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See detailImportin-8 could cause CAE/JME by delaying early neuroblast migration
Nganou, Gerry ULiege; Tanaka, Miyabi; Coumans, Bernard ULiege et al

Poster (2017, April 27)

Abstract : Childhood Absence Evolving to Juvenile Myoclonic Epilepsy (CAE/JME) is an uncommon form of genetic generalized epilepsy that appears as absence in childhood and evolves into generalized ... [more ▼]

Abstract : Childhood Absence Evolving to Juvenile Myoclonic Epilepsy (CAE/JME) is an uncommon form of genetic generalized epilepsy that appears as absence in childhood and evolves into generalized tonic–clonic seizures with myoclonic jerks during adolescence. In some family of patients affected by CAE/JME, mutations have been observed in the gene encoding for the transport protein importin-8 (IPO8). IPO8 could be at the origin of CAE/JME via its role in the transport of its targets (like Ago-2, Smad4, c-Jun). RT-qPCR has shown that IPO8 mRNA is expressed at all ages with no big difference in expression level. Using ISH, a clear expression of mIPO8 mRNA was observed in the sub-ventricular/ventricular zone (SVZ/VZ), the cortical plate (CP) and the ganglionic eminences (GE) of developing brain at E14. Both SVZ/VZ and GE are the “neurogenic niches” that generate glutamatergic and GABAergic neurons respectively. The implication of IPO8 in the generation of “glutamatergic neurons” was investigated by In Utero electroporation (IUE) and MGE Electroporation. Using shRNA, we observed that after 3 days, “glutamatergic neuroblasts” do not reach the CP in contrast to the control condition. This effect can be rescued by the co-expression of a form of IPO8 that is resistant to the shRNA. When overexpressing the pathological forms of hIPO8, but not a variant, migration of “glutamatergic neuroblasts” was also impaired. However, when the observation is made later, i.e. at P5, we observed that the neuroblasts finally reach their correct layer in the cortex, suggesting IPO8 only delayed but not blocked migration. Moreover, shRNA against IPO8 mRNA lead to alteration of interneurons (GABAergic neurons) migration same to overexpression of one mutated form of IPO8. Conclusion : IPO8 is expressed in mouse brain during development. It shows a clear expression during embryogenesis in the “neurogenic niches”. Moreover, IPO8 modulates neuroblasts (radial and tangential) migration in the developing brain. So, abnormal brain development due to IPO8 mutations could be at the origin of CAE/JME. [less ▲]

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See detailThiamine and benfotiamine prevent stress-induced suppression of hippocampal neurogenesis in mice exposed to predation without affecting brain thiamine diphosphate levels
Vignisse, Julie ULiege; Sambon, Margaux ULiege; Gorlova, Anna et al

in Molecular and Cellular Neuroscience (2017), 82

Thiamine is essential for normal brain function and its deficiency causes metabolic impairment, specific lesions, oxidative damage and reduced adult hippocampal neurogenesis (AHN). Thiamine precursors ... [more ▼]

Thiamine is essential for normal brain function and its deficiency causes metabolic impairment, specific lesions, oxidative damage and reduced adult hippocampal neurogenesis (AHN). Thiamine precursors with increased bioavailability, especially benfotiamine, exert neuroprotective effects not only for thiamine deficiency (TD), but also in mouse models of neurodegeneration. As it is known that AHN is impaired by stress in rodents, we exposed C57BL6/J mice to predator stress for 5 consecutive nights and studied the proliferation (number of Ki67-positive cells) and survival (number of BrdU-positive cells) of newborn immature neurons in the subgranular zone of the dentate gyrus. In stressed mice, the number of Ki67- and BrdU-positive cells was reduced compared to non-stressed animals. This reduction was prevented when the mice were treated (200 mg/kg/day in drinking water for 20 days) with thiamine or benfotiamine, that were recently found to prevent stress-induced behavioral changes and glycogen synthase kinase-3β (GSK-3β) upregulation in the CNS. Moreover, we show that thiamine and benfotiamine counteract stress-induced bodyweight loss and suppress stress-induced anxiety-like behavior. Both treatments induced a modest increase in the brain content of free thiamine while the level of thiamine diphosphate (ThDP) remained unchanged, suggesting that the beneficial effects observed are not linked to the role of this coenzyme in energy metabolism. Predator stress increased hippocampal protein carbonylation, an indicator of oxidative stress. This effect was antagonized by both thiamine and benfotiamine. Moreover, using cultured mouse neuroblastoma cells, we show that in particular benfotiamine protects against paraquat-induced oxidative stress. We therefore hypothesize that thiamine compounds may act by boosting anti-oxidant cellular defenses, by a mechanism that still remains to be unveiled. Our study demonstrates, for the first time, that thiamine and benfotiamine prevent stress-induced inhibition of hippocampal neurogenesis and accompanying physiological changes. The present data suggest that thiamine precursors with high bioavailability might be useful as a complementary therapy in several neuropsychiatric disorders. [less ▲]

