg/0.6 ml.h) or saline (SAL, 0.6 ml/h) was infused for 3 h (1100-1400 h) via an intrajugular cannula in estradiol-17 beta-primed ovariectomized rats. In accord with previous studies, NAL infusion significantly increased plasma LH levels at 1400 h. concomitant with this activation of LH release, NPY gene expression was also augmented. As compared with initial control levels at 1100 h, preproNPY mRNA levels in the medial basal hypothalamus increased at 1400 h in SAL (106%)- and NAL (202%)-infused rats, and at this time, preproNPY mRNA levels were significantly higher in NAL-infused rats than in SAL-infused rats. In situ hybridization studies showed that NAL infusion significantly increased the preproNPY mRNA signal at 1400 h mainly in the rostral and middle regions of the arcuate nucleus (ARC) as compared with that seen at 1100 and 1400 h in SAL-infused rats. To examine further the relationship between the NAL-induced increase in LH release and increase in NPY gene expression, the effects of NPY mRNA antisense oligonucleotides (oligos) on NPY levels in the ME-ARC and on plasma LH levels were studied in NAL-infused rats. In NAL-infused rats, intracerebroventricular administration of a NPY antisense oligo (25 micrograms/rat) at 1000, 1200, and 1300 h decreased NPY levels in the ME-ARC and blocked the increase in plasma LH levels at 1500 h, whereas control missense oligos had no effect. Collectively, these results show that a decrease in opioid influence rapidly augments NPY gene expression in a subpopulation of neurons in the ARC, and support the hypothesis that a disinhibition from opioid influence acutely promotes NPY synthesis and release, which is necessary for phasic LH discharge in rats.

Animals↗

PubMed1996

Morphological and pharmacological evidence for neuropeptide Y-galanin interaction in the rat hypothalamus.

Galanin (GAL) and neuropeptide Y (NPY) have been shown to play important roles in the regulation of pituitary hormone secretion, as well as ingestive and sexual behaviors, by acting within the hypothalamus. While the mechanism of action of these regulatory peptides is under intensive investigation, less attention has been paid to the possible interaction between them in influencing these central regulatory processes. Because NPY and GAL augment pituitary gonadotropin release, the present study was undertaken to evaluate the nature of morphological and functional relationships between these excitatory hypothalamic peptidergic systems. Double immunolabeling for NPY and GAL was carried out on vibratome sections taken from the hypothalamus of colchicine-pretreated female rats. Avidinbiotin peroxidase technique and a dark blue diaminobenzidine reaction was used to visualize NPY profiles, while the GAL neurons were labeled with a light brown diaminobenzidine reaction using either the avidin-biotin peroxidase or the peroxidase antiperoxidase technique. Light microscopic examination of the immunostained material showed that in the arcuate nucleus, paraventricular nucleus, supraoptic nucleus, anterior hypothalamus, and medial preoptic area, an abundant network of NPY-immunoreactive axons surrounded GAL-immunostained cells. Numerous dark blue NPY-containing putative boutons were observed in close proximity to GAL-immunolabeled cell bodies and dendrites. Correlated light and electron microscopic examination revealed that most of the immunoreactive NPY axon terminals established synaptic connections with GAL-expressing cells. Synaptic connections were most frequently found in the medial preoptic area and in the magnocellular region of the paraventricular nucleus and arcuate nucleus. Fewer connections were observed in the supraoptic nucleus. These morphological observations demonstrate the existence of a strong NPY input to hypothalamic GAL neurons, thereby suggesting a modulatory role for NPY in monitoring GAL release. To evaluate the functional relevance of this anatomical relationship, the effects of intraventricular injection of a GAL receptor antagonist, galantide, were examined on NPY-induced LH release in ovarian steroid-primed ovariectomized rats. As expected, intraventricular injection of NPY readily stimulated LH release. Although, while on its own, galantide was ineffective in altering basal LH release, it markedly attenuated the NPY-induced LH response, thereby suggesting that GAL released in response to NPY administration may, in part, mediate the excitatory effects of NPY. These experimental results, taken together with the morphological observations, document the involvement of an NPY --> GAL signaling modality in the release of gonadotropins and, likewise, raise the possibility of a similar signaling process in the release of other pituitary hormones and elicitation of behavioral effects attributed to NPY and GAL.

Analysis of Variance↗

PubMed1995

In vivo release of interleukin-1 beta into hypothalamic extracellular fluid in rats: effects of repeated sampling.

