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Biomedical subjects

S M Rybak

Publications and source records attributed to S M Rybak.

49 records · Page 3Linked to original sources
PubMed1988

Expression of human angiogenin in cultured baby hamster kidney cells.

Baby hamster kidney cells were transformed with DNA sequences derived from the gene for human angiogenin. Expression was under the transcriptional control of the inducible mouse metallothionein 1 promoter. Recombinant angiogenin was purified and shown to be chemically, biologically, and enzymatically indistinguishable from the natural product. The large-scale production of recombinant angiogenin achieved should facilitate detailed studies into the structure-function relationships of this potent angiogenic molecule.

Angiogenesis Inducing Agents↗
PubMed1988

Base cleavage specificity of angiogenin with Saccharomyces cerevisiae and Escherichia coli 5S RNAs.

The base cleavage specificity of angiogenin toward naturally occurring polyribonucleotides has been determined by using rapid RNA sequencing technology. With 5S RNAs from Saccharomyces cerevisiae and Escherichia coli, angiogenin cleaves phosphodiester bonds exclusively at cytidylic or uridylic residues, preferably when the pyrimidines are followed by adenine. However, not all of the existent pyrimidine bonds in the 5S RNAs are cleaved, likely owing to elements of structure in the substrate. Despite the high degree of sequence homology between angiogenin and ribonuclease A (RNase A), which includes all three catalytic as well as substrate binding residues, the cleavage patterns with natural RNAs are unique to each enzyme. Angiogenin significantly hydrolyzes certain bonds that are not appreciably attacked by RNase A and vice versa. The different cleavage specificities of angiogenin and RNase A may account for the fact that the former is angiogenic while the latter is not.

Adenocarcinoma↗
PubMed1988

Comparison of the effects of class 1 and class 2 heparin-binding growth factors on protein synthesis and actin mRNA expression in BALB/c-3T3 cells.

Biological effects of class 1 or class 2 heparin-binding growth factors (HBGFs) were compared in BALB/c-3T3 cells. Changes in protein synthesis, as monitored by two-dimensional gel electrophoresis, reveal that while both HBGFs induce the same changes in the synthesis of intracellular proteins, class 2 HBGF selectively increases the synthesis of a 43-kD extracellular protein. Heparin, which potentiates the mitogenic activity of class 1 but not class 2 HBGF, does not potentiate the changes in protein synthesis elicited by HBGF-1. Since each HBGF increases actin synthesis, regulation of actin mRNA expression was examined. Actin mRNA levels increase rapidly and transiently in response to either HBGF, and similar superinduction responses are observed in the presence of HBGF and cycloheximide. Although the maximum increase in actin mRNA stimulated by either HBGF is similar, the levels of mRNA induced by class 2 HBGF remain elevated up to 48 hours compared to the level induced by class 1 HBGF. These results imply that in the same cell type class 1 and class 2 HBGFs may modulate some biological effects differently.

Actins↗
PubMed1987

Angiogenin mRNA in human tumor and normal cells.

Angiogenin mRNA was characterized in HT-29 human colon adenocarcinoma cells and its distribution in other human cell types was studied. Several RNA species ranging from 800 to 6000 nucleotides hybridized to the angiogenin probes with the smallest being the major poly(A)-containing transcript. This transcript was detected in tumor cells of diverse cellular origin. Expression of angiogenin mRNA is not limited to neoplastic cells and is detected in normal epithelial cells, fibroblasts and peripheral blood cells. The latter, when induced by mitogens, expressed more mRNA than did unstimulated or HT-29 cells. Transformed fibroblasts did not contain higher levels of angiogenin mRNA than their normal counterparts, demonstrating that increased angiogenin mRNA expression does not necessarily occur upon neoplastic transformation. This study demonstrates that angiogenin mRNA is expressed in a wide spectrum of cells and is not correlated to a particular cell phenotype.

Angiogenesis Inducing Agents↗
PubMed1987

Angiogenin abolishes cell-free protein synthesis by specific ribonucleolytic inactivation of ribosomes.

