Biomedical subjects

Philippe Durand

Publications and source records attributed to Philippe Durand.

31 records · Page 2Linked to original sources

PubMed2004

Characterization of cell lines stably expressing human normal or mutated EGFP-tagged MC4R.

The melanocortin receptor type 4 (MC4-R) is involved in food intake and represents a potential target for the treatment of some forms of obesity. The fluorescent protein EGFP was fused to the wild-type or mutated coding sequence of the human MC4-R. After transfection in HEK 293, clones stably expressing hMC4-R-EGFP were selected. Wild-type chimeric hMC4-R was well addressed to the cell membrane as demonstrated using confocal microscopy and displayed the same pharmacological characteristics as native hMC4R. NDP-alpha MSH induced a time-dependent internalization of MC4-R that was partially prevented by AgRP. The two mutated chimeric receptors studied here (CTCT-deleted and C271A) showed a high alteration of their response to ligand and were retained inside the cells. In conclusion, we have developed a model of clones stably expressing EGFP-tagged-hMC4-R. This is the only such model available to date and it provides a useful tool to follow the trafficking of MC4-R inside living cells.

Agouti-Related Protein↗

PubMed2004

CCR4-associated factor CAF1 is an essential factor for spermatogenesis.

The CCR4-associated protein CAF1 has been demonstrated to play several roles in the control of transcription and of mRNA decay. To gain further insight into its physiological function, we generated CAF1-deficient mice. They are viable, healthy, and normal in appearance; however, mCAF1(-/-) male mice are sterile. The crossing of mCAF1(+/-) mice gave a Mendelian ratio of mCAF1(+/+), mCAF1(+/-), and mCAF1(-/-) pups, indicating that haploid mCAF1-deficient germ cells differentiate normally. The onset of the defect occurs during the first wave of spermatogenesis at 19 to 20 days after birth, during progression of pachytene spermatocytes to haploid spermatids and spermatozoa. Early disruption of spermatogenesis was evidenced by Sertoli cell vacuolization and tubular disorganization. The most mature germ cells were the most severely depleted, but progressively all germ cells were affected, giving Sertoli cell-only tubes, large interstitial spaces, and small testes. This phenotype could be linked to a defect(s) in germ cells and/or to inadequate Sertoli cell function, leading to seminiferous tubule disorganization and finally to a total disappearance of germ cells. The mCAF1-deficient mouse provides a new model of failed spermatogenesis in the adult that may be relevant to some cases of human male sterility.

Animals↗

PubMed2003

Key role for cyclin-dependent kinases in the first and second meiotic divisions of rat spermatocytes.

In all systems examined so far, the G2/M phase transition is controlled by the M-phase promoting factor (MPF), a complex of cdc2 (CDK1) and cyclin B1. Histone H1 kinase activity and MPF components are present in pachytene spermatocytes (PS). However, it has not been demonstrated yet that direct inhibition of MPF activity prevents the G2/M transition in these cells. When roscovitine, a potent inhibitor of CDK1, CDK2, and CDK5 activities, was added to cocultures of PS with Sertoli cells, the number of both secondary spermatocytes and round spermatids formed were lower than in control cultures, despite similar cell viability. This effect of roscovitine was reversible, did not involve the Sertoli cells, and was dependent on the concentration of the inhibitor. Roscovitine did not modify the amount of MPF in these germ cells but inhibited the CDK1- or CDK2-associated histone H1 kinase activity of PS. Hence a functional relationship between cyclin-dependent kinase activity and the spontaneous processing of the first meiotic division and, for the first time, of the second meiotic division of male germ cells is shown.

Animals↗

PubMed2003

Agouti-related protein antagonizes glucocorticoid production induced through melanocortin 4 receptor activation in bovine adrenal cells: a possible autocrine control.

