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Biomedical subjects

C Dupont

Publications and source records attributed to C Dupont.

At least 145 records · Page 8Linked to original sources
PubMed1997

Glucoamylase mutants in the conserved active-site segment Trp170-Tyr175 located at a distance from the site of catalysis.

To mimic the structure of the 1.8-fold more active (k(cat)) Rhizopus oryzae glucoamylase (GA), Aspergillus niger GA was subjected to site-directed mutagenesis in the Trp170-Tyr175 segment of the third of the six well-conserved alpha-->alpha connecting loops of the catalytic (alpha/alpha)6-barrel. While the Trp170-->Phe, Gln172-->Asn and Tyr175-->Phe mutants showed an up to 1.7-fold increased k(cat) and Gly174-->Cys GA and approximately 2-fold reduced k(cat) towards maltotriose and longer substrates, Asn171-->Ser, Thr173-->Gly and A.niger wild-type GA had very similar kcat and K(m) values for the hydrolysis of isomaltose and the malto-oligosaccharides of DP 2-7. Crystal structures of pseudotetrasaccharide inhibitor complexes of Aspergillus awamori var. X100 GA, which is 94% identical to A.niger GA, indicate that Tyr175 is located at binding subsite 4, while the preceding target residues and the high-mannose type unit on Asn171 are at a larger distance from the site of catalysis. The mutations had a modest effect on thermostability; the temperature for 50% inactivation, Tm, was thus unchanged for Tyr175 -->Phe GA and reduced by 0.2-2.9 degrees C for the other mutants. The deletion of the N-linked high-mannose unit-in Asn171 -->Ser and Thr173-->Gly GAs-appeared to be of minor importance for enzyme activity and thermostability, and did not increase the sensitivity to proteolysis.

Amino Acid Sequence↗
PubMed1997

Asparagine-127 of xylanase A from Streptomyces lividans, a key residue in glycosyl hydrolases of superfamily 4/7: kinetic evidence for its involvement in stabilization of the catalytic intermediate.

Site-directed mutagenesis of asparagine-127 (N127) of xylanase A (XlnA) from Streptomyces lividans, belonging to family 10 and superfamily 4/7 of glycosyl hydrolases, was chosen to study the role of this conserved residue. The isosteric mutation N127D introduced did not affect the fold of XlnA as revealed by circular dichroism. Comparison of the kinetic constants of N127D and wild-type XlnA revealed a 70-fold decrease in the specificity constant (kcat/K(M)) towards birchwood xylan, which is attributed solely to the difference in the kcat value and indicates a role of N127 in stabilization of the catalytic intermediate. N127 also plays a role in maintaining the ionization states of the two catalytic residues, as shown by the modified pH profile of XlnA-N127D. Characterization of XlnA-N127D and the analysis of the three-dimensional structure of XlnA converge towards a stabilization role for N127 in the catalytic site of XlnA.

Asparagine↗
PubMed1997

Multiple food allergy: a possible diagnosis in breastfed infants.

Six infants suspected of food allergy during breastfeeding were evaluated using prick tests, total IgE, RASTs and intestinal permeability measurements during fast and provocation with mother's milk. An elimination diet was undertaken in mothers, removing first cow's milk protein (CMP), then, when inefficient, all foods suspected on the clinical history or a positive prick test in the child, followed by oral challenges in mother's diet with the corresponding food. The sole CMP-free diet in mothers always proved insufficient. In four, an additional diet excluding two to three other foods cleared the symptoms. Oral provocations in mother's diet with those foods were positive in all. In two, mothers turned down a diet excluding more than four foods, symptoms cleared while feeding the child with an extensively hydrolysed formula, whereas challenges with mother's milk induced immediate reactions. Intestinal permeability was altered during provocation tests with mother's milk sampled before maternal diet. Food allergy during breastfeeding may be due to multiple foods and the inefficacy of the sole CMP elimination in mothers does not rule out food sensitization.

Breast Feeding↗
PubMed1997

Effect of terfenadine on TNF alpha release from peripheral blood mononuclear cells during cow's milk allergy.

