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Biomedical subjects

H Oppermann

Publications and source records attributed to H Oppermann.

At least 19 recordsLinked to original sources
PubMed1992

Osteogenic protein-2. A new member of the transforming growth factor-beta superfamily expressed early in embryogenesis.

Osteogenic protein-2, OP-2, a new member of the transforming growth factor-beta (TGF-beta) superfamily, closely related to the osteogenic/bone morphogenetic proteins, was discovered in mouse embryo and human hippocampus cDNA libraries. The TGF-beta domain of OP-2 shows 74% identity to OP-1, 75% to Vgr-1, and 76% to BMP-5, hence OP-2 may also have bone inductive activity. The genomic locus of OP-2 has seven exons, like OP-1, and spans more than 27 kilobases (kb). In the C-terminal TGF-beta domain, OP-2 has a unique additional cysteine. Mouse embryos express relatively high levels of OP-2 mRNA at 8 days, two species of 3 and 5 kb. A careful study of mRNA expression of the osteogenic proteins in specific organs revealed discrete mRNA species for BMP-3, BMP-4, BMP-5, and BMP-6/Vgr-1 in lung or liver of young and adult mice. OP-1 is expressed in kidney; however, OP-2 and BMP-2 mRNAs were not detected in any organs studied, suggesting an early developmental role.

Amino Acid Sequence↗
PubMed1991

Murine osteogenic protein (OP-1): high levels of mRNA in kidney.

The murine OP-1 gene (EMBL accession No. X56906) encoding the homolog of human osteogenic protein-1 was isolated from cDNA and genomic libraries using human OP-1 cDNA as probe. The deduced murine OP-1 amino acid sequence revealed 11 amino acids changes, three of them in the mature protein. Murine OP-1 probes were used for analysis of OP-1 mRNA in mouse embryo and organ tissues. Northern blot hybridization revealed multiple mRNA species: the major species of 2.2 kb, minor species of 1.8 and 2.4 kb and a large 4 kb species, which may represent alternative splices. Tissue specific expression was studied in brain, lung, heart, liver, spleen, kidney, adrenal and bladder tissue. Maximal levels of OP-1 mRNA were found in kidney which may explain the phenomenon of epithelial osteogenesis, first described by Huggins in 1931 using epithelium from the urinary tract. Moreover, our data suggest that kidneys may be the main site of OP-1 synthesis, even though it is distant from its physiological site of action, skeletal bone.

Amino Acid Sequence↗
PubMed1991

Biosynthetic antibody binding sites: development of a single-chain Fv model based on antidinitrophenol IgA myeloma MOPC 315.

The functional antigen binding region of antidinitrophenol mouse IgA myeloma MOPC 315 has been produced as a single-chain Fv (sFv) protein in E. coli. Recombinant 315 proteins included sFv alone, a bifunctional fusion protein with amino-terminal fragment B (FB) of staphylococcal protein A, and a two-chain 315 Fv fragment. Successful refolding of the 315 sFv required formation of disulfide bonds while the polypeptide was in a denatured state, as previously observed for the parent Fv fragment. Affinity-purified recombinant 315 proteins showed full recovery of specific activity, with values for Ka,app of 1.5 to 2.2 x 10(6) M-1, equivalent to the parent 315 Fv fragment. As observed for natural 315 Fv, the sFv region of active FB-sFv315 fusion protein was resistant to pepsin treatment, whereas inactive protein was readily degraded. These experiments will allow the application of protein engineering to the 315 single-chain Fv; such studies can advance structure-function studies of antibody combining sites and lead to an improved understanding of single-chain Fv proteins.

Amino Acid Sequence↗
PubMed1990

A bifunctional fusion protein containing Fc-binding fragment B of staphylococcal protein A amino terminal to antidigoxin single-chain Fv.

A bifunctional molecule was genetically engineered which contained an amino-terminal effector domain that bound immunoglobulin Fc (fragment B of staphylococcal protein A) and a carboxyl-terminal domain that bound digoxin [a single-chain Fv (sFv)]. Effector and sFv binding properties were virtually identical with those of the parent molecules, despite the proximity of the FB to the sFv combining site. This finding is unprecedented since in all molecules of the natural immunoglobulin superfamily, the antigen binding domain is amino terminal to the effector domain. The FB-sFv sequence was encoded in a single synthetic gene and expressed as a 33,106 molecular weight protein in Escherichia coli. After purification, renaturation, and affinity isolation, yield of active fusion protein were 110 mg/L of fermented cells (18.5-g cell paste). Bifunctionality was confirmed by the ability of FB-sFv to cross-link IgG to digoxin-bovine serum albumin, as measured by plate assays and by Ouchterlony analysis. Analysis of the expressed fusion protein suggests that the sFv holds promise for the development of multifunctional, targetable single-chain proteins.

