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C Coutelle

Publications and source records attributed to C Coutelle.

At least 91 records · Page 5Linked to original sources
PubMed1994

Cell binding and internalization by filamentous phage displaying a cyclic Arg-Gly-Asp-containing peptide.

Ligands that bind mammalian cell surface integrins with high affinity can mediate cellular internalization. We show that particles of the bacteriophage fd that display the cyclic integrin-binding peptide sequence GGCRGDMFGC in a proportion of their major coat protein subunits bind to cells and are efficiently internalized. In the displayed peptide the conformation of the RGD motif is restricted within a hairpin loop formed by a disulfide bridge between the 2 cysteine residues. Cellular internalization of phage was demonstrated by confocal and non-confocal immunofluorescence microscopy of tissue-cultured cells incubated with phage particles. The phage were contained in juxtanuclear vesicles in the same serial sections as transferrin receptor but were not colocalized with the cell surface marker alkaline phosphatase. Cell binding and internalization was inhibited by preincubation of cells with the integrin-binding peptide GRGDSP, whereas the control peptide GRGESP had no inhibitory effect. These results indicate that cyclic integrin-binding peptides can be used to target and enter cells and that it should be possible to exploit such peptides for the introduction of DNA, drugs, or other macromolecules.

Amino Acid Sequence↗
PubMed1994

Identification of three novel mutations in the CFTR gene using temperature-optimized non-radioactive conditions for SSCP analysis.

Optimal temperature conditions for the detection of 28 known mutations on 15 exons of the human cystic fibrosis transmembrane conductance regulator gene by single strand conformation polymorphism analysis using the Diagen TGGE Apparatus were established. This procedure was applied to the detection of unknown mutations in 58 non-deltaF508 chromosomes. Three novel mutations, -471del3 (5' flanking region), 3171insC (exon 17a) and 4700(T)8/9 (3' non-translated region) of the CFTR gene were found. Mutation 3171insC occurred in conjunction with the delta F508 mutation on the other allele of a child presenting with severe pathology. Mutation -471del3 has so far only been found in one healthy individual and her father, and 4700(T)8/9 is a DNA sequence polymorphism.

Base Sequence↗
PubMed1994

Acceptability of carrier screening for cystic fibrosis during pregnancy in a German population.

A pilot project offering voluntary heterozygote screening for the delta F508 mutation causing cystic fibrosis (CF) to 638 pregnant women attending two antenatal clinics in the eastern part of Berlin was carried out from 1990-1993. Participation was invited using an information leaflet and inclusion in the study was conditional on written informed consent. Of those invited to participate, only one refused to be tested, on the grounds of non-acceptance of prenatal diagnosis. Eighteen pregnant women were identified as carriers of the delta F508 mutation. All of them and their male partners accepted counselling in which the genetics of CF, its prognosis and treatment were explained, with emphasis on the meaning of heterozygosity, the fact that carriers are healthy, and the risk of an affected fetus when only one parent is identified as a heterozygote. All partners agreed to be tested for the delta F508 R553X and G551D mutations and a second counselling session was carried out after this test result was available. No problems were observed during initial testing but, as in other studies, we found considerable anxiety on being given the result in all couples where the woman tested positive; this was reduced substantially by counselling and when the partner tested negative. All probands found to be carriers stated that they found screening acceptable. In contrast to the cautious statement by the German Berufsverband Medizinische Genetik and the hostile reaction from a representative of the CF self-support organisation towards community-based heterozygote screening for CF, this study shows that CF screening is generally acceptable in this German population and that it is actively taken up by most pregnant women when offered.

Base Sequence↗
PubMed1994

Alcohol and aldehyde dehydrogenase genotypes, alcoholism and alcohol related disease.

There is evidence for a genetic component to the aetiology of alcohol related problems. An important way of elucidating this is to search for candidate genes which may show an association with these problems. We have examined for associations between ADH and ALDH polymorphisms, and alcoholism and alcohol related diseases. In Asian populations the ALDH2-2 allele appears protective against alcohol abuse and alcoholism, but may favour alcoholic liver disease. The ADH2-2 allele and the ADH3-1 allele seem to play a similar role. These findings deserve confirmation. Improved clinical methodology will be important in future studies.

Alcohol Dehydrogenase↗
PubMed1992

Polymorphism of alcohol dehydrogenase, alcohol and aldehyde dehydrogenase activities: implication in alcoholic cirrhosis in white patients. The French Group for Research on Alcohol and Liver.