BACKGROUND: In infants with cow's milk allergy and intestinal symptoms, peripheral blood mononuclear cells stimulated in vitro with cow's milk proteins, secrete large amounts of the proinflammatory cytokine TNF alpha thus altering intestinal barrier capacity. Terfenadine, an antihistaminic drug, inhibits the release of several inflammatory mediators, including histamine, prostaglandins and leukotrienes. OBJECTIVES: To test the potential ability of terfenadine to inhibit TNF alpha secretion by mononuclear cells from infants with cow's milk allergy. METHODS: Mononuclear cells from infants allergic to cow's milk proteins were stimulated in vitro for 6 days by a mixture of milk proteins (beta-lactoglobulin, alpha-lactalbumin and casein) with or without terfenadine (0.1-1 microM) and culture supernatants were assayed for TNF alpha by enzyme immunoassay. The effect of culture supernatants on intestinal barrier capacity was evaluated by measuring the electrical resistance (index of integrity) of filter-grown HT29-19 A intestinal cells in Ussing chambers. RESULTS: During active cow's milk allergy, mononuclear cells stimulated with cow's milk proteins secreted large amounts of TNF alpha which significantly reduced the electrical resistance of HT29-19 A intestinal cells. There was a dose-dependent decrease in TNF alpha secretion in the presence of terfenadine, with a maximal inhibition of 62% of this secretion at 1 microM. Accordingly, terfenadine-treated mononuclear cells supernatants did not alter the electrical resistance of intestinal HT29.19 A cells. CONCLUSION: These results indicate that in infants with intestinal dysfunction due to cow's milk allergy, terfenadine is a potent inhibitor of the TNF alpha secretion induced by sensitizing milk protein antigens. This inhibition prevents the degradation of intestinal function as measured in an intestinal cell line, in vitro.

Animals↗
PubMed1997

[Gastroesophageal reflux and dysphonia in children].

UNLABELLED: With the exception of congenital anomalies, the aetiology of dysphonia in children is often unknown. Yet, in adults, GOR has been shown to play an important role. 22 children (aged between 2 and 14 years, 14 boys and 8 girls), who had been suffering from a chronic dysphonia for more than six months were seen at consultation. After a clinical ENT examination including a fibreoptic laryngoscopy, each child had a long duration pH-study that lasted approximately 24 hours. Using the classical criteria for GOR, a pathological GOR was discovered in 14 children, ie 64%. Analysis of the pH traces revealed that the vast majority of refluxes occurred when the child was awake. CONCLUSION: more than 64% of children suffering from chronic dysphonia had pathological GOR. The pH traces highlighted that the majority of these refluxes occurred when the child was awake.

Child↗
PubMed1996

Substrate specificity and mode of action of acetylxylan esterase from Streptomyces lividans.

The substrate specificity of purified acetylxylan esterase (AcXE) from Streptomyces lividans was investigated on partially and fully acetylated methyl glycopyranosides. The enzyme exhibited deacetylation regioselectivity on model compounds which provided insights pertaining to its function in acetylxylan degradation. The enzyme catalyzed double deacetylation of methyl 2,3,4-tri-O-acetyl-beta-D-xylopyranoside and methyl 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranoside at positions 2 and 3. Two methyl xylopyranoside diacetates, which had a free hydroxyl group at position 2 or 3, i.e. the derivatives that most closely mimic monoacetylated xylopyranosyl residues in acetylxylan, were deacetylated 1 to 2 orders of magnitude faster than methyl 2,3,4-tri-O-acetyl-beta-D-xylopyranoside and methyl 2,3-di-O-acetyl-beta-D-xylopyranoside. These observations explain the double deacetylation. The second acetyl group is released immediately after the first one is removed from the fully acetylated methyl beta-D-xylo- and -glucopyranoside. The results suggest that in acetylxylan degradation the enzyme rapidly deacetylates monoacetylated xylopyranosyl residues, but attacks doubly acetylated residues much more slowly. Evidence is also presented that the St. lividans enzyme could be the first real substrate-specific AcXE.

Acetylation↗
PubMed1996

Purification and characterization of an acetyl xylan esterase produced by Streptomyces lividans.

