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Biomedical subjects

A Lemonnier

Publications and source records attributed to A Lemonnier.

At least 19 recordsLinked to original sources
PubMed1999

Improved fluorescent PCR-based assay for sizing CGG repeats at the FRAXA locus.

Fragile X syndrome is the most frequent heritable genetic disease involving mental retardation and is usually caused by an expanded CGG repeat in the first exon of the FMR1 gene. Therefore, searching for CGG expansion at the FRAXA locus among the mentally retarded has become a routine investigation in neuro-paediatric practice. Consequently, we have developed a fluorescent PCR-based assay for sizing repeats as an alternative to laborious and time-consuming Southern blot. The procedure utilises a reverse fluorescent labelled primer, and the Expand Long Template PCR system (Roche) with addition of dimethylsulfoxide and 7-deaza-dGTP It allows precise determination of the CGG repeat number in males and females for alleles from normal to premutation size range and detection of full mutations in males. We believe that this PCR protocol, allowing a high sample throughput, is useful for first-line screening among mentally retarded males, possibly complemented by Southern blot analysis to assess the methylation status of large mutated alleles.

Electrophoresis, Polyacrylamide Gel↗
PubMed1995

Rapid diagnosis of long chain and medium chain fatty acid oxidation disorders using lymphocytes.

A method based on the release of tritiated water from [9,10(n)-3H] palmitic and myristic acids previously described for fibroblasts, was adapted for lymphocytes for the rapid diagnosis of fatty acid oxidation disorders. Optimal concentrations for both substrates and linearity of the assay were established. Normal values were established in control subjects of different age groups (58 children and 117 adults) and 16 patients with known fatty acid oxidation disorders were tested. Tritiated water production from patients' lymphocytes was expressed as a ratio between residual oxidations of palmitate and myristate and the results show that this method allows good differentiation between long chain and medium chain fatty acid oxidation defects.

Acyl-CoA Dehydrogenase↗
PubMed1994

Regulatory effects of galactose on galactose-1-phosphate uridyltransferase activity on human hepatoblastoma HepG2 cells.

Galactose-1-phosphate uridyltransferase (GALT) deficiency results in galactosemia in man. We have studied the regulation of the GALT gene expression on the HepG2 cell line by growing the cells in glucose or galactose medium. No difference of Km values was observed in glucose or galactose media but the Vmax value with galactose was 50% higher than that with glucose. Also in galactose medium, an increased GALT specific activity was detected suggesting the production of more enzyme proteins. Yet, slot dot quantification of GALT mRNA revealed a decreased amount of these transcripts in cells cultured with galactose or inosine while Northern blot analysis revealed the normal 1.4 kb transcript in all culture media used. Finally, IEF gel analysis displayed different isozymic patterns for the GALT enzyme in cells grown in glucose, galactose or inosine media. With glucose-free media, the major band of GALT corresponds to that found in human liver. Altogether, these results suggest that the control of GALT gene expression in HepG2 cells is located at the post-transcriptional level and correlated to the growth rate of the cell.

Blotting, Northern↗
PubMed1994

Human kidney prolidase--purification, preincubation properties and immunological reactivity.

1. Prolidase I (EC 3.4.13.9) was purified from human kidney to SDS-PAGE homogeneity. The molecular weights of native and denatured purified enzyme were estimated to be 115,000 and 55,000, respectively. 2. Agarose electrophoresis revealed migration in the alpha 1 globulin region, and an isoelectric point (pl) of 4.65 was estimated by both isoelectric focusing (IEF) and the titration curve method. 3. Activation by preincubation for 24 hr at 37 degrees C with 1 mM MnCl2 was maintained throughout the purification steps, using gly-pro and phe-pro dipeptides as substrates. 4. Activation in the presence of gly-pro was higher (4.5- to 11-fold) than in the presence of phe-pro (1.3- to 2.3-fold). 5. Lineweaver-Burk plot consisted of one and two lines with gly-pro and phe-pro, respectively. Km, Vmax and the Vmax/Km ratio were increased and the two lines with phe-pro were conserved after prolonged preincubation. 6. A specific polyclonal antibody was raised in rabbits against the purified enzyme and immunoreactivity was investigated between rabbit antiserum and both prolidase I from various tissues and human kidney prolidase II. 7. Prolidase I from liver, erythrocytes and plasma was immunochemically identical to renal prolidase I. The polyclonal antibody did not react with prolidase II. 8. These results indicate that a specific immunoassay might be developed to investigate prolidase I protein in plasma and tissues from patients with prolidase deficiency and hepatic fibrosis.

Antibodies, Monoclonal↗
PubMed1994

Metabolic effects of galactose on human HepG2 hepatoblastoma cells.

HepG2 cells were used as a model system to study the effects of galactose overload on the liver, a target organ of galactose toxicity in patients suffering from transferase-deficient galactosemia. In the presence of galactose, HepG2 cell growth was slow and the pattern of gene expression remained characteristic of liver cells (secretion of alpha-fetoprotein [AFP] albumin, and transferrin). Galactose-1-phosphate (Gal-1-P) accumulated, as it does in galactosemic cells, but did not affect the energetic status of the cells (no adenosine triphosphate [ATP] depletion). However, the substitution of galactose for glucose as the sole hexose in the medium affected the specific activities of the galactose-metabolizing enzymes. Galactokinase (GALK) activity was decreased, and those of galactose-1-phosphate uridyltransferase (GALT), phosphoglucomutase, and glucose-6-phosphate dehydrogenase (G6PDH) were increased. The conversion of radiolabeled galactose to glucose (CO2 production and glycogen level) was greater in galactose medium than in glucose medium after a 7-day culture. Therefore, the culture of HepG2 cells in galactose medium indicates that the enhanced utilization of this hexose is due to the increased enzyme activities regulating its own metabolism. Hence, HepG2 cells constitute a good model for the study of modulation of galactose-metabolizing enzymes by galactose.

