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J Rivier

Publications and source records attributed to J Rivier.

At least 19 recordsLinked to original sources
PubMed1995

Potent, structurally constrained agonists and competitive antagonists of corticotropin-releasing factor.

Predictive methods, physicochemical measurements, and structure activity relationship studies suggest that corticotropin-releasing factor (CRF; corticoliberin), its family members, and competitive antagonists (resulting from N-terminal deletions) usually assume an alpha-helical conformation when interacting with the CRF receptor(s). To test this hypothesis further, we have scanned the whole sequence of the CRF antagonist [D-Phe12,Nle21,38]r/hCRF-(12-41) (r/hCRF, rat/human CRF; Nle, norleucine) with an i-(i + 3) bridge consisting of the Glu-Xaa-Xaa-Lys scaffold. We have found astressin [cyclo(30-33)[D-Phe12,Nle21,38,Glu30,Lys33]r/ hCRF(12-41)] to be approximately 30 times more potent than [D-Phe12,Nle21,38]r/hCRF-(12-41), our present standard, and 300 times more potent than the corresponding linear analog in an in vitro pituitary cell culture assay. Astressin has low affinity for the CRF binding protein and high affinity (Ki = 2 nM) for the cloned pituitary receptor. Radioiodinated [D-125I-Tyr12]astressin was found to be a reliable ligand for binding assays. In vivo, astressin is significantly more potent than any previously tested antagonist in reducing hypophyseal corticotropin (ACTH) secretion in stressed or adrenalectomized rats. The cyclo(30-33)[Ac-Pro4,D-Phe12,Nle21,38,Glu30,Lys33++ +]r/hCRF-(4-41) agonist and its linear analog are nearly equipotent, while the antagonist astressin and its linear form vary greatly in their potencies. This suggests that the lactam cyclization reinstates a structural constraint in the antagonists that is normally induced by the N terminus of the agonist.

Adrenalectomy↗
PubMed1995

A new family of Conus peptides targeted to the nicotinic acetylcholine receptor.

In this work, a new family of Conus peptides, the alpha A-conotoxins, which target the nicotinic acetylcholine receptor, is defined. The first members of this family have been characterized from the eastern Pacific species, Conus purpurascens (the purple cone); three peptides that cause paralysis in fish were purified and characterized from milked venom. The sequence and disulfide bonding pattern of one of these, alpha A-conotoxin PIVA, is as follows: [formula: see text] where O represents trans-4-hydroxyproline. The two other peptides purified from C. purpurascens venom are the under-hydroxylated derivatives, [Pro13]alpha A-conotoxin PIVA and [Pro7,13]alpha A-conotoxin PIVA. The peptides have been chemically synthesized in a biologically active form. Both electrophysiological experiments and competition binding with alpha-bungarotoxin demonstrate that alpha A-PIVA acts as an antagonist of the nicotinic acetylcholine receptor at the postsynaptic membrane.

Amino Acid Sequence↗
PubMed1995

Potent pituitary-gonadal axis suppression and extremely low anaphylactoid activity of a new gonadotropin releasing hormone (GnRH) receptor antagonist "azaline B".

