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Biomedical subjects

P Reynier

Publications and source records attributed to P Reynier.

At least 37 records · Page 2Linked to original sources
PubMed2001

[Multiple sclerosis and Leber's hereditary optic neuropathy mitochondrial DNA mutations].

Multiple sclerosis (MS) has long been known to be associated with Leber's hereditary optic neuropathy (LHON), a disease caused by mitochondrial DNA (mtDNA) mutations. We have investigated the possible involvement of LHON mtDNA point mutations in MS. The study covered a group of 75 unrelated Caucasian patients, with the relapse-remitting or primary progressive form of MS, and a control group of 75 volunteers (matched for age, gender and ethnic origin). Mitochondrial DNA from each subject was examined for 4 primary LHON mutations (at nucleotide positions 3460, 4160, 11778 and 14484) and 7 secondary LHON mutations (at nucleotide positions 4216, 4917, 5244, 7444, 13708, 15257 and 15812) by means of restriction site polymorphism and sequencing techniques. None of the primary LHON mutations were detected in the MS patients or in the controls, whereas the proportion of individuals with secondary LHON mutations was identical (27 p. cent) in the two groups. A combination of 2 or 3 homoplasmic mutations, defining mtDNA haplogroups, was found in the majority of cases. Haplogroups J, T and X were not particularly associated with MS. The frequency of the 13708 mutation alone (haplogroup X), or associated with the 4216 mutation (haplogroup J), was somewhat higher (p=0.059) in the subgroup of MS patients with optic neuritis (ON). ON was the initial symptom in all but one of the patients with haplogroups J or X. No other correlation was found between MS phenotypes and mtDNA genotypes. Our observations confirm previous reports that neither primary nor secondary LHON mutations are involved in the development of MS. However, MS patients with haplogroups J or X appear to have a moderately higher risk of developing optic neuritis. Thus, a specific mtDNA background may be a predisposing genetic factor for optic nerve damage in MS patients.

Adult↗
PubMed2000

Dietary fat clearance in type V hyperlipoproteinaemia secondary to a rare variant of human apolipoprotein E: the apolipoprotein E3 (Arg 136-->Ser)

This present case report describes two siblings with severe type V hyperlipoproteinaemia, diagnosed very early in life and due to the combination of the common apolipoprotein (Apo) E2 allele and rare mutant variant of ApoE, ApoE3 (Arg 136-->Ser). Phenotyping of ApoE falsely identified E2/E2 phenotype. The presence of mutated ApoE was suspected on an unusual restriction polymorphism of a Hha 1 restriction site and confirmed by sequence analysis of the cloned polymerase chain reaction fragment of exon 4 and familial segregation study. The severity of the hypertriacylglycerolaemia was modulated by the lipid content of the diet. A low-fat diet enriched in medium-chain triacylglycerol (TAG) decreased but did not normalize plasma TAG levels in both affected patients of the pedigree. A standardized lipid-enriched test meal showed a marked impairment of TAG-rich lipoprotein (TRL) clearance, especially the exogeneous TRL bearing ApoB-48 which still represented 79% of total TRL 7 h after the fat load. Finally, differences between the male and female siblings with the existence of a consanguine relationship in their parents suggested the involvement of other genetic factors in modulating the severity of phenotypic expression. This observation reinforces the usefulness of genotyping of ApoE for the characterization of genetic hypertriacylglycerolaemia and selection of the appropriate diet and treatment.

Adult↗
PubMed2000

No evidence of thyrotropin receptor and G(s alpha) gene mutation in high iodine uptake thyroid carcinoma.

Usually, thyroid carcinoma presents as a cold nodule on radioiodine scintigraphy. High-uptake nodules on iodine thyroid scans are associated with an exceedingly low incidence of malignancy. Only 29 cases of carcinomas appearing as hot or warm nodules have as yet been reported. From 1993 to 1999, we have observed eight similar cases (4 hot and 4 warm thyroid nodules) suggesting that thyroid carcinomas may not be as rare as usually considered in these circumstances. Four tumors were available for molecular analysis on paraffin-embedded sections. Because no mutations were found in the whole coding portions of thyrotropin-receptor (TSH-R) gene and fragments encompassing the mutational hot spots of the G(s alpha) gene, it is unlikely that activating mutations of the TSH-R or G(s alpha) genes were involved in these carcinomas.

Adenocarcinoma, Follicular↗
PubMed2000

Long-lasting hyperalgesia induced by fentanyl in rats: preventive effect of ketamine.

