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J J Montagne

Publications and source records attributed to J J Montagne.

15 recordsLinked to original sources
PubMed1999

Cloning of thyrotropin-releasing hormone precursor and receptor in rat thymus, adrenal gland, and testis.

TRH is a hypophysiotropic peptide that acts mainly via the hypothalamic-pituitary-thyroid axis, but TRH immunoreactivity is also detected in several peripheral tissues. PCR with two pairs of primers enabling amplification of three fragments of TRH complementary DNA (cDNA) was used to demonstrate local production of TRH. Products of the expected size were detected in the testis, adrenal gland, lymphoid organs, thymus, and spleen. The amplified cDNA fragments were cloned and sequenced to show that the TRH gene is expressed in the thymus, spleen, and adrenal gland. Competitive RT-PCR showed that the TRH messenger RNA content of the testis was about one third that of the hypothalamus, whereas the adrenal gland contained 2% and the thymus 6%. HPLC analysis of thymus and spleen extracts showed small amounts of TRH, with a particular processing pattern of pro-TRH in lymphoid organs. The expression of the TRH receptor gene in peripheral organs was investigated to determine whether TRH had an autocrine or a paracrine action. cDNA fragments that encompassed the coding region of the receptor were identified in the testis, adrenal gland and thymus. No signal was detected in the spleen. These findings indicate that TRH may have a biological activity in extrapituitary organs and may act locally in the testis, adrenal gland, and thymus.

Adrenal Glands↗
PubMed1997

Specific binding sites for rat prepro-TRH-(160-169) on C6 glioma and BN1010 clonal neural cells.

A connecting decapeptide corresponding to rat prepro-TRH-(160-169) (Ps4) displays several biological activities that are related or unrelated to TRH. We have previously characterized pituitary binding sites for this connecting peptide and elucidated structural determinants for high peptide binding affinity. In the current study, a series of cell lines was screened for the presence of specific binding sites with a highly potent derivative of Ps4, the monoiodinated radioligand [125I-Tyr0]Ps4. Neuroblastoma x glioma hybrid NG108-15, glioma C6 and neuroblastoma BN1010 cell lines were found to have high-affinity [125I-Tyr0]Ps4 binding sites containing 600, 9700 and 130000 sites/cell, respectively. The specific binding of [125I-Tyr0]Ps4 was rapid, time-dependent, reversible and proportional to the amount of C6 and BN1010 membrane preparation. Furthermore, Scatchard or Hill analysis revealed that [125I-Tyr0]Ps4 was bound by a single population of non-interacting sites with dissociation constants in the subnanomolar range. Competition studies made with Ps4 analogues indicated that [125I-Tyr0]Ps4 binding sites on C6 and BN1010 cells were similar to those previously described on rat pituitary membranes. It is concluded that C6 and BN1010 cells are suited for studies on the intracellular events following binding of the Ps4 and for the molecular characterization of the Ps4 binding sites.

Animals↗
PubMed1997

Thyrotropin-releasing hormone immunoreactivity in rat adrenal tissue is localized in mast cells.

Pro-thyrotropin-releasing hormone (pro-TRH) has been shown to be present throughout the central nervous system and in several peripheral tissues. In adrenals, TRH immunoreactivity has been reported but not characterized. We show here that two rat pro-TRH-derived peptides, TRH and prepro-TRH[160-169] (Ps4), were detected in extracts of rat adrenal glands by enzyme immunoassay. Endogenous TRH and Ps4 were purified by gel exclusion chromatography and reverse-phase HPLC. Structural identification of each peptide was achieved by chromatographic comparison with synthetic standards. By using the indirect immunofluorescence technique, TRH-immunoreactive cell bodies were found rather widely scattered outside the adrenal, in the brown adipose tissue in which the gland is embedded. These immunofluorescent cells have the typical appearance of mast cells and are metachromatic after histological staining with acidic Toluidine Blue. Our findings suggest that pro-TRH-derived peptides exist in rat mast cells.