We report here the biological characterization of azaline B, a new gonadotropin releasing hormone (GnRH) receptor antagonist, with the following amino acid sequence: [Ac-D-Nal1, D-Cpa2, D-Pal3, Aph5(atz), D-Aph6(atz), Ilys8, D-Ala10]-GnRH. Azaline B was shown to suppress several reproductive processes in rats including ovulation, and had very low anaphylactoid activity compared with other GnRH antagonists. Azaline B inhibited histrelin (a GnRH agonist)-mediated follicle stimulating hormone (FSH) and luteinizing hormone (LH) release from cultured rat pituitary cells. Three antagonists ([Nal-Glu]-GnRH, [Nal-Lys]-GnRH ("antide"), and azaline B) inhibited 0.1 nM histrelin-mediated gonadotropin release to baseline levels with EC50 values of approximately 0.6 nM. Azaline B, when injected s.c. into rats on the afternoon of proestrus, was more potent at inhibiting ovulation than either [Nal-Glu]-GnRH or [Nal-Lys]-GnRH. The relative order of antiovulatory potencies of the three antagonists was azaline B > [Nal-Glu]-GnRH > [Nal-Lys]-GnRH. Similar azaline B potency was shown by its ability to suppress gonadotropin levels in castrated rats. The improved selectivity of azaline B was demonstrated when it was compared with other GnRH antagonists in the cutaneous anaphylactoid assay (local wheal response) in rats. Results with azaline B were not significantly different from results with vehicle in this assay. [Nal-Glu]-GnRH was more than twice as potent as [Nal-Lys]-GnRH in stimulating a wheal response. Furthermore, the maximal wheal response produced by azaline B was only 0.6 times that of [Nal-Lys]-GnRH, currently one of the most selective antagonists identified. Finally, both azaline B and [Nal-Lys]-GnRH were much less potent than [Nal-Glu]-GnRH in the guinea pig cardiopulmonary anaphylactoid assay after i.v. administration. These data show that azaline B is a potent and selective GnRH receptor antagonist with little or no anaphylactoid activity in animal models, and therefore has potential for use in the treatment of many reproductive endocrine disorders, as well as for use as a contraceptive.

Amino Acid Sequence↗
PubMed1995

Ligand requirements of the human corticotropin-releasing factor-binding protein.

CRF-binding protein (CRF-BP), identified as a 37-kilodalton human serum protein, binds human (h) CRF (Kd = 0.17 +/- 0.01 nM) and blocks hCRF's ability to stimulate ACTH release by pituitary cells in vitro. The present study examines ligand requirements of CRF-BP by testing the affinity of recombinant CRF-BP for synthetic analogs of CRF and peptides in the CRF family. The relative affinities of various fragments of hCRF or related peptides for CRF-BP indicate that residues 9-28 are crucial for ligand binding. CRF-BP binds human/rat CRF and urotensin-I with high affinity, sauvagine with moderate affinity, and ovine (o) CRF with low affinity. The marked difference in the affinity of CRF-BP for oCRF (Ki = 1100 +/- 97 nM) compared to hCRF (Ki = 0.17 +/- 0.01 nM), when considered with the importance of the central domain, suggests that amino acids 22, 23, and/or 25 are critical for binding. Altering oCRF residues 22, 23, or 25 individually or collectively to match those of hCRF increases the affinity of CRF-BP for these ligands; [Ala22, Arg23, Glu25]oCRF, in which all three of these central amino acids are substituted by their hCRF counterparts, binds CRF-BP with an affinity equal to that of hCRF. CRF-BP has differential affinities for CRF receptor antagonists, binding alpha-helical CRF-(9-41) with high affinity and [D-Phe12, Nle21,38]hCRF-(12-41) with low affinity. Thus, the structural requirements for binding to CRF-BP can clearly be distinguished from those for CRF receptor recognition of both agonists and antagonists. Peptides such as hCRF-(9-33), with low biological activity but which retain high affinity for the binding protein, can competitively override the effects of CRF-BP to block CRF-induced ACTH secretion, raising the possibility that whereas endogenous CRF-BP serves to limit the distribution or duration of action of CRF, specific pharmacological inhibitors of the ligand-binding protein interaction might be used to therapeutically elevate free CRF levels.

Amino Acid Sequence↗
PubMed1994

Decreased release of gonadotropin-releasing hormone during the preovulatory midcycle luteinizing hormone surge in normal women.

