Thierry Grisar

Housekeeping genes as internal standards: use and limits

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 phsophoribosyltransferase (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 uses are discussed.

Thiamine status in humans and content of phosphorylated thiamine derivatives in biopsies and cultured cells.

Background Thiamine (vitamin B1) is an essential molecule for all life forms because thiamine diphosphate (ThDP) is an indispensable cofactor for oxidative energy metabolism. The less abundant thiamine monophosphate (ThMP), thiamine triphosphate (ThTP) and adenosine thiamine triphosphate (AThTP), present in many organisms, may have still unidentified physiological functions. Diseases linked to thiamine deficiency (polyneuritis, Wernicke-Korsakoff syndrome) remain frequent among alcohol abusers and other risk populations. This is the first comprehensive study on the distribution of thiamine derivatives in human biopsies, body fluids and cell lines. Methodology and Principal Findings Thiamine derivatives were determined by HPLC. In human tissues, the total thiamine content is lower than in other animal species. ThDP is the major thiamine compound and tissue levels decrease at high age. In semen, ThDP content correlates with the concentration of spermatozoa but not with their motility. The proportion of ThTP is higher in humans than in rodents, probably because of a lower 25-kDa ThTPase activity. The expression and activity of this enzyme seems to correlate with the degree of cell differentiation. ThTP was present in nearly all brain and muscle samples and in ∼60% of other tissue samples, in particular fetal tissue and cultured cells. A low ([ThTP]+[ThMP])/([Thiamine]+[ThMP]) ratio was found in cardiovascular tissues of patients with cardiac insufficiency. AThTP was detected only sporadically in adult tissues but was found more consistently in fetal tissues and cell lines. Conclusions and Significance The high sensitivity of humans to thiamine deficiency is probably linked to low circulating thiamine concentrations and low ThDP tissue contents. ThTP levels are relatively high in many human tissues, as a result of low expression of the 25-kDa ThTPase. Another novel finding is the presence of ThTP and AThTP in poorly differentiated fast-growing cells, suggesting a hitherto unsuspected link between these compounds and cell division or differentiation.

Thiamine triphosphate and thiamine triphosphatase activities: from bacteria to mammals

AbstractIn 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.

Sleep architecture of the melanin-concentrating hormone receptor 1-knockout mice.

Growing amounts of data indicate involvement of the posterior hypothalamus in the regulation of sleep, especially paradoxical sleep (PS). Accordingly, we previously showed that the melanin‐concentrating hormone (MCH)‐producing neurons of the rat hypothalamus are selectively activated during a PS rebound. In addition, intracerebroventricular infusion of MCH increases total sleep duration, suggesting a new role for MCH in sleep regulation. To determine whether activation of the MCH system promotes sleep, we studied spontaneous sleep and its homeostatic regulation in mice with deletion of the MCH‐receptor 1 gene (MCH‐R1–/– vs. MCH‐R1+/+) and their behavioural response to modafinil, a powerful antinarcoleptic drug. Here, we show that the lack of functional MCH‐R1 results in a hypersomniac‐like phenotype, both in basal conditions and after total sleep deprivation, compared to wild‐type mice. Further, we found that modafinil was less potent at inducing wakefulness in MCH‐R1–/– than in MCH‐R1+/+ mice. We report for the first time that animals with genetically inactivated MCH signaling exhibit altered vigilance state architecture and sleep homeostasis. This study also suggests that the MCH system may modulate central pathways involved in the wake‐promoting effect of modafinil.

EFHC1 interacts with microtubules to regulate cell division and cortical development

Mutations in the EFHC1 gene are linked to juvenile myoclonic epilepsy (JME), one of the most frequent forms of idiopathic generalized epilepsies. JME is associated with subtle alterations of cortical and subcortical architecture, but the underlying pathological mechanism remains unknown. We found that EFHC1 is a microtubule-associated protein involved in the regulation of cell division. In vitro, EFHC1 loss of function disrupted mitotic spindle organization, impaired M phase progression, induced microtubule bundling and increased apoptosis. EFHC1 impairment in the rat developing neocortex by ex vivo and in utero electroporation caused a marked disruption of radial migration. We found that this effect was a result of cortical progenitors failing to exit the cell cycle and defects in the radial glia scaffold organization and in the locomotion of postmitotic neurons. Therefore, we propose that EFHC1 is a regulator of cell division and neuronal migration during cortical development and that disruption of its functions leads to JME.

Glial contribution to seizure: K+ activation of (Na+, K+)-ATPase in bulk isolated glial cells and synaptosomes of epileptogenic cortex.