Adipose Tissue, Brown↗
PubMed1996

Covalent structure, synthesis, and structure-function studies of mesentericin Y 105(37), a defensive peptide from gram-positive bacteria Leuconostoc mesenteroides.

A 37-residue cationic antimicrobial peptide named mesentericin Y 105(37) was purified to homogeneity from cell-free culture supernatant of the Gram-positive bacterium Leuconostoc mesenteroides. The complete amino acid sequence of the peptide, KYYGNGVHCTKSGCSVNWGEAASAGIHRLANGGNGFW, has been established by automated Edman degradation, mass spectrometry, and solid phase synthesis. Mesentericin Y 105(37) contains a single intramolecular disulfide bond that forms a 6-membered ring within the molecule. Mesentericin Y 105(37) was synthesized by the solid phase method. The synthetic replicate was shown to be indistinguishable from the natural peptide with respect to electrophoretic and chromatographic properties, mass spectrometry analysis, automated amino acid sequence determination, and antimicrobial properties. At nanomolar concentrations, synthetic mesentericin Y 105(37) is active against Gram+ bacteria in the genera Lactobacillus and Carnobacterium. Most interestingly, the peptide is inhibitory to the growth of the food-borne pathogen Listeria. CD spectra of mesentericin Y 105(37) in low polarity medium, which mimic the lipophilicity of the membrane of target organisms, indicated 30-40% alpha-helical conformation, and predictions of secondary structure suggested that the peptide can be configured as an amphipathic helix spanning over residues 17-31. To reveal the molecular basis of the specificity of mesentericin Y 105(37) targetting and mode of action, NH2- or COOH-terminally truncated analogs together with point-substituted analogs were synthesized and evaluated for their ability to inhibit the growth of Listeria ivanovii. In sharp contrast with broad spectrum alpha-helical antimicrobial peptides from vertebrate animals, which can be shortened to 14-18 residues without deleterious effect on potency, molecular elements responsible for anti-Listeria activity of mesentericin Y 105(37) are to be traced at once to the NH2-terminal tripeptide KYY, the disulfide bridge, the putative alpha-helical domain 17-31, and the COOH-terminal tryptophan residue of the molecule. It is proposed that the amphipathic helical domain of the peptide interacts with lipid bilayers, leading subsequently to alteration of the membrane functions, whereas residues 1-14 form part of a recognition structure for a membrane-bound receptor, which may be critical for peptide targetting. Because mesentericin Y 105(37) is easy to synthesize at low cost, it may represent a useful and tractable tool as a starting point for the design of more potent analogs that may be of potential applicability in foods preservation.

Amino Acid Sequence↗
PubMed1996

Identification and cellular localization of thyrotropin-releasing hormone-related peptides in rat testis.

Endogenous TRH-like products were analyzed in rat testis using a TRH enzyme immunoassay coupled to molecular sieve filtration and HPLC identification. Of the three immunoreactive peptides detected, the two major forms exhibited the same chromatographic properties as synthetic TRH and pGlu-Phe-Pro-NH2. These peptides accounted respectively for 33% and 54% of the total TRH immunoreactivity in the testis. In rat serum, HPLC analysis showed the presence of only one immunoreactive peak with a retention time similar to that of authentic TRH. Ethylene dimethanesulfonate treatment of adult rat was used to assess the effect of Leydig cell destruction on the TRH immunoreactivity content. The concentration of the three TRH immunoreactive peptides fell gradually after treatment and reached a minimum at day 21, where a marked decrease (98%) was observed. At day 41, the regeneration of Leydig cells was achieved, as shown by histochemistry and measurements of serum testosterone and testicular weight. However, no restoration of the TRH immunoreactive content was achieved, the three TRH-related peptides being only detectable in trace amount. On the other hand, no significant change in hypothalamic levels of TRH was observed at any treatment time, indicating that hypothalamic TRH biosynthesis was not influenced by testosterone. By using the indirect immunofluorescence technique, TRH immunoreactive cells were found in the interstitial space of the testis. These results suggest that Leydig cells are the only source of authentic TRH and TRH-like peptides in the rat testis. A paracrine, or autocrine role for these products is suggested.

