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M Wellman

Publications and source records attributed to M Wellman.

53 records · Page 3Linked to original sources
PubMed1990

Gamma-glutamyltransferase from human hepatoma cell lines: purification and cell culture of HepG2 on microcarriers.

After screening different human hepatoma cell lines, we observed that both HepG2 and PLC/PRF/5 naturally produced large amounts of gamma-glutamyltransferase. We optimized HepG2 cell culture conditions and observed that higher cell densities were obtained when cells were cultured on microcarriers, particularly when Cytodex 3 was used and that cell growth was optimal when DMEM, the basic medium, was supplemented with 5% fetal calf serum and 6 mmol/l glutamine. These culture conditions allowed us to produce the highest amounts of GGT after about 150 h of culture. The GGT obtained from HepG2 cells was partially purified and some of its physico-chemical properties characterized. Successive Con A gel chromatography separated the activity into two peaks, suggesting that GGT from HepG2 is not uniformly glycosylated. Papain-treated HepG2 GGT showed a Mr of about 120 kDa and migrated as a single-chain protein in SDS-PAGE. Immunological and kinetic properties of the GGT were similar to other human GGTs (liver, kidney and serum). It appears that HepG2 GGT could be a source for the preparation of a human enzyme reference material.

Blotting, Western↗
PubMed1990

Enzyme immunoassay for flecainide.

In order to develop an enzyme immunoassay (ELISA) for measurement of plasma flecainide levels, we first synthesized flecainide-hemisuccinate-BSA as immunogen. A highly specific antiserum was then raised in rabbits. Enzyme-labelled flecainide was prepared by the mixed anhydride method using hemifumarate-flecainide and horse radish peroxidase. The limit of detection was 10 ng/ml, the method was linear up to 1,000 micrograms/l. The intra and inter-assay variation was 5.1-7.8%. Cross reactivity with various metabolites of flecainide or some chemically related drugs was less than 0.1%. By contrast the cross reaction with some structural analogues achieved 25%. The importance of parts of the aromatic ring structure in immunogenic properties of this drug is discussed.

Antibodies↗
PubMed1990

An avidin-biotin ELISA for the measurement of mitochondrial aspartate aminotransferase in human serum.

We describe a new double-antibody ELISA system using avidin-biotin amplication for the mass measurement of mitochondrial aspartate aminotransferase (m-AST) in human serum. The assay is very sensitive and as little as 0.5 ng/ml of m-AST may be detected. The method is linear up to 100 micrograms/l. The within-day and day-to-day coefficients of variation were found to be 8.9% and 11.5% respectively for a low m-AST concentration (2.7 micrograms/l), and 5.9% and 8.0% respectively for a high level of m-AST (30 micrograms/l). The assay requires 100 microliters of serum and can be completed within 5 h. The ELISA procedure and a classical immunoprecipitation technique measuring the catalytic activity of the isoenzyme were applied simultaneously to the sera of 189 subjects. The protein levels determined by ELISA correlated poorly (r = 0.66) with the catalytic activity of m-AST.

Adolescent↗
PubMed1990

Production, cross reactivity, and epitope analysis of monoclonal antibodies against rat kidney gamma glutamyltransferase.

Eighteen IgG1 monoclonal antibodies (Mabs) have been produced against gamma-glutamyl transferase (GGT) from rat kidney. They were specific to the light subunit of the enzyme with affinity constants ranging from 0.3 to 7.5 10(8) M-1, while they did not react with GGT from other sources i.e. human and pig kidney, rat and guinea pig liver, suggesting species and organ specificity. Two of the Mabs (No 11 and 21) lost their immunoreactivities towards rat kidney GGT in the presence of N-acetyl-neuraminic acid, while immunoreactivities of the other Mabs were unchanged. Furthermore, Mabs No 11 and 21 did not react with desialylated rat kidney GGT. These findings suggest that N-acetyl-neuraminic acid is involved in the epitopes recognized by these two Mabs.

Animals↗
PubMed1990

Pattern of expression of gamma-glutamyl transpeptidase in rat liver and kidney during development: study by immunochemistry and in situ hybridization.

We used an in situ hybridization technique using single-stranded RNA probes to study the expression pattern of gamma-glutamyl transpeptidase (GGT) in rat liver and kidney during development. The results were compared to those obtained with an immunoperoxidase technique and with Northern blot analysis of GGT mRNA. In the kidney, northern blot revealed a 20-fold increase of GGT mRNA between day 18 of gestation and adulthood. Protein and mRNA localization clearly identified the proximal tubules as the site of synthesis of GGT. In the liver, the expression was lower than in the kidney and Northern blot showed a dramatic decrease of expression after birth. Using immunohistochemistry, the protein was detected within parenchymal cells in embryo and hepatocyte membranes and bile ducts in adults. Using in situ hybridization, GGT mRNA was only detected on days 1 and 2 after birth and exclusively in hepatocytes. Immunoperoxidase may be more sensitive than in situ hybridization to study the expression of minor liver protein such as GGT. However, the study of GGT expression using in situ hybridization is possible in cases of increased expression such as alcoholism, cholestasis, and carcinogenesis.

