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Biomedical subjects

F Vincent

Publications and source records attributed to F Vincent.

At least 55 records · Page 3Linked to original sources
PubMed1999

A simple procedure for polymerase chain reaction of the PSBA gene in algae: application to the screening of mutations conferring atrazine resistance and discrimination of natural populations of Porphyra linearis.

A simplified procedure is described for polymerase chain reaction (PCR) of a partial sequence (bp 601-893) of the plastid gene psbA in the rhodophyte Porphyra linearis and the diatoms Haslea ostreria and Skeletonema costatum. This procedure involves the use of all tissues of P. linearis and live cell suspensions of H. ostreria or S. costatum, as DNA templates, without any further purification of DNA. As in the case of PCR with DNA extracts, a single major band of the expected size (292 bp) was obtained after PCR for the three species. Sequences of the amplified fragments were aligned, confirming that the amplified products were part of the psbA gene. The method was then used to screen mutations in partial psbA genes of 23 samples of P. linearis collected at four different stations along the mid-Atlantic coast of France. An alignment was obtained indicating the existence of mutations, though not in codons known for herbicide resistance. All mutations found were silent. However, genetic polymorphism discriminated between samples collected from two stations. The method employed allows rapid amplification of the herbicide target gene and simplifies the procedure for screening mutations or populations in algae. Its application to other genes and species is considered.

Atlantic Ocean↗
PubMed1999

Thioridazine lengthens repolarization of cardiac ventricular myocytes by blocking the delayed rectifier potassium current.

Proarrhythmia has been observed with the antipsychotic agent thioridazine (THIO). The mechanisms underlying these effects are unknown. The objectives of this study were 1) to characterize the effects of THIO on cardiac repolarization and 2) to determine whether lengthening of the Q-T interval could be explained by blocking major K+-repolarizing currents. Isolated, buffer-perfused guinea pig hearts (n = 32) were stimulated at various pacing cycle lengths (150-250 ms) and exposed to THIO at concentrations ranging from 300 nM to 3 microM. THIO increased monophasic action potential duration at 90% repolarization (MAPD90) in a concentration-dependent manner from 14.9 +/- 1.8 at 300 nM to 37.1 +/- 3.2 ms at 3 microM. Increase in MAPD90 was also reverse frequency-dependent; THIO (300 nM) increased MAPD90 by 14.9 +/- 1.8 ms at a pacing cycle length of 250 ms, but by only 7.7 +/- 1.2 ms at a pacing cycle length of 150 ms. Patch-clamp experiments demonstrated that THIO decreases the time-dependent outward K+ current elicited by short depolarizations (250 ms; IK250) in a concentration-dependent manner. Estimated IC50 for IK250, which mostly underlies IKr, was 1.25 microM. Time-dependent outward K+ current elicited in tsA201 cells expressing high levels of HERG protein was also decreased approximately 50% by 1.25 microM THIO. On the other hand, THIO was less potent (IC50 of 14 microM) to decrease time-dependent K+ current elicited by long pulses (5000 ms; IK5000). Under the latter conditions, IK5000 corresponds mainly to IKs. Thus, these results demonstrate block of K+ currents and lengthening of cardiac repolarization by THIO in a concentration-dependent manner. This may provide an explanation of Q-T prolongation observed in some patients treated with THIO.

Action Potentials↗
PubMed1999

[Simplified electroretinography protocol and diagnosis of retinal dystrophies in children].

PURPOSE: To report results of a simplified electroretinogram in children. PATIENTS: 124 children under 6 years of age with nystagmus, blindness, neurological disease, cone rod dystrophy in the family, or abnormal fundus appearance were examined. METHOD: The electroretinogram was recorded by corneal electrodes in an awake state without sedation. A light-emitting orange diode stimulator was used. Stimulation was performed subsequently after 3 minutes of light adaptation and after 8 minutes of darkness. If the electroretinogram was abnormal, a second recording was done a few weeks later using the same method. In some cases, the children were re-examined, and a ganzfeld stimulation ERG was recorded. RESULTS AND DISCUSSION: This method allowed a reliable diagnosis of photoreceptor dystrophy: Leber's congenital amaurosis with or without rare metabolic diseases, X link pigmentary retinopathy or retinal dystrophy with general disease in children. It helped to differentiate isolated retinal pigment changes with normal electroretinogram from functional retinal impairment and rod-cone dystrophy from other retinal dystrophies.