Animals↗
PubMed1994

Distribution of TRH-potentiating peptide (Ps4) and its receptors in rat brain and peripheral tissues.

TRH-potentiating peptide (Ps4) is a peptide originally isolated from bovine hypothalamus, which corresponds to the deduced rat prepro-TRH-(160-169) sequence. We have examined the distribution of Ps4 and [125I-Tyr0]Ps4 binding sites in various rat tissues. Very high specific binding of [125I-Tyr0]Ps4 was observed in pituitary membranes. Almost identical relative magnitude of binding was found in spinal cord, hypothalamus, hippocampus and olfactory lobe. In addition, a high density of Ps4 binding sites was demonstrated in urogenital organs. The observed distribution pattern of Ps4 in brain tissues was similar to that reported for TRH, with high concentrations in the hypothalamus and significant amount in spinal cord and olfactory lobe. These findings support the hypothesis that the TRH-potentiating peptide could act as neuromodulator or neurotransmitter in the central nervous system (CNS). In peripheral tissues, very low concentrations of Ps4 were detected except in testis which seems to be the major locus for Ps4 biosynthesis. This study demonstrated for the first time that TRH-potentiating peptide and its receptors are widely distributed in the CNS and peripheral tissues.

Animals↗
PubMed1994

Molecular cloning, sequencing, and functional expression of a cDNA encoding human coproporphyrinogen oxidase.

Coproporphyrinogen oxidase (EC 1.3.3.3) catalyzes the sixth step in the heme biosynthetic pathway, the oxidation of coproporphyrinogen III to protoporphyrinogen IX. The activity of this enzyme is deficient in the disease hereditary coproporphyria. The sequence of the cDNA and predicted amino acid sequence of the human coproporphyrinogen oxidase are presented. The human protein sequence contains a region completely homologous to that we obtained by sequencing an 11-amino acid peptide fragment from purified murine liver coproporphyrinogen oxidase. Results of Southern blotting were consistent with the presence of a single human coproporphyrinogen oxidase gene, and Northern blotting demonstrated one transcript of similar size in erythroid and nonerythroid cell lines. Expression of the cDNA coding for the putative mature human coproporphyrinogen oxidase in Escherichia coli resulted in a 17-fold increase in coproporphyrinogen activity over endogenous activity.

Amino Acid Sequence↗
PubMed1994

Analysis of structural requirements for TRH-potentiating peptide receptor binding by analogue design.

Previous studies established that the [125I-Tyr0]Ps4 derivative of TRH-potentiating peptide (Ps4), Ser-Phe-Pro-Trp-Met-Glu-Ser-Asp-Val-Thr, displays high affinity and selectivity for an orphan membrane receptor in rat anterior pituitary. To identify the sites in Ps4 that determine receptor binding affinity, we have synthesized and screened a panel of 15 single-point substituted analogues for their ability to displace bound [125I-Tyr0]Ps4. The affinity of [Tyr0]Ps4 for rat anterior pituitary membranes [inhibitory constant (Ki), approximately 5 nM] was drastically reduced by the substitution of either Tyr0 with Gly or Asp8 with Asn (Ki approximately 95 and approximately 51 nM, respectively). Deamination of [Tyr0]Ps4 also sharply reduced affinity (Ki approximately 1100 nM). In contrast, Ser1-->Ala, Pro3-->Ala and Thr10-->Val substitutions led to analogues showing a tenfold increase in binding affinity relative to the parent peptide. The change of Phe2-->Leu, Trp4-->Ala, Glu6-->Gln, Val9-->Thr and carboxamidation of Thr10 had no effect on binding affinity. The data suggest that substitutions of the amino-terminal group, Asp8 and Tyr0, have a marked effect on the ability of [Tyr0]Ps4 to compete with [125I-Tyr0]Ps4 for binding to TRH-potentiating peptide pituitary receptor.