BACKGROUND: It has been reported that mu-opioid receptor activation leads to a sustained increase in glutamate synaptic effectiveness at the N-methyl-D-aspartate (NMDA) receptor level, a system associated with central hypersensitivity to pain. One hypothesis is that postoperative pain may result partly from the activation of NMDA pain facilitatory processes induced by opiate treatment per se. The authors tested here the effectiveness of the opiate analgesic fentanyl for eliciting a delayed enhancement in pain sensitivity. METHODS: The consequences of four bolus injections (every 15 min) of fentanyl (20-100 microg/kg per injection, subcutaneously) on immediate (for several hours) and long-term (for several days) sensitivity to nociceptive stimuli in the rat (paw-pressure vocalization test) were evaluated. The effects of the combination of the NMDA-receptor antagonist ketamine (10 mg/kg, subcutaneously) with fentanyl also were assessed. RESULTS: Fentanyl administration exhibited a biphasic time-dependent effect: first, an early response (for 2-5 h) associated with a marked increase in nociceptive threshold (analgesia), and second, a later response associated with sustained lowering of the nociceptive threshold (5 days for the longest effect) below the basal value (30% of decrease for the maximal effect) indicative of hyperalgesia. The higher the fentanyl dose used, the more pronounced was the fentanyl-induced hyperalgesia. Ketamine pretreatment, which had no analgesic effect on its own, enhanced the earlier response (analgesia) and prevented the development of long-lasting hyperalgesia. CONCLUSIONS: Fentanyl activates NMDA pain facilitatory processes, which oppose analgesia and lead to long-lasting enhancement in pain sensitivity.

Analgesics, Opioid↗
PubMed1999

mtDNA haplogroup J: a contributing factor of optic neuritis.

Optic neuritis frequently occurs in multiple sclerosis (MS), and shares several similarities with the optic neuritis of Leber's hereditary optic neuropathy (LHON), which is mainly due to maternally transmitted mitochondrial DNA (mtDNA) mutations. Our report shows for the first time that a mitochondrial DNA background could influence the clinical expression of MS. One European mtDNA haplogroup was found only in MS patients with optic neuritis but not in MS patients without visual symptoms. Therefore, we hypothesize that mtDNA haplogroup J might constitute a risk factor for optic neuritis occurrence when it is coincidentally associated with MS, but not be a risk factor for developing MS per se as suggested previously.

Alleles↗
PubMed1998

Long PCR analysis of human gamete mtDNA suggests defective mitochondrial maintenance in spermatozoa and supports the bottleneck theory for oocytes.

The long PCR and the Southern blot techniques were used to study mitochondrial DNA (mtDNA) in 94 sperm samples, and in 35 oocytes collected from 12 women. The sperm samples were classified in two sets: 37 samples from normal subjects, and 57 samples from patients with oligoasthenospermia. In both sets, most of the spermatozoan mitochondria had multiple mtDNA deletions. The rate of mtDNA mutation, which appears unexpectedly high, considering the short life span of the spermatozoa, may be due to impaired maintenance during differentiation. In contrast, despite the long life span of oocytes and the extended meiotic period, oocyte mitochondria showed few mtDNA rearrangements. However, mitochondria in oocytes from a given donor revealed considerable mutational heterogeneity. This supports the bottleneck theory of rapid segregation of mtDNA genotypes during early oogenesis. The long PCR technique, which allows analysis of the entire mitochondrial genome, provides new information on mtDNA instability in human gametes. Our findings suggest that mtDNA maintenance differs in the two types of gametes.

Base Sequence↗
PubMed1997

Cloning and initial characterization of human and mouse Spot 14 genes.

The intricate regulation of Spot 14 expression in rat lipogenic tissues has provided a useful tool in studying nutritional and hormonal factors involved in transcription. To gain insight into its function and its possible involvement in human lipid disorders, we cloned human and mouse Spot 14 genes that shared with the rat gene a strong homology concerning the deduced amino acid sequence (81 and 94%, respectively) as well as the promoter region. The mouse promoter was characterized by transfection studies, while quantitative RT-PCR and in situ hybridization experiments showed that Spot 14 is expressed in human liver and, at a high level, in multiple symmetric lipomatosis nodules.

Amino Acid Sequence↗
PubMed1997

Male infertility associated with multiple mitochondrial DNA rearrangements.