Amino Acid Sequence↗
PubMed1990

Bovine osteogenic protein is composed of dimers of OP-1 and BMP-2A, two members of the transforming growth factor-beta superfamily.

A bone-inductive protein has been purified from bovine bone and designated as osteogenic protein (OP). The purified OP induces new bone at less than 5 ng with half-maximal bone differentiation activity at about 20 ng/25 mg of matrix implant in a subcutaneous bone induction assay. The purified osteogenic protein is composed of disulfide-linked dimers that migrate on sodium dodecyl sulfate gels as a diffuse band with an apparent molecular weight of 30,000. Upon reduction, the dimers yield two subunits that migrate with molecular weights of 18,000 and 16,000. Both subunits are glycosylated. After chemical or enzymatic deglycosylation, the dimers migrate as a diffuse 27-kDa band that upon reduction yields two polypeptides that migrate at 16 kDa and 14 kDa, respectively. The carbohydrate moiety does not appear to be essential for biological activity since the deglycosylated proteins are capable of inducing bone formation in vivo. Amino acid sequences of peptides generated by proteolytic digestion show that the subunits are distinct but related members of the transforming growth factor-beta super-family. The 18-kDa subunit is the protein product of the bovine equivalent of the human OP-1 gene and the 16-kDa subunit is the protein product of the bovine equivalent of the human BMP-2A gene.

Alkaline Phosphatase↗
PubMed1990

OP-1 cDNA encodes an osteogenic protein in the TGF-beta family.

Amino acid sequences of two tryptic peptides derived from enriched bovine osteogenic protein preparations revealed considerable homology to two members of the TGF-beta (transforming growth factor beta) supergene family, DPP (decapentaplegic protein) of Drosophila and Vg-1 (vegetal protein) of Xenopus. Building upon this information we constructed a synthetic consensus gene to use as a probe to screen human genomic libraries. This resulted in the isolation of three interrelated genes. Among these were BMP-2b and BMP-3 which have recently been described by others. The third gene, termed OP-1 (osteogenic protein one), is new and was subsequently shown to encode the human homolog of a major component of bovine osteogenic protein. The genomic clones were used to isolate the corresponding complementary DNA (cDNA) clones. Sequence analysis indicates that OP-1 is a relative of the murine Vgr-1 (Vg-1 related gene). This report describes the cDNA structure and putative amino acid sequence of OP-1.

Amino Acid Sequence↗
PubMed1990

Hormonal and renal responses to arterial chemoreceptor stimulation by almitrine in healthy and normotensive men.

Healthy and normotensive men (n = 11) were hospitalized and kept under controlled fluid and sodium intake (120 mequ/d) for 5 days. Their systemic arterial blood pressures as well as heart and breathing rates were measured, and venous blood and urine samples were collected at intervals of 1-4 h. Diuresis was induced by scheduled drinking of tea (150 ml/h). Electrolytes, osmolality, and creatinine were determined in both plasma and urine samples. Aldosterone, cortisol, and vasopressin concentrations were measured only in the plasma. On the 2nd and 3rd day of the experiments the participants received orally either a placebo-pill or 100 mg almitrine bismesylate (Vectarion). Each subject was tested in a placebo- and an almitrine experiment. The subjects responded to the almitrine treatment with a suppression of the plasma aldosterone content, a transient rise of glomerular filtration rate, a natriuresis and an increase of renal concentrating ability. In the placebo-experiments, only the transient rise of filtration rate was significant. The data indicate that almitrine, by stimulating the peripheral arterial chemoreceptors, suppresses plasma aldosterone and inhibits renal proximal sodium reabsorption by so far unknown mechanisms. They also suggest that oral and intravenous almitrine administrations, respectively, might differently affect renal hemodynamics and excretory function.

Adult↗
PubMed1988

Preparation of raf-oncogene-specific antiserum with raf protein produced in E. coli.

In this report we describe the expression of v-raf protein in E. coli using a tryptophan-promoter-driven expression vector and its immunological characterization by anti-peptide sera. The purified recombinant protein was used to produce raf-specific antibodies which are suitable for studying v-raf and c-raf proteins in vitro and in vivo in a variety of species ranging from mouse to man.

Amino Acid Sequence↗
PubMed1979

Uninfected vertebrate cells contain a protein that is closely related to the product of the avian sarcoma virus transforming gene (src).

