Effect of ppMCH derived peptides on PBMC proliferation and cytokine expression

in Regulatory Peptides (2007), 143(1-3), 104-108

The mRNA encoding prepro-Melanin concentrating hormone (ppMCH) is mainly expressed in the central nervous system but has also been detected at lower amount in many peripheral tissues including spleen and ... [more ▼]

The mRNA encoding prepro-Melanin concentrating hormone (ppMCH) is mainly expressed in the central nervous system but has also been detected at lower amount in many peripheral tissues including spleen and thymus. At the peptide level however, several forms of the precursor can be detected in these tissues and are sometimes expressed at similar levels compared to brain. In the present work, we have studied the in vitro action of a wide range of concentration (1 nM to 1 microM) of the different peptides encoded by ppMCH i.e. neuropeptide glycine-glutamic acid (NGE), neuropeptide glutamic acid-isoleucine (NEI), Melanin concentrating hormone (MCH) and the dipeptide NEI-MCH on peripheral blood mononuclear cells (PBMC) proliferation and cytokine production following anti-CD3 stimulation. Among them only MCH decreased PBMC proliferation with a maximal effect of 35% at 100 nM. Moreover as demonstrated by using ELISA, MCH significantly decreases IL-2 production by 25% but not IL-4, INF-gamma or TNF-alpha expression. Interestingly, exogenous IL-2 decreases significantly MCH-mediated inhibition, suggesting that it is an important downstream mediator of MCH action. Finally, we showed that after 7 to 9 days of incubation, MCH also inhibits proliferation of non-stimulated PBMC. Altogether, these data demonstrate that fully mature MCH modulates proliferation of anti-CD3 stimulated PBMC partially through regulation of IL-2 production. [less ▲]

Mice lacking the melanin-concentrating hormone receptor-1 exhibit an atypical psychomotor susceptibility to cocaine and no conditioned cocaine response

in Behavioural Brain Research (2006), 173(1), 94-103

The present study aimed at characterizing the acute and intermittent psychomotor responsiveness to cocaine in mice lacking the MCHR1 receptor, which is thought to modulate the mesocorticolimbic sytem ... [more ▼]

The present study aimed at characterizing the acute and intermittent psychomotor responsiveness to cocaine in mice lacking the MCHR1 receptor, which is thought to modulate the mesocorticolimbic sytem functioning [Smith DG, Tzavara ET, Shaw J, Luecke S, Wade M, Davis R, et al. Mesolimbic dopamine super-sensitivity in melanin-concentrating hormone-1 receptor deficient mice. J Neurosci 2005;25:914-22]. On a first free-drug session, MCHR1-deficient mice exhibited significantly higher levels of locomotor activity elicited by the novelty of the test chambers than their wild-type counterparts. On the following day session, a first injection of 6 or 12mg/kg cocaine induced comparable dose-related psychomotor activations in both genotypes, without significant difference in the relative increase in locomotion. Over the following eight once-daily test sessions, the slight psychomotor increase induced by 6mg/kg was equivalent in both genotypes and constant over the sessions. At 12mg/kg, cocaine induced a clear-cut incremental responsiveness to cocaine in both genotypes on the three first sessions; on the following sessions, only the wild-types displayed an incremental responsiveness until the last session, a sensitized effect that was confirmed for the wild-types but not for the knockouts on a subsequent sensitization test (cocaine challenge). Finally, the knockouts did not exhibit any sign of cocaine-conditioning (saline challenge), contrarily to the wild-types. It is speculated that MCHR1 may contribute to the neurobiological mechanisms of conditioned cocaine-induced psychomotor effects, possibly to those underpinning sensitization, and to a lesser extent to those sub-serving acute pharmacological cocaine action. [less ▲]

Some genetic and biochemical aspects of myoclonus

in Neurophysiologie Clinique = Clinical Neurophysiology (2006), 36(5-6, Sep-Dec), 271-279

Can a gene defect be responsible for the occurrence in an individual, at a particular age, of such a muscle twitch followed by relaxation called: "myoclonus" and defined as sudden, brief, shock-like ... [more ▼]