Abstract Glial and neuronal (Na+, K+)-ATPase have dissimilar apparent affinities for K+ ions. Glial (Na+, K+)-ATPase is maximally activated by 20 mM K+ while neuronal (Na+, K+)-ATPase is maximally stimulated by 3–5 mM K+. Because this glial property may play an important role in the clearance of [K+]0 during seizures, we investigated the K+ activation of (Na+, K+)-ATPase within bulk isolated glial cells and synaptosomes isolated from epileptogenic brains. The primary focus (F), the homolateral brain area around the focus (PF) and the mirror (M) or secondary focus induced by freezing lesions were studied. Results show that both glial and synaptosomal enzyme activities in the epileptogenic state change in comparison with controls, i.e. sham-operated cats. In F and M., glial enzyme decreased reaction velocities between 3 and 18 mM K+. In PF, maximum velocities increased in glial (Na+, K+)-ATPase. These results indicate that in actively firing epileptogenic tissue (F, M), glial (Na+, K+)-ATPase decreased rate reactions while the catalytic activity was increased in cortical tissues surrounding the focus. These phenomena appeared early, i.e. 1–3 days after production of the freezing lesion, and was associated with a sharp decrease in absolute levels of enzyme activity. Synaptosomal (Na+, K+)-ATPase from controls always exhibited a saturation curve at 3–6 mM K+. Synaptosomal enzyme activities in the primary (F) lesion increased slightly 24 h after lesion production, then progressively decreased 3 days after lesion production. No significant changes were seen in M and PF.

Partial Cloning and Distribution of Estrogen Receptor Beta in the Avian Brain

A partial estrogen receptor beta (ER-β) cDNA was isolated from testicular quail RNA by RT-PCR with degenerate primers specific to the rat ER-β sequence. A high expression of ER-β was demonstrated by RT-PCR in the telencephalon, diencephalon, pituitary, testis and kidneys of male quail but little or no expression was detected in the cerebellum, pectoral muscle and adrenal gland. In situ hybridization with a 35S-labelled oligoprobe in sections through the preoptic area-rostral hypothalamus identified high expression in the medial preoptic nucleus, bed nucleus striae terminalis and nucleus taeniae. These data demonstrate the presence of an ER-β in brain areas implicated in the control of reproduction in a non-mammalian species.

Human immune cells express ppMCH mRNA and functional MCHR1 receptor

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.

Characteristics of putrescine uptake and subsequent GABA formation in primary cultured astrocytes from normal C57BL/6J and epileptic DBA/2J mouse brain cortices

Brain maturation and GABA metabolism are known to play a key role in epileptogenesis. The metabolism of the polyamines (putrescine, spermidine and spermine) is closely linked to the process of brain maturation. Putrescine has been shown to be catabolized to GABA in brain tissue and astrocytes. In order to better understand the importance of glial putrescine transport and metabolism, a model of age-dependent epilepsy was used to study the kinetic properties of [14C]putrescine uptake into cultured astrocytes from normal C57/BL and audiogenic DBA/2 newborn mice, and the subsequent GABA formation. (1) Putrescine uptake exhibited non-Michaelian allosteric kinetics with positive co-operativity (Hill factor = 2), suggesting a physiological importance of putrescine uptake by astrocytes. (2) The Vmax of putrescine uptake was significantly higher in C57/BL astrocytes than in DBA/2J, but the uptake affinity for putrescine was higher in DBA/2J than in C57/BL. (3) Higher K+ concentrations (18 mM) had little effect on putrescine uptake in either strain. (4) Ten-micromolar N-acetylputrescine, the first putrescine metabolite, stimulated putrescine uptake into astrocytes of both strains, but to a different degree: +46% in C57/BL and + 102% in DBA/2J. (5) The specific radioactivity of the GABA formed from labelled putrescine was four times higher in astrocytes from DBA/2J than from C57/BL mice. (6) The molar ratio of glutamate/GABA in the cerebral cortex of the DBA/2J mice was significantly higher during the period of audiogenic seizure susceptibility than in age-matched C57/BL mice. Our results show characteristics of putrescine uptake into astrocytes; we demonstrated distinct kinetic properties between normal and epileptic strains of mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of nicotine on rat gingival fibroblasts in vitro

Tobacco smoking is considered a major risk factor for the development and progression of periodontal diseases (Haber, J. and Wattles, J. (1994). J. Periodontol. 64, 16-23). The purpose of this study was to determine the effects of nicotine on rat gingival fibroblasts (RGF) cultured in vitro. After ether anesthesia, rat gingival tissues were obtained from the attached gingiva of a Wistar rat. Small fragments of gingiva were maintained in culture in Petri dishes. Fibroblasts developing from these explants were collected to obtain monolayer cultures. After the fourth passage (T4), cells were supplemented with nicotine at various concentrations. Control and treated cells were examined under phase contrast or transmission electron microscopy. They were compared as regards their DNA content, mitochondrial activity, collagen and protein synthesis, and cell death by apoptosis or necrosis. Nicotine from 0.05 uM to 1 miVl did not affect the DNA content or protein and collagen synthesis. At concentrations between 3 and 5 niM. growth was significantly diminished and the survival rate reduced. Ultrastructural analysis revealed dilated mitochondria and vacuolization in treated cells, suggestive of necrosis, but increased apoptosis was also revealed by cytometry. On the basis of this in vitro study, it appears that tobacco, through its component nicotine, may directly affect various functions of RGF.