Two types of factors can theoretically modulate alcohol metabolism toward increased acetaldehyde production. These factors are the following: (a) individual, genetically determined isoenzymes with distinct catalytic properties, and (b) modifications of enzyme activity induced by alcohol itself or liver damage. To investigate the respective roles of these factors in white individuals, we studied the alcohol dehydrogenase phenotype, together with liver alcohol dehydrogenase and aldehyde dehydrogenase activities, in 161 patients. Patients with alcoholic cirrhosis (n = 31) were compared with three types of controls: patients with nonalcoholic cirrhosis (n = 25) and excessive (n = 62) and moderate drinkers (n = 43) without liver disease. No association between alcohol dehydrogenase-3 phenotype and alcoholic cirrhosis was found. The prevalence of atypical alcohol dehydrogenase in the four groups was less than 1%. Patients with cirrhosis, regardless of its cause, had significantly lower alcohol dehydrogenase activity than the patients without cirrhosis (p less than 0.05 and p less than 0.01 vs. excessive and moderate drinkers, respectively). Among the noncirrhotic patients, alcohol dehydrogenase activity was significantly lower in the excessive drinkers than in the moderate drinkers (p less than 0.001). Aldehyde dehydrogenase activity was not different between cirrhosis-free excessive and moderate drinkers; in contrast, compared with these two groups, it was significantly lower in the two cirrhosis groups (p less than 0.01). These results suggest that no phenotypic pattern of alcohol dehydrogenase-3 associated with alcoholic cirrhosis in white patients exists, that liver alcohol dehydrogenase activity falls as a consequence of both alcohol abuse and cirrhosis and that liver aldehyde dehydrogenase activity is unaffected by alcohol abuse and only falls after the onset of cirrhosis.

Adult↗
PubMed1992

Prevalence of cystic fibrosis mutations in the East German population.

A representative multicenter cystic fibrosis (CF) mutation analysis on about half of all known cystic fibrosis patients of the 5 East German Länder is reported. Analyses for 17 mutations, among them Delta F508, R553X, G542X, S549R,N,I, G551D, S1255X, R347P,H, and Y122X, were performed. As expected, the delta F508 mutation in exon 10 of the CFTR gene is the major gene alteration causing CF in our patients. However, in comparison to studies from Western Germany, a significantly lower percentage of just over 60% is found in our patients, resembling data obtained from slavonic populations. The severe phenotype of cystic fibrosis is most frequently associated with homozygosity for the delta F508 mutation. No particular allele association could be found with the intermediate and mild phenotypes of this disease. The next most frequent of the investigated mutations is R553X (13.3% of non-delta F chromosomes) followed by R347P (9.2%) and G542X (4.4%).

Cystic Fibrosis↗
PubMed1992

[Molecular biological analysis of cystic fibrosis--a model example for the strategy of "reverse genetics"].

The elucidation of the basic defect causing cystic fibrosis (CF) is a paradigm for the application of "reverse genetics" to the analysis of human genetic disease. Following this strategy, linkage analysis localized the responsible gene for CF on chromosome 7. Chromosome mediated gene transfer and chromosome walking and jumping led to the isolation of the CFTR-gene and its cDNA. A major 3 bp deletion mutation (DeltaF508) and more than 100 other mutations of this gene have been identified as molecular basis of cystic fibrosis. The CFTR-amino acid sequence, obtained by conversion of the cDNA-sequence, indicates that CFTR belongs to a group of integral membrane transport proteins (ABC-proteins). The normal cAMP-stimulated chloride-transport, lacking in CF-cells is restored by transfer and expression of CFTR-cDNA-recombinants in these cells. CFTR is most likely itself a chloride channel. The molecular identification of this gene has already led to substantial advances in diagnosis and prevention of this disease. New therapeutic approaches by pharmacological means or gene therapy are expected from the further molecular and functional analysis of the CFTR-gene.

Child↗
PubMed1992

[Optimization of a spectrophotometry assay of total and oxidized blood glutathione: comparison with a fluorimetric method].

We developed a method for the enzymatic assay of glutathione which is easy to practice, rapid, specific, based on the reaction of the thiol group of glutathione with dithiobis-nitrobenzoic acid after the action of glutathione reductase in the presence of NADPH. This spectrophotometric technique allowed, on the one hand, the determination of total glutathione and on the other hand, that of oxidized glutathione (disulfide), after the blockage of reduced glutathione by 2-vinyl-pyridine. The improvements of the assay of blood glutathione concerned the sample preparation, the reaction sensitivity, thanks to a better definition of the optimal pH and a reduction ot the blockage time by 2-vinyl-pyridine in well defined operating conditions. We compared the performances of our technique with a fluorimetric method. We used our method for the determination of total and oxidized blood glutathione in a control population.

