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Biomedical subjects

D Irwin

Publications and source records attributed to D Irwin.

At least 55 records · Page 3Linked to original sources
PubMed1984

Application of SDS gradient polyacrylamide slab gel electrophoresis to analysis of apolipoprotein mass and radioactivity of rat lipoproteins.

An application of SDS gradient polyacrylamide slab gel electrophoresis to the analysis of lipoprotein polypeptides is described. The 10-15% polyacrylamide gradient provides a high degree of resolution and sensitivity resulting in a single separation of the major apoproteins which can be easily visualized. When combined with autofluorography, individual protein mass and radioactivity can be determined densitometrically while still retaining excellent resolution. Examples of rat lymph and plasma apolipoproteins are shown, and apparent heterogeneity of certain apoprotein subgroups is described.

Absorptiometry, Photon↗
PubMed1984

Glycosaminoglycan synthesis by rat fibroblast monolayers: effects of serum and solubilized bone matrix fractions.

Glycosaminoglycan synthesis by fibroblasts, from skeletal muscle of the neonatal rat, is stimulated by a bone matrix preparation, soluble in isotonic medium, obtained by extracting decalcified rat bone with 4 M guanidine HCl. The stimulation in glycosaminoglycan synthesis is dependent on the presence of serum in the culture, but the stimulatory effect can be clearly distinguished from that of serum. The stimulatory activity in the bone matrix has been fractionated by ion-exchange chromatography and coelutes largely with anionic, non-collagenous matrix glycoproteins.

Animals↗
PubMed1984

The effect of solubilized bone matrix fractions from different mammalian species on glycosaminoglycan synthesis by cultured fibroblasts.

Human and bovine bone matrices were extracted with salt solutions of different composition and the extracts tested for stimulation of incorporation of radioactivity from [3H]glucosamine and [35S]SO4 into the hyaluronic acid and chondroitin sulfate of the cell pellet, the cell surface and the medium fractions of human synovial cells in culture. Stimulatory activity was extracted with a solution of 0.3 M EDTA in 2.5 M NaCl from bovine but not human bone. Subsequent extraction of the residues with 4 M guanidinium hydrochloride yielded activity from both matrices. A major stimulation of incorporation of radioactivity was observed in the cell surface fractions. Human synovial cells constitute a more sensitive assay system for the stimulatory activity than rabbit synovial cells.

Animals↗
PubMed1983

Evidence for sterol carrier protein2-like activity in hepatic, adrenal and ovarian cytosol.

Purified sterol carrier protein2 (SCP2) from rat liver stimulated utilization of endogenous cholesterol for pregnenolone synthesis by adrenal mitochondria. Cytosolic preparations of rat liver, adrenal and luteinized ovary were also stimulatory in mitochondrial pregnenolone synthesis to different extents. Treatment of all preparations with rabbit anti-rat SCP2 IgG neutralized the stimulatory effects, and immunoprecipitated proteins gave similar patterns on SDS-gradient polyacrylamide gel electrophoresis. Treatment with rabbit pre-immune IgG had no effect on these parameters. Thus, proteins which are immunochemically compatible with hepatic SCP2 appear to be present in steroidogenic tissues and may play a role in control of mitochondrial cholesterol side chain cleavage activity.

Adrenal Glands↗
PubMed1983

Sterol carrier protein2. Identification of adrenal sterol carrier protein2 and site of action for mitochondrial cholesterol utilization.

Addition of homogeneous rat liver sterol carrier protein2 (SCP2) or an adrenal cytosolic fraction enhanced pregnenolone production by adrenal mitochondria. Pretreatment of SCP2 or adrenal cytosol with anti-SCP2 IgG abolished the stimulatory effect of both preparations on mitochondrial pregnenolone output. Incubation of mitochondria with aminoglutethimide, which blocks interaction of cholesterol with inner membrane cytochrome P-450scc, resulted in decreased pregnenolone production and a decreased level of mitoplast cholesterol. Addition of SCP2 to the incubation media caused an almost 2-fold increase in cholesterol associated with the mitoplast, but did not enhance mitochondrial pregnenolone production. Studies with reconstituted cytochrome P-450scc in phospholipid vesicles also suggested that SCP2 did not affect interaction of cholesterol with the hemoprotein. Treatment of rats with cycloheximide alone or with adrenocorticotropic hormone resulted in a dramatic increase in mitochondrial cholesterol. However, these mitochondria did not exhibit increased levels of pregnenolone output under control incubation conditions. When SCP2 was included in the mitochondrial incubation media, pregnenolone production was significantly increased over that observed with adrenal mitochondria from untreated or adrenocorticotropic hormone-treated rats. The results imply that SCP2 enhances mitochondrial pregnenolone production by improving transfer of mitochondrial cholesterol to cytochrome P-450scc on the inner membrane, but does not directly influence the interaction of substrate with the hemoprotein.

Adrenal Glands↗
PubMed1983

Pesticide-induced complete and partial chromosome loss in screens with repair-defective females of Drosophila melanogaster.

Commercial preparation of fungicides (captan and maneb), herbicides (bromacil, paraquat, picloram, and 2,4-D), and insecticides (carbaryl, chlorpyrifos, dimethoate, DDT, diazinon, carbofuran, and permethrin) were tested for their ability to induce complete and partial chromosome losses in Drosophila melanogaster males. In an attempt to identify the mutagenic activity of pesticides that are toxic in low concentrations in Drosophila, these males were mated with mus-302 repair-defective females. The rationale for this mating scheme is based on the repair of genetic damage in Drosophila sperm by maternal enzymes in the zygote, and on the reports that there may be increases in the frequency of recovery of chemically induced chromosome losses in crosses of treated males with mus-302 females. Verification of the sensitivity of this screen in this study came from significant increases in the frequency of chromosome loss induced by low concentrations of the positive controls, N-nitrosodimethylamine and methyl methanesulfonate. Of the 13 pesticides, the insecticide chlorpyrifos induced a significant amount of ring-X chromosome loss. No pesticide induced a significant increase in partial chromosome loss. These results are discussed in relation to the usefulness of repair-defective mutants in screens for genetic damage in Drosophila and other higher eukaryotes by chemicals that are toxic or cause sterility at low concentrations.

