Quantitative studies are commonly realised in the biomedical research to compare RNA expression in different experimental or clinical conditions. These quantifications are performed through their comparison to the expression of the housekeeping gene transcripts like glyceraldehyde-3-phosphate dehydrogenase (G3PDH), albumin, actins, tubulins, cyclophilin, hypoxantine phosphoribosyltransferase (HRPT), L32. 28S and 18S rRNAs are also used as internal standards. In this paper, it is recalled that the commonly used internal standards can quantitatively vary in response to various factors. Possible variations are illustrated using three experimental examples. Preferred types of internal standards are then proposed for each of these samples and thereafter the general procedure concerning the choice of an internal standard and the way to manage its used are discussed. © 1999 Elsevier Science B.V. All rights reserved.Keywords: Internal standards; Housekeeping genes; RNase protection; RT-PCR www.elsevier.com/locate/jbiotec Quantitative assays widely use housekeeping gene transcripts as b-actin, glyceraldehyde-3-phosphate dehydrogenase (G3PDH) or L32 whose presumed stable expression allows quantification of other expressions, for example those of cytokines, by comparison to this internal standard. In this paper, a series of in vivo and in vitro models are presented using housekeeping genes showing in certain cases the limits at the use of such internal standards. Different possible methods enabling the management of this problem will be discussed.The study of biological regulations is very often correlated to quantification assays, which can be related to proteins or RNA. This paper will discuss the problem of mRNA quantification.Abbre6iations: G3PDH, glyceraldehyde-3-phosphate dehydrogenase; HPRT, hypoxantine phosphoribosyltransferase; PMA, phorbol 10-myristate 13-acetate.

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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.

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.

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 (200mg/kg/day in drinking water for 20days) 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.