To investigate the contribution of hypothalamic gonadotropin-releasing hormone (GnRH) secretion to the midcycle gonadotropin surge in the human, the response of luteinizing hormone (LH) to competitive GnRH receptor blockade achieved by administration of a range of doses of a pure GnRH antagonist was used to provide a semiquantitative estimate of endogenous GnRH secretion. The LH response to 5, 15, 50, and 150 micrograms/kg s.c. of the NAL-GLU GnRH antagonist ([Ac-D-2Nal1,D-4ClPhe2,-D-Pal3,Arg5,D-4-p-met hoxybenzoyl-2-aminobutyric acid6,D-Ala10]GnRH, where 2Nal is 2-naphthylalanine, 4ClPhe is 4-chlorophenylalanine, and 3Pal is 3-pyridylalanine) was measured in normal women in the early and late follicular phases of the menstrual cycle, at the time of the midcycle LH surge and in the early luteal phase. LH decreased in a dose-response fashion after administration of the GnRH antagonist in all cycle phases (P < 0.0001). When this suppression was expressed as maximum percent inhibition, there was no difference in response during the early and late follicular and early luteal phases. However, at the midcycle surge, there was a leftward shift of the dose-response curve with significantly greater suppression of LH at the lower antagonist doses in comparison to the other cycle phases (P < 0.005), but no difference at the highest dose. Thus, we draw the following conclusions. (i) There is a consistently greater degree of LH inhibition by GnRH antagonism at the midcycle surge at submaximal degrees of GnRH receptor blockade than at other phases of the menstrual cycle in normal women. (ii) This leftward shift of the dose-response relationship to GnRH receptor blockade suggests that the overall amount of GnRH secreted at the midcycle surge is less than at other cycle stages. (iii) These data confirm the importance of pituitary augmentation of the GnRH signal at the time of the midcycle gonadotropin surge in the human.

Adolescent↗
PubMed1994

Conformationally restricted competitive antagonists of human/rat corticotropin-releasing factor.

Corticotropin releasing factor (CRF) is a 41-peptide amide which stimulates the release of ACTH (Vale et al. Science 1981, 213, 1394). CRF has been postulated to assume an alpha-helical conformation upon binding to its pituitary receptor (Hernandez et al. J. Med. Chem. 1993, 36, 2860). We have exploited this hypothesis in the design of a limited series of cyclic analogues and have taken into consideration the effects of side-chain deletion (Alanine scan, Kornreich et al. J. Med. Chem. 1992, 35, 1870) as well as of changes in chirality (Rivier et al. J. Med. Chem. 1993, 36, 2851), with the rationale that side chains necessary for binding could also be replaced by side-chain bridges. In particular, we have used computer modeling to predict likely side chain bridging opportunities and evaluated the effects of such replacements by correlating biological results with those derived from CD spectroscopy. We have synthesized 38 monocyclic peptide amides, competitive antagonists of human/rat CRF, using solid-phase methodology on MBHA resin. After purification by preparative RP-HPLC, the peptides were analyzed by RP-HPLC and capillary zone electrophoresis and characterized by mass spectroscopy and amino acid analysis. CRF antagonists were tested for their ability to interfere with CRF-induced release of ACTH by rat anterior pituitary cells. In most cases, one of the bridge heads was located at a position where substitution by a D-residue was tolerated (i.e., positions 12 and 20). It has become clear that careful optimization of bridge length and chirality is critical. This is best exemplified by the fact that out of the 38 analogues that were synthesized and tested, only two, [cyclo(20-23)[DPhe12,Glu20,Lys23, Nle21,38]h/rCRF12-41 and cyclo(20-23)[DPhe12,Glu20,Orn23,Nle21,38] h/rCRF12-41], were found to be more potent (3 and 2 times, respectively) than [DPhe12,Nle21,38]h/rCRF12-41, the parent compound. Six analogues belonging to two different families were found to be half as potent as the standard, 18 had 2-20% of the potency of the standard, and the others were significantly less potent. CD results of all analogues in 50% TFE (a concentration of TFE that induced nearly maximum helicity of [DPhe12,Nle21,38]h/rCRF12-41) suggest that while helicity may be an important factor for CRF analogue recognition, little correlation is found between percent helicity as determined by spectral deconvolution and biological activity in vitro.

Adrenocorticotropic Hormone↗
PubMed1994

Characterization of a novel and potent corticotropin-releasing factor antagonist in rats.