Amino Acid Sequence↗
PubMed1994

Modulation of the biological activity of thyrotropin-releasing hormone by alternate processing of pro-TRH.

Thyrotropin-releasing hormone prohormone contains multiple copies of TRH linked together by connecting sequences. Like other plurifunctional prohormone proteins, pro-TRH undergoes differential proteolytic processing in various tissues to generate, beside authentic TRH, several other novel peptides corresponding to C-terminally extended forms of TRH and connecting fragments. The pro-TRH connecting peptides are, together with TRH, predominant storage forms of TRH-precursor related peptides in the rat hypothalamus. Connecting peptides are co-localized with TRH in the median eminence nerve endings and co-released through a mechanism involving voltage-operated Ca2+ channels. The connecting peptide Ps4 is involved in potentiation of the action of TRH on thyrotropin hormone release by pituitary in vitro and in vivo through interactions with a specific pituitary cell receptor coupled to dihydropyridine and omega-connotoxin sensitive Ca2+ channels of the L-type. It also causes dose-dependent increases in the steady state levels of mRNAs of TSH and prolactin through stimulation of the respective gene promoter activities. These findings indicate that Ps4 and TRH, two peptides which originate from a single multifunctional biosynthetic precursor, can function on the same target tissues in a coordinate manner to promote hormonal secretion. This suggests that differential processing of the TRH prohormone may have the potential to modulate the biological activities of TRH.

Amino Acid Sequence↗
PubMed1993

New mechanism-based inactivators of trypsin-like proteinases. Selective inactivation of urokinase by functionalized cyclopeptides incorporating a sulfoniomethyl-substituted m-aminobenzoic acid residue.

In order to obtain selective suicide substrates of trypsin-like proteases including plasminogen activators, plasmin, and thrombin, a series of cyclopeptides cyclo[Arg or Lys-aB(CH2X)-Gly4], in which a substituted o- or m-aminobenzoyl group constitutes a latent electrophile, have been prepared. Treatment of the corresponding phenyl ethers cyclo[P1-aB(CH2OC6H5)-Gly4] with HBr/HOAc or R1R2S/TFA gives the bromides (X = Br) or the sulfonium salts (X = +SR1R2 with R1 = R2 = Me or R1 = Me and R2 = C6H5), respectively. These water-soluble cyclopeptides behave as time-dependent inhibitors of bovine trypsin and human urokinase (u-PA) but have no effect on tissue plasminogen activator (t-PA) and no or poor effect on plasmin and thrombin. The compounds containing a m-aminobenzoic acid residue are more efficient inactivators than their anthranilic analogues. The kinetic criteria expected for a suicide inhibition are met. A mechanism of inhibition involving the formation of a quinonimmonium methide intermediate is proposed. The activity of the inhibitors is very sensitive to the nature of the X benzylic substituent. An increased efficiency for the inactivation of human urokinase is observed with the sulfonium salts. The selectivity of the inactivation of u-PA compared to t-PA could be of therapeutical significance in controlling cell proliferation and invasion.

Aminobenzoates↗
PubMed1992

Isolation and amino acid sequence of the TRH-potentiating peptide from bovine hypothalamus.

A neuropeptide termed TRH-potentiating peptide, which potentiates TRH-evoked thyrotropin secretion by antehypophysis in vitro, was isolated from an acetonic powder of bovine hypothalamus. The peptide was purified to homogeneity by a 3-step protocol involving molecular sieve filtration, ion-exchange chromatography and reverse phase high performance liquid chromatography. The complete amino acid sequence of the decapeptide was determined as Ser-Phe-Pro-Trp-Met-Glu-Ser-Asp-Val-Thr by automated Edman degradation with a solid-phase sequencer. Bovine TRH-potentiating peptide is structurally identical to Ps4, a decapeptide which was deduced from the cDNA encoding the rat TRH precursor. This study provides for the first time a direct chemical evidence for the existence of non-TRH peptides originating from posttranslational processing of the TRH precursor in vivo.