The acetyl xylan esterase cloned homologously from Streptomyces lividans [Shareck, Biely, Morosoli and Kluepfel (1995) Gene 153, 105-109] was purified from culture filtrate of the overproducing strain S. lividans IAF43. The secreted enzyme had a molecular mass of 34 kDa and a pI of 9.0. Under the assay conditions with chemically acetylated birchwood xylan the kinetic constants of the enzyme were: specific activity, 715 units/mg, Km 7.94 mg/ml and Vmax 1977 units/mg. Optimal enzyme activity was obtained at 70 degrees C and pH 7.5. Hydrolysis assays with different acetylated substrates showed that the enzyme is specific for deacetylating the O-acetyl group of polysaccharides and is devoid of N-deacetylation activity. Sequential hydrolysis shows that its action is essential for the complete degradation of acetylated xylan by the xylanases of S. lividans.

Acetylesterase↗
PubMed1996

Factors associated with the release of cardiac troponin T following percutaneous transluminal coronary angioplasty.

BACKGROUND: Recent studies have suggested that immunoassay of cardiac troponin T (cTnT) provides a more sensitive measurement of myocardial necrosis than creatine kinase MB (CK-MB) mass concentration. HYPOTHESIS: The purpose of this study was to compare the release of cTnT and CK-MB isoenzyme in patients undergoing percutaneous coronary angioplasty, and to investigate the clinical, procedural, and angiographic correlates of abnormal elevations of both of these markers. METHODS: Total creatine kinase (total CK), CK-MB, and cTnT levels were measured immediately before and 12 h following intervention in 110 patients, including 100 consecutive patients undergoing coronary angioplasty and 10 control patients undergoing diagnostic cardiac catheterization. All patients had normal levels of all three markers at baseline. A postintervention total CK level > 225 U/l, an increase in CK-MB > 5.0 ng/ml, and/or an increase in cTnT > 0.04 ng/ml were considered indicative of myocardial injury. RESULTS: Coronary angioplasty was successfully performed in all 100 patients without emergency bypass surgery or death, although six patients required emergent placement of an intracoronary stent for threatened closure. Eight patients demonstrated an abnormal increase in total CK, including six who were undergoing primary angioplasty for an acute myocardial infarction. One of these patients sustained a Q-wave infarction. Post angioplasty, 18 patients had elevations of both CK-MB and cTnT, 23 had elevations of only cTnT, and the remaining 59 patients had elevations of neither. All patients with CK-MB elevation also had cTnT elevation. Neither serologic marker increased in the diagnostic catheterization control patients. In comparison with patients without postintervention cTnT rise, patients with abnormal cTnT levels had a higher incidence of complex lesion morphology (p < 0.01) and intracoronary thrombus (p < or = 0.0001) prior to coronary angioplasty, and a higher incidence of coronary dissection (p < or = 0.01), abrupt closure (p < or = 0.05), and side-branch occlusion (p < or = 0.01) during angioplasty. In patients with elevation of both cTnT and CK-MB, postintervention CK-MB levels were 12-fold higher and cTnT levels were 21-fold higher than in patients with isolated elevation of only cTnT (p < 0.01). CONCLUSIONS: These data indicate that > 40% of patients undergoing coronary angioplasty have evidence of minor degrees of myocardial damage, as evidenced by cTnT release. High-risk coronary lesions and both minor and major complications of angioplasty are associated with cTnT release. cTnT appears to be a more sensitive marker of myocardial injury than CK-MB under these circumstances. In comparison with isolated cTnT rise, elevation of both CK-MB and cTnT may be indicative of greater levels of myocardial injury.

Aged↗
PubMed1996

Evidence for lysozyme-type mechanism of hydrolysis in xylanases.

In the last year several new xylanase three-dimensional structures were solved. Examination of these new structures in combination with recently obtained data from site-directed mutagenesis and kinetic analysis provided insights into the catalytic mechanism of xylanases. It is now possible to determine the type of mechanism by which xylanases hydrolyse a complex substrate such as xylan.

Amino Acid Sequence↗
PubMed1996

Evaluation of a serological test for diagnosis of Helicobacter pylori infection in children.

A serological test for the diagnosis of Helicobacter pylori infection (Cobas Core Roche, IgG, 2nd Generation; Roche, France) was compared with the examination of biopsy samples (culture and histology) obtained after endoscopy in 115 children to assess its value. In 94 children (42 positive and 52 negative), results were concordant. In 10 children a positive serological test was associated with an absence of Helicobacter pylori, while in 11 others a negative serological test was associated with a positive culture. Sensitivity of the test was 79.2% and specificity 83.9%. A relationship between IgG titers and age (r = 0.31, p < 0.05) was found. Serological tests could be useful for the diagnosis of Helicobacter pylori infection, but a negative test result does not rule out infection, particularly in children under 10 years of age.

