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Biomedical subjects

R Miskin

Publications and source records attributed to R Miskin.

At least 37 records · Page 2Linked to original sources
PubMed1984

DNA repair and induction of plasminogen activator in human fetal cells treated with ultraviolet light.

We have tested human fetal fibroblasts for development associated changes in DNA repair by utilizing nucleoid sedimentation as an assay for excision repair. Among skin fibroblasts the rate of excision repair was significantly higher in non-fetal cells than in fibroblasts derived from an 8 week fetus; this was evident by a delay in both the relaxation and the restoration of DNA supercoiling in nucleoids after irradiation. Skin fibroblasts derived at 12 week gestation were more repair proficient than those derived at 8 week gestation. However, they exhibited a somewhat lower rate of repair than non-fetal cells. The same fetal and non-fetal cells were also tested for induction of the protease plasminogen activator (PA) after u.v. irradiation. Enhancement of PA was higher in skin fibroblasts derived at 8 week than in those derived at 12 week gestation and was absent in non-fetal skin fibroblasts. These results are consistent with our previous findings that in human cells u.v. light-induced PA synthesis is correlated with reduced DNA repair capacity. Excision repair and PA inducibility were found to depend on tissue of origin in addition to gestational stage, as shown for skin and lung fibroblasts from the same 12 week fetus. Lung compared to skin fibroblasts exhibited lower repair rates and produced higher levels of PA after irradiation. The sedimentation velocity of nucleoids, prepared from unirradiated fibroblasts, in neutral sucrose gradients with or without ethidium bromide, indicated the presence of DNA strand breaks in fetal cells. It is proposed that reduced DNA repair in fetal cells may result from alterations in DNA supercoiling, and that persistent DNA strand breaks enhance transcription of PA gene(s).

Cell Line↗
PubMed1983

Plasminogen activator in the developing rat cerebellum: biosynthesis and localization in granular neurons.

The histochemical localization of plasminogen activator (PA) and the level of the translatable mRNA species coding for active PA were analyzed during ontogenesis of normal and of irradiation-agranulated rat cerebellum. Autoradiographic localization of PA activity was performed by plasminogen-dependent fixation of [125I]fibrin degradation products to frozen sections of developing rat cerebellum. Both the immature external and the adult internal granular layers were intensely labeled, in addition to labeling of meninges. In the irradiation-aggranulated cerebellum, PA labeling could be observed in residual granular neurons which went through their final division prior to the irradiation protocol. The concentration of the mRNA species directing the synthesis of catalytically active PA (PAmRNA) was monitored by an in ovo bioassay, using Xenopus oocytes as a translation system. A major species of 80,000 and a minor species of 50,000 apparent molecular weight of active PA were translated by mRNA from either control or X-irradiated cerebellum throughout ontogenesis. These could be detected by electrophoretic analysis of extracts and incubation media of microinjected oocytes. Both the content and the concentration of PAmRNA were found to be the highest at the stage of cerebellar development when granular neurons proliferate and migrate. These observations suggest that a major portion of the PA activity in the rat cerebellum is synthesized and localized in granular neurons through cerebellar ontogenesis, and that PA activity in the developing cerebellum is largely determined by the level of translatable mRNA coding for this enzyme.

Animals↗
PubMed1982

Plasminogen activator is enriched in the synaptosomal plasma membranes.

The intracellular localization of the serine protease plasminogen activator was analyzed in homogenates of bovine brain cortex using differential fractionation procedures. The distribution of the enzyme was clearly different from that of cytosol and mitochondrial markers, and was similar to that of plasma membrane proteins and of the muscarinic acetylcholine receptor, which is a specific marker for the synaptic membrane. The specific activity of plasminogen activator was increased in fractions enriched in intact synaptosomes. Most of the enzyme in intact synaptosomes was found to be firmly associated with the synaptosomal membrane, and could be solubilized by high concentrations of salt or by non-ionic detergent. Purified synaptic vesicles, however, did not contain large amounts of plasminogen activator. Bovine brain synaptosomes were shown to contain two species of the enzyme, having apparent molecular weights of 80,000 and 55,000. The presence of plasminogen activator in the synaptosomal membrane may indicate its possible involvement in the functioning of nerve terminals.

Animals↗
PubMed1982

Acetylcholine receptor: effects of proteolysis on receptor metabolism.

Previous studies (Miskin, R., T. G. Easton, and E. Reich, 1970, Cell. 15:1301-1312) have shown that sarcoma virus transformation and tumor promoters reduced the cell surface concentration of acetylcholine receptors (AChR) in differentiating chick embryo myogenic cultures. Both of these agents also induced high rates of plasminogen activator (PA) synthesis in myogenic cultures (Miskin, R., T. G. Easton, A. Maelicke, and E. Reich, 1978, Cell. 15:1287-1300), and the present work was performed to establish whether proteolysis might significantly affect receptor metabolism. Proteolysis in myogenic cultures was modulated by one or more of the following: stimulation of PA synthesis, direct addition of plasmin, removal of plasminogen, or addition of plasmin inhibitors. The results were: (a) When the rates of proteolysis were raised either by addition of plasmin or by stimulating PA synthesis in the presence of plasminogen, both the steady-state concentration and the half-life of surface AChR decreased, but the rate of receptor synthesis was unaffected. (b) The magnitude of these effects, and their dependence on added plasminogen, indicated that proteolysis initiated by plasminogen activation could account almost entirely for the reduction in receptor half-life produced by sarcoma virus transformation and phorbol ester. (c) The rate of receptor synthesis, which is also reduced by viral transformation and tumor promoters, was not modified by proteolysis; hence plasmin action may be responsible for a large part, but not all of the change in surface receptor under these conditions. (d) The plasmin catalysed changes in receptor parameters appear to occur in response to modified membrane metabolism resulting from proteolysis of surface components other than AChR itself.