Can a gene defect be responsible for the occurrence in an individual, at a particular age, of such a muscle twitch followed by relaxation called: "myoclonus" and defined as sudden, brief, shock-like movements? Genetic defects could indeed determine a subsequent cascade of molecular events (caused by abnormal encoded proteins) that would produce new aberrant cellular relationships in a particular area of the CNS leading to re-builded "myoclonogenic" neuronal networks. This can be illustrated reviewing some inherited neurological entities that are characterized by a predominant myoclonic picture and among which a clear gene defect has been identified. In the second part of this chapter, we will also propose a new point of view on how some structural genes could, under certain conditions, when altered, produced idiopathic generalized epilepsy with myoclonic jerks, taking juvenile myoclonic epilepsy (JME) and the myoclonin (EFHC-1) gene as examples. (c) 2007 Elsevier Masson SAS. All rights reserved. [less ▲]

Pig tissues express a catalytically inefficient 25-kDa thiamine triphosphatase: Insight in the catalytic mechanisms of this enzyme

in Biochimica et Biophysica Acta - General Subjects (2005), 1725(1), 93-102

Thiamine triphosphate (ThTP) is found in most organisms and may be an intracellular signal molecule produced in response to stress. We have recently cloned the cDNA coding for a highly specific mammalian ... [more ▼]

Thiamine triphosphate (ThTP) is found in most organisms and may be an intracellular signal molecule produced in response to stress. We have recently cloned the cDNA coding for a highly specific mammalian 25-kDa thiamine triphosphatase. The enzyme was active in all mammalian species studied except pig, although the corresponding mRNA was present. In order to determine whether the very low ThTPase activity in pig tissues is due to the absence of the protein or to a lack of catalytic efficiency, we expressed human and pig ThTPase in E. coli as GST fusion proteins. The purified recombinant pig GST-ThTPase was found to be 2-3 orders of magnitude less active than human GST-ThTPase. Using site-directed mutagenesis, we show that, in particular, the change of Glu85 to lysine is responsible for decreased solubility and catalytic activity of the pig enzyme. Immunohistochemical studies revealed a distribution of the protein in pig brain very similar to the one reported in rodent brain. Thus, our results suggest that a 25-kDa protein homologous to hThTPase but practically devoid of enzyme activity is expressed in pig tissues. This raises the possibility that this protein may play a physiological role other than ThTP hydrolysis. [less ▲]

Promoter characterization of the mouse melanin-concentrating hormone receptor 1

in Biochimica et Biophysica Acta-Gene Structure and Expression (2004), 1678(1), 1-6

The gene encoding the mouse melanin-concentrating hormone receptor 1 was isolated and its structural organization and flanking regions were characterized. The 3' flanking region is marked by the presence ... [more ▼]

The gene encoding the mouse melanin-concentrating hormone receptor 1 was isolated and its structural organization and flanking regions were characterized. The 3' flanking region is marked by the presence of two polyadenylation signals but used with different frequencies. RNase protection and 5' rapid amplification of cDNA ends (RACE) identified multiple transcription initiation sites between -150 and -203 bp upstream of the ATG initiation codon. Functional analysis of deletion mutants reveals a cell independent transcriptional activity localized between nucleotide -305 and -589. The proximal 1.5 kb region does not possess consensus TATA or CAAT boxes but has several consensus sequences for regulatory elements including USF, GATA, AP1, AP4, MyoD, GKLF and Ikaros that could explain the broad expression of the receptor. [less ▲]

Neuronal localization of the 25-kDa specific thiamine triphosphatase in rodent brain

in Neuroscience (2004), 125(4), 833-840

Thiamine triphosphate (ThTP) is found in small amounts in most organisms from bacteria to mammals, but little is known about its physiological role. In vertebrate tissues, ThTP may act as a phosphate ... [more ▼]

Thiamine triphosphate (ThTP) is found in small amounts in most organisms from bacteria to mammals, but little is known about its physiological role. In vertebrate tissues, ThTP may act as a phosphate donor for the phosphorylation of certain proteins; this may be part of a new signal transduction pathway. We have recently characterized a highly specific 25-kDa thiamine triphosphatase (ThTPase) that is expressed in most mammalian tissues. The role of this enzyme may be the control of intracellular concentrations of ThTP. As the latter has been considered to be a neuroactive form of thiamine, we have studied the distribution of ThTPase mRNA and protein in rodent brain using in situ hybridization and immunohistochemistry. With both methods, we found the strongest staining in hippocampal pyramidal neurons, as well as cerebellar granule cells and Purkinje cells. Some interneurons were also labeled and many ThTPase mRNA-positive and immunoreactive cells were distributed throughout cerebral cortical gray matter and the thalamus. White matter was not significantly labeled. ThTPase immunoreactivity seems to be located mainly in the cytoplasm of neuronal perikarya. Immunocytochemical data using dissociated cultured cells from hippocampal and cerebellum showed that the staining was more intense in neurons than in astrocytes. The protein was rather uniformly located in the perikarya and dendrites, suggesting that ThTP and ThTPase may play a general role in neuronal metabolism rather than a specific role in excitability. There was no apparent correlation between ThTPase expression and selective vulnerability of certain brain regions to thiamine deficiency. (C) 2004 IBRO. Published by Elsevier Ltd. All rights reserved. [less ▲]

