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Biomedical subjects

C Thibault

Publications and source records attributed to C Thibault.

At least 19 recordsLinked to original sources
PubMed2006

Apoptosis and differentiation commitment: novel insights revealed by gene profiling studies in mouse embryonic stem cells.

Mouse embryonic stem (ES) cells remain pluripotent in vitro when grown in the presence of leukemia inhibitory factor (LIF). LIF starvation leads to apoptosis of some of the ES-derived differentiated cells, together with p38alpha mitogen-activated protein kinase (MAPK) activation. Apoptosis, but not morphological cell differentiation, is blocked by a p38 inhibitor, PD169316. To further understand the mechanism of action of this compound, we have identified its specific targets by microarray studies. We report on the global expression profiles of genes expressed at 3 days upon LIF withdrawal (d3) compared to pluripotent cells and of genes whose expression is modulated at d3 under anti-apoptotic conditions. We showed that at d3 without LIF cells express, earlier than anticipated, specialized cell markers and that when the apoptotic process was impaired, expression of differentiation markers was altered. In addition, functional tests revealed properties of anti-apoptotic proteins not to alter cell pluripotency and a novel role for metallothionein 1 gene, which prevents apoptosis of early differentiated cells.

Animals↗
PubMed2005

Direct microcontact printing of oligonucleotides for biochip applications.

BACKGROUND: A critical step in the fabrication of biochips is the controlled placement of probes molecules on solid surfaces. This is currently performed by sequential deposition of probes on a target surface with split or solid pins. In this article, we present a cost-effective procedure namely microcontact printing using stamps, for a parallel deposition of probes applicable for manufacturing biochips. RESULTS: Contrary to a previous work, we showed that the stamps tailored with an elastomeric poly(dimethylsiloxane) material did not require any surface modification to be able to adsorb oligonucleotides or PCR products. The adsorbed DNA molecules are subsequently printed efficiently on a target surface with high sub-micron resolution. Secondly, we showed that successive stamping is characterized by an exponential decay of the amount of transferred DNA molecules to the surface up the 4th print, then followed by a second regime of transfer that was dependent on the contact time and which resulted in reduced quality of the features. Thus, while consecutive stamping was possible, this procedure turned out to be less reproducible and more time consuming than simply re-inking the stamps between each print. Thirdly, we showed that the hybridization signals on arrays made by microcontact printing were 5 to 10-times higher than those made by conventional spotting methods. Finally, we demonstrated the validity of this microcontact printing method in manufacturing oligonucleotides arrays for mutations recognition in a yeast gene. CONCLUSION: The microcontact printing can be considered as a new potential technology platform to pattern DNA microarrays that may have significant advantages over the conventional spotting technologies as it is easy to implement, it uses low cost material to make the stamp, and the arrays made by this technology are 10-times more sensitive in term of hybridization signals than those manufactured by conventional spotting technology.

Journal Article↗
PubMed2005

Microbiological characterization of winery effluents: an inventory of the sites for different treatment systems.

In a more and more regulated and socially pressured environment, the durable management of winery effluents must take into account their characteristics and their potential impact on their natural setting. The object of this exploratory study is to establish an inventory of the microbiological composition of winery effluents coming from different treatment systems. We have observed that winery effluents are charged with micro-organisms, by a factor that ranges from 10(5) to 10(8) UFC/ml, and that the level of "microbiological pollution" is independent of the type of system. The composition of the flora is closely tied to the time of year and therefore to winery activities, so certain micro-organisms will be favoured in certain periods and others will have a tendency to decrease. We have seen that from one year to another our observations remain identical; the flora equilibrium therefore occurs systematically and naturally. Faecal germs are found in very small quantities in winery effluent treatment systems. They represent minor sanitary risks. Good correlations were observed between some micro-organisms and some physical-chemical parameters (COD). It is, however, difficult to use these "easy-to-measure" parameters as reliable markers of certain microbial populations.

Agriculture↗
PubMed2003

Effect of feeding prepubertal heifers with a high oil diet on mammary development and milk production.