Animals↗
PubMed1989

Electrophoretic mobility of gamma-glutamyltransferase in rat liver subcellular fractions. Evidence for structure difference from the kidney enzyme.

Adult rat liver gamma-glutamyltransferase (GGT) has been poorly characterized because of its very low concentration in the tissue. In contrast with the kidney, the liver enzyme is inducible by some xenobiotics, and its relationship to hepatic ontogeny and carcinogenesis seems to be important. Liver GGT polypeptides were identified by immunoblot analysis in subcellular fractions (rough endoplasmic reticulum, smooth endoplasmic reticulum, Golgi membranes and plasma membranes). Rat liver GGT appeared as a series of polypeptides corresponding to different maturation steps. Polypeptides related to the heavy subunit of GGT were detected in rough endoplasmic reticulum at 49, 53 and 55 kDa, and in Golgi membranes at 55, 60 and 66 kDa. Two polypeptides related to the light subunit of GGT were also observed in Golgi membranes. In plasma membranes GGT was composed of 100 kDa, 66 kDa and 31 kDa polypeptides. The 66 kDa component could correspond to the heavy subunit of the rat liver enzyme, and if so has a molecular mass higher than that of the purified rat kidney form of GGT (papain-treated). These data suggest different peptide backbones for the heavy subunits of liver GGT and kidney GGT.

Animals↗
PubMed1989

Expression of rat renal gamma-glutamyltransferase cDNA in Escherichia coli.

To obtain the expression of rat kidney gamma-glutamyltransferase (GGT) cDNA in E. coli, plasmids containing the cDNA sequences coding for various parts of GGT were constructed. Transformation of E. coli cells by these hybrid vectors results in a production of unglycosylated recombinant proteins, immunologically recognized by specific antirat kidney GGT antibodies. Plasmid, expressing the complete coding sequence of GGT cDNA, allows the production of enzymatically active proteins localized in the periplasmic space, while the same sequence without the N-terminal hydrophobic region results in a production of cytoplasmic proteins. These recombinant proteins present a very basic isoelectric point (pI greater than 9). These results suggest that the presence of the N-terminal region seems to be necessary to direct the expressed proteins enzymatically active in the periplasmic space.

Animals↗
PubMed1989

Serum mitochondrial aspartate aminotransferase activity: not useful as a marker of excessive alcohol consumption in an unselected population.

Using an immunochemical method, we measured the activity of the mitochondrial isoenzyme (mAST) of aspartate amino-transferase (EC 2.6.1.1, AST) in the serum of 687 subjects attending the Centre for Preventive Medicine for a health examination. The distributions of the activities were asymmetrical, with mean values of 1.8 U/L (SD 2.0) for men and 1.4 U/L (SD 1.6) for women. The average ratio of mitochondrial to total AST activity was 0.051 (range 0-0.42). In this unselected population we found no change in the mitochondrial activity or in the mitochondrial-to-total ratio attributable to alcohol consumption, even in subjects who consumed more than 88 g per day. Of 35 men with an alcohol consumption greater than 88 g/d, 19 had a serum gamma-glutamyltransferase activity of greater than or equal to 60 U/L, 17 had glutamate dehydrogenase values greater than or equal to 5 U/L, and only nine had an mAST activity greater than or equal to 3 U/L (values corresponding to the 80th percentiles of the total population). We conclude that the test is not particularly useful as a screening procedure in an unselected population under present-day conditions of measurement.

Adult↗
PubMed1988

Monoclonal antibodies to human kidney gamma-glutamyltransferase.

Eight hybridoma clones secreting large amounts of monoclonal antibodies against purified human kidney gamma-glutamyltransferase (GGT) were isolated and produced in ascites. None of them inhibits the catalytic activity of GGT. They all bind to the heavy subunit of this dimeric enzyme. Immunoblot analysis showed that these antibodies react with the catalytically active GGT. The monoclonal antibodies also recognize the heavy subunit of the human liver enzyme. This is of interest, as serum GGT is known to originate from the liver. None of the monoclonals reacts with GGTs from rat or pig kidney. After identification of epitopes specificities, the antibodies will be used for the development of immunoassays of GGT especially in human serum.

Animals↗
PubMed1988

gamma-Glutamyltransferase reference material: an example of international cooperation.

The different steps in the preparation of a certified reference material for gamma-glutamyltransferase are described. A preparation of pig kidney gamma-glutamyltransferase, partly hydrolyzed with papain, was prepared and lyophilized in a matrix containing bovine serum albumin. The between-ampoule variability of gamma-glutamyltransferase was estimated to be 0.6%. The predicted degradation at -20 degrees C is 0.00% per year. The certification procedure involved 15 participating laboratories. The certified gamma-glutamyltransferase catalytic concentration of the reconstituted material, using the IFCC proposed method, is 86.8 U/1 with a 0.95 confidence interval of +/- 2.1 U/1.