Agouti-related protein (Agrp), primarily expressed in the hypothalamus, is an endogenous antagonist of alphaMSH at the level of melanocortin 3 receptor (MC3-R) and MC4-R, but the adrenal gland represents the second major Agrp-expressing tissue. In adrenal fasciculata cells, the glucocorticoid secretion is under the control of ACTH, which binds specifically MC2-R, the only functional melanocortin receptor described in these cells to date. Nevertheless, using cultured bovine fasciculata adrenal cells, we report that Agrp has no antagonistic properties against ACTH at the level of MC2-R. In our studies, (Nle4, d-Phe7)-alphaMSH (NDP-alphaMSH) stimulated the production of cortisol in a dose-dependent manner, and these effects were abolished by Agrp or SHU9119, a synthetic antagonist of MC3-R and MC4-R. Using a more specific antagonist (JKC-363) and RT-PCR analysis, we can postulate that the effects of NDP-alphaMSH were mediated via MC4-R. These results are suggestive that adrenal glucocorticoid production could be regulated through MC4-R that may have some relevance in the physiology of adrenal cells. Moreover, Agrp might exert an autocrine control on adrenal cells because a protein with biological Agrp-like activity is secreted by these cells. This peptide could then modulate locally the functions of some peripheral tissues such as adrenals.

Adrenal Cortex↗

PubMed2003

Stable expression of human melanocortin 3 receptor fused to EGFP in the HEK293 cells.

Among the melanocortins alpha-MSH is known to be involved in feeding behavior. These hormones mediate their effects through G protein-coupled receptors by stimulating adenylate cyclase. In this study, we have developed an in vitro expression model for human melanocortin 3 receptor (hMC3R) tagged at its C terminus with EGFP. The corresponding chimeric cDNA was stably expressed in HEK293 cells. The selected clones expressing the hMC3R-EGFP exhibited cell surface fluorescence and responded to NDP-MSH stimulation by producing cAMP in a dose-dependent manner (EC(50): 0.3 nM). Binding studies revealed a single class of binding sites with a K(D) of 2.24 nM. Moreover, Agouti-related protein was also demonstrated to be an antagonist of the hMC3R-EGFP. Thus, the hMC3R tagged with EGFP stably expressed in HEK293 cells, exhibiting the same characteristics than the wild-type hMC3R, is the only model of expression of this receptor allowing its direct localization inside living cells.

Base Sequence↗

PubMed2003

Development of the meiotic step in testes of pubertal rats: comparison between the in vivo situation and under in vitro conditions.

The present work aimed to compare some features of the meiotic process which develops in the testis of pubertal rats, in vivo and in vitro, paying special attention to the time-course of the phenomenon. The differentiation of spermatocytes was assessed in testes of 20- to 46-day-old rats and in tubule segments of 20- or 28-day-old rats cultured over a 4-week period. Very similar results were obtained in vivo and in vitro, during the first week of culture, when considering the changes in the cell populations of different ploidy, the gene expression of germ cells, the kinetics of differentiation of BrdU-labeled early or middle pachytene spermatocytes and the levels of apoptosis in the different cell populations. However, during the second week of culture, the decrease in the proportion of the 4C cell population which was only slightly more marked than that observed in vivo between 27 and 34 days, was not associated with an increase in the 1C cell population as large as in vivo. This result could be explained partly by a high proportion of apoptotic 1C cells beyond one week of culture. Concomitantly, the rate of in vitro differentiation of BrdU-labeled spermatocytes slowed down when reaching the stage of middle pachytene spermatocytes and BrdU-labeled round spermatids were observed 6-11 days later than when BrdU-labeled spermatocytes differentiated in vivo. Taken together, our results indicate that the bottleneck for the development of the meiotic cells in vitro is at the transition from middle to late pachytene spermatocytes. Hence, comparing the expression of locally produced regulatory molecules in vivo and in vitro at different days of culture should allow to identify key regulators of the meiotic step of spermatogenesis.

Animals↗

PubMed2003

Quantitation by image analysis of global DNA methylation in human spermatozoa and its prognostic value in in vitro fertilization: a preliminary study.