The present study examined the ability of two analogs of human/rat corticotropin-releasing factor (h/rCRF), [Met18, Lys23, Glu27,29,40, Ala32,41, Leu33,36,38] h/rCRF9-41 (alpha-hel CRF9-41) and [D-Phe12, Nle21,38, C alpha MeLeu37] h/rCRF12-41 (D-Phe CRF12-41), to antagonize CRF- and stress-induced behavioral changes in rats. The potency and duration of action of D-Phe CRF12-41 in vivo was compared with that of alpha-hel CRF9-41, the most effective CRF antagonist studied to date. When administered i.c.v., both CRF antagonists dose-dependently reduced the locomotor activity induced by rat CRF (0.5 microgram/rat i.c.v.) and attenuated the social stress-induced anxiogenic-like effect, as measured by the elevated plus-maze. However D-Phe CRF12-41 was 5 times more potent and remained effective for a longer period (> 1.5 hr) than alpha-hel CRF9-41. High doses of alpha-hel CRF9-41 (25 micrograms/rat), by itself, exhibited weak agonist effects, which were not observed with D-Phe CRF12-41 at the same doses. These results demonstrate that a N-terminus-shortened CRF antagonist that encompasses the substitution D-Phe12 has significantly higher biological potency and an extended duration of action without intrinsic agonist effects in these in vivo systems. The availability of potent and effective CRF antagonists will provide valuable tools for exploring the functional role of brain CRF and may ultimately lead to an understanding of the biological basis of stress-related illnesses.

Amino Acid Sequence↗
PubMed1994

Pharmacological comparison of two corticotropin-releasing factor antagonists: in vivo and in vitro studies.

The present study compared the effects of two analogs of corticotropin-releasing factor (CRF), [D-Phe12,Nle21,38, C alpha MeLeu37]CRF12-41 (D-PheCRF12-41) and alpha helical CRF9-41, as antagonists of CRF in in vivo and in vitro assays. In halothane-anesthetized rats, intracerebroventricular (i.c.v.) administration of both analogs inhibited the activation of locus coeruleus (LC) neuronal discharge produced by CRF (3.0 micrograms, i.c.v.). LC activation by hypotensive stress elicited by intravenous (i.v.) infusion of nitroprusside was antagonized by the same doses of the CRF antagonists that were effective in antagonizing CRF, suggesting that the receptors involved in LC activation by CRF and by hypotensive stress are similar. However, D-PheCRF12-41 was approximately 100 times more potent than alpha helical CRF9-41 when administered i.c.v. The IC50 values for D-PheCRF12-41 as an antagonist of CRF and of nitroprusside were 0.16 and 0.14 microgram, i.c.v., respectively. The IC50 values for alpha helical CRF9-41 as an antagonist of CRF and of nitroprusside were 18 and 27 micrograms, i.c.v., respectively. In contrast, D-PheCRF12-41 was only slightly more potent than alpha helical CRF9-41 in antagonizing CRF-stimulated cyclic AMP production in rat brain homogenates, with IC50s of 78 +/- 15 and 260 +/- 30 nM for D-PheCRF12-41 and alpha helical CRF9-41, respectively. Moreover, the antagonists had similar affinities for CRF binding sites in rat brain homogenates, with Kis of 15.5 +/- 4 nM and 10.3 +/- 6 nM for D-PheCRF12-41 and alpha helical CRF9-41, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗
PubMed1993

Role of CRF in stress-related alterations of gastric and colonic motor function.

Major advances have been made in the understanding of the pathophysiology of stress-related alteration of gut function. A wealth of information indicates that CRF is involved in the central mechanisms by which stress inhibits gastric emptying while stimulating colonic motor function. CRF acts in the PVN to trigger both the inhibition of gastric emptying and the stimulation of colonic motor function in response to stress, in addition to previously established endocrine and behavioral responses. Preliminary evidence exists that CRF acts in the locus coeruleus to induce a selective stimulation of colonic transit without influencing gastric emptying. The central actions of CRF to alter gastric and colonic motor function are conveyed by autonomic pathways and are unrelated to the associated stimulation of pituitary hormone secretion. The demonstration that central CRF plays a role in mediating gastric stasis resulting from surgery, peritonitis or high levels of central interleukin-1 provides new insight into the mechanisms involved in gastric ileus induced postoperatively or by infectious disease. Likewise, the demonstration that CRF in the PVN and locus coeruleus induce the anxiogenic and colonic motor responses to stress and that colonic distention activates neurons in the locus coeruleus opens new avenues for the understanding of the pathogenesis of a subset of IBS patients with colonic hypersensitivity associated with psychopathological disturbance and diarrhea-predominant symptoms.

Animals↗
PubMed1993

Molecular and functional characterization of GnRH receptors cloned from rat pituitary and a mouse pituitary tumor cell line.