European Union↗
PubMed1985

Distribution of G + C-rich regions in varicella-zoster virus DNA.

The distribution of G + C-rich sequences in the genome of varicella-zoster virus (VZV) was investigated by partial denaturation, equilibrium sedimentation and Southern blot analyses. Portions of the IRS and TRS repeat sequences bounding the US region of the DNA were found to have a G + C content 10 to 20% greater than the overall 47% G + C content of the VZV genome. A stretch of DNA (approx. 1500 base pairs) at the UL-IRS junction and repeated at the terminus of the TRS sequences was found to be about 64% G + C, based on sedimentation equilibrium measurements. We also report the cloning of a novel fragment containing sequences from both the UL and TRS termini of the VZV genome. Our ability to clone this fragment suggests that unusual forms of VZV DNA including closed circular molecules and molecules with an inverted UL region can be packaged into nucleocapsids.

Base Composition↗
PubMedDate not supplied

Rapid liquid chromatographic assay of glutathione in cultured cells.

A rapid, sensitive and selective method for the assay of glutathione in cultured cells has been developed using ion-pair reversed phase rapid high performance liquid chromatography. The use of a 4 micron particle, 5 cm long column (Superspher 100 RP 18 end-capped) allowed complete analysis of glutathione within 3 min. A postcolumn derivatization reaction with o-phthalaldehyde and fluorometric detection made the assay fully selective with regard to other endogenous thiols and sensitive (the detection limit was 0.5 ng of glutathione injected). The linearity range was between 0.1 and 2.0 microgram/mL with good repeatability (relative standard deviation less than 5% for the lowest concentration quantitated). Recoveries of GSH from cultured cell samples were above 98%. The rapid analysis enabled the processing of a large number of samples in a short time (up to 20 per hour). The method was applied to the measurement of the intracellular glutathione amount in V79 fibroblasts along cell growth in culture.

Animals↗
PubMedDate not supplied

Leukoerythroblastosis and normoblastemia in the cat.

Over a six-month period, 6% of 313 cats evaluated hematologically had either leukoerythroblastosis or normoblastemia. Diseases associated with these hematological conditions included haemobartonellosis, hepatic lipidosis, trauma, viral and bacterial infections, myeloproliferative disorders, and hemangiosarcoma. The finding of leukoerythroblastosis or normoblastemia may aid in diagnosing cats presenting with nonspecific signs.

Anemia, Myelophthisic↗
PubMedDate not supplied

[Changes of plasma and tissue gamma-glutamyltransferase under the influence of drugs].

Measurement of gamma-glutamyltransferase (GGT) activity in plasma is widely used in clinical biology (in order to detect hepatic diseases or to monitor treatment for alcoholism), and also in pharmacology (since this test is the only plasmatic marker for hepatic induction in human). However, the correct interpretation of a high plasmatic activity should take into account the various analytical factors which can affect results, as well as the physiological parameters known to modify this activity. It also requires its comparison to defined reference values. Several mechanisms may be involved in the increase of plasmatic activity as an index of hepatic induction, such as an increase in the protein synthesis, a release of the enzyme from the membrane or a modification in the biliary flux.

Chemical Phenomena↗
PubMedDate not supplied

Sensitivity to taxoid derivatives of a newly established human endometrioid ovarian adenocarcinoma radioresistant cell line.

An endometrioid ovarian adenocarcinoma cell line CAVEOC-2 was characterized. Maintained in monolayered culture, CAVEOC-2 cells exhibited a 33-hr doubling time. When xenografted into nude mice, these cells produced fast growing tumors. Colony-forming efficiency in agar was 50%. DNA index was 1.5 and cytogenetic analysis showed a triploid karyotype. CAVEOC-2 cells did not express mdr-1 gene and were chemosensitive to doxorubicin (IC50 = 1.82 +/- 0.76 mumol/l), paclitaxel (IC50 = 3.33 +/- 0.26 nmol/l) and docetaxel (IC50 = 0.68 +/- 0.28 nmol/l), while they showed an intermediate sensitivity to cisplatin (IC50 = 9.40 +/- 1.02 mumol/l). CAVEOC-2 cells seemed highly radioresistant (SF2 = 0.81, alpha = 0.02 Gy-1, beta = 0.025 Gy2, and MID = 4.31 Gy). Activities of glutathione S transferase and gamma-glutamyl transpeptidase were respectively 23.5- and 3.4- fold higher than those of sensitive A2780 cell line. These characteristics make the CAVEOC-2 cells a suitable model for the study of human endometrioid ovarian adenocarcinoma.

ATP Binding Cassette Transporter, Subfamily B, Mem↗