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Biomedical subjects

J R Hartman

Publications and source records attributed to J R Hartman.

35 records · Page 2Linked to original sources
PubMed1991

Recombinant human superoxide dismutases: production and potential therapeutical uses.

In many pathological situations, tissue damage is caused by cellular generation of superoxide free radicals (O2-). These active species are generated during post-ischemic reperfusion of organs, in hyperoxic tissue, during acute and chronic inflammation and during exposure to ionizing radiation. Exogenous superoxide dismutase (SOD) was shown to significantly prevent such damage. The genes for human cytosolic Cu/ZnSOD and mitochondrial MnSOD were cloned and introduced into an E. coli expression system. The proteins were expressed in high yields and purified to homogeneity, yielding pharmaceutical-grade materials. These enzymes were used in a variety of in vivo animal models for the demonstration of their protective effects against oxidative damage. Comparative pharmacokinetic studies in rats have revealed that the half-life of Cu/ZnSOD was 6-10 min., while that of MnSOD was 5-6 hours, thus indicating that MnSOD may be superior to Cu/ZnSOD for the treatment of chronic diseases. Indeed, MnSOD was found to be effective as an anti-inflammatory agent in the rat carrageenan induced paw edema acute inflammation model. Both enzymes were also effective in ameliorating post-irradiation damage in mice exposed to whole-body or localized chest X-ray radiation.

Animals↗
PubMed1986

High-level expression of enzymatically active human Cu/Zn superoxide dismutase in Escherichia coli.

Expression of human Cu/Zn superoxide dismutase (SOD) with activity comparable to the human erythrocyte enzyme was achieved in Escherichia coli by using a vector containing a thermoinducible lambda PL promoter and a beta-lactamase-derived ribosomal binding site. The recombinant human SOD was found in the cytosol of disrupted bacteria and represented greater than 10% of the total bacterial protein. The enzyme was purified to homogeneity by salt precipitation, gel filtration chromatography, and ion exchange chromatography. The active enzyme was obtained in high yield only when 1 mol of copper and 1 mol of zinc were incorporated into each mol of subunit during bacterial growth or by reconstitution of the apoenzyme. Human Cu/Zn SOD produced in bacteria has an apparent subunit molecular mass of 19 kDa on NaDodSO4/polyacrylamide gels. The native enzyme behaves as a dimer of 32 kDa as determined by gel filtration. Sequence analysis of the NH2 terminus revealed that the first 14 amino acids corresponded to authentic human SOD except that the NH2-terminal alanine was not acetylated. Thus, the bacterial processing system readily removes the NH2-terminal methionine residue from recombinant human SOD.

Amino Acid Sequence↗
PubMed1982

Human influenza virus hemagglutinin is expressed in monkey cells using simian virus 40 vectors.

We have cloned and expressed the hemagglutinin (HA) gene of a human influenza virus (A/WSN/33) in monkey kidney cells by linking it to deleted simian virus 40 (SV40) genomes that contain the entire early gene region, the origin of replication, and late leader sequences. The HA gene (1775 base pairs long) was originally inserted by the dG . dC tailing technique into the multicopy plasmid of Escherichia coli, pBR322, using cDNA made from viral RNA. The cloned gene was further modified by treatment with nuclease Bal 31 to remove the dG . dC tails and some of the untranslated sequences and recloned in E. coli after addition of BamHI restriction endonuclease linkers. A number of SV40 and HA recombinants (SV--HA) were constructed by inserting recloned HA DNA into the late gene region of SV40. The SV--HA recombinants, when complemented in a lytic infection of monkey cells by the helper function of SV40 early deletion mutants expressed influenza HA as detected by immunofluorescence and immunoprecipitation of in vivo-labeled proteins using either heterogeneous anti-influenza rabbit antibodies or monoclonal antibodies against HA. Furthermore, the WSN HA expressed by the SV--HA recombinants was also glycosylated and possessed the same molecular weight (approximately 70,000) as the uncleaved HA of WSN virus in monkey cells.

Animals↗
PubMed1977

Inhibition of viral protein synthesis in monkey cells treated with interferon late in simian virus 40 lytic cycle.

We have investigated the effect of interferon on SV40 gene expression late in the lytic cycle, after early functions have been expressed and viral DNA replication has been initiated. Whereas pretreatment with interferon prior to infection reduces the amount of early SV40 RNA, post-infection treatment does not inhibit viral RNA synthesis. Viral 19S and 16S RNA species are found undiminished in quantity and poly(A) content. Despite the apparent normalcy of viral RNA classes, however, there is a marked reduction in the synthesis of their protein products, both T antigen and capsid polypeptides. The association of viral RNA with heavy polyribosomes is strongly reduced. On the other hand, there is no degradation of nonviral polyribosomes and the synthesis of most cellular proteins continues. These experiments demonstrate that late in infection, interferon treatment results in an inhibition of viral mRNA translation.

Antigens, Viral↗
PubMed1973

In vitro production of mouse mammary tumor virus in a mouse mammary tumor ascites line.

An ascites line derived from a spontaneous mouse mammary carcinoma produces, on explantation and cultivation in vitro, large amounts of oncornavirus particles. The biochemical, biophysical, and electron microscopic characteristics of the virions are described. Molecular hybridization and immunological methods identify these virions as mouse mammary tumor virus.

Animals↗