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M Wellman

Publications and source records attributed to M Wellman.

At least 37 records · Page 2Linked to original sources
PubMed1996

Cloning and expression of a novel type (III) of human gamma-glutamyltransferase truncated mRNA.

We report the characterization of a novel human gamma-glutamyltransferase mRNA type. This type III mRNA differs from type I and type II mRNAs previously described by several point mutations and the presence of an unspliced 81 bp intron in the open reading frame. Further, type III mRNAs are truncated ones and are tissue and pathology specifically expressed. In fact, type III mRNAs are present in human placenta, sigmoid, lung and in 50% of acute lymphoblastic leukemia blood cells but they are never found in healthy lymphocytes.

Adult↗
PubMed1995

gamma-Glutamyltransferase expression during all-trans retinoic acid-induced differentiation of hematopoietic cell lines.

gamma-Glutamyltransferase activity, genes transcripts and differentiation by all-trans retinoic acid have been investigated in cultured HL-60, U937, and K562 cells. Acquisition of morphological and functional characteristics confirmed the terminal differentiation of HL-60 and U937 cells. All-trans retinoic acid increased gamma-glutamyltransferase activity in a cell type- and time dependent manner. Treatments with all-trans retinoic acid isomers and structurally analogs showed that only retinoids with carboxylic acid group were able to induce enzyme activity in terminal differentiated cells. Additionally, the analysis of gamma-glutamyltransferase genes transcription products demonstrated clearly that, both in untreated and in RA treated cells, only mRNA type I transcribed from the gene 6, was expressed.

Base Sequence↗
PubMed1995

Influence of sample preparation on cellular glutathione recovery from adherent cells in culture.

During the last decade, the unbound glutathione content of cultured adherent cells has become a very important biological marker for many pharmacological and toxicological in vitro studies with regard to the protective role of the tripeptide in its reduced form (GSH). However, the literature does not provide extensive information on the influence of sample preparation on cellular GSH and thiol analyses. Using the fibroblast-like V79 cell line as model, we undertook a comparative study of the efficiency of different procedures reported in the literature with respect to GSH recovery. Depending on the preanalytical step, up to 10-fold discrepancies could be observed in the recovery of intracellular GSH. Different parameters that must be controlled in order to maximize GSH recovery are discussed. The optimal strategy consisted in rapid perchloric acid deproteinization performed directly in the dish, which was extremely valuable for preparing GSH samples from adherent cells, and especially from cells expressing elevated gamma-glutamyl transferase activity.

Animals↗
PubMed1995

Blood and plasma glutathione measured in healthy subjects by HPLC: relation to sex, aging, biological variables, and life habits.

We report an HPLC method for measuring the concentrations of reduced (GSH) and total (GSHt) free glutathione in human plasma and whole blood. The chromatographic step was coupled with a postcolumn derivatization reaction and fluorometric detection. The linear range was 0.81-13.02 mumol/L, and the detection limit was 0.13 mumol/L. In healthy adults (ages 18-73 years), mean concentrations were 941 +/- 155 mumol/L for GSHt and 849 +/- 63 mumol/L for GSH in blood (107 men, 94 women), and 3.39 +/- 1.04 mumol/L for GSH in plasma (66 men, 58 women). Blood GSHt but not GSH was significantly lower in children (32 boys, 32 girls: 872 +/- 157 mumol/L) than in adults. Blood GSHt and GSH appeared to be correlated positively with the number of cigarettes smoked per day and the regular practice of physical exercise, and negatively with alcohol abstinence. We observed positive correlations between blood GSHt and cholesterol and calcium concentrations, and between blood GSH and cholesterol concentration.

Adolescent↗
PubMed1995

Characterization and regulatory effect of gamma-glutamyltransferase messenger RNA untranslated regions in human leukemia.

In this paper we report the presence and function of the 5' untranslated region (5'UTR) from the mRNA encoding human gamma-glutamyltransferase (GGT) in three different hematopoietic cell lines (HL-60, U-937 and K-562) as well as in the RNA of the leukocyte fraction from six acute lymphoblastic leukemias (ALL). Results obtained by RNase protection analysis demonstrate the presence of a unique form of 5'UTR expressed in most human tissues. In order to investigate the possible role of this type of sequence on regulation of GGT in hematopoietic cells, plasmid constructs carrying human hepatoma GGT 5'UTR and a luciferase reporter gene were transfected into the three blood cell lines. Compared to control untransfected cells, transfected HL-60 and K-562 showed a decrease in reporter gene activity of 51 and 73%, respectively. In contrast, transfected U-937 showed a 139% increase of reporter gene activity. Results were compared to GGT activity in the relevant cells and we concluded that the 5'UTR appears to have a regulatory role in GGT expression as a tissue-specific modulator of translation.