Interleukin-1 beta (Il-1 beta) concentrations in extracellular fluid (ECF) withdrawn at 10-min intervals through a push-pull cannula (PPC) located in the hypothalamus were studied in freely behaving male rats for 1 h at 24 and 72 h and again at 7 days after PPC implantation. Il-1 beta concentrations in ECF were similar in the latter. However, when ECF was sampled at 3 h and again 7 days after PPC implantation, Il-1 beta concentrations were greatly elevated at 7 days when compared to all other intervals. These results demonstrate how the relationships between Il-1 beta measured in ECF and the conditions of measurement appear to be integral parts of a whole intracerebral system: cytokine concentrations appear to be inextricably bound to intrahypothalamic conditions created by the sampling device presence and frequency of use.

Animals↗

PubMed1995

Agmatine, a novel hypothalamic amine, stimulates pituitary luteinizing hormone release in vivo and hypothalamic luteinizing hormone-releasing hormone release in vitro.

Agmatine, a clonidine displacing substance and imidazoline receptor agonist, was recently isolated from bovine brain and shown to be present in the rat hypothalamus. Since clonidine can stimulate the release of pituitary luteinizing hormone (LH), we tested the hypothesis that agmatine may similarly act in the rat to stimulate the hypothalamic luteinizing hormone-releasing hormone (LHRH)-pituitary LH axis. Administration of agmatine intracerebroventricularly rapidly augmented the release of LH in a dose-related fashion in ovariectomized, ovarian steroid-primed rats. Additionally, agmatine enhanced the in vitro efflux of LH releasing hormone from the median eminence-arcuate nucleus of the hypothalami of rats similarly pretreated with steroids. These studies imply that the endogenous imidazoline receptor agonist, agmatine, may serve as an excitatory neurotransmitter/neuromodulator in the hypothalamic control of LH release and we suggest that the previously reported excitatory effects of clonidine on LH release may be attributed to stimulation by clonidine of imidazoline receptors.

Agmatine↗

PubMed1995

Tyrosine is a potential site for covalent attachment of activated complement component C3.

Activation of C3 results in the generation of metastable C3b, which has been shown to preferentially react with the hydroxyl groups of carbohydrates and with specific serine and threonine residues in proteins. In this study we have examined the reactivity of metastable C3b with the third type of hydroxyl group present in proteins, tyrosine (Tyr). The results demonstrated that Tyr reacts with the thioester of metastable C3b and that this reactivity was 11-fold better than that of threonine, 47-fold better than serine and 50-fold better than the reactivity of carbohydrates. Model peptides containing Tyr showed even higher reactivity than free Tyr, demonstrating that incorporation into peptide structures does not block C3b attachment. The site of attachment was found to be in the alpha'-chain of C3b and the bond was hydroxylamine sensitive, indicating an ester linkage. The stability of the C3b-Tyr complex was measured under physiological conditions (pH 7.4, 37 degrees C) and compared to the stability of other C3b complexes. C3b-Tyr decayed 50% in 19 hr at 37 degrees C, but C3b bound to a Tyr-containing peptide was more stable, exhibiting a t1/2 of 53 hr. The ester linked complexes C3b-IgG and C3b-glycerol were less stable each exhibiting a t1/2 of approximately 8 hr. As yet, only two specific C3b attachment sites on proteins have been identified, Ser1217 in C4b and Thr144 in IgG1. The present evidence demonstrates that Tyr residues are highly reactive and that the C3b-Tyr linkage is stable. The findings suggest that complexes involving tyrosine residues as the site of attachment will also be found.

Amino Acid Sequence↗

PubMed1995

Evidence that hypothalamic neuropeptide Y gene expression increases before the onset of the preovulatory LH surge.

Neuropeptide Y (NPY), a 36 amino acid residue peptide, is involved in stimulation of LHRH and LH surges on proestrus and those induced by ovarian steroids in ovariectomized (ovx) rats. Recently, we observed that NPY gene expression in the medial basal hypothalamus (MBH) was increased before the onset of the LH surge in the ovarian steroid-primed ovx rats. Since the ovarian steroidal milieu during the estrous cycle is markedly different from that prevailing after ovarian steroid injections in ovx rats, we evaluated in cycling rats the temporal relationship between MBH preproNPY mRNA levels and the preovulatory LH surge on the day of proestrus and compared that with diestrus II, concomitant with basal LH levels. PreproNPY mRNA levels in the MBH were measured by solution hybridization/RNAse protection assay, using a cRNA probe. On the day of diestrus II, preproNPY mRNA levels changed little between 1000 and 1800 h. Quite unexpectedly, preproNPY mRNA levels at 1000 h on proestrus were similar to diestrus II levels, despite additional exposure to ovarian steroids during this interval. However, from these low levels at 1000 h, the preproNPY mRNA profile displayed a biphasic rise. During the first phase, preproNPY mRNA rose significantly at 1200 h and remained elevated at 1300 and 1400 h concomitant with basal serum LH levels. Thereafter, a second rise in preproNPY mRNA began at 1500 h, peaked rapidly at 1600 h and declined significantly at 1800 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of Variance↗