The objective of this study was to evaluate the effect of feeding prepubertal heifers a diet containing a high level of polyunsaturated fatty acids on mammary development and milk production. A total of 116 Holstein heifers were either fed a conventionally formulated concentrate or a high oil (HO) concentrate, using the same formulation but including 20% soybean oil, from birth to 6 mo of age. After 6 mo of age, all heifers were managed identically. Mammary gland development was evaluated on heifers slaughtered at 4 mo (n = 10) and 12 mo (n = 30) of age. Other heifers were bred when they reached 15 mo of age and milk production and feed intake were recorded every day from wk 4 to 18 of lactation. Feeding the high oil concentrate increased the concentration of linoleic acid in blood plasma (176%) and mammary fat pad (78%) at 4 mo of age and mammary fat pad (93%) at 12 mo of age. At 4 mo of age, mammary development was similar in both treatments. At 12 mo of age, total, parenchyma, and stroma weights of the mammary gland were not affected by treatments. However, lipid content was lower and concentration of DNA was higher in the parenchyma of heifers fed the high oil diet. Nevertheless, total parenchymal DNA and dry fat free tissue content did not reach statistical significance despite the fact that they were, respectively, 15 and 21% higher in HO heifers. Milk production and composition was not affected by treatments. In conclusion, feeding prepubertal heifers with a high oil concentrate slightly improved the mammary development but effects were too small to be translated into better lactating performances.

Aging↗
PubMed2001

Ovarian steroids and selective estrogen receptor modulators activity on rat brain NMDA and AMPA receptors.

Glutamate and glutamate receptors are well known to play a major excitatory role in the brain. Recent findings on ovarian steroids and selective estrogen receptor modulators (SERMs) activity on rat brain AMPA and NMDA receptors are reviewed. Ovarian steroid withdrawal by ovariectomy is without effect on NMDA and AMPA receptors in most brain regions, except in hippocampus, where it decreases NMDA receptor specific binding, compared to intact rat values. Estradiol treatment increases hippocampal NMDA receptor specific binding of ovariectomized rats while it decreases this binding in frontal cortex and striatum. Estradiol treatment has no effect on AMPA receptor specific binding in hippocampus, but decreases binding in frontal cortex, striatum and nucleus accumbens. Progesterone and estradiol+progesterone treatments decrease NMDA, but not AMPA receptors specific binding in frontal cortex compared to ovariectomized rats. No effect was observed in other brain regions. Tamoxifen and raloxifene are SERMs with varying effects on estrogen responses in mammary, bone and uterine tissues. Tamoxifen and raloxifene have estrogenic activity upon modulation of brain NMDA and AMPA receptors. Using specific ligands for binding autoradiography of NMDA receptor subunits and specific probes for subunits measured by in situ hybridization, it was shown that estradiol and SERMs modulate NR1 and NR2B subunits whereas the NR1/2A subunit remains unchanged. In summary, regional agonist estrogenic activity on brain AMPA and NMDA receptors of tamoxifen and raloxifene, like that of estradiol, is observed, whereas progesterone has limited effects or opposes the estradiol effect.

Animals↗
PubMed2001

Estrogen-like activity of tamoxifen and raloxifene on NMDA receptor binding and expression of its subunits in rat brain.

Hormonal specificity of modulation of N-methyl- D-aspartate (NMDA) receptors was investigated by comparing the effects of estradiol with tamoxifen or raloxifene, which display different responses in breast, bone, and uterus. Two weeks ovariectomy in rats decreased uterine weight, which was prevented by a two-week estradiol treatment; tamoxifen and raloxifene had weaker uterine stimulation than estradiol. Ovariectomy in rats decreased L-[3H]glutamate specific binding to NMDA receptors in CA1 and dentate gyrus but not CA2/3 regions of hippocampus and was without effect in cortex, striatum, nucleus accumbens, and olfactory tubercle. [3H]Ro 25-6981 (an NMDA antagonist selective for NR1/NR2B assembly) specific binding and mRNA levels of NMDA receptor subunits 1 and 2B decreased in CA1 after ovariectomy. Estradiol, tamoxifen, and raloxifene decreased L-[3H]glutamate specific binding to NMDA receptors and [3H]Ro 25-6981 specific binding in cortical area of ovariectomized rats and prevented the decrease of [3H]glutamate specific binding to NMDA receptors in CA1 and dentate gyrus, as well as [3H]Ro 25-6981 specific binding in CA1. Estradiol prevented the decrease of NMDA receptor subunits 1 and 2B mRNA levels in CA1 only; tamoxifen and raloxifene prevented the decrease of NMDA receptor subunit 1 mRNA levels in CA1. No effect of ovariectomy or treatments on L-[3H]CGP 39653 (an NMDA antagonist selective for NR1/NR2A assembly) specific binding and NMDA receptor subunit 2A mRNA levels was observed in all brain regions assayed. Our results showed brain regional and subunits specific agonist estrogenic activity of tamoxifen and raloxifene on NMDA receptors.