Human recombinant thiamine triphosphatase: purification, secondary structure and catalytic properties

in International Journal of Biochemistry & Cell Biology (2004), 36(7), 1348-1364

Thiamine triphosphate (ThTP) is found in most living organisms and it may act as a phosphate donor for protein phosphorylation. We have recently cloned the cDNA coding for a highly specific mammalian 25 ... [more ▼]

Thiamine triphosphate (ThTP) is found in most living organisms and it may act as a phosphate donor for protein phosphorylation. We have recently cloned the cDNA coding for a highly specific mammalian 25 kDa thiamine triphosphatase (ThTPase; EC 3.6.1.28). As the enzyme has a high catalytic efficiency and no sequence homology with known phosphohydrolases, it was worth investigating its structure and catalytic properties. For this purpose, we expressed the untagged recombinant human ThTPase (hThTPase) in E. coli, produced the protein on a large scale and purified it to homogeneity. Its kinetic properties were similar to those of the genuine human enzyme, indicating that the recombinant hThTPase is completely functional. Mg2+ ions were required for activity and Ca2+ inhibited the enzyme by competition with Mg2+. With ATP as substrate, the catalytic efficiency was 10(-4)-fold lower than with ThTP, confirming the nearly absolute specificity of the 25 kDa ThTPase for ThTP. The activity was maximum at pH 8.5 and very low at pH 6.0. Zn2+ ions were inhibitory at micromolar concentrations at pH 8.0 but activated at pH 6.0. Kinetic analysis suggests an activator site for Mg2+ and a separate regulatory site for Zn2+. The effects of group-specific reagents such as Woodward's reagent K and diethylpyrocarbonate suggest that at least one carboxyl group in the active site is essential for catalysis, while a positively charged amino group may be involved in substrate binding. The secondary structure of the enzyme, as determined by Fourier-transform infrared spectroscopy, was predominantly beta-sheet and alpha-helix. [less ▲]

A general method for the chemical synthesis of gamma-P-32-labeled or unlabeled nucleoside 5 '-triphosphates and thiamine triphosphate

in Analytical Biochemistry (2003), 322(2), 190-197

Several methods for the chemical synthesis of gamma-P-32-labeled and unlabeled nucleoside 5'-triphosphates and thiamine triphosphate (ThTP) have been described. They often proved unsatisfactory because of ... [more ▼]

Several methods for the chemical synthesis of gamma-P-32-labeled and unlabeled nucleoside 5'-triphosphates and thiamine triphosphate (ThTP) have been described. They often proved unsatisfactory because of low yield, requirement for anhydrous solvents, procedures involving several steps or insufficient specific radioactivity of the labeled triphosphate. In the method described here, all these drawbacks are avoided. The synthesis of [gamma-P-32]TbTP was carried out in one step, using 1,3-dicyclohexyl carbodiimide as condensing agent for thiamine diphosphate and phosphoric acid in a dimethyl sulfoxide/pyridine solvent mixture. Anhydrous solvents were not required and the yield reached 90%. After purification, [gamma-P-32]ThTP had a specific radioactivity of 11 Ci/mmol and was suitable for protein phosphorylation. The method can also be used for the synthesis Of [gamma-P-32]ATP of the desired specific radioactivity. It can easily be applied to the synthesis of unlabeled ThTP or ribo- and deoxyribonucleoside 5'-triphosphates. In the latter case, inexpensive 5'-monophosphate precursors can be used as reactants in a 20-fold excess of phosphoric acid. Deoxyribonucleoside 5'-triphosphates were obtained in 6 h with a yield of at least 70%. After purification, the nucleotides were found to be suitable substrates for Taq polymerase during polymerase chain reaction cycling. Our method can easily be scaled up for industrial synthesis of a variety of labeled and unlabeled triphosphoric derivatives from their mono- or diphosphate precursors. (C) 2003 Elsevier Inc. All rights reserved. [less ▲]