Angiogenin is a potent inhibitor of cell-free protein synthesis. When incubated with rabbit reticulocyte lysate at a concentration of 40-60 nM, it completely abolishes the capacity of the lysate to support protein synthesis. The inhibition appears to be due to its ribonucleolytic activity since it (i) generates limited cleavage products from reticulocyte RNA and (ii) is prevented from both cleaving RNA and inhibiting protein synthesis by placental RNase inhibitor. The ribonucleolytic activity of angiogenin toward the reticulocyte RNA system is highly specific. Thus, under conditions where angiogenin totally abolishes protein synthesis, an equivalent concentration of pancreatic RNase A inhibits it only partially. In contrast, RNase A is a much more effective enzyme than angiogenin using isolated RNA as substrate. Angiogenin inhibits protein synthesis by cleaving rRNA, thereby inactivating the protein synthesis machinery. Addition of isolated reticulocyte ribosomes to an angiogenin-treated lysate restores the capacity for protein synthesis, whereas addition of tRNA or mRNA does not. This potent effect on protein synthesis suggests a possible physiological function of angiogenin whose overall relevance and implications should become evident as the mechanisms of neovascularization are deciphered. The use of angiogenin may also further elucidate ribosome structure and its role in protein synthesis.

Cell-Free System↗
PubMed1986

Lysates of two established human tumor lines contain heparin-binding growth factors related to bovine acidic brain fibroblast growth factor.

Cell lysates of two established human tumor lines, a medulloblastoma (TE671), and a rhabdomyosarcoma (RD), contain mitogenic activity which elutes from heparin-Sepharose under conditions typical of class 1 heparin-binding growth factors, such as acidic brain fibroblast growth factor. The presence of this class of mitogen in both cell lines was confirmed by their chromatographic behavior on reversed-phase C3 columns, and by the ability of heparin to enhance their mitogenic activity. Using a specific synthetic DNA probe, RNA's were isolated from both cell lines by hybridization-selection, translated in vitro, and translated proteins affinity fractionated on heparin-Sepharose. The results demonstrate that TE671 and RD cell lysates contain mRNA's for mitogens related to acidic brain fibroblast growth factor, and also suggest that high molecular weight proteins exist that are closely related to, or are precursor forms of, the class 1 mitogens.

Animals↗
PubMed1982

Mechanism of induction of delta 5-3 beta-hydroxysteroid dehydrogenase-isomerase activity in rat adrenocortical cells by corticotropin.

The regulation of delta 5-3 beta-hydroxysteroid dehydrogenase-isomerase (3 beta-HSD) was studied in primary cultures of rat adrenocortical cells. In the absence of ACTH, this enzymic activity was found to decay with a half-life of 3.1 days, which was similar to the half-life of the enzyme activity induced by ACTH in vitro (3.5 days). The increase in 3 beta-HSD activity was highly specific for ACTH and dibutyryl cAMP; the activity was not increased by other hormones known to affect adrenocortical growth or function. The induction of 3 beta-HSD activity by ACTH or dibutyryl cAMP required a lag period of approximately 4 h and was dependent on RNA and protein syntheses. The increase in 3 beta-HSD activity observed after ACTH treatment was not a result of ACTH-induced inhibition of degradation of the enzyme, nor was it due to the synthesis of a soluble intermediate which could directly activate the enzyme. ACTH stimulated the incorporation of [35S]methionine into a protein associated with 3 beta-HSD activity detected on polyacrylamide gels after electrophoresis of Triton X-100 extracts of adrenocortical cells. The induction of this protein by ACTH was inhibited by actinomycin D. A protein band of a partially purified preparation of rat adrenal 3 beta-HSD was found to comigrate with the ACTH-induced protein on sodium dodecyl sulfate-polyacrylamide gel. These results suggest that ACTH caused the de novo synthesis of 3 beta-HSD by a mechanism dependent on RNA synthesis.

3-Hydroxysteroid Dehydrogenases↗
PubMed1981

Primary culture of normal rat adrenocortical cells. I. Culture conditions for optimal growth and function.

Rat adrenocortical cells retained their differentiated characteristics over 2 wk in culture without a specific requirement for additives other than inorganic salts, amino acids, vitamins, and fetal bovine serum. The cells were maintained free from fibroblast overgrowth by substitution of D-value in place of L-valine in the medium. Corticotropin (ACTH) inhibited the growth of adrenocortical cells in this medium and the effect was reversible. The adrenocortical cells had a limited capacity for growth as reflected by total cell counts and [3H]thymidine uptake with cells from young animals demonstrated a greater potential for DNA synthesis than cells obtained from mature animals. A very sensitive assay for ACTH using a small number of cells in primary culture also is described.