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See detailImplication of Importin-8 in mouse brain development
Nganou, Gerry ULiege; Coumans, Bernard ULiege; De Nijs, Laurence et al

Poster (2016, December 06)

Importin-8 (IPO8) is a protein that regulates the nucleocytoplasmic transport of some proteins important for cerebral development (Ago2, c-Jun, Smad4). In HEK cells, IPO8 seems to not plays others roles ... [more ▼]

Importin-8 (IPO8) is a protein that regulates the nucleocytoplasmic transport of some proteins important for cerebral development (Ago2, c-Jun, Smad4). In HEK cells, IPO8 seems to not plays others roles (like mitotic spindle formation, primary cilium transport). In situ hybridization performed in mouse brains slices shows that IPO8 is already expressed at E14. Moreover, inhibition of its action (IPO8) in embryonic mouse brain leads to impairment of neuroblast migration to upper cortical layers. IPO8 could then be at the origin of some pathologies with neuronal migration deficit. [less ▲]

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See detailImportin-8 mutations could cause Jeavons Syndrome, CAE and JME by altering early neuroblast migration
Nganou, Gerry ULiege; Tanaka, Miyabi; Coumans, Bernard ULiege et al

Poster (2016, December)

Jeavons syndrome (JS) is an uncommon form of juvenile epilepsy. In some family affected by JS, the gene of importin-8 (IPO8) a member of karyopherin superfamily of proteins, has been found mutated ... [more ▼]

Jeavons syndrome (JS) is an uncommon form of juvenile epilepsy. In some family affected by JS, the gene of importin-8 (IPO8) a member of karyopherin superfamily of proteins, has been found mutated. Karyopherin are known to regulate nucleo-cytoplasmic transport of many proteins. IPO8, a member of the β-karyopherin sub-family, is reported to control the transport Ago-2, c-Jun and Smad-4 for example, three proteins important for brain development. Here, we show that IPO8 is well expressed in mouse brain at embryonic stage. Moreover inhibition of IPO8 mRNA by shRNA in-utéro-electroporation (IUE), impairs early neuroblast migration as weel as IUE overexpression of mutated form of human IPO8. So, abnormal brain development due to IPO8 mutations could be at the origin of Jeavons Syndrome, Chilhood abscence evolving (CAE) and/or juvenile myoclonic epilepsy (JME). [less ▲]

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See detailImplication of Importin-8 in mouse brain development
Nganou, Gerry ULiege; Denijs, Laurence; Lakaye, Bernard ULiege et al

Poster (2016, October 08)

Regulation of nucleocytoplasmic transport of proteins by the karyopherin superfamily is critical for cell physiology as it controls mainy fundamental processes such as division, differenciation, migration ... [more ▼]

Regulation of nucleocytoplasmic transport of proteins by the karyopherin superfamily is critical for cell physiology as it controls mainy fundamental processes such as division, differenciation, migration, adaptation to external environment etc. Beside this fundamental role, ß1 and ß2 members of this superfamily are also implicated in mitosis and ciliary entry respectively. Importin-8 (IPO8), a member of the β-karyopherin family, is reported to control the transport Ago-2, c-Jun and Smad-4 for example, three proteins important for brain development. First, we have verify the subcellular localisation of IPO8 in HEK and hTert cells. No colocalisation with either the mitotic spindle or the primary cilia could be observed. So it seems IPO8 only plays a role in nuclear transport of proteins. Then we have assessed the expression of IPO8 in mouse brain by In Situ Hybridization at various embryonic (E12, E14, E18) and post natal age (P5, P60). A strong expression was observed during embryonic stages, and especially in the ventricular zone and the cortical plate of the cerebral cortex and the ganglionic eminences both at E14. Therefore, the implication of IPO8 in the radial migration has been assessed by in utero electroporation of shRNA at E14. Three days after IUE, we observed that neuroblast accumulates in the Intermediate zone (IZ) and do not reach the cortical plate (CP) in constrast to the control condition. This effect can be corrected by coexpressing a form of IPO8 that is not targeted by the shRNA, demonstrating the specificity of the effect. In conclusion, regarding its role in transport, IPO8 could modulate neurons migration in the developing brain and could be also at the origin of some diseases associated with neurons migration defects. [less ▲]