OBJECTIVE: To determine the relationship between sperm DNA methylation level and sperm characteristics and pregnancy rates. DESIGN: Prospective study. Quantitation by image analysis of DNA methylation in sperm nucleus. SETTING: Department of Reproduction Biology, Edouard Herriot Hospital, Lyon, France. PATIENT(S): Infertile couples undergoing IVF-ET. INTERVENTION(S): The immunostaining of 5 methyl-cytosine was performed on the spare sperm suspension that was used for an assisted reproduction technology procedure. MAIN OUTCOME MEASURE(S): Sperm characteristics according to World Health Organization criteria, sperm motility parameters with computer-assisted semen analysis, sperm DNA methylation level, and heterogeneity index (HI). RESULT(S): Sperm DNA methylation level and HI are correlated with sperm DNA characteristics. HI is negatively correlated with fertilization rate; sperm DNA methylation level is correlated with pregnancy rate. CONCLUSION(S): The DNA methylation level in human spermatozoa could be a new approach to evaluating the ability of spermatozoa to fertilize and lead to normal embryo development.

3,3'-Diaminobenzidine↗

PubMed2003

Whole-body cooling after perinatal asphyxia: a pilot study in term neonates.

In order to test the practicability and safety of whole-body cooling in term neonates with moderate-to-severe hypoxic-ischaemic encephalopathy (HIE) and to report outcomes, a prospective pilot study was carried out in 25 term infants (median postmenstrual age 38 weeks, range 36 to 41 weeks; 20 males, five females). Whole-body cooling, to a target core temperature of 33 to 34 degrees C, started within 6 hours of birth and was maintained for 72 hours. Of the 25 newborn infants (19 Sarnat II and six Sarnat III, 18 outborn), 18 survived, including 13 (72%) with normal cerebral signal by MRI. Temperature instability occurred during cooling in 15 infants, but neither severe haemodynamic instability nor renal failure was seen. Thrombocytopenia developed in 12 infants, including seven with biological disseminated intravascular coagulation. One patient had hypoxaemia with right-to-left shunting through the ductus arteriosus, and seven had limited meningeal or subdural bleeding. Whole-body cooling is feasible in term neonates, with no life-threatening adverse events. Improvements are needed to obtain stable hypothermia for 72 hours.

Asphyxia Neonatorum↗

PubMed2003

[Infantile and maternal choriocarcinoma].

Infantile or congenital choriocarcinoma is a very uncommon complication of gestational choriocarcinoma. We report such a case with fatal outcome in a 3-week-old newborn, admitted for a hemorrhagic syndrome. Lungs, liver and brain masses were discovered and suggested an angiomatous process. The diagnosis was made later on gingival biopsy with necropsic confirmation. The mother's B-HCG level was elevated. She had asymptomatic pulmonary nodules and a uterine mass. This case report highlights characteristic but non specific clinical findings leading to the diagnosis. Chemotherapy must be undertaken as soon as possible to be effective. It is also necessary to assay maternal serum B-HCG when infantile choriocarcinoma is disclosed.

Choriocarcinoma↗

PubMed2002

(+)-Catechin inhibits platelet hyperactivity induced by an acute iron load in vivo.

Reactive oxygen species and platelets are thought to be involved in the pathogenesis of cardiovascular disease. Epidemiological data have indicated that high consumption of fruits and vegetables is associated with a lower incidence of vascular events. Polyphenols were proposed to provide such a protection. In the present study performed in rats, we investigated the influence of (+)-catechin (Cat), a polyphenol identified in tea, cocoa, and red wine, on an acute iron load-induced model of platelet hyperactivity. We found that platelet function was significantly enhanced in iron-loaded rats. These changes were associated with impairment of the antioxidative defense including ex vivo free radical-induced hemolysis. Pretreatment with Cat (10 mg/kg, ip, 4 d) normalized biomarkers of antioxidative status and platelet hyperactivity. The benefits of Cat treatment were only observed in iron-loaded animals and not in control animals. In light of the known antioxidant properties of Cat (or its metabolites), we suggest that oxidative injury-induced modification of platelet calcium homeostasis may have explained the iron load-induced platelet hyperactivity. The protective effect of Cat appears to work probably through normalization of the antioxidative status.

Animals↗

PubMed2002

Linkage of one gene for familial glucocorticoid deficiency type 2 (FGD2) to chromosome 8q and further evidence of heterogeneity.