Adaptation, Ocular↗
PubMed1999

[Acute respiratory Chlamydia pneumoniae infections in adults. Value of direct gene amplification diagnosis].

Chlamydia pneumoniae has been established recently as an important human respiratory pathogen. The aim of this study was to define the incidence of Chlamydia pneumoniae in acute respiratory infections by evaluating its presence in posterior nasopharyngeal aspirates or broncho-alveolar lavage specimens by polymerase chain reaction-hybridization (PCR-EIA) as well as the titres of specific antibodies in serum by a rELISA test and a micro-immunofluorescence (MIF) test. 68 adults patients were investigated. Eight patients (11.8%) were positive by either rELISA or PCR-EIA or both, with an infection rate of 5 patients with community-acquired pneumonia, 2 asthmatic patients and 1 patients with an exacerbation of chronic obstructive pulmonary disease. Serological evidence of acute infection was found in four of these patients with rELISA test and in three others with MIF test. PCR-EIA detected Chlamydia pneumoniae DNA in four patients, but there were concordant results with rELISA and PCR-EIA in only one patient. In conclusion, Chlamydia pneumoniae appears to be a common etiologic agent of acute respiratory infections in adults. The discrepancy between serological test and PCR-EIA results reflects the difficulties in routine laboratory diagnosis of Chlamydia pneumoniae. The ambiguous results of serological tests from a single serum sample assess the utility of PCR for prompt diagnosis. When PCR is negative or no feasible, a second serology to 15/21 days of interval is necessary. Further studies with optimised techniques must be developed.

Acute Disease↗
PubMed1999

p53-independent regulation of cyclin B1 in normal human fibroblasts during UV-induced G2-arrest.

Recently we demonstrated, using normal human fibroblasts (NHFs), that UVc radiation induces a G2/M arrest which was even more pronounced when p53 expression was inhibited. So, the aim of this study was to evaluate in NHFs the relationship between UV-induced G2/M arrest and cyclin B1 regulation and to investigate if p53 could contribute to the cyclin B1 regulation in these conditions. Following exposure of asynchronous NHFs to UV light, we showed that the induced G2/M arrest was accompanied by a dose-dependent down-regulation of cyclin B1 mRNA as evaluated by RT-PCR. Concomitantly, using flow cytometric analysis, we observed a strong accumulation of cyclin B1 protein which was correlated to the apparition of the G2/M arrest. In order to study the contribution of p53 to the cyclin B1 accumulation in response to UV exposure, we inhibited p53 induction using p53 antisense oligonucleotides. We found that the inhibition of p53 protein induction after UV exposure had no effect on the level of cyclin B1 mRNA. Moreover, although inhibition of p53 protein induction increased the number of the cells in the G2-M phase, the mean content of cyclin B1 protein was not augmented in these cells. These results indicate clearly that the induction of p53 protein following UV exposure does not regulate the level of cyclin B1 mRNA or protein in normal cells.

Cell Culture Techniques↗
PubMed1998

hSmad5 gene, a human hSmad family member: its full length cDNA, genomic structure, promoter region and mutation analysis in human tumors.