PubMed1995

Insulin and insulin-like growth factor II suppress neuropeptide Y release from the nerve terminals in the paraventricular nucleus: a putative hypothalamic site for energy homeostasis.

It has been recently recognized that a distinct signaling pathway in the hypothalamus is involved in the stimulation of feeding in mammals. Neuropeptide Y (NPY), a member of the pancreatic polypeptide family, is the most potent orexigenic signal, and its secretion in discrete hypothalamic sites increases in response to insulinopenia produced by food deprivation or experimental diabetes. To establish the site of interaction between the hypothalamus and the pancreas, we examined the effects of insulin on NPY release in vivo and in vitro from hypothalamic sites known to be involved in feeding behavior. In the first study we evaluated the effects of peripheral insulin injections (1 U/kg.day, sc) on NPY levels in seven hypothalamic nuclei in food-deprived (FD) and ad libitum-fed rats. Whereas food deprivation for 3 days increased NPY levels in the medial preoptic area, paraventricular nucleus (PVN), and arcuate nucleus, insulin injections, which did not alter blood glucose levels, returned NPY levels to the control range selectively in the PVN. NPY levels in the hypothalamic nuclei remained unchanged after insulin injections in ad libitum-fed rats. The in vivo NPY release in the PVN of FD rats, evaluated by the push-pull cannula technique, also decreased in response to peripheral insulin injections. Finally, the effects of insulin, insulin-like growth factor I (IGF-I), and IGF-II on NPY release in vitro from the microdissected PVN and two central neighboring sites, the ventromedial nucleus and the median eminence-arcuate nucleus, of FD rats were evaluated. Both insulin (0.67 or 6.7 nM) and IGF-II (0.7 or 7.0 nM) decreased the release of NPY in a dose-dependent manner only from the PVN. On the other hand, IGF-I (0.07 or 7.0 nM) failed to alter the basal PVN NPY efflux. As the PVN is richly innervated by NPY-containing nerve terminals, the results of these in vivo and in vitro studies suggest that the site of insulin action on the hypothalamic NPY network may reside at the level of PVN nerve terminals or at the interneurons in contact with NPY nerve terminals. Although insulin may have a direct effect in reducing NPY release from the PVN, the effectiveness of IGF-II in decreasing NPY release from the PVN raises the possibility that insulin's action may also be mediated via hypothalamic IGF-II neuronal pathways.

Animals↗

PubMed1994

Covalent attachment of human complement C3 to IgG. Identification of the amino acid residue involved in ester linkage formation.

C3 (native complement component 3) plays a central role in the activation of complement and in the transport and processing of immune complexes. Proteolytic activation of C3 exposes a highly reactive thioester bond which preferentially reacts with the hydroxyl groups of acceptor molecules on activators such as immune complexes or carbohydrates on microorganisms. Recently, a C3 attachment site has been localized on the CH1 domain of IgG1 between Val134 and Lys156. We have synthesized a series of peptide analogs of this region to identify the preferred residue for C3b (the proteolytically activated form of C3) attachment. The parent peptide included all 6 hydroxyl-containing amino acids present in the proposed binding site and was highly reactive with activated C3. The C3b-peptide complex was sensitive to hydroxylamine as was C3b-IgG indicating that both were ester-linked. The kinetic profile of hydrolysis of the C3b-peptide complex under physiologic conditions was found to be nearly identical to the profiles of C3b-IgG, C3b-IgG1, and C3b-glycerol complexes. Site-specific amino acid substitution of threonine and serine residues in the peptide indicated that, in contrast to the attachment site in C4b, little or no attachment occurred at serine residues. The threonine corresponding to Thr144 in the CH1 domain of IgG was found to be the major acceptor site for C3b. Thr148 was the second most reactive site on the peptide, but this residue is buried in native IgG.

Amino Acid Sequence↗