Biomedical subjects

F R Collart

Publications and source records attributed to F R Collart.

27 records · Page 2Linked to original sources

PubMed1991

Amplification and molecular cloning of the IMP dehydrogenase gene of Leishmania donovani.

A mutant (MPA100) strain of Leishmania donovania was generated from a wild type (D1700) population by virtue of its ability to survive the selective pressure of gradually increasing concentrations of mycophenolic acid (MPA), an inhibitor of IMP dehydrogenase (IMPDH) activity. Comparative growth experiments revealed that the MPA100 strain was 100-fold more resistant to MPA toxicity and cross-resistant to ribavarin, another inhibitor of IMPDH. A direct comparison of IMPDH levels in D1700 and MPA100 cells showed that the latter expressed at least 20-fold higher enzyme activity. In order to evaluate the mechanism by which MPA100 cells overexpressed IMPDH, the leishmanial gene encoding IMPDH was isolated from a genomic library in EMBL3 by cross-hybridization to a mouse IMPDH cDNA, and a 2.3-kilobase EcoRV-PstI fragment was subcloned into a Bluescript vector and sequenced. The EcoRV-PstI fragment contained an open reading frame of 514 amino acids that encompassed the entire leishmanial IMPDH coding sequence. The predicted amino acid sequence showed a 52.5% identity with that of the corresponding human IMPDH. The codon usage of the leishmanial IMPDH gene reflected a strong bias toward codons containing either G or C in the wobble position. The EcoRV-PstI fragment hybridized to a 3.0-kilobase mRNA that was expressed at 10-20-fold greater levels in the MPA100 cells. Using the EcoRV-PstI fragment as a probe, the increased amount of IMPDH activity and IMPDH mRNA in the MPA100 cells could be attributed to an approximately 10-20-fold amplification of the leishmanial IMPDH gene.

Amino Acid Sequence↗

PubMed1991

Regulation of IMP dehydrogenase gene expression by its end products, guanine nucleotides.

To study the regulation of IMP dehydrogenase (IMPDH), the rate-limiting enzyme of guanine nucleotide biosynthesis, we examined the effects of nucleosides, nucleotides, nucleotide analogs, or the IMPDH inhibitor mycophenolic acid (MPA) on the steady-state levels of IMPDH mRNA. The results indicated that IMPDH gene expression is regulated inversely by the intracellular level of guanine ribonucleotides. We have shown that treatment with guanosine increased the level of cellular guanine ribonucleotides and subsequently reduced IMPDH steady-state mRNA levels in a time- and dose-dependent manner. Conversely, MPA treatment diminished the level of guanine ribonucleotides and increased IMPDH mRNA levels. Both of these effects on the steady-state level of IMPDH mRNA could be negated by cotreatment with guanosine and MPA. The down regulation of IMPDH gene expression by guanosine or its up regulation by MPA was not due to major changes in transcriptional initiation and elongation or mRNA stability in the cytoplasm but rather was due to alterations in the levels of the IMPDH mRNA in the nucleus. These results suggest that IMPDH gene expression is regulated by a posttranscriptional, nuclear event in response to fluctuations in the intracellular level of guanine ribonucleotides.

Animals↗

PubMed1990

Expression of IMP dehydrogenase in differentiating HL-60 cells.

Addition of mycophenolic acid to cultures of HL-60 cells results in a decreased cellular level of guanine nucleotides and the induction of cell differentiation. During the early stages of this induction, steady-state levels of cellular IMP dehydrogenase (IMPDH), messenger RNA (mRNA), and protein are increased, perhaps because of cellular compensation for the inhibition of IMPDH activity. The subsequent decrease in IMPH mRNA and protein levels after several days of treatment suggests a change in the control of IMPDH expression. In contrast to the pattern of increased IMPDH expression observed in the mycophenolic acid-treated cells, treatment of HL-60 cells with two other inducers of differentiation, namely retinoic acid and phorbol 12-myristate 13-acetate, resulted in stable or decreased levels of cellular IMPDH mRNA and protein. However, the kinetics of this expression were different. These results suggest that a number of factors influence the regulation of IMPDH expression during the induction of HL-60 cell differentiation, including the nature of the inducer. A decrease in the cellular IMPDH activity was observed for all of the inducers, suggesting that this decreased activity may be a determining factor in the acquisition of a mature phenotype in the HL-60 cells.