A cDNA for a GnRH receptor (mtGnRH-R) was obtained from a mouse gonadotropic pituitary cell line (alpha T3-1) by expression cloning. This full-length cDNA was subsequently used as a probe to clone a rat pituitary GnRH receptor (rGnRH-R). The two receptors differ by 13 amino acids and are 100% identical to those recently reported. The analysis of the cloned receptors by photoaffinity-labeling followed by SDS-PAGE reveals a major band of approximately 70 kDa. This is in contrast to the native rat pituitary and mouse alpha T3-1 receptors whose major labeled species migrate with an apparent size of approximately 45 kDa. Functional studies reveal that both receptors, when transiently expressed in COSM6 cells, can bind GnRH with high affinity and transduce the stimulation of IP3 accumulation in response to GnRH.

Affinity Labels↗
PubMed1993

Evidence for a common neural mechanism mediating growth hormone-releasing factor-induced and somatostatin-induced feeding.

We have previously reported that the somatotropin regulatory peptides, growth hormone-releasing hormone (GRF) and somatostatin (SS) increase feeding when applied centrally in low picomole-range doses. The similarity between the dose-response characteristics and photoperiod sensitivities of these two peptide's feeding effects, together with indirect evidence suggesting that these peptides interact functionally within the hypothalamus, suggests that GRF and SS may induce feeding via a common neural mechanism. The present studies sought to investigate this possibility. In the first experiment combined intracerebroventricular (icv) injections of optimal doses of GRF and SS were found to be less potent than icv injections of GRF or SS alone, suggesting that the mechanisms mediating GRF-induced and SS-induced feeding are not totally independent. In further studies, icv injections of antiserum raised against SS blocked GRF-induced feeding. In contrast, icv injections of antiserum raised against GRF were ineffective in blocking SS-induced feeding. These results suggest that SS may act downstream to GRF in a putative neural feeding mechanism which utilizes both peptides. The possible anatomical basis for such a feeding mechanism involving GRF and SS is discussed.

Animals↗
PubMed1992

Gonadotropin-releasing hormone antagonists with N omega-triazolylornithine, -lysine, or -p-aminophenylalanine residues at positions 5 and 6.

In order to be used as fertility regulators in humans, gonadotropin releasing hormone (GnRH) antagonists must be extremely potent and long acting and exhibit negligible side effects such as stimulating histamine release. To this aim, we have recently synthesized a series of analogues with the standard Ac-DNal1-DCpa2-DPal3 substitutions, where the N omega-amino function of ornithine, lysine, or p-aminophenylalanine (Aph) was converted to the aminotriazolyl (atz) derivatives at positions 5 and 6 with further modifications at positions 7 and 10. The analogues were tested for their ability to bind to pituitary cell membranes, to release histamine in a mast cell assay, to inhibit luteinizing hormone (LH) secretion by castrated male rats or cultured pituitary cells, and to interfere with the ovulation in intact female rats. While the subcutaneous (sc) injection of 50 micrograms of Azaline A (7, [Ac-DNal1,DCpa2,DPal3,Lys5(atz),DLys6++ +(atz),ILys8,DAla10]GnRH) dissolved in 0.2 mL of an aqueous media significantly inhibited LH release in the castrated male rat for 24 h, the same dose of Azaline B (11), [Ac-DNal1,DCpa2,DPal3,Aph5(atz),DAph6++ +(atz),ILys8,DAla10]GnRH, inhibited LH release for 72 h. A similar long duration of action was observed for Antide ([Ac-DNal1,DCpa2,DPal3,Lys5(Nic),DLys6(Nic ),ILys8,DAla10]GnRH) but not for Nal-Glu ([Ac-DNal1,DCpa2,DPal3,Arg5,4-(pmethoxybenzoy l)-D-2-Abu6,DAla10]GnRH). In the same paradigm, a 5-fold dilution of the peptide (50 micrograms in 1 mL) and the use of three injection sites rather than one resulted in significantly shorter duration of action for most of the peptides tested. This suggested that long duration of action might be the result of slow release from the injection site(s). In order to investigate this possibility, Nal-Glu and Azaline B were injected intravenously (i.v.) at three doses (10, 50, 250 micrograms) to castrated male rats. At all doses, both peptides significantly lowered LH levels for 8 h. By 24 h, Nal-Glu (250 micrograms) and Azaline B (50 and 250 micrograms) still measurably inhibited LH secretion. Finally, only Azaline B (250 micrograms) was still active at 48 h. These findings demonstrate that subtle structural modifications will yield peptides with different half-lives after iv administration. These findings led us to investigate the effects of other structural modifications on duration of action. We observed that systematic substitutions at positions 7 (NMeLeu) and 10 (Pro9-NHEt, and Gly-NH2) were found to be deleterious. Of interest was the observation that only the DAla10-NH2 substitution led to long duration of action and enzymatic stability under the conditions tested.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Alanine series of ovine corticotropin releasing factor (oCRF): a structure-activity relationship study.

