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Biomedical subjects

P Reynier

Publications and source records attributed to P Reynier.

At least 19 recordsLinked to original sources
PubMed2006

[Spermatozoon mitochondrial DNA].

Mitochondria play a primary role in cellular energetic metabolism, homeostasis and death. In spermatozoa, in particular, mitochondria produce the ATP necessary for motility. Mitochondrial functions depend, at least partially, on mitochondrial DNA (mtDNA). The mitochondrial genome, the transmission of which is exclusively maternal contributes to cytoplasmic heredity. Qualitative and quantitative mtDNA abnormalities have been associated with male infertility. This review focuses on the role of mtDNA in human fertility.

DNA, Mitochondrial↗
PubMed2005

[An OPA3 gene mutation is responsible for the disease associating optic atrophy and cataract with extrapyramidal signs].

INTRODUCTION: In 1961, Garcin et al. described a family with several members affected with optic atrophy associated with cataract, and neurological symptoms. The authors believed this condition to be distinct from other diseases known at that time, e.g. the Behr syndrome, Marinesco-Sjogren syndrome and Friedreich's ataxia. METHOD: This family was followed over a period of 40 years and genes known to be responsible for optic atrophy were sequenced. RESULTS: The G277A mutation of OPA3 gene was responsible for this familial disease. DISCUSSION: A new clinical entity is identified: autosomal dominant optic atrophy and cataract, due to a heterozygous mutation of the OPA3 gene, a nuclear gene encoding a mitochondrial protein.

Adult↗
PubMed2004

Low oocyte mitochondrial DNA content in ovarian insufficiency.

BACKGROUND: Mitochondrial biogenesis and bioenergetics play an important role in oocyte maturation and embryo development. We have investigated the relationship between defective mitochondrial biogenesis and the lack of oocyte maturity observed during IVF procedures with patients suffering from ovarian dystrophy and ovarian insufficiency. METHODS: We used real-time quantitative PCR to quantify mitochondrial DNA (mtDNA) in 116 oocytes obtained from 47 women undergoing the ICSI procedure. We compared the mtDNA content of oocytes from women with a normal ovarian profile with that of oocytes from women with ovarian dystrophy and ovarian insufficiency. RESULTS: We found an average of 256,000 +/- 213,000 mitochondrial genomes per cell. The mean mtDNA copy number was not significantly different in ovarian dystrophy compared with controls, but it was significantly lower in oocytes from women with ovarian insufficiency (100,000 +/- 99,000, P < 0.0001). CONCLUSIONS: Our results suggest that low mtDNA content is associated with the impaired oocyte quality observed in ovarian insufficiency.

Case-Control Studies↗
PubMed2004

A hydroxyethylated cholesterol-based cationic lipid for DNA delivery: effect of conditioning.

We have synthesised a novel cholesterol-based cationic lipid to promote DNA transfer in cells. This lipid, dimethyl hydroxyethyl aminopropane carbamoyl cholesterol iodide (DMHAPC-Chol) contains a biodegradable carbamoyl linker and a hydroxyethyl group in the polar amino head moiety and is characterised by NMR. Liposomes prepared from this lipid and dioleoyl phosphatidyl ethanolamine (DOPE) in equimolar proportion showed a weak cytotoxicity as revealed by MTT assays and are efficient to deliver plasmids DNA evaluated by the expression of reporter genes in vitro and in vivo. In this paper, we present an original method to determine the lipid concentration based on the colorimetric detection of the colipid DOPE and the measure of the molar ratio DOPE/cationic lipid in the liposome by FTIR spectroscopy. The liposomes and lipid/DNA complexes structures were characterized by transmission electron microscopy (TEM) and by quasi-elastic light scattering (QLS). TEM indicated that the complexes correspond to aggregates containing globular substructures with liposomes size. The method of immuno-gold labelling was used to detect plasmid in the complex and reveals the presence of DNA inside the aggregates. Transfection results showed efficient DNA transfer depending on the charge ratio and liposomes conditioning. Gel retardation results indicated that at a molar charge ratio between X = 1.5 and X = 2.5 (depending on the liposome conditioning), all DNA was taken by liposomes. We showed that conditioning by freeze-drying (lyophilization) facilitates storage and improves transfection efficiency. When the liposomes were lyophilized prior to DNA addition or when the complexes were subjected to freeze-thawing cycles, the obtained complexes showed a transfection with levels enhanced up to four and five-fold respectively for the lyophilized liposomes and freeze-thawed complexes. NMR was used to characterize the modifications under freezing which showed an effect on 31P spectra.

