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Biomedical subjects

J M Weber

Publications and source records attributed to J M Weber.

At least 109 records · Page 6Linked to original sources
PubMed1992

Adenovirus transformation revertant resistant to retransformation by E1 but not by SV40-T and HPV16-E7 oncogenes.

We have previously described a revertant cell line which expresses a dominant tumor suppressor phenotype to E1 but not to heterologous oncogenes such as c-myc, N-ras, or polyoma middle t (Sircar et al. (1988) Oncogene 3, 725-728). DNA tumor virus oncogenes have been suggested to transform cells via the common mechanism of sequestering the Rb-105 antioncoprotein. This paradigm would predict that our revertant cell line, which is resistant to retransformation by E1a, should also be resistant to the other members of the Rb-105 binding family of oncogenes. To test this hypothesis we transfected the revertant cell line with plasmids bearing SV40-T or the HPV16-E7 oncogenes. Because transformation was obtained by both oncogenes at efficiencies similar to the transformation of a related revertant cell line, the results suggest that the resistance phenotype is specific to E1a. This specificity was further confirmed by cell fusion experiments.

Adenoviridae↗
PubMed1992

Reduction of c-myc expression correlated with E1a expression but not with the transformed phenotype.

The adenovirus E1a oncogene has both positive and negative regulatory effects on the expression of a variety of host genes. Both type of effects have been reported for certain cell cycle genes such as c-myc. To study the potential role of c-myc in adenovirus transformation, we have assessed the steady-state levels of c-myc mRNA after serum stimulation in genetically related transformed or non-transformed cell lines in the presence or absence of E1a. Serum stimulated the accumulation of stable c-myc mRNA only in cell lines which did not express E1a. Therefore under the present assay conditions, E1a had a negative effect on the steady-state level of c-myc mRNA. Surprisingly, this effect was independent of the transformed phenotype.

Adenoviridae↗
PubMed1991

An erythromycin derivative produced by targeted gene disruption in Saccharopolyspora erythraea.

Derivatives of erythromycin with modifications at their C-6 position are generally sought for their increased stability at acid pH, which in turn may confer improved pharmacological properties. A recombinant mutant of the erythromycin-producing bacterium, Saccharopolyspora erythraea, produced an erythromycin derivative, 6-deoxyerythromycin A, that could not be obtained readily by chemical synthesis. This product resulted from targeted disruption of the gene, designated eryF (systematic nomenclature, CYP107), that apparently codes for the cytochrome P450, 6-deoxyerythronolide B (DEB) hydroxylase, which converts DEB to erythronolide B (EB). Enzymes normally acting on EB can process the alternative substrate DEB to form the biologically active erythromycin derivative lacking the C-6 hydroxyl group.

Amino Acid Sequence↗
PubMed1991

Complementation of adenovirus early region 1a and 2a mutants by Epstein-Barr virus immortalized lymphoblastoid cell lines.

Human B-lymphocytes may be infected by both adenoviruses and the Epstein-Barr virus (EBV). Some of the immediate early and early proteins in the two viruses are similar in function even though their primary structures are different. As these viruses might infect the same B-cells in man, we asked if complementation could take place. The adenovirus mutant H5ts125 has a thermolabile DNA-binding protein and is defective in DNA replication at 39 degrees. Several EBV-transformed human lymphoblastoid cell lines and a tamarin cell line B95-8 were infected with H5ts125 and incubated at either the nonpermissive or the permissive temperatures. Adenoviral DNA replication and assembly of new virions were observed at both temperatures, suggesting complementation by the resident EBV gene products. The adenovirus E1a region is deleted in the mutant d1312. Complementation of this mutant was only obtained in the EBV producer B95-8 cells. Immortalization by EBV was apparently not sufficient for effective complementation. This supports an earlier observation that one of the EBV early proteins (MS-EA) behaves like adenovirus E1a and can transactivate the E4 promoter in a CAT assay. The complementation of mutant adenoviruses in EBV-transformed lymphocytes may help the rescue of new adenovirus serotypes in immunosuppressed patients.

Adenovirus Early Proteins↗
PubMed1991

Effect of endurance swimming on the lactate kinetics of rainbow trout.

The lactate turnover rate of rainbow trout (Oncorhynchus mykiss) was measured by bolus injection of [U-14C]lactate at rest and during prolonged swimming at 85% Ucrit to determine the importance of this metabolic fuel for endurance locomotion in fish, to assess whether lactate exchange between white and red muscle could be a possible mechanism for supplying oxidizable fuel to their lateral red muscle, and to compare the contribution of lactate to total energy provision between teleost and mammalian species. Turnover rate only increased from 4.41 +/- 0.33 to 9.71 +/- 1.69 mumol kg-1 min-1 between rest and prolonged swimming, and the contribution of lactate oxidation to total metabolism declined during exercise. Lactate exchange between white and red muscle is, therefore, not a significant mechanism to fuel the active lateral red musculature during prolonged swimming. The lactate turnover rate of teleosts is one or two orders of magnitude lower than in mammals of equivalent size, but lactate has the same importance as a fuel in both vertebrate groups. However, lactate turnover rate and oxidation rate do not scale with body mass in the same fashion as does metabolic rate. The slope of the mammalian relationship for whole-body lactate turnover and oxidation is much lower (0.58) than the slope of the classic relationship for metabolic rate (0.75), indicating that lactate is a much more important oxidative substrate for small than for large animals.

Animals↗
PubMed1991

Dominant suppression of adenovirus mediated transformation and insufficiency of p105Rb binding as a condition for oncogenic transformation.