Previous structure-activity relationship studies of CRF have shown that residues 1-4 were not necessary for receptor binding or transduction, that residues 4-8 were important for activation, and that residues 12-41 were mostly responsible for binding. Finally it was proposed that CRF assumed an alpha-helical structure when interacting with its receptor. By systematic substitution of each residue (except residues 1-4) in ovine CRF (oCRF) by Ala, we have investigated the role played by individual side chains in receptor recognition and activation. Out of 33 analogues (synthesized using SPPS on an MBHA resin, purified by RPHPLC and characterized by amino acid and mass spectral analyses), a significant loss of biological potency (less than 1% potency of native) was observed for 6 analogues ([Ala6], [Ala8], [Ala10], [Ala12], [Ala14], and [Ala38]); 12 analogues had biological potencies ranging from 1% to 60% and ranked as follows: [Ala35] less than [Ala16] less than [Ala9] less than [Ala19] less than [Ala15] less than [Ala13] less than [Ala7] less than [Ala23] less than [Ala11] less than or equal to [Ala21] less than [Ala27] less than or equal to [Ala18]; 8 analogues were found to be equipotent (greater than 60% and less than 150%) ([Ala5], [Ala17], [Ala26], [Ala29], [Ala30], [Ala34], [Ala36], and [Ala37]; and 7 analogues were found to be approximately 2-5 times more potent than native oCRF ([Ala25] = [Ala40] less than or equal to [Ala39] less than or equal to [Ala33] less than [Ala20] less than [Ala22] less than [Ala32], in an in vitro pituitary cell culture assay. In summary, the Ala substitutions which showed the greatest loss of potency (less than 1% of native oCRF) were those replacing hydrophobic residues while those showing the greatest increase in potency were replacing hydrophilic residues. Of the 22 Ala-containing analogues in the C-terminal half of the molecule, 17 analogues have equal or greater potencies than native oCRF. Substitution of Ala in the N-terminal region (residues 5-19) on the other hand is generally detrimental to biological activity. These results suggest that the side chains of residues 5-19 are very important for receptor binding and activation while, in the C-terminal region, the amino acid side chains may be more responsible for structural conservation than for functional expression.

Adrenocorticotropic Hormone↗
PubMed1992

Immunoreactivities for antisera to three putative neuropeptides of the rat melanin-concentrating hormone precursor are coexpressed in neurons of the rat lateral dorsal hypothalamus.

Antisera (AS) raised against rat melanin-concentrating hormone (rMCH) and against two additional peptides sequences derived from the rat MCH precursor (neuropeptide glutamic acid-isoleucineamide (NEI), and neuropeptide glycine-glutamic acid (NGE)) exclusively stained the hypothalamic neurons previously described using AS to salmon MCH, human somatocrinin 1-37 (GRF37) and alpha-MSH. Liquid phase and dot-blot controls for specificity indicated that rMCH-, NEI- and NGE-AS bound epitopes recognized by sMCH-, alpha-MSH- and GRF-37-AS, respectively. The distinct intracellular patterns of immunoreactivity obtained in control animals with rMCH-, NGE- and NEI-AS, as well as the changes observed after intracerebroventricular injection of colchicine matched previous findings using sMCH-, GRF37- and alpha-MSH-AS.

Animals↗
PubMed1992

Total chemical synthesis, characterization, and immunological properties of an MHC class I model using the TASP concept for protein de novo design.