Neoplastic transformation of cell by avian sarcoma virus is mediated by a single viral gene (src), which encodes a phosphoprotein (pp60src) with the enzymatic activity of a protein kinase. The DNAs of vertebrate species contain a highly conserved homologue of src that is also represented in the polysomal RNA of uninfected cells and, hence, may specify a normal cellular protein. We have used antisera directed against pp60src to isolate a closely related phosphoprotein (denoted vertebrate pp60) from uninfected chicken, quail, rat, and human cells. Our data indicate that vertebrate pp60 is a homologue of pp60src, highly conserved both antigenically and chemically. Moreover, the cellular protein may possess protein kinase activity similar to that associated with pp60src. We conclude that the product of src is a slightly modified analogue of a normal cellular protein.

Alpharetrovirus↗
PubMed1978

Effect of antabuse (disulfiram) on Rous sarcoma virus and on eukaryotic cells.

Antabuse (disulfiram) is widely used in the treatment of chronic alcoholism. We have examined the effect of this drug on malignant transformation by Rous sarcoma virus, on eukaryotic cell synthesis, and on nucleic acid binding. It was found that: (1) Disulfiram inhibits the activity of the RNA dependent DNA polymerase of Rous sarcoma virus and inactivates the ability of the virus to malignantly transform chick embryo cells. The monomer of disulfiram, diethyldithiocarbamate does not affect the virus. (2) Disulfiram induced the synthesis of four proteins in normal chick embryo and human foreskin cells. The monomer diethyldithiocarbamate, induced these proteins also. Cellular DNA synthesis is more sensitive to disulfiram than are RNA and protein synthesis. (3) Disulfiram binds to neither DNA or RNA in the presence or absence of copper. However, diethyldithiocarbamate in the presence of, but not in the absence of, copper binds to HeLa cell DNA and to Rous sarcoma virus 70 S genome RNA. These results indicate that this compound, which causes no symptoms in people who do not consume alcohol, may have significant effects on a cellular level.

Animals↗
PubMed1978

Induction of four proteins in eukaryotic cells by kethoxal bis(thiosemicarbazone).

Kethoxal bis(thiosemicarbazone) induces the synthesis of four proteins (100 000, 70 000, 35 000 and 25 000 daltons) in normal chick embryo cells. The 70 000 dalton species is produced at the fastest rate 2 hr after exposure to the compound. Pulse-chase experiments revealed neither precursors nor products of these proteins and both actinomycin and cycloheximide inhibited their synthesis. Neither of the two substituents of the inducer, kethoxal or thiosemicarbazide, were active. The four proteins were induced in several other species, but human cells produced only three proteins (100 000, 70 000 and a different 30 000 dalton form).

Animals↗
PubMed1978

Evidence that the transforming gene of avian sarcoma virus encodes a protein kinase associated with a phosphoprotein.

Avian sarcoma virus (ASV) induces sarcomas in animals and transforms fibroblasts to a neoplastic state in cell culture. A single viral gene (src) is responsible for both the induction and maintenance of neoplastic transformation. Recent work has identified a protein with a molecular weight of 60,000 daltons that is apparently encoded in src and may be the effector molecule for the gene (Brugge and Erikson, 1977; Purchio et al, 1978). The putative product of src can be immunoprecipitated by antisera obtained from rabbits bearing tumors induced by ASV. We have used this approach to isolate the protein to characterize further its genetic origins and possible function. Our rabbit tumor antisera precipitated a protein with a molecular weight of 60,000 daltons; according to serological, biochemical and genetic criteria, this protein is encoded in src. We found that this protein is phosphorylated and therefore denoted it pp60. Phosphorylation of pp60 could be accomplished in vitro with extracts of ASV-infected cells. A temperature-sensitive conditional mutation in src had no demonstrable effect on either the production or stability of pp60 in the infected cell, but phosphorylation of the protein was temperature-sensitive. Since the mutant src is not expressed at the restrictive temperature, our findings raise the possibility that phosphorylation of pp60 is required for its function as the putative effector of src. Immunoprecipitates prepared with extracts of ASV-infected cells and the rabbit tumor antisera contained a protein kinase activity that catalyzed phosphorylation of the heavy chains of immunoglobulin molecules, using either ATP or GTP as phosphate donor. The kinase activity immunoprecipitated in parallel with pp60 was obtained only from cells that contained a functioning product of src and could not be precipitated with antisera directed against structural proteins of ASV. A temperature-sensitive conditional mutation in src caused the kinase activity to be thermally inactivated in vitro far more rapidly than the activity from cells infected with wild-type virus. We conclude that both the protein kinase and pp60 are encoded in src, and that the enzymatic activity may be an intrinsic property of pp60. Phosphorylation of pp60 in cellular extracts was inhibited by calcium ion, whereas the immunoprecipitable kinase activity was not, suggesting that the kinase responsible for pp60 phosphorylation may be distinct from that encoded in src. Collett and Erikson (1978) have also identified a protein kinase activity associated with pp60. These findings raise the possibility that phosphorylation of specific cellular targets might account for transformation of the host cell by src.

Adenosine Triphosphate↗