Animals↗
PubMed2004

In vitro embryo production efficiency in cattle and its association with oocyte adenosine triphosphate content, quantity of mitochondrial DNA, and mitochondrial DNA haplogroup.

Mitochondria have a broad range of functions that affect reproduction, and structural as well as quantitative variation in mtDNA has been associated with gamete quality and reproductive success. To investigate the mitochondria effect on in vitro embryo production, we collected oocytes by ultrasound-guided follicular aspiration from donor cows known to differ in the developmental capacity, measured by the blastocyst formation rate, of their oocytes. To evaluate the potential effects of mtDNA and mitochondrial function on oocyte quality, the donor cows' mtDNA control region was sequenced and, after pairwise comparisons of polymorphisms, animals were grouped into two major haplogroups. The number of mtDNA molecules per oocyte was quantified by real-time PCR, and the adenosine triphosphate (ATP) content was measured in each oocyte to identify variations between haplogroups. Overall, ATP stocks in oocytes of the two haplogroups differed significantly (P < 0.05; means +/- SEM) both at the germinal vesicle and metaphase II stages (2.8 +/- 0.06 pmol vs. 2.6 +/- 0.07 pmol and 2.9 +/- 0.1 pmol vs. 2.3 +/- 0.06 pmol, respectively). The proportion of development to blastocyst was significantly different between haplogroups (22.3 +/- 2.1 % vs. 36.7 +/- 2.9 %). The number of mtDNA molecules per oocyte was highly variable (377 327 +/- 14 104, ranging from 2.0 x 10(3) to 1.2 x 10(6)) but not significantly different between the two haplogroups; significant differences were observed between animals without any apparent relationship to blastocyst production. These data suggest that mitochondria and mtDNA haplogroup affect the developmental capacity of bovine oocytes in vitro.

Adenosine Triphosphate↗
PubMed2004

Modifications in the head group and in the spacer of cholesterol-based cationic lipids promote transfection in melanoma B16-F10 cells and tumours.

A series of four cationic lipids derived from cholesterol was synthesised and their efficiencies to vectorise nucleic acids were compared. The investigation concerns the effects of systematic chemical modifications in the polar head and in the spacer. The cationic lipid molecules used are in the same family of 3beta[N-(N',N',N'-trimethylaminoethane)-carbamoyl] cholesterol iodide (TMAEC-Chol), presenting a spacer of two or three carbons and a quaternary ammonium polar head ramified with methyl or ethyl groups. These lipids formed stable liposomes sizing from 100 to 200 nm when prepared with the colipid dioleoyl phosphatidylethanolamine (DOPE). The goal of this work was to investigate the effect of the chemical structure of these cationic lipids on lipofection. Their ability to form complexes with DNA, their cytotoxicity and their transfection efficiency in vitro and in vivo were studied. Results were compared with those obtained from the well known cholesterol-based cationic lipid DC-Chol. In a melanoma cell line (B16-F10), results showed that either the polar head or the spacer affected the cytotoxicity. Cationic lipids with three ethyl groups in the head are more toxic than those with three methyl groups while cationic lipids with three carbons in the spacer are less toxic than those with two carbons in the spacer. The best transfection level was obtained in vitro and in vivo with cationic lipids having 3C in the spacer. Data indicated that among these lipids, in vivo gene transfer is advantaged by the methylated polar head while in vitro the best level was obtained with the ethylated one. Finally, it was observed that the chemical structure influences the transfection in the presence of serum while the complex charge and the DOPE ratios in liposomes preferentially affect the interaction with erythrocytes. Argumentations are proposed to explain the discrepancies between in vitro and in vivo transfection results concerning the optimal charge ratio and the chemical nature of the cationic lipid head group.

Animals↗
PubMed2003

Transfer into a mesothelioma cell line of tumor suppressor gene p16 by cholesterol-based cationic lipids.

In this work, the tumor suppressor gene p16 was efficiently transferred into FR cells isolated from a patient with malignant mesothelioma using cationic liposomes prepared from trimethyl aminoethane carbamoyl cholesterol (TMAEC-Chol) and triethyl aminopropane carbamoyl cholesterol (TEAPC-Chol). This transfer was performed after preliminary assays were undertaken to find the optimal transfection conditions. Results showed that an efficient transfer of plasmids containing the reporter gene pCMV-beta galactosidase vectorized by TMAEC-Chol/DOPE and TEAPC-Chol/DOPE liposomes into mesothelioma FR cells was obtained as assessed by luminometric measurements of beta-galactosidase activity. Cytotoxicity studied by MTT test showed that at concentrations used for this study, the cationic liposomes have no effect on cell growth. Transfer into mesothelioma FR cells of a plasmid construct containing the tumor suppressor gene p16 was carried out with these liposomes. Western blotting and immunofluorescence showed the presence of p16 in treated cells. An inhibition of cell growth was observed, indicating that efficient tumor suppressor gene transfer can be performed by using cationic liposomes.