Male sterility results from a number of characterized exogenous or genetic dysfunctions preventing normal differentiation into mobile spermatozoa. This may now be overcome by intra cytoplasmic sperm injection (ICSI). This practice does not require mobile, or even mature spermatozoa for in vitro fecondation. However, a functional respiratory chain, partly encoded by the mitochondrial DNA (mtDNA), is required for the mobility of the spermatozoa. We report the case of an infertile patient who wished to procreate. ICSI was proposed but he displayed multiple mtDNA deletions of possible nuclear origin in the spermatozoa and in the deltoid muscle. Even though mtDNA is maternally inherited, the possibility of a nuclear-driven mutation affecting the integrity of the mtDNA should be taken into account when ICSI is to be performed. Together with recent genetic in vitro manipulations in mammals, our data point to the importance of studying the mtDNA structure in human spermatozoa, and the potential risks of these non-natural practices for procreation.

Adult↗
PubMed1997

Oligoasthenospermia associated with multiple mitochondrial DNA rearrangements.

A patient who wished to be treated for infertility by intracytoplasmic sperm injection (ICSI) was referred to our group for assessment. Upon clinical examination, a ptosis (partial closure of the eyelid) was noted, and histology revealed ragged red fibres in the skeletal muscle. Southern blot analysis of spermatozoa and skeletal muscle revealed the presence of multiple mitochondrial DNA deletions. This kind of rearrangement may be of nuclear origin since three nuclear loci have been ascribed to multiple mitochondrial DNA deletions in humans. Since mitochondrial DNA is maternally transmitted, the use of ICSI was feasible. However, an alteration of nuclear gene product affecting the integrity of mitochondrial DNA, and thus sperm mobility, might be transmitted to the offspring with the risk of developing a mitochondrial DNA disease.

Adult↗
PubMed1996

Quantitative multistandard RT-PCR assay using interspecies polymorphism.

The use of RT-PCR to quantify mRNA is often compromised by the variability of reverse-transcription and amplification reactions as well as by the difficulty of assessing the amount and/or the integrity of input RNA. Use of a competitor RNA or the coamplification of an endogenous standard are widespread methods of monitoring these steps. Taking advantage of both sequence conservation between homologous genes in related animal species and interspecific polymorphism, a protocol that may be regarded as a compromise between these two methods is described here. Total RNA samples, extracted from even minute amounts of tissue belonging to a first animal species, were supplemented with a constant amount of total RNA prepared from a second animal species, which thus acts as a multistandard source. The mixture was reverse-transcribed using hexa-random primers. Separate PCRs were then undertaken so that, for each mRNA of interest, products from both origins could be distinguished. Since the ratio between amplified mRNAs is constant in the standard preparation, an accurate normalization in the assay samples of most variations inherent to PCR is obtained. This protocol allows quantification of several mRNAs species, whose amounts may be very different, in a single cDNA preparation.

Actins↗
PubMed1996

Impact of various handling and storage conditions on quantitative detection of hepatitis C virus RNA.

BACKGROUND/AIMS: Both HCV RNA viral load and HCV genotype have been described as important predicting factors determining the response to interferon in chronic hepatitis C. To investigate whether processing and storage conditions might influence the stability and could alter the concentration of the HCV RNA in serum, quantification of HCV RNA was performed by branched DNA assay. METHODS: We studied serum samples obtained from seven patients with histologically proven chronic hepatitis C. These were subjected to the following physical conditions: (1) immediate quantification, (2) storage at room temperature for 5 days, (3) storage at 4 degrees C for 5 days, (4) storage at -20 degrees C for 5 days, (5) storage at -80 degrees C for 5 days, (6) five freeze-thaw cycles, (7) blood unspun for 4 h at room temperature then centrifuged and stored at -80 degrees C for 5 days, (8) storage at 4 degrees C for 6 months, (9) storage at -20 degrees C for 6 months, (10) storage at -80 degrees C for 6 months. RESULTS: A loss of 100% HCV RNA titers was observed after storage at RT for 5 days and then storage at 4 degrees C for 6 months. A surprising decrease of HCV RNA titer (15.6%) was observed in sera stored for 5 days at -20 degrees C. Five freeze-thaw cycles resulted in a 16% decrease of the HCV RNA level. When centrifugation was performed after a 4 h delay at room temperature, a significant loss of HCV RNA titers of 29.5% was observed. Long-term stability (6 months) was observed at -80 degrees C with a slight loss of about of 10% HCV RNA titers, but a significant decrease in HCV RNA of 23% was observed at -20 degrees C. The reproducibility of the bDNA assay on five patient samples was performed eight times in duplicate and showed an average coefficient of variation of 9.1%. CONCLUSIONS: These data confirm the importance of storage and handling in measuring the amount of HCV RNA in clinical samples.