Blotting, Northern↗

PubMed1990

Cell differentiation and altered IMP dehydrogenase expression induced in human T-lymphoblastoid leukemia cells by mycophenolic acid and tiazofurin.

The IMP dehydrogenase inhibitors mycophenolic acid (MPA) and tiazofurin (TZ) induce a time- and dose-dependent inhibition of cell growth, as well as differentiation in T-lymphoid CEM-2 leukemia cells. The differentiated cells have acquired a suppressor/cytotoxic T-lymphocyte phenotype characterized by reactivity with maturation-specific monoclonal antibodies. Coadministration of guanosine and hypoxanthine reduces the growth inhibition and diminishes the induction of differentiation by either MPA or TZ. No such reduction was observed for differentiation induced by phorbol 12-myristate 13-acetate (PMA), another inducer of a suppressor/cytotoxic phenotype in CEM-2 cells. During the first 2 days of treatment with MPA or TZ, a pattern of stable IMPDH mRNA levels and increased amounts of cellular enzyme was observed, perhaps, because of compensation for the inhibitor-mediated decrease in cellular IMPDH activity or a MPA- or TZ-mediated decrease in proteolysis of IMPDH. PMA treatment decreased the levels of IMPDH mRNA, protein, and activity. In addition, treatment of CEM-2 cells with either IMPDH inhibitors or PMA caused different alterations of the ribonucleotide pools. The lack of a consistent pattern of IMPDH expression in CEM-2 cells treated with IMPDH inhibitors or PMA indicates that no general association exists between the induction of cell differentiation and the expression of IMPDH. Nevertheless, our results indicating that IMPDH inhibitors can induce differentiation in CEM-2 cells suggest that this treatment may provide a useful approach to circumvent the differentiation block in some tumor cells.

Antineoplastic Agents↗

PubMed1990

Induction of cell differentiation in melanoma cells by inhibitors of IMP dehydrogenase: altered patterns of IMP dehydrogenase expression and activity.

To study the induction of differentiation in human melanoma cells, we treated 12 melanoma cell lines with mycophenolic acid and tiazofurin, inhibitors of IMP dehydrogenase (IMPDH). In all cell lines studied, both agents inhibited cell growth and increased melanin content. However, the degree of growth inhibition did not necessarily correspond to the increase in melanin content. A detailed analysis of the HO and SK-MEL-131 cell lines indicated that mycophenolic acid and tiazofurin caused a time- and dose-dependent increase in the expression of a series of other maturation markers, including formation of dendrite-like structures, tyrosinase activity, and reactivity with the CF21 monoclonal antibody. The growth inhibition and melanogenesis induced by the IMPDH inhibitors was abrogated by the addition of exogenous guanosine. No such effect was observed after treatment of the cells with phorbol 12-myristate 13-acetate or retinoic acid, two other inducers of differentiation in these cells. The mycophenolic acid- and tiazofurin-treated cells also showed an increased level of IMPDH mRNA and protein, perhaps because of compensation for the inhibitor-mediated decrease in IMPDH activity. In contrast, treatment with phorbol 12-myristate 13-acetate or retinoic acid resulted in decreased levels of IMPDH mRNA and protein. The lack of a consistent pattern of IMPDH expression in the cells treated with IMPDH inhibitors and phorbol 12-myristate 13-acetate or retinoic acid suggests that the altered expression of IMPDH is not a general requirement for the induction of cell differentiation in these cells. Our results also suggest that IMPDH inhibitors may provide a useful approach to circumvent the differentiation block in melanoma.

Biomarkers↗

PubMed1989

A protein containing the cystic fibrosis antigen is an inhibitor of protein kinases.