Adolescent↗
PubMed1996

Expression of functional HIV-1 integrase in the yeast Saccharomyces cerevisiae leads to the emergence of a lethal phenotype: potential use for inhibitor screening.

The integrase of the human immunodeficiency virus type 1 (HIV-1) has been expressed in yeast in order to investigate its potential lethal effect mediated by DNA damage. To this end, we have constructed an expression plasmid containing the retroviral integrase gene under the control of the inducible promotor ADH2/GAPDH which is regulated by the glucose concentration of the medium. Haploid yeast strain W303-1A did not appear to be clearly sensitive to HIV-1 integrase expression. However, disruption of the RAD 52 gene, which is involved in the repair of double-strand DNA breaks, strongly increased the deleterious effects of the retroviral enzyme in this yeast strain. The diploid strain constructed with W303-1A and an isogenic strain of the opposite mating type also showed a strong sensitivity to the HIV-1 integrase. Under yeast culture conditions allowing moderate integrase synthesis, the deleterious effect was totally abolished by missense integrase mutations, which are known to abolish HIV-1 integrase activities in vitro. We conclude that the lethal phenotype due to HIV-1 integrase expression in yeast may be closely related to the HIV-1 integration reaction in infected human cells, and that yeast may be a useful tool to study the HIV-1 integration process and to screen drugs capable of inhibiting HIV-1 integration in vivo.

Antiviral Agents↗
PubMed1996

Expression and secretion of beta-glucuronidase and Pertussis toxin S1 by Streptomyces lividans.

Streptomyces lividans IAF18, obtained by homologous cloning, is capable of over-producing XlnA. To investigate the possibility of the expression of foreign genes, various coding regions of the xylanase A gene (xlnA) were analysed. Expression/secretion vectors were constructed containing the regulatory elements of xlnA with the coding region of the leader peptide with or without the truncated structural gene encoding the first 310 amino acids of the XlnA. The genes coding for the Escherichia coli beta-glucuronidase and subunit 1 of the Bordetella pertussis toxin (S1) were used and their expression analysed. S. lividans transformants where the beta-glucuronidase gene was fused with the leader sequence produced up to 30 mg beta-glucuronidase/culture filtrate whereas only fused XlnA/S1 was detected and its yield was estimated to be 1 mg/1. The disappearance of the B. pertussis toxin S1 and beta-glucuronidase from the culture medium was due to the concomitant appearence of secreted proteases from S. lividans.

Bordetella pertussis↗
PubMed1996

Genotyping of Helicobacter pylori isolates by sequencing of PCR products and comparison with the RAPD technique.

Two genotyping methods were performed on bacterial suspensions of the human pathogen Helicobacter pylori. A total of 29 clinical isolates were analysed by sequencing of a 294-bp PCR-derived internal segment of the essential ureC/glmM gene of H. pylori, and by random amplified polymorphic DNA (RAPD) using a single 11-bp oligonucleotide made up of an arbitrary nucleotide sequence. Each isolate exhibited a distinct sequence over a 210-bp stretch of the ureC/glmM gene. Similarly, the isolates bore different profiles when tested by RAPD fingerprinting. Successive strains arising from patients who relapsed following antibiotic treatment and strains isolated from two patients institutionalized in the same care centre had identical ureC/glmM gene sequences and RAPD profiles. Both methods were found to be discriminatory. However, PCR sequencing of the ureC/glmM gene appeared to be more reproducible and more reliable for distinguishing between strains than the RAPD technique.

Adolescent↗
PubMed1996

[Sleeping position, prevention of sudden death syndrome and gastroesophageal reflux].

Based on results of epidemiological studies, dorsal or lateral sleeping positions are now recommanded in the prevention of sudden infant death syndrome (SIDS). This raises an ethical question about the attitude towards the ventral positioning therapy for gastroesophageal reflux (GOR). The consensus conference considers that the ventral position should only be recommanded in GOR when the benefit appears to outweigh the risk of SIDS that it induces. The conference proposes that for infants with simple uncomplicated reflux, sleeping in the prone position should not be introduced in the first line treatment. Prone positioning should be restricted to complicated cases resistant to dietary and medical measures.

Gastroesophageal Reflux↗