Animals↗
PubMed2000

Expression profiling of neural cells reveals specific patterns of ethanol-responsive gene expression.

Adaptive changes in gene expression are thought to contribute to dependence, addiction and other behavioral responses to chronic ethanol abuse. DNA array studies provide a nonbiased detection of networks of gene expression changes, allowing insight into functional consequences and mechanisms of such molecular responses. We used oligonucleotide arrays to study nearly 6000 genes in human SH-SY5Y neuroblastoma cells exposed to chronic ethanol. A set of 42 genes had consistently increased or decreased mRNA abundance after 3 days of ethanol treatment. Groups of genes related to norepinephrine production, glutathione metabolism, and protection against apoptosis were identified. Genes involved in catecholamine metabolism are of special interest because of the role of this pathway in mediating ethanol withdrawal symptoms (physical dependence). Ethanol treatment elevated dopamine beta-hydroxylase (DBH, EC 1.14.17.1) mRNA and protein levels and increased releasable norepinephrine in SH-SY5Y cultures. Acute ethanol also increased DBH mRNA levels in mouse adrenal gland, suggesting in vivo functional consequences for ethanol regulation of DBH. In SH-SY5Y cells, ethanol also decreased mRNA and secreted protein levels for monocyte chemotactic protein 1, an effect that could contribute to the protective role of moderate ethanol consumption in atherosclerotic vascular disease. Finally, we identified a subset of genes similarly regulated by both ethanol and dibutyryl-cAMP treatment in SH-SY5Y cells. This suggests that ethanol and cAMP signaling share mechanistic features in regulating a subset of ethanol-responsive genes. Our findings offer new insights regarding possible molecular mechanisms underlying behavioral responses or medical consequences of ethanol consumption and alcoholism.

Animals↗
PubMed1999

Cloning and characterization of the rat and human phosducin-like protein genes: structure, expression and chromosomal localization.

We isolated and characterized the rat gene encoding phosducin-like protein (PhLP), a putative heterotrimeric G protein modulator. The transcription start site was mapped by primer extension. The putative promoter region lacked a TATA sequence but contained a potential initiator element. Two splice variants were identified by RT-PCR of rat brain RNA, potentially generating either the full length or an amino-truncated protein. Only the full-length protein was immunodetected in all mouse tissues surveyed. Comparison of the conceptual translation product of the rat PhLP gene with those from human and Drosophila clones shows a striking conservation in the amino-terminal region of PhLP from these species. This contrasts with the relatively low degree of homology between PhLP and phosducin in this region, suggesting a functional role for this portion of the PhLP protein. Finally, we mapped the human PhLP gene by PCR analysis of somatic cell hybrids and the Stanford G3 radiation hybrid panel. The human PhLP gene (PDCL) is located on chromosome 9, linked to the polymorphic markers D9S1876 and D9S1674 (66-71 cM).

Amino Acid Sequence↗
PubMed1999

[Ovulation].

This review summarizes our knowledge on ovulation process. After gonadotropin surge or LH injection, 13 factors are involved in the follicular rupture. All of them plays a role since the inhibition of their synthesis or activity prevents the rupture of preovulatory follicles or reduces the number of ovulations. Most of them are involved in all inflammatory reactions. At the apex of the follicle, blood vessels constriction, free TNF alpha and probably factors from the ovarian epithelium cells are involved in the full dissociation of follicle layers and cell death, allowing the localized rupture of the follicle wall. Gonadotrophins and angiotensine II are the only factors able to induce both follicle rupture and meiotic resumption.

Animals↗
PubMed1998

Inhibitors of messenger RNA and protein synthesis affect differently serotonin arylalkylamine N-acetyltransferase activity in clock-controlled and non clock-controlled fish pineal.