Amino Acid Sequence↗
PubMed1991

Functionalized N-aryl azetidinones as novel mechanism-based inhibitors of neutrophil elastase.

A functionalized N-aryl azetidinone has been shown to inactivate human leukocyte elastase (HLE) and porcine pancreatic elastase (PPE) by an enzyme-mediated process. The inactivation is characterized by the following kinetic constants at pH 8.0 and 37 degrees C: kinact = 0.035 s-1, KI = 1.2 x 10(-4) M for HLE, 0.08 s-1 and 2.7 x 10(-4) M for PPE, respectively. Two parent molecules devoid of the latent leaving group failed to inactivate HLE and PPE and behaved as substrates of these enzymes. A suicide mechanism is postulated involving the formation of an acyl-enzyme and the simultaneous unmasking of a latent quinonimmonium methide ion which irreversibly reacts with an active site nucleophile. Moreover, the inhibitor is still effective at inhibiting elastase preabsorbed onto elastin.

Animals↗
PubMed1990

Evidence for Pro-dermorphin processing products in rat tissues.

Dermorphin (Tyr-D.Ala-Phe-Gly-Tyr-Pro-Ser-NH2) and dermenkephalin (Tyr-D.Met-Phe-His-Leu-Met-Asp-NH2), two powerful opioid peptides issued from a common biosynthetic precursor, were recently isolated from the skin of the frog phyllomedusa sauvagii. Since many amphibian's skin secretory peptides or their homologues are found in the mammalian central nervous system and gastrointestinal tract, dermorphin and dermenkephalin may have counterparts in mammals. We have prepared antibodies directed against dermorphin, dermenkephalin and the spacer sequence lying between them in the progenitor form and developed sensitive enzyme immunoassays that detect under the picogram level to verify the occurrence of these peptides in rat tissues. Immunocytochemical studies of rat brain sections revealed a similar distribution of immunoreactivities both at the regional and the cellular levels when either one of these three anti-sera was used. Characterization of the immunoreactive peptides was achieved with molecular sieve filtration followed with Reverse Phase High Performance Liquid Chromatography of various rat tissues extracts. Identification was achieved by immunological analysis and chromatographic comparison with synthetic peptides. Immunoreactive materials corresponding to dermorphin, dermenkephalin and the spacer peptide were detected in either brain, stomach or intestine, indicating processing of the dermorphin precursor in these tissues. Immunoreactive species of higher Mr were also detected in all three tissues and may represent extended forms or homologous peptides.

Amino Acid Sequence↗
PubMed1989

Biological effects of asbestos fibres on rat lung maintained in vitro.

The in vitro systems used to determine whether asbestos acts as an initiator or as a promoter have failed to give definitive answers. We studied the effect of chrysotile and crocidolite in an initiation-promotion model on the Fischer rat embryo lung. Two assay systems were used in succession: organ culture of the lung cultured for 24 days and epithelial cell culture derived from treated or untreated explants cultured for 25 passages. Apart from the control groups, three major groups were analysed: (1) fibres with complete carcinogenic potency: explants and/or cells treated with fibres alone; (2) fibres with initiating potency; short treatment with fibres, followed by treatment with the classical promoter TPA; (3) fibres with promoting potency: short benzo[a]pyrene treatment followed by treatment with the fibres. In organ culture, fibres alone induce only cytotoxic lesions; in the 'fibres with promoting potency' group, precancerous lesions were observed. In epithelial cell culture, several transformation criteria are analysed. Our results with the cell system confirm that fibres act as a promoter, but also as a complete carcinogen. However, for equal doses, crocidolite needs a longer treatment time than chrysotile. These different assays failed to demonstrate any initiating activity of the fibres. The use of organ and cell culture in succession makes it possible to demonstrate the in vitro promoting effect of chrysotile and crocidolite.

Animals↗