Cell Division↗
PubMed2003

Increased sperm mitochondrial DNA content in male infertility.

BACKGROUND: There is increasing evidence that mitochondrial DNA (mtDNA) anomalies in sperm may lead to infertility. Point mutations, deletions and the presence of a specific mtDNA haplogroup have been associated with poor sperm quality, but little attention has been paid to the role of mtDNA content. METHODS: Using density gradient separation and swim-up methods, we selected motile sperm from 32 normal and 35 abnormal sperm samples. The mtDNA/beta-globin gene ratio was determined by real-time quantitative PCR. RESULTS: The average mtDNA/beta-globin ratio of sperm collected from 100% density layers was 1.4 for normal sperm, 6.1 for sperm samples presenting at least one abnormal criterion [among the three criteria established by World Health Organization (1999), i.e. sperm count, motility and morphology], and 9.1 for sperm samples presenting two or more of these abnormal criteria. These differences are very highly significant (P < 0.0001). The mtDNA numbers were also much greater in sperm collected from the 40% density gradient layers (mean: 17.1, P < 0.001), known to contain the most abnormal sperm of the sperm samples, than in those collected from the 100% layers known to contain sperm with the best fertilizing ability. CONCLUSION: Our results showed significant mtDNA amplification in sperm collected from abnormal sperm samples.

Computer Systems↗
PubMed2002

West syndrome and mitochondrial diabetes: relationship or coincidence?

West syndrome occurs in infancy and in early childhood. It is characterized by intractable seizures occurring almost daily, severe psychomotor retardation, poor prognosis and EEG abnormalities, known as hypsarrhythmia. We report here the case of a 28-year-old patient, who was diagnosed with West syndrome when he was 8 months old and with diabetes mellitus when he was 25 years old. Sequencing analyses and restriction analyses were suggestive of mitochondrial diabetes. Four years after the diagnosis of diabetes, this patient's diabetes is still controlled by diet and biguanides.

DNA, Mitochondrial↗
PubMed2002

In vitro and in vivo transfection of melanoma cells B16-F10 mediated by cholesterol-based cationic liposomes.

In vitro and in vivo transgene expression in B16-F10 melanoma cells has been investigated using an original cationic liposome prepared with triethyl aminopropane carbamoyl cholesterol iodide (TEAPC-Chol) as carrier. TEAPC-Chol/DOPE (dioleoyl phosphatidyl ethanolamine) liposomes are unilamellar, very stable and not toxic in the used concentration range. The yield in complexation with plasmid DNA can reach 100% even in the presence of fetal calf serum. The transfection level has been evaluated by luminometric measurements of luciferase expression. With TEAPC-Chol/DOPE (1:1) liposomes, a relatively high transfection level in B16-F10 cells has been observed comparing to commercial reagents. For in vivo assays, the transfection level in tumors induced in Nude mice has been optimized by studying the effects of charge ratio, of the helper lipid and of the injection volume. Results showed that TEAPC-Chol/DOPE (1:1) liposomes have improved 10-fold transfection level versus direct gene transfer of free DNA.

Animals↗
PubMed2002

Analysis of Tg transcripts by real-time RT-PCR in the blood of thyroid cancer patients.

Serum Tg (sTg) assays are sometimes unsatisfactory for monitoring thyroid cancer because interference caused by anti-Tg antibodies may reduce the sensitivity of the tests during thyroid hormone therapy. We have therefore developed a complementary method using real-time quantitative RT-PCR based on the amplification of Tg mRNA. Two different pairs of primers were used for the determination of the frequency of one of the variants of the alternative splicing of Tg mRNA. The frequency of this variant was as high in patients (n = 40) as in controls (n = 30), accounting for about 33% of the total Tg mRNA. Using appropriate primers, we observed that Tg mRNA values in controls varied according to the volume of thyroid tissue and the TSH concentration. The Tg mRNA values allowed the definition of a positive cutoff point at 1 pg/microg total RNA. This cutoff point, tested on the group of patients treated for thyroid cancer, produced fewer false negative results than those obtained with sTg assays. The standardized, highly sensitive real-time RT-PCR technique may therefore prove useful as a complement to sTg assays, particularly for patients with recurrent thyroid cancer receiving T(4) therapy.