Base Sequence↗
PubMed1995

Developmental- and tissue-specific DNA methylation patterns and expression of rat gamma-glutamyltransferase.

In order to investigate the mechanism controlling gamma-glutamyltransferase (GGT) gene expression in adult and fetal rat tissues, as well as in H5-6 and Fao hepatoma cells, the relationship between DNA methylation and GGT expression was studied. Southern blot analysis of genomic DNA digested with MspI and HpaII showed that highly methylated DNA patterns observed especially in adult liver, heart and Fao cells as well as in fetal kidney, pancreas, lung and heart correlate well with low GGT activity determined in these tissues. In contrast, hypomethylated GGT DNA was found in fetal liver, adult kidney and lung, the tissues highly expressing GGT activity. So, observed developmental- and tissue-specific changes in the GGT gene methylation seem to play an important role in the control of GGT gene transcription.

Animals↗
PubMed1994

Localization of a regulatory region on the 5'-untranslated region of human hepatoma HepG2 gamma-glutamyltransferase mRNA and response to dexamethasone and antisense oligonucleotide treatment.

We are reporting the functional analysis of the 5'-untranslated region (5'UTR) of human hepatoma HepG2 gamma-glutamyltransferase (GGT) mRNA. Transient expression of hybrid GGT-luciferase reporter gene mutants in HepG2 shows that this 5'UTR acts as a tissue-specific translational enhancer. A domain of 173 bases containing a steroid hormone response element (HRE) is responsible for the enhancing effect, which can be amplified by addition of dexamethasone at 10(-6) M. Furthermore, the regulatory role of the 5'UTR is demonstrated by interaction with sense and antisense oligonucleotides.

Base Sequence↗
PubMed1994

Molecular and functional characterization of recombinant human gamma-glutamyltransferase. Coupling of its activity to glutathione levels in V79 cells.

We previously described the establishment of a transfected cell line (V79HGGT) that stably produces the highest recombinant human gamma-glutamyltransferase (GGT) activity. We now report the utilization of V79HGGT as a model system for studying human GGT. The papain-solubilized recombinant enzyme has been highly purified from cultured cells by a new procedure. Studies on the purified enzyme, either by N-terminal sequencing or by characterization of its enzymic activities, confirmed that recombinant GGT shares structural and catalytic identity with native human enzymes. The circular dichroism analysis indicated an alpha-helical content of 19%. Based on these data, we have undertaken a study on the functional consequences of elevated GGT activity on the reduced glutathione (GSH) content. GSH status was followed in V79 and V79HGGT cells throughout growth. A particular pattern was observed for each cell line, depending on, but differentially affected by, alteration of the culture medium. Elevated GGT activity was associated with a 2.5-fold reduced GSH content, clearly suggesting a negative influence of the highly expressed enzyme on the GSH level under normal growth conditions. Possible mechanisms involved are proposed. Our findings pointed out that, among the GSH-related enzymes, GGT could constitute an important factor determining the steady-state content of GSH.

Amino Acid Sequence↗
PubMed1993

The 5' untranslated region of the human gamma-glutamyl transferase mRNA contains a tissue-specific active translational enhancer.

We report the functional and structural analysis of the 5' untranslated region (5'UTR) of human hepatoma HepG2 gamma-glutamyltransferase (GGT) mRNA. Transient expression of a hybrid GGT-luciferase gene in HepG2, MIA-Pa-Ca-2 and MG 63 cell lines shows that this 5'UTR acts as a tissue-specific translational enhancer. Evidence for transcripts with multiple 5'UTR coding for HepG2 GGT was obtained by RNase protection. Computer analysis of this 5'UTR detected the existence of a stable stem and loop structure containing multiple steroid modulatory elements.

Base Sequence↗
PubMed1993

Establishment of a V79 transfected cell line highly producing recombinant human gamma-glutamyltransferase.

gamma-Glutamyltransferase (GGT) is a glutathione-metabolizing enzyme whose activity variations in serum and organs are valuable markers of preneoplastic processes, alcohol abuse and induction by drugs. To elucidate the implication of GGT in various metabolic pathways, we established a stable transfected V79 cell line highly producing the human GGT. A full length cDNA, encoding the human hepatoma Hep G2 GGT, was subcloned into an expression vector under the control of the SV40 early promoter and was used to transfect V79 cells. A cell line was selected, exhibiting a GGT activity of 2 units per mg of protein, one of the highest levels reported to date. The recombinant GGT purified from this cell line showed the expected heterodimeric structure, with two subunits existing as sialylated and differentially glycosylated isoforms, with mean molecular masses of 80 and 29 kDa. Catalytic features were found to be identical to those of human serum and Hep G2 GGTs. Thus, the newly engineered cell line should be useful for the production of human GGT and as a potential alternative model for pharmacological studies.