Analysis of Variance↗
PubMed1993

Effects of alpha-tocopherol on antioxidant enzyme activity in human fibroblast cultures.

The activities of superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (Cat) were determined in human fibroblast cultures at four concentrations of exogenous alpha-tocopherol: 0.2, 2.5, 10 and 50 micrograms/ml of culture medium, or without alpha-tocopherol. Relationships between alpha-tocopherol levels and the activities of SOD and GPx were identified. The cellular alpha-tocopherol level correlated with GPx activity (p < or = 0.01) and inversely correlated with SOD activity (p < or = 0.003), but only when alpha-tocopherol was added to the culture medium. The variations in the cellular GPx/SOD ratio depended on the level of cellular alpha-tocopherol (p < or = 0.001). Furthermore, there was a strong inverse correlation between SOD and GPx activity (p < or = 0.0001). Cat activity did not correlate either with cellular alpha-tocopherol concentration, or with SOD or GPx activity. These results underline the complex interplay between alpha-tocopherol and other antioxidant systems in human fibroblast cultures.

Catalase↗
PubMed1992

Amino acid transport systems in the human hepatoma cell line Hep G2.

The human hepatoma cell line Hep G2 was used to investigate amino acid transport systems in human liver tissue. The ubiquitous transport systems responsible for the uptake of most neutral amino acids (systems A, ASC and L) were found to be present. Transport system A was predominant for proline uptake but system ASC was the major Na(+)-dependent transport system, particularly for glutamine. The specific hepatic system N was functional, but only partially mediated glutamine uptake. The study of Na(+)-independent arginine uptake demonstrated the presence of the cationic transport system Y+, reflecting the transformed nature of Hep G2 cells.

Alanine↗
PubMed1992

Only prolidase I activity is present in human plasma.

1. After ion exchange chromatographic separation, liver prolidase exhibits two isoforms (prolidase I and II). 2. The activity of both was explored in human and rat tissues, and in normal and cytolytic human plasma. 3. The activity of prolidase I, eluted at the lowest ionic strength, was stimulated by 24 hr of preincubation with 1 mM MnCl2, but prolidase II activity was strongly inhibited by this long preincubation. In both normal and cytolytic human plasma, chromatographic separation also disclosed that only prolidase I activity was present. 4. This isoform displayed properties resembling those of liver and kidney prolidase I. 5. To explain the absence of prolidase II activity from the plasma, we tested the possibility that its tissue distribution differed. 6. However, this was not substantiated by the distribution found, or by the location, molecular weight and behavior of human liver prolidase II after neuraminidase treatment. 7. We also explored the hypothesis that plasma proteins inhibit prolidase II activity, and found that albumin almost abolished this activity after 6 hr incubation.

Animals↗
PubMed1991

Biochemical diagnosis of hepatic glycogen storage diseases: 20 years French experience.

French experience of 242 cases of liver glycogenoses is reported. Screening tests based on serum biochemical data and glucagon tolerance tests are briefly reviewed. The diagnosis of types I glycogen storage disease (GSD) was ascertained in 73 patients' liver biopsies by measurement of glycogen content and by studying the glucose-6-phosphatase system. Liver biopsies were also required at the beginning for the diagnosis of other hepatic GSDs; later on, the possibilities of diagnosis using peripheral blood cells were investigated. Eighty-four cases of type III GSD were confirmed by measurement of debranching enzyme activity and glycogen content using either liver biopsies (78 cases) and/or erythrocytes (37 cases); enzyme determination was also performed in leukocytes and/or fibroblasts for 18 patients. Twenty-four cases of type VI GSD underwent liver biopsies, and the diagnosis could be confirmed using mononuclear or polymorphonuclear cells for 11 of these patients. Sixty-one patients were identified as type IX GSD; phosphorylase kinase deficiency was demonstrated in erythrocytes for all patients, and a liver biopsy was analyzed for 26 of these cases. From this experience, the possibilities of diagnosis of liver GSD using peripheral blood cells are emphasized.

Blood Chemical Analysis↗
PubMed1991

Diagnostic value of serum gamma-glutamyl transpeptidase activity in liver diseases in children.

The clinical usefulness of serum gamma-glutamyl transpeptidase (gamma GT) assay for the diagnosis of liver disease in children was assessed retrospectively in 398 children investigated from 1981 to 1986, in whom diagnosis was ascertained according to currently accepted criteria including liver histology in each case. Serum gamma GT activity was within normal limits in 10 controls, in 19 children with portal vein obstruction, and in 10 of 12 children with congenital hepatic fibrosis. Serum gamma GT was raised in all children with biliary atresia, sclerosing cholangitis, paucity of interlobular bile ducts, and alpha 1-antitrypsin deficiency with jaundice. Serum gamma GT was normal in spite of patent clinical signs of cholestasis in 3 patients with benign recurrent intrahepatic cholestasis, 1 infant with post-hemolytic neonatal cholestasis, and in 22 of 28 patients with progressive idiopathic cholestasis akin to Byler disease. In the latter group, children with raised serum gamma GT displayed extensive portal fibrosis and bile duct proliferation on liver histology, while this was not a prominent feature in children with normal serum gamma GT. These results indicate (a) the value and limits of the assay for serum gamma GT activity in children with liver disease, (b) that raised serum gamma GT may be considered a fairly reliable index of bile duct damage, and (c) that serum gamma GT may prove a useful tool in separating two forms of progressive idiopathic cholestasis, with or without bile duct involvement.

Adolescent↗