The pineal organ of fish contains photoreceptor cells. In some species (e.g., pike) each photoreceptor is a cellular circadian system which contains a photoreceptive unit, the clock and an output unit. In others (e.g., trout) the clock is lacking. The main rhythmic output of the pineal photoreceptor is melatonin, an internal 'zeitgeber' of the organisms. The nocturnal rise in melatonin secretion results from an increase in the activity of the arylalkylamine-N-acetyltransferase (AA-NAT) which converts serotonin to N-acetylserotonin. In the present study we investigated the effects of transcription and translation inhibitors on AA-NAT activity in pike and trout pineal organs in culture. Cycloheximide, anisomycin, and puromycin inhibited the rise in AA-NAT activity observed during the first 2, 4 or 6 h of the dark phase, in both species. Actinomycin D was active only in the pike. Six hours of treatment during the first half of the night induced inhibition of AA-NAT activity, providing that forskolin (an adenylyl cyclase stimulator) was present in the culture medium. When the treatment was run for 3, 6 or 12 h, starting at midday of a 12L/12D cycle, basal and forskolin-stimulated AA-NAT activity (measured at midnight) were dramatically reduced. Such a treatment had no effect on trout AA-NAT activity. It is concluded that: (1) the dark-induced rise in AA-NAT activity and melatonin secretion are dependent on newly synthesized protein in both pike and trout pineal; (2) AA-NAT regulation takes place at the translational and post-translational levels in both species; (3) AA-NAT regulation occurs also at the transcriptional level in the pike, but not in the trout; and (4) the cAMP-dependent activation of AA-NAT requires transcription in the pike, not in the trout. The presence of a cell population acting as a circadian clock in the pike pineal, but not in the trout pineal, can explain the difference between these two species. Thus, we suggest that the clock mechanism operates at the genetic level in these cells. Further comparative studies between clock-controlled and non-clock-controlled pineals might prove interesting to demonstrate the difference between these two regulatory pathways.

Acetylserotonin O-Methyltransferase↗
PubMed1998

Phosducin-like protein (PhLP), a regulator of G beta gamma function, interacts with the proteasomal protein SUG1.

Phosducin-like protein (PhLP) and phosducin are highly homologous proteins that interact with the beta gamma subunits of guanine nucleotide binding proteins. While phosducin has a well-characterized role in retinal signal transduction, PhLP function remains unclear. To further understand the function of PhLP, we have examined other potential protein:protein interactions with PhLP using the yeast two-hybrid system. PhLP was found to interact with a mouse homologue of the yeast SUG1, a subunit of the 26S proteasome which may also indirectly modulate transcription. This interaction was further confirmed by an in vitro binding assay and co-immunoprecipitation of the two proteins in overexpression studies. Inhibition of proteasome function by lactacystin led to accumulation of high molecular weight, ubiquitin-immunoreactive protein precipitated by PhLP antiserum. We suggest that PhLP/SUG1 interaction may target PhLP for proteasomal degradation.

Adenosine Triphosphatases↗
PubMed1998

Pancreatic beta-cell regeneration after 48-h glucose infusion in mildly diabetic rats is not correlated with functional improvement.

We investigated the effect of glucose infusion on beta-cell regeneration in rats made mildly diabetic by a single injection of low dosage (35 mg/kg) streptozotocin (STZ). Nondiabetic (ND) and STZ rats were submitted to a 48-h glucose infusion (hyperglycemia approximately 22 mmol/l in both groups: ND and STZ hyperglycemic-hyperinsulinemic [ND HG-HI and STZ HG-HI rats]). Before infusion, beta-cell mass was 65% lower in STZ rats than in ND rats (2.0 +/- 0.02 vs. 5.5 +/- 0.6 mg), 1.6-fold increased in ND HG-HI rats (8.7 +/- 1.7 mg), and 2.7-fold increased in STZ HG-HI rats (5.4 +/- 0.9 mg). In ND HG-HI rats, beta-cell enlargement was related to an increase in beta-cell responsiveness to nutrient secretagogues both in vivo and in vitro, whereas in STZ HG-HI rats, no significant improvement in insulin secretion could be noticed. To determine the respective role of hyperglycemia and hyperinsulinemia on beta-cell area changes, ND and STZ rats were submitted to a 48-h hyperinsulinemic-euglycemic clamp. No modification of beta-cell mass was detected in either group. In conclusion, 48-h superimposed hyperglycemia was enough to restore beta-cell mass previously reduced by STZ injection. This effect seemed to be due to hyperglycemia rather than hyperinsulinemia alone. The data stress the dissociation between beta-cell regeneration and improvement in islet function in diabetic rats. Our model seems suitable for studying factors that can improve the plasticity and function of the pancreas in NIDDM.