Animals↗
PubMed1993

Eosinophilic pleural or peritoneal effusions in dogs and cats: 14 cases (1986-1992).

Case records of 9 dogs and 5 cats with eosinophilic effusions were reviewed. The animals ranged from 11 months to 13 years old. Seven animals had pleural effusions, 5 had peritoneal effusions, and 2 had pleural and peritoneal effusions. Neoplasia was confirmed in 6 animals and suspected in 1. Eosinophilic pleural effusion was diagnosed 2 days after pneumothorax developed as a consequence of thoracic tube placement in a cat, and pneumothorax was diagnosed in another cat with eosinophilic peritoneal effusion. Other abnormalities seen in 1 or 2 animals associated with eosinophilic effusion were radiographic signs of interstitial or peribronchial pulmonary infiltrates, a history of allergic respiratory tract and skin disease, intestinal lymphangiectasia and lung lobe torsion, chylothorax, bite wounds causing intestinal perforation, and feline leukemia virus infection. Based only on the protein concentration of the effusion, 7 effusions were classified as transudates and 7 were classified as exudates. Five of the 14 animals had eosinophilia (> 1,200 eosinophils/microliters); 3 of these animals had neoplastic disease. Mean eosinophil count in blood samples was not significantly different between animals with neoplasia and those without. Eosinophil counts in blood samples were not linearly related to counts in effusions; however, in some animals the number of eosinophils in the effusion was much higher than the eosinophil count in blood, suggesting concentration of eosinophils in the effusion.

Animals↗
PubMed1993

Induction of recombinant human gamma-glutamyl transferase by sodium butyrate in transfected V79 and CHO Chinese hamster cells.

Sodium butyrate was used to enhance biosynthesis rates of recombinant human gamma-glutamyl transferase (GGT) expressed under the control of the SV40 or the cytomegalovirus immediate early promoter, respectively, in transfected V79 and CHO Chinese hamster cell lines. Maximal induction of GGT specific activity in butyrate-treated cells ranged from 3 to 5-fold and resulted from a strong increase in the GGT mRNA ratio. We also observed that maximal transcription level in V79 cells occurred within 12 hr of treatment, whilst the cell proliferation was transiently arrested. Despite its processing requirements, induced GGT exhibited unchanged catalytic and physico-chemical features relative to human serum or hepatoma enzyme, thus appearing as an excellent model for further studies on human GGT.

Animals↗
PubMed1993

Application of gene transfer technologies to the production of enzyme reference materials: example of gamma-glutamyltransferase.

Protein reference materials are traditionally prepared by purification from mammalian or human tissues. The supply of these tissues is limited; consequently, there is a growing need for applied molecular and cellular biology technologies for the production of human recombinant proteins. This is especially true when only small amounts of the proteins are available in the tissues. We review the current knowledge necessary for high-level production of such proteins in different heterologous expression systems, using our data on gamma-glutamyltransferase (EC 2.3.2.2) as an example. We describe the steps required to achieve the expression of enzymes and other proteins in Escherichia coli, yeast, or mammalian cells. We list many of the problems investigators may face in preparing recombinant proteins, and provide information on selecting the most appropriate system as well as the most favorable experimental conditions. Depending on the expression system, recombinant proteins can potentially be obtained for most, if not all, enzymes of interest in clinical chemistry, and such proteins should possess characteristics very similar to those of the corresponding human native proteins. Studies suggest that these products can be used as reference materials in clinical chemistry laboratories.

Animals↗
PubMed1992

Gamma-glutamyltransferase: nucleotide sequence of the human pancreatic cDNA. Evidence for a ubiquitous gamma-glutamyltransferase polypeptide in human tissues.

gamma-Glutamyltransferase (GGT, EC 2.3.2.2) is an enzyme involved in glutathione metabolism and drug and xenobiotic detoxification. Using human hepatoma Hep G2 GGT cDNA as probe, we isolated a cDNA from a human pancreatic cDNA library. Analysis of the nucleotide sequences revealed a 2244-bp insert that includes an open reading frame of 1710 bp, encoding a protein identical to the Hep G2 and human placenta GGTs. Similarly, the 5' untranslated region, though shorter, is highly homologous to that of Hep G2 cDNA. These data suggest strongly that the same gene encodes human GGT in the placenta, Hep G2 and the pancreas. We further studied the distribution of the corresponding mRNA, called type I mRNA, in different human tissues. Using a highly sensitive method associating reverse transcription with specific amplification by polymerase chain reaction, cDNA was synthesized from total RNA isolated from the tissues and GGT specific fragments were amplified. We observed the presence of a specific cDNA fragment corresponding to the type I mRNA in the human tissues and cells tested, providing the evidence for a ubiquitous expression of this GGT mRNA in human tissues.