hSmad (mothers against decapentaplegic)-related proteins are important messengers within the Transforming Growth Factor-beta1 (TGF-beta1) superfamily signal transduction pathways. To further characterize a member of this family, we obtained a full length cDNA of the human hSmad5 (hSmad5) gene by rapid amplification of cDNA ends (RACE) and then determined the genomic structure of the gene. There are eight exons and two alternative transcripts; the shorter transcript lacks exon 2. We identified the hSmad5 promoter region from a human genomic YAC clone by obtaining the nucleotide sequence extending 1235 base pairs upstream of the 5' end of the cDNA. We found a CpG island consistent with a promoter region, and we demonstrated promoter activity in a 1232 bp fragment located upstream of the transcription initiation site. To investigate the frequency of somatic hSmad5 mutations in human cancers, we designed intron-based primers to examine coding regions by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis. Neither homozygous deletions or point mutations were found in 40 primary gastric tumors and 51 cell lines derived from diverse types of human cancer including 20 cell lines resistant to the growth inhibitory effects of TGF-beta1. These results suggest that the hSmad5 gene is not commonly mutated and that other genetic alterations mediate the loss of TGF-beta1 responsiveness in human cancers.

Alternative Splicing↗
PubMed1998

cDNA cloning and expression analysis of flounder p53 tumour suppressor gene.

The screening of a flounder cDNA library with a rainbow trout p53 probe allowed the isolation of a 2.8-kb fragment homologous to human (50%) and rainbow trout (57%) p53 coding sequences. The fragment contains a single open reading frame coding for a 366-amino acid protein. The predicted amino acid sequence is relatively divergent from other p53 proteins but it displays the main p53 features: five highly conserved domains, an acidic N-terminus, a hydrophilic and charged C-terminus, a penultimate serine residue and a putative nuclear localization signal. Furthermore, conservation of critical amino acids and comparable distribution of charge and hydrophobicity suggest that flounder p53 properties could be similar to those in mammals. Northern blot analysis revealed a single transcript of about 3 kb in the flounder ovary tissues. In fact, RT-PCR showed an ubiquitous but very low expression of p53 gene in all flounder tissues.

Amino Acid Sequence↗
PubMed1998

Production of a polyclonal antibody raised against recombinant flounder p53 protein.

The cDNA encoding the wild type p53 protein from flounder, Platichthys flesus, was expressed in Escherichia coli using the GST fusion protein system. Several milligrams of recombinant p53 protein were purified. This protein displayed an apparent molecular weight of 45,000 Da, a value which is very similar to Xenopus p53, but significantly greater than was expected based on the length of the open reading frame. Immunization of rabbits against purified p53 protein allowed the production of high titre polyclonal antiserum. This new polyclonal antibody recognized recombinant flounder p53 protein in Western blot. Cross reaction was also observed with recombinant Xenopus p53 protein but not with human p53 protein. Immunoblotting of the total protein extract from normal flounder ovaries did not reveal any p53 expression.

Animals↗
PubMed1998

Comparison of two serological methods and a polymerase chain reaction-enzyme immunoassay for the diagnosis of acute respiratory infections with Chlamydia pneumoniae in adults.

Chlamydia pneumoniae is a common respiratory tract pathogen. Serological methods currently used for the diagnosis of C. pneumoniae infection lack specificity, give ambiguous results from a single serum sample and often provide only a retrospective diagnosis. A prospective study was undertaken to assess whether PCR could be a useful addition to the serological techniques routinely practised for diagnosis. This study investigated 68 adult patients with a diagnosis of acute respiratory infection. Acute and convalescent serological determination of antibodies to C. pneumoniae were performed by means of an rELISA test and a micro-immunofluorescence (MIF) test. Nasopharyngeal aspirates or bronchoalveolar lavage specimens and bronchial aspirates obtained from the 68 patients were evaluated by PCR-enzyme immunoassay (PCR-EIA) for the presence of C. pneumoniae and by immunofluorescence assay and cell culture for virus identification. Mycoplasma pneumoniae serology was also performed. Eight patients (11.8%) were positive by either rELISA or PCR-EIA, or both, with an infection rate of 5 (18.5%) of 27 in patients with community-acquired pneumonia, 2 (9%) of 22 in asthmatic patients and 1 (5%) of 19 in patients with an exacerbation of chronic obstructive pulmonary disease. Serological evidence of acute infection was found in four of these patients with the rELISA test and in three others with the MIF test. PCR-EIA detected C. pneumoniae DNA in four specimens, but there were concordant results with both rELISA and PCR-EIA in only one patient A positive PCR-EIA was also obtained in a patient who did not show an antibody response in acute serum. The discrepancy between serological and PCR-EIA results reflects the difficulties in routine laboratory diagnosis of C. pneumoniae infection and the necessity for further studies with optimised techniques.