An adenovirus-specific transformation resistant cell line (G2) expressing biologically active E1a proteins and originally isolated as a revertant from Ad2-transformed rat cells (F4), was shown to form stable Rb-E1a and 300K-E1a complexes in immunoprecipitation experiments. Consistent with the transformation resistant phenotype, cell hybrids between G2 and F4 were all nontumorigenic. Retrovirus insertion mutagenesis resulted in tumorigenic cell lines and identified a common locus responsible for the E1a-specific dominant tumor suppressor phenotype of G2 cells.

Adenoviruses, Human↗
PubMed1990

Cloning of genes involved in erythromycin biosynthesis from Saccharopolyspora erythraea using a novel actinomycete-Escherichia coli cosmid.

Two plasmids were constructed that replicate in Saccharopolyspora (Sac.) erythraea, Escherichia coli and Streptomyces (S.) lividans, and used for the cloning of a locus involved in the synthesis of the macrolide antibiotic erythromycin (Er). Plasmid pAL7002 contains the thiostrepton-resistance gene (tsr), a replicon-containing fragment from pJVI and pUC9. Plasmid pNJI contains the lambda cos site but is otherwise similar to pAL7002. A library of total DNA from Sac. erythraea was constructed in pNJI and probed in colony hybridizations with a DNA fragment containing ermE, the Sac. erythraea ErR-encoding gene. Plasmids obtained were subsequently introduced into EryA mutants of Sac. erythraea blocked in synthesis of Er (Ery-) and transformants were screened for restoration of Er production (Ery+). Several plasmids were found to convert two mutants to Ery+, but a third EryA strain could not be restored to Ery+ by any of the plasmids employed. A 5-kb segment, designated eryAI, responsible for restoring the Ery+ phenotype in the EryA strains, was identified and mapped in the segment 12 to 17 kb downstream from ermE. Gene disruption experiments indicated that the 5-kb length of eryAI is fully internal to an eryAI-containing transcript. In Southern blots it was shown that one of the EryA strains carried a small deletion in eryAI and that, in at least some of the transformants restored to Ery+, the deletion had been replaced by the wild-type eryAI allele.(ABSTRACT TRUNCATED AT 250 WORDS)

Actinomycetaceae↗
PubMed1990

Adenovirus proteinases: comparison of amino acid sequences and expression of the cloned cDNA in Escherichia coli.

Adenoviruses (Ad) synthesize serine-center endoproteinases (AdEPs) responsible for maturation cleavages within the virus particle. Many questions regarding these enzymes remain unanswered because previous studies utilized crude cells or viral lysates as the enzyme source. Here, we report on the comparison of the amino acid (aa) sequences of several AdEPs and on the expression of the cDNA of the Ad2Ep in Escherichia coli. The AdEPs consist of about 200 aa and their size is around 23 kDa. Among the seven sequences known, 60% of aa were strictly conserved. The usual serine proteinase active site sequence, GDSGG, is absent. The recombinant Ad2EP, produced by an inducible vector as a protein-A fusion product is capable of autocatalytic cleavage, and of cleaving its natural viral substrates as well as foreign proteins. Therefore, other viral proteins or mammalian specific post-translational modifications are not required for enzyme activity.

Adenoviridae↗
PubMed1990

Lanosterol 14 alpha-demethylase-encoding gene: systematic analysis of homologous overexpression in Saccharomyces cerevisiae using strong yeast promoters.

The Saccharomyces cerevisiae 14DM gene, encoding cytochrome P450 lanosterol 14 alpha-demethylase (14DM), was overexpressed in various S. cerevisiae strains under the control of three strong heterologous yeast transcription promoters (pADC1, pGPD, pPHO5) and under the control of its own promoter. Striking, strain-specific differences in 14DM transcription and in 14DM contents have been observed. The relative abundances of 14DM-specific mRNA and protein derived from a series of different expression plasmids were compared. It was found that the inducible PHO5 promoter in combination with the JL745 host led to the highest expression levels. 14DM-specific RNA reached up to 2% of the total cellular mRNA in this strain and approx. 3% of the total soluble yeast-cell protein was determined to be 14DM by quantitative Western blotting. By comparing the abundances of the different fusion transcripts with the transcript originating from the corresponding endogenous gene from which the promoter was derived, it could be concluded that the expression levels of the different 14DM fusion genes were far below the theoretically attainable values.

Base Sequence↗
PubMed1990

Retransformation of a revertant cell line with the adenovirus E1 oncogenes and vanadate.

We have isolated a revertant cell line (G5) from an adenovirus transformed rat cell line (F4) which failed to express the integrated viral oncogenes. To determine whether the reversion mutation was acting in cis or trans the G5 cells were co-transfected with an E1 gene bearing expression plasmid and a neomycin phosphotransferase bearing plasmid. G418-resistant colonies were picked and shown to express the E1 proteins and to be tumorigenic. This re-transformation could be partially mimicked by treatment with vanadate, an inhibitor of phosphotyrosine phosphatases. These results show that the continued presence of the E1 proteins was required to maintain the transformed phenotype, and that the reversion mutation was a cis-acting event affecting directly the integrated E1 genes.

Adenoviridae↗
PubMed1990

Adenovirus type 2 precursor proteins are cleaved by proteinases of other adenoviruses.

A virus-encoded endoproteinase is responsible for the cleavage of adenovirus type 2 (Ad2) precursor proteins. By analyzing the genetic maps of previously characterized interserotypic recombinants between Ad2 and Ad5 and by means of an in vitro proteinase assay using Ad1, 2, 3, 4, 5, 6, 7, 9 as the source of enzyme, it was demonstrated that (i) the enzyme was present in these serotypes and by extension it is likely to be present in all adenoviruses, and (ii) that proteolytic cleavage can take place efficiently even between widely different serotypes. These attributes suggest a high degree of sequence conservation of the enzyme and substrate cleavage sites, thus designating proteolytic processing as an excellent target for chemotherapy.

Adenoviridae↗