Adrenal Cortex↗
PubMed1981

Primary culture of normal rat adrenocortical cells. II. Quantitation of steroid dehydrogenase stain.

A simple, rapid procedure for the quantitation of the histochemical stain for delta 5-3beta-hydroxysteroid dehydrogenase (3beta-HSD) in cultured cells is described. Adrenocortical cells were stained for 3beta-HSD and then solubilized with 0.5 N NaOH. The absorbance of the solubilized cell solution was measured at 570 nm and a linear relationship was obtained between the number of cells and the intensity of the absorption. It is shown that the regulation of 3beta-HSD activity in primary cultures of normal rat adrenocortical cells can be monitored by the quantitative histochemical assay. The results show a good correlation with the data obtained by a biochemical assay for the enzyme.

3-Hydroxysteroid Dehydrogenases↗
PubMed1981

Comparison of the structural features of corticotropin required for stimulation of steroidogenesis and cAMP production in rat and rabbit adrenocortical cells.

The steroidogenic activities of ACTH, alpha-MSH, beta-MSH as well as analogs of the hormones have been compared in rat and rabbit adrenocortical cells. ACTH is equally active in both species and the melanotropins have very low steroidogenic potency in either species. The steroidogenic potencies of the peptide analogs are strikingly similar in the two species, suggesting that the structural requirements for eliciting steroidogenesis are the same in rat and rabbit adrenocortical cells. The analog NPS-ACTH has low, comparable steroidogenic activity in both species. NPS-ACTH is a potent antagonist of ACTH-induced cAMP production in rat adrenocortical cells but acts as a weak partial agonist in rabbit adrenocortical cells. These results suggest that steroidogenesis may be mediated by receptors different from those involved in the cAMP response observed at supraphysiological concentrations of ACTH.

Adrenal Cortex↗
PubMedDate not supplied

Rational immunotherapy with ribonuclease chimeras. An approach toward humanizing immunotoxins.

Members of the pancreatic ribonuclease (RNase) family have diverse activities toward RNA that could cause them to function during host defense and physiological cell death pathways. This activity could be harnessed by coupling RNases to cell binding ligands for the purpose of engineering them into cell-type specific cytotoxins. Therefore, the cytotoxic potential of RNase was explored by linking bovine pancreatic ribonuclease A via a disulfide bond to human transferrin or antibodies to the transferrin receptor. The RNase hybrid proteins were cytotoxic to K562 human erythroleukemia cells in vitro with an IC50 around 10(-7) M, whereas > 10(-4) M of native RNase was required to inhibit protein synthesis. Cytotoxicity required both components of the conjugate since excess transferrin or ribonuclease inhibitors added to the medium protected the cells from the transferrin-RNase toxicity. Importantly, the RNase conjugates were found to have potent antitumor effects in vivo. Chimeric RNase fusion proteins were also developed. F(ab')2-like antibody-enzyme fusions were prepared by linking the gene for human RNase to a chimeric antitransferrin receptor heavy chain gene. The antibody enzyme fusion gene was introduced into a transfectoma that secreted the chimeric light chain of the same antibody, and cell lines were cloned that synthesized and secreted the antibody-enzyme fusion protein of the expected size at a concentration of 1-5 ng/mL. Culture supernatants from clones secreting the fusion protein caused inhibition of growth and protein synthesis toward K562 cells that express the human transferrin receptor but not toward a nonhuman derived cell line. Since human ribonucleases coupled to antibodies also exhibited receptor mediated toxicities, a new approach to selective cell killing is provided. This may allow the development of new therapeutics for cancer treatment that exhibit less systemic toxicity and, importantly, less immunogenicity than the currently employed ligand-toxin conjugates.

Amino Acid Sequence↗
PubMedDate not supplied

Specifically targeting the CD22 receptor of human B-cell lymphomas with RNA damaging agents.

Targeting CD22 on human B-cells with a monoclonal antibody conjugated to a cytotoxic RNAse causes potent and specific killing of the lymphoma cells in vitro. This translates to anti-tumor effects in human lymphoma models in SCID mice. RNA damage caused by RNAses could be an important alternative to standard DNA damaging chemotherapeutics. Moreover, targeted RNAses may overcome problems of toxicity and immunogenicity associated with plant or bacterial toxin containing immunotoxins.

Animals↗