Animals↗
PubMed1997

Interaction of phosducin-like protein with G protein betagamma subunits.

Phosducin-like protein (PhLP), a widely expressed ethanol-responsive gene (Miles, M. F., Barhite, S., Sganga, M., and Elliott, M. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 10831-10835), is a homologue of phosducin, a known major regulator of Gbetagamma signaling in retina and pineal gland. However, although phosducin has a well characterized role in retinal phototransduction, function of the PhLP remains unclear. In this study we examine the ability of PhLP to bind Gbetagamma dimer in vitro and in vivo. Using PhLP glutathione S-transferase fusion proteins, we show that PhLP directly binds Gbetagamma in vitro. Studies with a series of truncated PhLP fusion proteins indicate independent binding of Gbetagamma to both the amino- and C-terminal halves of PhLP. Protein-protein interactions between Gbetagamma and PhLP are inhibited by the alpha subunit of Go and Gi3, suggesting that PhLP can bind only free Gbetagamma. Finally, we show that PhLP complexes, at least partially, with Gbetagamma in vivo. Following overexpression of epitope-tagged PhLP together with Gbeta1gamma2 proteins in COS-7 cells, a PhLP-Gbetagamma complex is co-immunoprecipitated by monoclonal antibody directed against the epitope tag. Similarly, polyclonal anti-PhLP antibody co-precipitates endogenous PhLP and Gbetagamma proteins from NG108-15 cell lysates. These data are consistent with the hypothesis that PhLP is a widely expressed modulator of Gbetagamma function. Furthermore, because alternate forms of the PhLP transcript are expressed, there may be functional implications for the existence of two Gbetagamma-binding domains on PhLP.

Animals↗
PubMed1997

Enhanced expression of Gi-protein precedes the development of blood pressure in spontaneously hypertensive rats.

In the present studies we have investigated if the increased expression of Gi alpha proteins reported earlier in heart and aorta from SHR (spontaneously hypertensive rats) is the cause or effect of hypertension. The SHRs at various ages of the development of blood pressure (3-5 days, 2 weeks, 4 weeks and 8 weeks) and their age-matched WKY were used for these studies. The expression of Gi alpha-2 and Gi alpha-3 (inhibitory guanine nucleotide regulatory protein and Gs alpha (stimulatory guanine nucleotide regulatory protein) at protein and mRNA level was determined by immunoblotting and Northern blotting technique using specific antibodies and cDNA probes. The SHR at early ages up to 2 weeks did not show any increase in blood pressure, however it started to go up from 4 weeks. The levels of Gi alpha 2 and Gi alpha 3 at protein and mRNA in heart from SHR were not different in 3-5 days old SHR as compared to WKY (Wistar-Kyoto rats), however, the expression of Gi alpha-2 and Gi alpha-3 protein and mRNA was significantly increased in 2 weeks and older SHR. The mRNA level of the catalytic subunit type V enzyme was significantly decreased in SHR 2 weeks and later ages as compared to their age-matched WKY. On the other hand, the expression of Gs alpha was not different in SHR as compared to WKY at all the ages studied. In addition, the oxotremorine and C-ANF4-23 (a ring deleted analog of atrial natriuretic factor) mediated inhibitions of adenylyl cyclase in hearts and aorta were also significantly enhanced in 2 weeks and older SHRs as compared to WKY rats, whereas, at younger age of SHR (3-5 days old), no change in the percent inhibition of adenylyl cyclase by C-ANF4-23 was observed and oxotremorine was unable to inhibit adenylyl cyclase activity. Furthermore, the basal enzyme activity and the stimulatory responses of isoproterenol, NECA (N-ethylcarboxamideadenosine), glucagon and forskolin on adenylyl cyclase were significantly decreased at all ages of SHR as compared to WKY. These results suggest that the increased expression of genes for Gi alpha-2 and Gi alpha-3, decreased expression of type V enzyme mRNA and decreased cAMP levels precedes the development of blood pressure and may participate in the pathogenesis of hypertension.

Adenylyl Cyclase Inhibitors↗