Adult↗
PubMed1991

A pooling strategy for heterozygote screening of the delta F508 cystic fibrosis mutation.

A theoretical and practical approach to economize the analysis of large DNA sample numbers for identifying heterozygosity of the delta F508 mutation causing cystic fibrosis is presented. Sample pooling can reduce the number of polymerase chain reaction (PCR) tests for this mutation by up to 77%. Based on a mathematical model, the optimal number (n) of samples to be united in one pool is 24 for a German population with a delta F508 heterozygosity incidence of about 1/35. We show that the PCR method is sufficient to detect one heterozygote for the delta F508 mutation in a pool of up to 49 non-delated DNA samples.

Computer Simulation↗
PubMed1991

Glutathione S-transferase class mu in French alcoholic cirrhotic patients.

The lack of glutathione S-transferase mu (GST mu) was examined in 45 healthy French Caucasians and 45 alcoholic cirrhotic French Caucasians: microsamples of blood were taken and DNA amplified by the polymerase chain reaction. We have concluded that there is no relationship between this genotype and the development of alcoholic cirrhosis in these heavy consumers of ethanol.

Adult↗
PubMed1991

New DNA-analysis techniques (minireview).

The increasing knowledge about specific human genes and their alterations causing genetic diseases, requires new strategies and methods for mutation-detection. However, the large molecular heterogeneity of such mutations in each gene sets serious limitations to this approach. The polymerase chain reaction has become the basis of several new developments aiming to overcome this problem. Recently developed methods to search for unknown mutations are the denaturing gradient electrophoresis, chemical mismatch cleavage, single stranded conformation polymorphisms and direct genomic sequencing. Known mutations in a particular gene can be detected by the allele specific hybridization, the amplification refractory mutation system, the competitive oligonucleotide priming reaction or the oligonucleotide ligation assay. As spin-off of the human genome sequencing projects, automated DNA-sequencing may in the future become the easiest, fastest, most reliable and most informative technique and a powerful day-to-day method in every genetics and clinical chemistry laboratory.

Base Sequence↗
PubMed1990

Ultrasensitive enzymatic radioimmunoassay using a fusion protein of protein A and neomycin phosphotransferase II in two-chamber-well microtiter plates.

A new sensitive method for antigen detection employing a phosphorylation reaction is described using human serum albumin as a model. The antigen is initially bound to the surface of polystyrene microtiter plates and reacted with an antibody (rabbit). A microbiologically produced bifunctional fusion protein of protein A and neomycin phosphotransferase II (NPT II) serves as a second immunological reagent by virtue of its protein A component. The detection is based on the phosphorylation of an aminoglycoside antibiotic by the NPT II moiety of the fusion protein using [gamma-32P]ATP as a cosubstrate. This reaction is performed in solution and the evaluation is accomplished by dotting aliquots of the reaction mixture onto phosphocellulose paper, washing with water, and autoradiography. Microtiter plates with a specially designed 10 microliter-volume reaction chamber are particularly advantageous for this procedure. The sensitivity of detection is currently 10 fg (1 pg/ml) of antigen.

Animals↗
PubMed1990

High-sensitivity protein detection by a new "contact-copy" method using a protein A-neomycin phosphotransferase II fusion protein.

A new system for high-sensitivity protein detection by an immunoenzymatic "contact-copy" procedure is described. It is based on two components: (i) a microbiologically produced bifunctional fusion protein of protein A and neomycin phosphotransferase II (protein A-NPT II) in which the protein A moiety acts as a second immunological reagent while NPT II catalyzes the detection reaction and (ii) a novel kanamycin-loaded substrate matrix (kanamycin-cyanuric chloride-activated and sulfanilic acid-derivatized paper) brought into direct contact with a protein-carrying matrix after blot or dot application and initial immunoreaction--the NPT II enzyme reaction with [gamma-32P]ATP as cosubstrate leads to phosphorylation of the substrate kanamycin on the substrate matrix, which is used for further analysis. The contact-copy method has at least the same detection sensitivity as procedures employing 125I-protein A, but allows extremely short exposure times and avoids probe prelabeling. Twenty-five picograms of specific protein blotted from sodium dodecyl sulfate-polyacrylamide gels onto nitrocellulose is detected after 15 min of autoradiography. The limit of detection in dot tests was found to be 10 pg per dot (3 mm2). The method is suitable for quantitative determination of antigens in the range down to 100 pg. Several contact copies of the same original protein-carrying matrix can be produced and used for detection or quantitative analysis without destroying the original matrix.

Collodion↗