Adenocarcinoma, Follicular↗
PubMed2001

A conserved N-terminal sequence targets human DAP3 to mitochondria.

Human DAP3 (death-associated protein-3) has been identified as an essential positive mediator of programmed cell death. Structure-function studies have shown previously the N-terminal extremity of the protein to be required in apoptosis induction. Analysis of human DAP3 gene structure predicted 13 exons and subsequent targeting prediction by two software packages (MITOPROT and TargetP) gave a high probability for mitochondrial targeting. The predicted N-terminal targeting structure was also found in the mouse, Drosophila, and C. elegans orthologues with a strong sequence homology between mouse and human. Secondary structure analyses identified alpha-helical structures typical of mitochondrial target peptides. To confirm experimentally this targeting we constructed a fusion protein with N-terminal human DAP3 upstream of enhanced green fluorescent protein (EGFP). Confocal analysis of transfected human fibroblasts clearly demonstrated EGFP localization exclusive to mitochondria. The positioning of this key apoptotic factor at the heart of the mitochondrial pathway provides exciting insight into its role in programmed cell death.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Mitochondrial activity in XTC.UC1 cells derived from thyroid oncocytoma.

Thyroid oncocytoma is characterized by the presence of oncocytes containing abnormally large numbers of mitochondria. However, the relationship between the abundance of mitochondria and the pathogenesis of the tumors is unknown. Recently, a new cell line, named XTC.UC1, has been derived from a metastasis of thyroid oncocytoma. We have studied the metabolism and the gene expression profile of the mitochondria in XTC.UC1 cells, using B-CPAP cells as controls. There were no signs of mitochondrial respiratory chain defects or uncoupling between the respiratory chain and adenosine triphosphate (ATP) production. In XTC.UC1 cells, mtDNA transcripts were increased more than fivefold than in controls, in parallel with a 3.6-fold increase in mtDNA content. Finally, in spite of the glycolytic metabolism induced by the culture medium, the mitochondria of XTC.UC1 cells possess the phenotype of oncocytic cells with hypertrophic mitochondria, higher respiratory enzyme activity and higher mtDNA content than in controls. XTC.UC1 cells may therefore offer a useful model for investigating the coordination of the nuclear and mitochondrial genomes, in the context of thyroid tumors.

Adenoma, Oxyphilic↗
PubMed2001

Mitochondrial DNA content affects the fertilizability of human oocytes.

Mitochondrial DNA content varies considerably in oocytes, even when collected from the same patient. In the present study, real-time quantitative polymerase chain reaction analysis of 113 unfertilized oocytes obtained from 43 patients revealed an average of 193,000 (range: 20,000 to 598,000) mitochondrial genomes per cell. We compared several groups of oocytes to investigate the relationship between mitochondrial DNA content and fertilizability. The average mitochondrial DNA copy number was significantly lower in cohorts suffering from fertilization failure compared to cohorts with a normal rate of fertilization. In addition, the mitochondrial copy number of oocytes from patients with fertilization failure due to unknown causes was significantly lower than that of oocytes from patients in which IVF failure was due mainly to a severe sperm defect. The lower mtDNA copy number could be due to defective cytoplasmic maturation of oocytes. We conclude that low mitochondrial DNA content, due to inadequate mitochondrial biogenesis or cytoplasmic maturation, may adversely affect oocyte fertilizability.

Adult↗
PubMed2001

Defective mitochondrial ATP synthesis in oxyphilic thyroid tumors.

Oxyphilic tumors (oncocytomas or Hürthle cell tumors) form a rare subgroup of thyroid tumors characterized by cells containing abundant mitochondria. The relationship between the mitochondrial proliferation and the pathogenesis of these tumors is unknown. We have assessed the expression of the mitochondrial ND2 and ND5 (subunits of the nicotinamide adenine dinucleotide dehydrogenase complex) genes and the nuclear UCP2 (uncoupling protein 2) gene in 22 oxyphilic thyroid tumors and matched controls. The consumption of oxygen in mitochondria from tumors was determined by polarography. ATP assays were used to explore the mitochondrial respiratory chain activity and the oxidative phosphorylation coupling in seven fresh thyroid tumors and controls. Adenosine triphosphate synthesis was significantly lower in all the tumors, compared with controls, suggesting that a coupling defect in oxidative phosphorylation may be a cause of mitochondrial hyperplasia in oxyphilic thyroid tumors.

Adenoma, Oxyphilic↗