Amino Acid Sequence↗
PubMed1991

High-level expression of enzymatically active mature human gamma-glutamyltransferase in transgenic V79 Chinese hamster cells.

gamma-Glutamyltransferase [GGT; (5-glutamyl)-peptide:amino-acid 5-glutamyltransferase, EC 2.3.2.2] is a glutathione-metabolizing enzyme, whose activity variations in serum and organs are valuable markers of preneoplastic processes, alcohol abuse, and induction by xenobiotics. To elucidate the implication of GGT in various metabolic pathways, we established a stable transgenic V79 cell line, highly producing the human GGT. A full-length cDNA, encoding the human hepatoma HepG2 GGT, was subcloned in an expression vector under the control of the simian virus 40 early promoter and was used to transfect V79 cells. We selected a cell line exhibiting a GGT activity of 2 units per mg of protein, the highest GGT expression level reported to date. As described for the human kidney and liver enzymes, the recombinant GGT purified from this cell line showed a heterodimeric structure. Its two subunits existed as sialylated and differentially glycosylated isoforms, with mean molecular masses of 80 and 29 kDa. However, catalytic features were found to be identical to those of human serum and HepG2 GGTs. The newly engineered cell line thus should be useful for the production of human GGT and as a potential alternative model for pharmacological studies.

Animals↗
PubMed1991

Effects of RP 52028 and phenobarbital on mRNA levels of inducible and constitutive sex-specific cytochrome P450 isozymes in rat liver.

Sex-related differences in basal levels of mRNA coding for various cytochrome P450 isozymes and their inducibility by 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3- isoquinoline carboxamide (RP 52028) in comparison to phenobarbital (PB) were investigated in Sprague-Dawley rats. We observed that the inducible isozymes, namely cytochromes P450IIB1/2 and P450IIIA1/2 were barely detectable in non-induced animal livers. On the contrary, mRNAs coding for two constitutive forms of cytochrome, P450IIC7 and IIC11, were expressed at a high level in untreated rats in a sex-dependent manner. Cytochrome P450IIC11 mRNA was present in male rats only whereas P450IIC7 was expressed in both sexes but at a higher level in female rats. RP 52028 had a dose-dependent inducing effect on the P450IIB1/2 and IIIA1/2 isoforms in both sexes. After administration of a high dose (500 mg/kg), this molecule exhibited a pattern of induction similar to that of PB. Increases in the accumulation of these IIB1/2 and IIIA1/2 messengers were correlated with protein data, suggesting that RP 52028, like PB, induces these isozymes mainly through a pretranslational regulatory mechanism. On the other hand, PB and RP 52028 caused only a slight increase, less pronounced than in Wistar rats, in the mRNA level of the constitutive female-predominant P450IIC7, indicating a strain-related difference in inducibility of this isozyme. RP 52028 had no effect on P450IIC11 mRNA level in male rat liver, in contrast to the decreasing effect obtained with PB. Furthermore, the non-correlated changes in P450IIC11 mRNA level and microsomal testosterone 2 alpha-hydroxylase activity after treatment with RP suggests that this molecule modulates the expression of P450IIC11 at a posttranscriptional level only.

Animals↗
PubMed1991

Different constructs for the expression of mammalian gamma-glutamyltransferase cDNAs in Escherichia coli and in Saccharomyces cerevisiae.

To prepare a reference material for gamma-glutamyltransferase (GGT; EC 2.3.2.2) measurements in clinical chemistry, we constructed different vectors containing either the rat kidney or the human hepatoma Hep G2 GGT cDNA downstream from an inducible promoter for expression in Escherichia coli and Saccharomyces cerevisiae. Transformed bacterial and yeast cells were tested for GGT production by use of Western blot analysis and enzymatic activity measurements. Both rat renal and Hep G2 GGT cDNAs were expressed in E. coli, producing active and nonglycosylated enzymes localized in the periplasmic space. Recombinant Hep G2 GGT was synthesized as a single-chain protein, unlike rat renal GGT, which presented two polypeptides of 62 and 30 kDa, identified as the precursor and a GGT heavy-subunit-like peptide, respectively. Rat renal GGT was produced in S. cerevisiae as two polypeptides, 55 and 30 kDa, detected by antisera against rat renal GGT. These results suggest maturation mechanisms such as glycosylation and cleavage steps, enhancing the interest of S. cerevisiae as a useful expression system for producing active mammalian proteins as reference materials.

Animals↗