Animals↗
PubMed1983

Control of glycosaminoglycan synthesis and connective tissue differentiation in culture by solubilized bone matrix fractions.

Implantation of decalcified bone matrix in rodents induces the formation of new cartilage and bone by responding cells. Extraction of the bone matrix with high ionic strength solutions, followed by solubilization of part of the extract in isotonic salt solution, yielded material which stimulated glycosaminoglycan (GAG) synthesis by monolayers of fibroblastic cells. Bone is greatly enriched in this stimulatory activity compared to other connective tissues. However, fibroblastic cells from different sources respond to the stimulatory effect of low concentrations of the solubilized material. Kinetic studies of the GAG synthesized and secreted by the cultured cells revealed that a major stimulation of GAG secretion occurred at the cell surface as an early event following stimulation with the bone matrix extract. GAG accumulation in the culture medium occurs at a greater rate in cultures that have been stimulated with the bone matrix extract than in controls.

Animals↗
PubMed1982

Inhibition of initiation of translation in L1210 cells by 8-azaguanine.

The inhibition of protein synthesis by 8-azaguanine (azaG) in L1210 cells in culture was investigated. AzaG selectively inhibited protein synthesis at concentrations where viability was decreased significantly. AzaG altered the polyribosome sedimentation profile, increasing the numbers of monosomes and smaller polysomes and decreasing the number of larger polysomes. The reversal by cycloheximide of the alterations in the polysome profile suggested that azaG inhibited the initiation of translation. This was confirmed by the demonstration of inhibition of the formation of the 43S and 80S initiation complexes.

Animals↗
PubMed1982

Purine analogs revisited: interference in protein formation.

We have examined in L1210 cells in vitro the effects of three guanine analogs (8-azaguanine, 3-deazaguanine, and 6-thioguanine) on protein biosynthesis and have compared this action with respect to effects on macromolecular synthesis and mechanisms of tumor growth inhibition and cell viability. The major site of action of azaG was the inhibition of protein synthesis. TG seemed to inhibit RNA synthesis more than DNA and protein synthesis. In contrast, DG inhibited both protein and DNA synthesis but not RNA synthesis. Whereas the LD50 and ID50 for macromolecular synthesis for DG and also for azaG were quite similar, for TG the LD50 was one-tenth that of the ID50 for macromolecular synthesis. Since azaG, DG, and TG had different effects on protein and RNA synthesis, the effects of the analogs on the process of translation were examined. DG and azaG, but not TG, altered the polyribosome sedimentation profile, increasing the numbers of monosomes and smaller polysomes and decreasing the number of larger polysomes. This shift in the polysome profile was reversed by low concentrations of cycloheximide, suggesting that DG and azaG inhibited the initiation of translation. This was examined directly by the incorporation of 35S-met-tRNA into the initiation complexes. DG and azaG inhibited the formation of the 43S and 80S initiation complexes and this inhibition correlated closely with the inhibition of total protein synthesis. It is likely that the inhibition of tumor growth by azaG is due to the inhibition of initiation of translation, perhaps through actions on mRNA. The mechanism of growth inhibition for DG is not yet known but may similarly involve actions at this level.

Animals↗
PubMed1982

An integrative approach to the electrophoresis of the reductase and dehydrogenase isozymes in respiring and fermenting yeasts.

A method is described for qualitatively monitoring changes in the starch gel electrophoresis isozyme patterns of key yeast dehydrogenase and reductase activities involved in the transition from aerobic to anaerobic conditions during batch fermentations. The hypoxic transition was accompanied by the disappearance of catalase, superoxide dismutase, mitochondrial dehydrogenases and the appearance of a novel 6-phosphogluconic acid dehydrogenase activity. Significant genetic differences were noted in the banding patterns of wine yeasts, baker's yeast and a commonly used laboratory yeast strain.

Alcohol Oxidoreductases↗
PubMed1982

3-Deazaguanine: inhibition of initiation of translation in L1210 cells.

In L1210 cells in culture, 3-deazaguanine (DG), a relatively new purine analog, was found to inhibit DNA and protein synthesis but not total RNA synthesis. The effect of the drug on protein synthesis was therefore further examined. Polyadenylic acid-containing RNA synthesis was not decreased by DG treatment, suggesting that the inhibiton of protein synthesis was a function of an alteration in the process of translation. DG altered the polyribosome sedimentation profile in a dose-dependent manner, increasing the numbers of monosomes and smaller polysomes and decreasing the number of larger polysomes. The nascent polypeptides in DG-treated cells were labeled with [3H]leucine, and the increased number of monosomes was not associated with a proportionate amount of [3H]leucine when compared to the polysomes. This indicated that the monosomes had not been derived directly from the breakdown of active polysomes. The shift in the polysome profile was reversed by cycloheximide, suggesting that DG profile inhibited the initiation of translation. This was confirmed by the demonstration of the inhibition of DG of the formation of the 43S preinitiation complex. The inhibition of the initiation of protein synthesis by DG may contribute to the antitumor actions of this new purine analog.

Animals↗