Acute Disease↗
PubMed1998

Two cases of ras mutation associated with liver hyperplasia in dragonets (Callionymus lyra) exposed to polychlorinated biphenyls and polycyclic aromatic hydrocarbons.

ras gene activation in a naturally exposed feral fish population may prove to be a particularly sensitive genetic marker of malignancy. The aim of this study was to relate our current knowledge of polychlorinated biphenyl (PCB) bioactivation and polycyclic aromatic hydrocarbon (PAH)-induced DNA damage to ras gene activation in liver tumors from dragonets exposed to these environmental carcinogens. We identified a member of the ras gene family in the marine fish dragonet (Callionymus lyra). The first two exons of this new sequence showed a very high degree of homology with the human ras genes (81-86%) at the nucleic acid level and perfect homology at the amino-acid level. In a pilot study, we collected dragonets from the Seine estuary, an area highly contaminated with PAHs and PCBs. An increase in DNA adducts and an accumulation of ortho- and non-ortho-substituted chlorobiphenyls (CB-77, CB-126, and CB-169) were observed in the livers, a finding that correlates well with the levels of PAHs and PCBs in the sediment. Although liver neoplasia was uncommon, a codon 11 mutation was found in two fish with liver cell hyperplasia, suggesting a possible correlation between hepatic precancerous lesions and ras gene activation in dragonets.

Animals↗
PubMed1997

Mutation analysis of the transforming growth factor-beta type II receptor in human cell lines resistant to growth inhibition by transforming growth factor-beta.

The transforming growth factor-beta (TGF-beta) binds the type II TGF-beta growth factor receptor (RII) to inhibit the growth of most epithelial tissues. Most human colon and gastric cancers with microsatellite instability (MI) have frameshift mutations in polynucleotide repeats within the RII coding region; these mutations truncate the receptor protein and disable the serine/threonine kinase to produce TGF-beta resistance. To further investigate the type, frequency and tissue distribution of RII mutations, we selected 24 human cancer cell lines from various tissues which were previously reported to be resistant to the inhibitory effects of TGF-beta. We developed protocols for non-isotopic SSCP analysis of PCR products from genomic DNA samples, and we tested them for microsatellite instability. PCR-SSCP analysis followed by DNA sequencing identified deletion mutations in the exon 3 poly-adenine tract in three colon tumor cell lines: LS174T and SW48 had a single base deletion and LS411 had a two base deletion. Among the 24 previously unreported cell lines, only these three demonstrated microsatellite instability. These and other recent data indicate that RII mutations are essentially confined to colon and gastric cancers with microsatellite instability. The narrow spectrum of tissues containing RII mutations illustrates the complexity of genetic checkpoints in human carcinogenesis.

Cell Division↗
PubMed1997

[Combination of induction chemotherapy with surgery and/or radiotherapy in locally advanced head and neck cancers. A retrospective analysis of a series of 125 patients].

The prognosis of locally advanced cancers of the head and the neck is pejorative, particularly when nodal involvement is present. In order to improve local control and to reduce distant failures, we have treated stages III and IV patients with induction chemotherapy. From May 1986 to November 1992, 125 patients with squamous cell carcinoma of the head and neck were treated by induction chemotherapy: cisplatine (100 mg/m2 at J1) and 5FU (1 g/m2 from J1 to J5 in continuous infusion) every 21 days subsequent local therapy consisted of surgery for patients with resectable disease, and/or radiotherapy. One hundred and nineteen patients were assessable (110 men and 9 women) with a median age of 57 years (range: 36-78). All patients had performance status inferior or equal to 2. According to the TNM of UICC classification 50 patients were stage IV (42%), 61 stage III (51%), 7 stage II (6%) and a stage I (1%). One hundred (84%) patients have received at least 3 cycles of chemotherapy. Seventy-four patients (62%, IC: 60.4-63.5) had clinical objective response (complete response (CR) or partial response (PR)) with 24 patients (20%) CR and 50 patients (42%) PR. Local therapy included surgery in 81 patients (68%) and radiotherapy alone in 42 patients (35%). Overall, 103 patients (87%) were rendered clinically disease-free by treatment on this protocol. The toxicities of cisplatine and 5-FU chemotherapy consisted predominantly of myelosuppression (5%) and renal toxicities (4%) and were moderate as described for this combination. At a median follow-up of 32 months, the median survival is 38 months (CI 95%, 18-54 months), and the median time to progression is 62 months. The oropharynx localization reached statistical significance for survival rates (Log-rank test, p = 0.02).