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See detailMyoclonin-1 modulates the post-translational modification of microtubules
Medard, Laurie ULiege; Coumans, Bernard ULiege; Lakaye, Bernard ULiege et al

Poster (2016, June)

Juvenile myoclonic epilepsy is one of the most common forms of generalized genetic epilepsy. Genetic studies have shown that heterozygous mutations in Myoclonin1 are responsible for 3-9% of clinical cases ... [more ▼]

Juvenile myoclonic epilepsy is one of the most common forms of generalized genetic epilepsy. Genetic studies have shown that heterozygous mutations in Myoclonin1 are responsible for 3-9% of clinical cases worldwide. This protein contains three DM10 domains of unknown function and an EF-hand domain. We have previously demonstrated that Myoclonin1 is a microtubule-associated protein involved in cell division and radial migration during neocortex development. In cells, this protein co-localized with specific structures rich in microtubules (MTs) such as the centrosome, the poles of the mitotic spindle or the motile cilia but not with cytoplasmic MTs. This suggests post-translational modifications (PTM) of MTs may be important for the interaction between Myoclonin1 and MTs. We have co-express the different enzymes catalyzing PTM of MTs with Myoclonin1 in U2OS cell line. With one of these enzymes, we observed a strong increase in PTM in the presence of Myoclonin-1. This suggests that Myoclonin1 may interact with and modulate the activity of this enzyme. By using luciferase complementation assay and pull down experiments, we could demonstrate that it is indeed the case. Interestingly, the effect is observed even when a DM10 domain alone is co-expressed with the enzyme, suggesting for the first time a role for this domain. In conclusion our data suggest myoclonin-1 modulates specific PTM of MTs. This is of prime importance for microtubule dynamic and notably for neuroblast precursor migration during neocortex development. This could be the mechanism that explains why pathological forms of myoclonin-1 affect brain development. [less ▲]

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See detailEFHC1/Myoclonin-1 modulates the post-translational modification of microtubules
Medard, Laurie ULiege; Godin, Juliette; Coumans, Bernard ULiege et al

Poster (2015, December)

Rationale: Juvenile myoclonic epilepsies (JME) are one of the most common forms of genetic generalized epilepsy. Genetic studies have shown that heterozygous mutations in EFHC1/Myoclonin1 are responsible ... [more ▼]

Rationale: Juvenile myoclonic epilepsies (JME) are one of the most common forms of genetic generalized epilepsy. Genetic studies have shown that heterozygous mutations in EFHC1/Myoclonin1 are responsible for 3-22% of JME cases worldwide. The Myoclonin1 protein contains three DM10 domains of unknown function and an EF-hand domain. We have previously demonstrated that Myoclonin1 is a microtubule-associated protein involved in cell division and radial migration during neocortex development. In cells, this protein co-localized with specific structures rich in microtubules (MTs) such as the centrosome, the poles of the mitotic spindle or the motile cilia but not with cytoplasmic MTs. This suggests post-translational modifications (PTM) of MTs may be important for the interaction between Myoclonin1 and MTs Methods: We co-expressed the different enzymes catalyzing PTM of MTs with Myoclonin1 in U2OS cell line, and then performed immunocytochemistry and western blot analysis. We next performed pulldown and luciferase complementation assays to test protein interaction Results: With one of these enzymes, we observed a strong increase in PTM in the presence of Myoclonin- 1.Interestingly, the effect is observed even when a DM10 domain alone is co-expressed with the enzyme, suggesting for the first time a role for this domain. This suggests that Myoclonin1 may interact with and modulate the activity of this enzyme. By using luciferase complementation assay and pull down experiments, we could demonstrate that both proteins interact. Conclusions: Our data suggest Myoclonin-1 modulates specific PTM of MTs. This is of prime importance for microtubule dynamic and notably for neuroblast precursor migration during neocortex development. This could be the mechanism that explains why pathological forms of myoclonin-1 may affect brain development. [less ▲]

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See detailMyoclonin 1 modulates the post-translational modification of microtubules
Medard, Laurie ULiege; Lakaye, Bernard ULiege; Godin, Juliette ULiege et al

Poster (2015)

Juvenile myoclonic epilepsy is one of the most common forms of generalized genetic epilepsy. Genetic studies have shown that heterozygous mutations in Myoclonin1 are responsible for 3-9% of clinical cases ... [more ▼]