In several cases of familial glucocorticoid deficiency (FGD), referred to as FGD type 1, mutations have been described in the coding exon of the adrenocorticotropin receptor (melanonocortin receptor type 2, MC2R) gene. However, for the majority of cases (FGD type 2), no mutations were found in this gene. In the more informative families, the involvement of the MC2R locus could be excluded by linkage or sequencing analysis and, as there was no obvious candidate gene, a genome linkage scan was performed. Fourteen families were studied in this report. Evidence of linkage was found with markers on chromosome 8q in three out of the 14 families (maximum heterogeneity LOD score of 2.81 at D8S1763). These three families were consanguineous and the gene could be located by homozygosity mapping between markers D8S285 and D8S1718 in a 8.8-cM region. No potential candidate genes were apparent in the region. Linkage to this region could be excluded in some families from our sample giving highly negative LOD scores with the markers of the region. This result suggests that at least one other gene, located on a different region, must be responsible for FGD in these families and provides new evidence of genetic heterogeneity of this disorder.

Adrenal Cortex Diseases↗

PubMed2002

Assessment of lifestyle effects on the overall antioxidant capacity of healthy subjects.

Oxidative damage is increasingly recognized as playing an important role in the pathogenesis of several diseases such as cancer and cardiovascular diseases. Using a biologic test based on whole blood resistance to free-radical aggression, we sought to evaluate lifestyle factors that may contribute to the normal variability of the overall antioxidant status. We assessed this global antiradical defense capacity in 88 men and 96 women in relation to information on lifestyle obtained by questionnaire. In our relatively young, healthy population, we found a weak negative relation between male sex or aging and the resistance to oxidant stress. Among the factors studied, nonsmoking, vitamin and/or mineral supplementation, and regular physical activity were closely associated with an increased overall antioxidant capacity. Conversely, the antioxidant potential was negatively related to tobacco smoking; psychologic stress; alcohol consumption; moderate vegetable, low fruit, and low fish consumption; and, to a lesser extent, high natural ultraviolet light exposure. Thus, we were able to determine "unhealthy" and "healthy" lifestyle patterns that truly contributed to the variation of individual antioxidant capacity. We conclude that lifestyle determinants of cancer and cardiovascular risks were associated with a decreased overall antioxidant status as dynamically measured by means of a biologic test. Thus, the evaluation of the total human resistance against free-radical aggression, taking into account nutritional habits, lifestyle, and environmental factors, may be useful in preventive medicine as a precocious diagnosis to identify healthy subjects who are at risk for free-radical-mediated diseases.

Adult↗

PubMed2002

Osteopetrosis, lymphedema, anhidrotic ectodermal dysplasia, and immunodeficiency in a boy and incontinentia pigmenti in his mother.

A child with X-linked osteopetrosis, lymphedema, anhidrotic ectodermal dysplasia, and immunodeficiency (OL-EDA-ID) was recently reported. We report the clinical features of a second boy with this novel syndrome and his mother, who presented with signs of incontinentia pigmenti (IP). The child had mild osteopetrosis without neurosensory complications, unilateral lymphedema of the left leg, and characteristic features of anhidrotic ectodermal dysplasia with sparse hair, facial dysmorphy, delayed eruption of teeth, and sweat gland abnormalities. He died at 18 months of severe immunodeficiency with multiple infections caused by Gram-negative (Salmonella enteritidis) and Gram-positive (Streptococcus pneumoniae) bacteria, nontuberculous mycobacteria (Mycobacterium kansasii), and fungi (Pneumocystis carinii). His 30-year-old mother's medical history, together with residual cutaneous lesions, was highly suggestive of IP without neurologic impairment. In this patient with OL-EDA-ID, we detected the same NF-kappaB essential modulator stop codon hypomorphic mutation identified in the previous patient. The occurrence of the same clinical features in 2 unrelated patients with the same genotype demonstrates that OL-EDA-ID is a genuine clinical syndrome. The clinical and biological descriptions of the proband and his mother further corroborate the relationship between IP and EDA. Both syndromes are allelic and are associated with mutations in NF-kappaB essential modulator, with a genotype-phenotype correlation in hemizygous males. In contrast, loss-of-function mutations and hypomorphic mutations may cause IP in females.

Abnormalities, Multiple↗