Method for estimation of low outer membrane permeability to beta-lactam antibiotics

in Antimicrobial Agents and Chemotherapy (2002), 46(9), 2901-2907

The outer membrane of gram-negative bacteria plays a major role in beta-lactam resistance as it slows down antibiotic entry into the periplasm and therefore acts in synergy with beta-lactamases and efflux ... [more ▼]

The outer membrane of gram-negative bacteria plays a major role in beta-lactam resistance as it slows down antibiotic entry into the periplasm and therefore acts in synergy with beta-lactamases and efflux systems. Up to now, the quantitative estimation of low outer membrane permeability by the method of Zimmermann and Rosselet was difficult because of the secreted and cell surface-associated beta-lactamases. The method presented here uses the acylation of a highly sensitive periplasmic penicillin-binding protein (PBP) (BlaR-CTD) to assess the rate of beta-lactam penetration into the periplasm. The method is dedicated to measurement of low permeability and is only valid when the diffusion rate through the outer membrane is rate limiting. Cytoplasmic membrane associated PBPs do not interfere since they are acylated after the very sensitive BlaR-CTD. This method was used to measure the permeability of beta-lactamase-deficient strains of Enterobacter cloacae and Enterobacter aerogenes to benzylpenicillin, ampicillin, carbenicillin, cefotaxime, aztreonam, and cephacetrile. Except for that of cephacetrile, the permeability coefficients were equal to or below 10(-7) cm/s. For cephacetrile, carbenicillin, and benzylpenicillin, the outer membrane of E. cloacae was 20 to 60 times less permeable than that of Escherichia coli, whereas for cefotaxime, aztreonam, and ampicillin it was, respectively, 400, 1,000, and 700 times less permeable. The permeability coefficient for aztreonam is the lowest ever measured (P = 3.2 X 10(-9) cm/s). Using these values, the MICs for a beta-lactamase-overproducing strain of E. cloacae were successfully predicted, demonstrating the validity of the method. [less ▲]

Steroid receptor coactivator SRC-1 exhibits high expression in steroid-sensitive brain areas regulating reproductive behaviors in the quail brain.

in Neuroendocrinology (2002), 76(5), 297-315

The steroid receptor coactivator SRC-1 modulates ligand-dependent transactivation of several nuclear receptors, including the receptors for sex steroid hormones. Reducing the expression of SRC-1 by ... [more ▼]

The steroid receptor coactivator SRC-1 modulates ligand-dependent transactivation of several nuclear receptors, including the receptors for sex steroid hormones. Reducing the expression of SRC-1 by injection of specific antisense oligonucleotides markedly inhibits the effects of estrogens of the sexual differentiation of brain and behavior in rats and inhibits the activation of female sexual behavior in adult female rats. SRC-1 thus appears to be involved in both the development and activation of sexual behavior. In the Japanese quail brain, we amplified by RT-PCR a 3,411-bp fragment extending from the HLH domain to the activating domain-2 of the protein. The quail SRC-1 is closely related to the mammalian (m) SRC-1 and contains a high proportion of GC nucleotides (62.5%). Its amino acid sequence presents 70% identity with mammalian SRC-1 and contains the three conserved LXXLL boxes involved in the interaction with nuclear receptors. In both males and females, RT-PCR demonstrates a similarly high level of expression in the telencephalon, diencephalon, optic lobes, brain stem, spinal cord, pituitary, liver, kidney, adrenal gland, heart, lung, gonads and gonoducts. Males express significantly higher levels of SRC-1 in the preoptic area-hypothalamus than females. In both sexes, lower levels of expression are observed in the cerebellum and muscles. In situ hybridization utilizing a mixture of four digoxigenin-labeled oligonucleotides confirms at the cellular level the widespread distribution of SRC-1 mRNA in the brain and a particularly dense expression in steroid-sensitive areas that play a key role in the control of male sexual behavior. These data confirm the presence and describe for the first time the SRC-1 distribution in the brain of an avian species. They confirm its broad, nearly ubiquitous, distribution in the entire body including the brain as could be expected for a coactivator that regulates to the action of many nuclear receptors. However this distribution is heterogeneous in the brain and sexually differentiated in at least some areas. The very dense expression of SRC-1 in limbic and mesencephalic nuclei that are associated with the control of male sexual behavior is consistent with the notion that this coactivator plays a significant role in the activation of this behavior. [less ▲]