Blood Preservation↗
PubMed1995

Accumulation of deletions in MtDNA during tissue aging: analysis by long PCR.

Multiple deletions of mtDNA have not only been implicated in aging, but also in a wide variety of pathological conditions. The enzyme system used in long-PCR makes it possible to synthesize the entire mitochondrial genome (16.5 kb), exposing the multiple deletions in mtDNAs implicated in and, at least partially, responsible for these pathologies. But it is not the number or type of anomalous mtDNA that is crucial, rather it is their frequency relative to the number of intact copies of the mitochondrial genome. Our work exposes the necessity of quantitating the number of normal mitochondrial DNAs. The accuracy of the technique and the small sample size required permit one to detect multiple deletions, located in a specific organ, and simultaneously measure the fraction of intact molecules. This fraction can then be correlated with mitochondrial dysfunction to serve both as an indicator of tissue aging and a monitor of an impending myopathy.

Aged↗
PubMed1994

Multiple deletions of the mitochondrial DNA in polymyalgia rheumatica.

We analyzed the mitochondrial DNA of patients with polymyalgia rheumatica, a disease frequently associated with mitochondrial myopathy. In an attempt to study the deletions, we have developed a qualitative PCR method using a highly thermostable polymerase in order to amplify multiple mitochondrial DNA large fragments (up to 12 kb). PCR serves to observe both deleted and normal fractions of the mitochondrial DNA. We found multiple deletions of the mitochondrial DNA in all of the patient muscles. Although these muscles harbored many ragged red fibers, we found no point mutations of the tRNA(Leu)(UUR)) and the mutation at nucleotide position 8344 was not present.

Aged↗
PubMed1994

Association of deletion and homoplasmic point mutation of the mitochondrial DNA in an ocular myopathy.

The mitochondrial DNA of a 41 year old patient with ocular myopathy was explored. We found a deletion of 3540 base pair in about 50% of the mitochondrial genomes associated with a homoplasmic point mutation. The mutation at nucleotide pair 7444 converts stop codon AGA into lysine codon AAA (human mitochondrial genetic code). The synergistic effect between two point mutations has already been described in mitochondrial pathology but this is the first time that an association between a deletion and a point mutation is shown.

Adult↗
PubMed1993

Isolation and characterization of a repetitive DNA sequence from Leishmania infantum: development of a visceral leishmaniasis polymerase chain reaction.

To construct a DNA probe specific for protozoa that cause visceral leishmaniasis, we cloned Pst I fragments of Leishmania infantum genomic DNA into a Bluescript II SK vector. A clone of 4.3 kb that contained a highly repetitive sequence was isolated and cut with three restriction enzymes: Hae III, Rsa I, and Sau 3A. After a new molecular cloning step, we isolated and sequenced a 140-basepair (bp) fragment. Two oligonucleotides were synthesized to be used as primers for a polymerase chain reaction. Using this probe, we detected an amount of DNA equivalent to one promastigote of L. infantum. This probe showed a high specificity; all protozoa tested that cause visceral leishmaniasis and L. major (one of the causative agents of Old World cutaneous leishmaniasis) showed a 100-bp amplified sequence, whereas other Leishmania strains showed a signal of a different size or else no signal. Moreover, no amplified sequence was obtained with other pathogenic parasites tested (Trypanosoma brucei, T. cruzi, Plasmodium falciparum, Pneumocystis carinii, and Toxoplasma gondii).

Animals↗
PubMed1991

[Ocular toxoplasmosis. Comparison between two biological methods to study aqueous humor].

To diagnose ocular toxoplasmosis with certainty is often difficult and requires anti Toxoplasma gondii antibodies screening in the aqueous humour. We evaluated the results obtained in 30 controls and 5 patients investigated, using the ELISA and indirect immunofluorescence techniques to determine Desmonts's C coefficient and the sera/aqueous humour densities ratio obtained with ELISA. None of the chorioretinitis-free controls showed a false positive result. Negative serum antibodies levels indicated an absence of ocular toxoplasmosis. The sensitivity of this method depends on several biological parameters, notably the extent of ocular inflammation. Evaluating the C coefficient by the ELISA technique increases this sensitivity.

Albumins↗