To investigate myeloid cell maturation, we established a panel of monoclonal antibodies that recognize myeloid cell nuclear antigens. One of these monoclonal antibodies was used to purify a specific protein complex (PC) from a human spleen. This PC, which is present at high levels in peripheral blood monocytes and granulocytes, contains a protein that is the cystic fibrosis (CF) antigen. The purified PC was shown to inhibit the activity of casein kinase I and II but not cAMP-dependent protein kinase, protein kinase C, v-abl tyrosine kinase, or insulin receptor tyrosine kinase. The observed Ki values for casein kinases I and II purified from several sources were 1 microM or less. Furthermore, the addition of the purified PC to a nuclear extract from human cells was able to prevent protein kinase-mediated stimulation of RNA polymerase activity. The unique inhibitory character of the PC and its elevated levels in monocytes and granulocytes and of the CF antigen in CF patients implies that this complex may be associated with myeloid cell functions and perhaps with the cause or consequence of the clinical manifestations of CF.

Amino Acid Sequence↗

PubMed1988

Cloning and sequence analysis of the human and Chinese hamster inosine-5'-monophosphate dehydrogenase cDNAs.

Inosine-5'-monophosphate dehydrogenase, a key enzyme in the regulation of guanine nucleotide biosynthesis, was purified to homogeneity; and a polyclonal antibody directed against the purified protein was used to isolate human and Chinese hamster IMP dehydrogenase cDNA clones. These clones were sequenced and found to contain an open reading frame of a protein containing 514 amino acids. A sequence of 35 amino acids obtained by analysis of the purified protein is identical to a segment of the protein sequence deduced from the IMP dehydrogenase cDNA. The molecular mass of the deduced protein is 56 kDa, which is the observed molecular mass of the purified protein and of the immunoprecipitated in vitro translation product. Comparison of the protein sequences deduced from the human and Chinese hamster cDNA clones indicates only eight amino acid differences, suggesting that IMP dehydrogenase is a highly conserved protein.

Amino Acid Sequence↗

PubMed1987

Amplification of the IMP dehydrogenase gene in Chinese hamster cells resistant to mycophenolic acid.

The regulation of IMP dehydrogenase (IMPDH) was analyzed in Chinese hamster V79 cell variants that exhibit different degrees of resistance to the cytotoxic effect of mycophenolic acid, a specific inhibitor of IMPDH. Western blot (immunoblot) analysis with an IMPDH antiserum revealed a 14- to 27-fold increase in the amount of enzyme in the mycophenolic acid-resistant cells. The antiserum was also used to screen for a phage containing the IMPDH cDNA sequence from a lambda gt11 expression library. Northern blot (RNA blot) analyses of total cellular and poly(A)+ RNA showed that an IMPDH cDNA probe hybridized to a 2.2-kilobase transcript, the amount of which was associated with increased resistance. Southern blotting with the probe indicated an amplification of the IMPDH gene in the mycophenolic acid-resistant cells. Our findings suggest that the acquired mycophenolic acid resistance of the V79 cell variants is associated with increases in the amount and activity of IMPDH and the number of IMPDH gene copies.

Animals↗

PubMedDate not supplied

Efficient recognition of protein fold at low sequence identity by conservative application of Psi-BLAST: application.

Based on a study involving structural comparisons of proteins sharing 25% or less sequence identity, three rounds of Psi-BLAST appear capable of identifying remote evolutionary homologs with greater than 95% confidence provided that more than 50% of the query sequence can be aligned with the target sequence. Since it seems that more than 80% of all homologous protein pairs may be characterized by a lack of significant sequence similarity, the experimental biologist is often confronted with a lack of guidance from conventional homology searches involving pair-wise sequence comparisons. The ability to disregard levels of sequence identity and expect value in Psi-BLAST if at least 50% of the query sequence has been aligned allows for generation of new hypotheses by consideration of matches that are conventionally disregarded. In one example, we suggest a possible evolutionary linkage between the cupredoxin and immunoglobulin fold families. A thermostable hypothetical protein of unknown function may be a circularly permuted homolog to phosphotriesterase, an enzyme capable of detoxifying organophosphate nerve agents. In a third example, the amino acid sequence of another hypothetical protein of unknown function reveals the ATP binding-site, metal binding site, and catalytic sidechain consistent with kinase activity of unknown specificity. This approach significantly expands the utility of existing sequence data to define the primary structure degeneracy of binding sites for substrates, cofactors and other proteins.

Algorithms↗