Adult↗
PubMed1997

[Pheochromocytoma mimicking septic shock].

A tumour arising from chromaffin cells, the pheochromocytoma may be associated or present with hypotension and result in a lethal shock. The authors report a rare case of pheochromocytoma simulating septic shock. They review the mechanisms by which hypersecretion of catecholamines may cause shock. The detection of pheochromocytoma in its atypical presentation is essential for early diagnosis and the reduction of mortality related to this condition.

Adrenal Gland Neoplasms↗
PubMed1997

[Relationships between p53 induction, cell cycle arrest and survival of normal human fibroblasts following DNA damage].

It is now well established that in response to genotoxic stresses mammalian cells show an increased p53 protein levels and undergo cell cycle arrest at G1/S and G2/M checkpoints. But, the consequences of these cell cycle arrests on cell survival are not yet elucidated. In this study, we have analysed the relationships between p53 protein induction, cell cycle arrest and cell survival following exposure of normal human fibroblasts (NHFs) to various genotoxic agents such as cisplatin, UV radiation and gamma radiation. p53 protein accumulation and G2/M arrest arose at the same time following exposure to DNA damaging agents, suggesting that p53 is responsible for the G2/M block. However, following inhibition of p53 induction by an antisense oligonucleotide, this G2/M arrest is even more important and correlates with an enhanced sensitivity of NHFs to UV radiation. In addition, there appears to be a threshold in the response of NHFs to DNA damaging agents, p53 induction and cell cycle arrest being observed only with lethal UV doses. We show that: 1) there appears to be a threshold in the cellular response to genotoxic agents, below which neither p53 induction, nor cell cycle arrest, nor cell survival alteration occur and beyond which p53 induction is accompanied by cell cycle arrest and decreased cell survival; 2) although there is a tight temporal relationship, the onset of which depends of the DNA damaging agent used, between the start of p53 induction and the occurrence of G2/M arrest, this latter is independent of p53; 3) p53 inhibition enhances NHFs' sensitivity to DNA damaging agents, the extent of the G2/M arrest correlating with decreased cell survival. Finally, the lack of obligatory correlation between p53 inactivation, apoptosis and radio- or chemoresistance is discussed.

Apoptosis↗
PubMed1996

Mutation analysis of the transforming growth factor beta type II receptor in sporadic human cancers of the pancreas, liver, and breast.

The transforming growth factor beta (TGF beta) binds the type II TGF beta growth factor receptor (TGF beta RII) to inhibit growth of most epithelial tissues. Most human colon cancers with microsatellite instability have frameshift mutations in two microsatellites within the TGF beta RII coding region; such mutations truncate the receptor to produce resistance to TGF beta. To investigate this pathway in other tissues, we surveyed sporadic human cancers of the pancreas, liver and breast to determine the frequency of microsatellite mutations in the TGF beta RII. We amplified genomic DNA segments containing two microsatellites plus 72% of domain XI of the serine-threonine kinase region. SSCP analysis showed no evidence of mutation in 32 sporadic cancers (12 pancreas, 10 liver, and 10 breast). We conclude that microsatellite mutations in TGF beta RII are uncommon in sporadic tumors of the pancreas, liver and breast.

Base Sequence↗