Juvenile myoclonic epilepsy is one of the most common forms of generalized genetic epilepsy. Genetic studies have shown that heterozygous mutations in Myoclonin1 are responsible for 3-9% of clinical cases worldwide. This protein contains three DM10 domains of unknown function and an EF-hand domain. We have previously demonstrated that Myoclonin1 is a microtubule-associated protein involved in cell division and radial migration during neocortex development. In cells, this protein co-localized with specific structures rich in microtubules (MTs) such as the centrosome, the poles of the mitotic spindle or the motile cilia but not with cytoplasmic MTs. This suggests post-translational modifications (PTM) of MTs may be important for the interaction between Myoclonin1 and MTs. We have co-express the different enzymes catalyzing PTM of MTs with Myoclonin1 in U2OS cell line. With one of these enzymes, we observed a strong increase in PTM in the presence of Myoclonin-1. This suggests that Myoclonin1 may interact with and modulate the activity of this enzyme. By using luciferase complementation assay and pull down experiments, we could demonstrate that it is indeed the case. Interestingly, the effect is observed even when a DM10 domain alone is co-expressed with the enzyme, suggesting for the first time a role for this domain. In conclusion our data suggest myoclonin-1 modulates specific PTM of MTs. This is of prime importance for microtubule dynamic and notably for neuroblast precursor migration during neocortex development. This could be the mechanism that explains why pathological forms of myoclonin-1 affect brain development. [less ▲]

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See detailMelanin-concentrating hormone and immune function
Lakaye, Bernard ULiege; Coumans, Bernard ULiege; Harray, Sophie ULiege et al

in Peptides (2009), 30

To date,melanin-concentrating hormone (MCH) has been generally considered as peptide acting almost exclusively in the central nervous system. In the present paper, we revise the experimental evidence ... [more ▼]

To date,melanin-concentrating hormone (MCH) has been generally considered as peptide acting almost exclusively in the central nervous system. In the present paper, we revise the experimental evidence, demonstrating that MCH and its receptors are expressed by cells of the immune system and directly influence the response of these cells in some circumstances. This therefore supports the idea that, as with other peptides, MCH could be considered as a modulator of the immune system. Moreover, we suggest that this could have important implications in several immune-mediated disorders and affirm that there is a clear need for further investigation [less ▲]

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See detailEffect of ppMCH derived peptides on PBMC proliferation and cytokine expression
Coumans, Bernard ULiege; Grisar, Thierry ULiege; Nahon, J. L. et al

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 ▲]

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See detailEFHC1, a protein mutated in juvenile myoclonic epilepsy, associates with the mitotic spindle through its N-terminus
de Nijs, Laurence ULiege; Lakaye, Bernard ULiege; Coumans, Bernard ULiege et al

in Experimental Cell Research (2006), 312(15), 2872-2879

A novel gene, EFHC1, mutated in juvenile myoclonic epilepsy (JME) encodes a protein with three DM10 domains of unknown function and one putative EF-hand motif. To study the properties of EFHC1, we ... [more ▼]

A novel gene, EFHC1, mutated in juvenile myoclonic epilepsy (JME) encodes a protein with three DM10 domains of unknown function and one putative EF-hand motif. To study the properties of EFHC1, we expressed EGFP-tagged protein in various cell lines. In interphase cells, the fusion protein was present in the cytoplasm and in the nucleus with specific accumulation at the centrosome. During mitosis EGFP-EFHC1 colocalized with the mitotic spindle, especially at spindle poles and with the midbody during cytokinesis. Using a specific antibody, we demonstrated the same distribution of the endogenous protein. Deletion analyses revealed that the N-terminal region of EFHC1 is crucial for the association with the mitotic spindle and the midbody. Our results suggest that EFHC1 could play an important role during cell division. (c) 2006 Elsevier Inc. All rights reserved. [less ▲]

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See detailSome genetic and biochemical aspects of myoclonus
Grisar, Thierry ULiege; de Nijs, Laurence ULiege; Chanas, G. et al

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 ▲]

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See detailDisrupting the melanin-concentrating hormone receptor 1 in mice leads to cognitive and NMDA response deficit
Grisar, Thierry ULiege; Adamantidis, Antoine ULiege; Thomas, Elizabeth et al

in Journal of the Neurological Sciences (2005, November 15), 238(Suppl. 1), 288

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See detailPig tissues express a catalytically inefficient 25-kDa thiamine triphosphatase: Insight in the catalytic mechanisms of this enzyme
Szyniarowski, Piotr; Lakaye, Bernard ULiege; Czerniecki, Jan ULiege et al

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 ▲]