The design, total chemical synthesis, and immunological properties of a four-alpha-helix bundle template-assembled synthetic protein (TASP) mimicking some of the structural features of the major histocompatibility complex (MHC) class I is described. In a first approach, the native sequence 58-74 of the alpha 1 heavy chain domain of HLA-A2 was modeled in order to increase helix stability and amphiphilicity of the 17-mer peptide, preserving the residues for potential T-cell receptor (TcR) binding properties. According to the TASP concept, these helical segments were covalently attached to a cyclic template molecule designed for the induction of a four-helix-bundle topology of the assembled peptide blocks. After extensive HPLC purification, stepwise solid-phase synthesis resulted in a TASP molecule of high chemical purity as demonstrated by analytical HPLC, mass spectrometry, and amino acid analysis. CD spectroscopic investigations are consistent with the onset of a partial alpha-helical conformation in aqueous buffer as well as in TFE. Antibodies raised directly against this four-alpha-helix bundle TASP molecule (without prior conjugation to a carrier molecule) were detected by ELISA. Flow cytometry studies showed that these antibodies recognize the native MHC class I molecule on the surface of HLA-A2-positive cells. The results indicate that the TASP approach represents a versatile tool for mimicking conformational epitopes.

Amino Acid Sequence↗
PubMed1992

Abdominal surgery-induced delayed gastric emptying in rats: role of CRF and sensory neurons.

The role of peripheral corticotropin-releasing factor (CRF) and afferent pathways in mediating abdominal surgery-induced delayed gastric emptying was investigated using an intravenous injection of the competitive CRF antagonist alpha-helical CRF9-41 and capsaicin pretreatment in adult fasted rats. Gastric emptying of a non-caloric solution was measured by the phenol red method. Intravenous CRF (0.13 nmol) inhibited by 65% gastric emptying. CRF effect was completely reversed by simultaneous intravenous injection of alpha-helical CRF9-41 (13 nmol). Abdominal surgery under enflurane anesthesia inhibited gastric emptying by 60% measured 3 h postoperatively. Enflurane anesthesia alone had no effect on gastric emptying 3 h after exposure. Abdominal surgery-induced delay of gastric emptying was reversed by 46, 60, and 100% by capsaicin pretreatment (125 mg/kg, -3 wk), intravenous injection of alpha-helical CRF9-41 (13 nmol), and combined pretreatments with capsaicin and CRF antagonist, respectively. Capsaicin and alpha-helical CRF9-41 given alone or in combination did not influence gastric emptying. These results indicate that peripheral CRF receptors along with capsaicin-sensitive afferent neurons contribute to somatovisceral stress (abdominal surgery)-induced delayed gastric emptying.

Abdomen↗
PubMed1992

Isolation and characterization of hypothalamic growth-hormone releasing factor from common carp, Cyprinus carpio.

A growth hormone-releasing factor (GRF)-like peptide was isolated from the hypothalamus of common carp, Cyprinus carpio, by acid extraction, gel filtration chromatography, immunoaffinity chromatography using antiserum directed against rat GRF, and multiple steps of HPLC using octadecyl columns. Based on Edman degradation and peptide mapping, this teleost GRF was established to be a 45-residue peptide with the following primary structure: His-Ala-Asp-Gly-Met-Phe-Asn-Lys-Ala-Tyr-Arg-Lys-Ala-Leu-Gly-Gln-Leu-Ser- Ala-Arg - Lys-Tyr-Leu-His-Thr-Leu-Met-Ala-Lys-Arg-Val-Gly-Gly-Gly-Ser-Met-Ile-Glu- Asp-Asp-Asn-Glu-Pro-Leu-Ser. Carp GRF is closely related structurally to peptides of the glucagon-secretin superfamily, and more particularly to mammalian vasoactive intestinal peptide (VIP) precursors and the N-terminal portion of mammalian GRFs. A synthetic replicate of this peptide is highly potent [50% effective dose (ED50) approximately 0.08 nM] in stimulating GH release from cultured goldfish pituitary glands and in elevating serum GH levels 30 min after injection (0.1 micrograms/g) in goldfish.

Amino Acid Sequence↗