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See detailDisrupting the melanin-concentrating hormone receptor 1 in mice leads to cognitive deficits and alterations of NMDA receptor function.
Adamantidis, Antoine ULiege; Thomas, Elizabeth; Foidart, Agnès ULiege et al

in European Journal of Neuroscience (2005), 21(10), 2837-44

In order to investigate the physiological properties of the melanin-concentrating hormone (MCH) we have generated and used mice from which the MCH receptor 1 gene was deleted (MCHR1(Neo/Neo) mice ... [more ▼]

In order to investigate the physiological properties of the melanin-concentrating hormone (MCH) we have generated and used mice from which the MCH receptor 1 gene was deleted (MCHR1(Neo/Neo) mice). Complementary experimental approaches were used to investigate alterations in the learning and memory processes of our transgenic model. The ability of the knockout strain to carry out the inhibitory passive avoidance test was found to be considerably impaired although no significant differences were observed in anxiety levels. This impaired cognitive property prompted us to explore modifications in N-methyl D-aspartate (NMDA) responses in the hippocampus. Intracellular recordings of CA1 pyramidal neurons in hippocampal slices from the MCHR1(Neo/Neo) mice revealed significantly decreased NMDA responses. Finally, using in situ hybridization we found a 15% reduction in NMDAR1 subunit in the CA1 region. These results show for the first time a possible role for MCH in the control of the function of the NMDA receptor. [less ▲]

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See detailPromoter characterization of the mouse melanin-concentrating hormone receptor 1
Lakaye, Bernard ULiege; Adamantidis, Antoine ULiege; Coumans, Bernard ULiege et al

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 ▲]

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See detailHuman recombinant thiamine triphosphatase: purification, secondary structure and catalytic properties
Lakaye, Bernard ULiege; Makarchikov, Alexander F; Wins, Pierre et al

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 ▲]

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See detailThiamine triphosphate and thiamine triphosphatase activities: from bacteria to mammals
Makarchikov, Alexander F; Lakaye, Bernard ULiege; Gulyai, I. E. et al

in Cellular and Molecular Life Sciences : CMLS (2003), 60(7), 1477-1488

In most organisms, the main form of thiamine is the coenzyme thiamine diphosphate. Thiamine triphosphate (ThTP) is also found in low amounts in most vertebrate tissues and can phosphorylate certain ... [more ▼]

In most organisms, the main form of thiamine is the coenzyme thiamine diphosphate. Thiamine triphosphate (ThTP) is also found in low amounts in most vertebrate tissues and can phosphorylate certain proteins. Here we show that ThTP exists not only in vertebrates but is present in bacteria, fungi, plants and invertebrates. Unexpectedly, we found that in Escherichia coli as well as in Arabidopsis thaliana, ThTP was synthesized only under particular circumstances such as hypoxia (E. coli) or withering (A. thaliana). In mammalian tissues, ThTP concentrations are regulated by a specific thiamine triphosphatase that we have recently characterized. This enzyme was found only in mammals. In other organisms, ThTP can be hydrolyzed by unspecific phosphohydrolases. The occurrence of ThTP from prokaryotes to mammals suggests that it may have a basic role in cell metabolism or cell signaling. A decreased content may contribute to the symptoms observed during thiamine deficiency. [less ▲]

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See detailHuman immune cells express ppMCH mRNA and functional MCHR1 receptor
Verlaet, Myriam ULiege; Adamantidis, Antoine ULiege; Coumans, Bernard ULiege et al

in FEBS Letters (2002), 527(1-3), 205-210

Melanin-concentrating hormone (MCH) is highly expressed in the brain and modulates feeding behavior. It is also expressed in some peripheral tissues where its role remains unknown. We have investigated ... [more ▼]

Melanin-concentrating hormone (MCH) is highly expressed in the brain and modulates feeding behavior. It is also expressed in some peripheral tissues where its role remains unknown. We have investigated MCH function in human and mouse immune cells. RT-PCR analysis revealed a low expression of prepro-MCH and MCH receptor 1 (MCHR1) but not of MCHR2 transcript in tissular and peripheral blood immune cells. FACS and in vitro assay studies demonstrated that MCHR1 receptor expression on most cell types can trigger, in the presence of MCH, cAMP synthesis and calcium mobilization in peripheral blood mononuclear cells (PBMCs). Moreover, MCH treatment decreases the CD3-stimulated PBMC proliferation in vitro. Accordingly, our data indicate for the first time that MCH and MCHR1 may exert immunomodulatory functions. (C) 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved. [less ▲]

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