Animals↗
PubMed1981

Microinjected Xenopus oocytes synthesize active human plasminogen activator.

Induction of synthesis of the protease plasminogen activator (PA) by hormones, oncogenic viruses and tumor promoters occurs at the transcription level. A novel bioassay for PA messenger RNA was developed to study the regulation of PA synthesis and the genetic elements involved in it. Poly(A)-containing RNA from HEp-3, a PA-rich tumor of human origin, was found to direct the synthesis of a new proteolytic activity when microinjected into Xenopus oocytes. Newly synthesized protease can be detected within a few hours after microinjection of minute quantities of unfractionated mRNA. The new enzymatic activity is indistinguishable from human PA: it is absolutely dependent on human plasminogen; it is neutralized by serum raised against urokinase, the human urinary PA; and it comigrates with urokinase and HEp-3 PA in gel electrophoresis, exhibiting a molecular weight of 60,000.

Animals↗
PubMed1981

Plasminogen activator in the rodent brain.

The cellular origin(s), the biochemical properties and the developmental pattern of the protease plasminogen activator (PA) were investigated in the rodent brain. PA activity was localized in frozen brain sections by a novel autoradiography technique. PA levels and electrophoretic mobility were determined in homogenates prepared from major regions of the developing and the mature brain, and both the localization and the specific activity of the enzyme were examined in X-irradiated brain regions. PA activity was shown to be correlated with cell bodies in neuronal-enriched regions and also with endothelial, meningeal and ependymal layers. PA levels increased in a transient manner and at different rates and time periods in the various brain regions that were analyzed. PA in neuronal, but not in epithelial cell layers was affected by X-irradiation and one of the brain PA species had a similar molecular weight of that of neuroblastoma cells. Our findings suggest that in the brain PA is produced by neurons and by epithelial cells, and that it may have additional functions to that of thrombolysis both in the developing and the mature brain.

Aging↗
PubMed1981

Induction of plasminogen activator by UV light in normal and xeroderma pigmentosum fibroblasts.

Normal and DNA repair-deficient human fibroblasts have been used to study induction of plasminogen activator (PA) by DNA damage. UV light induced the synthesis of PA in skin fibroblasts of all types of xeroderma pigmentosum (XP) in XP heterozygotes and in human amniotic cells. Enzyme induction was, however, not observed in fibroblasts of normal adults. In classical XP, which are deficient in excision repair, PA synthesis occurred in a narrow range of low-UV fluences. In such strains, the level of enzyme produced was correlated with the extent of repair deficiency. UV fluences required for PA induction in XP variants and XP heterozygotes were at least 10 times those inducing enzyme synthesis in excision-deficient XP. Maximum enzyme induction occurred 48 hr after irradiation, and the highest levels of enzyme produced were 15-20 times those of PA baseline levels. Electrophoretic analysis showed that UV irradiation enhances the synthesis of the Mr 60,000 human urokinase-type PA, which is present in low amounts in untreated cells. Our results suggest that PA induction in human cells is caused by unrepaired DNA damage and represents a eukaryotic SOS-like function. In addition, PA induction may provide a sensitive assay for detection of cellular DNA repair deficiencies and identification of XP heterozygotes.

Amniotic Fluid↗
PubMed1981

Plasminogen activator: analysis of enzyme induction by ultraviolet irradiation mapping.

Ultraviolet irradiation mapping techniques have previously been used to study the organization of eucaryotic gene classes and transcription units. We used the same method to probe some regulatory phenomena observed in the induction of plasminogen activator (PA) biosynthesis: PA synthesis in chicken embryo fibroblasts is induced by tumor-promoting phorbol esters and by retinoic acid; furthermore, PA induction by phorbol esters is synergistic with transformation, being 10- to 20-fold greater in virus-transformed cells than in normal cells. We found that the ultraviolet irradiation inactivation cross sections for PA induction by phorbol esters and by retinoate differed significantly, suggesting that these agents induce PA biosynthesis by different mechanisms. On the other hand, the ultraviolet irradiation sensitivity of phorbol ester induction in normal chicken embryo fibroblasts was the same as in transformed cells, indicating that the synergism of transformation and phorbol esters is probably not due to different pathways of PA induction.

Animals↗
PubMed1980

Antibodies from myasthenic patients that compete with cholinergic agents for binding to nicotinic receptors.

We have purified immunoglobulins from sera of myasthenic patients and have identified antibodies directed against the cholinergic ligand-binding site of the nicotinic acetylcholine receptor. In the serum of one patient analyzed in detail these antibodies belonged to the IgG3 class, and their effects were as follows: (i) In chicken embryo myogenic cultures, antibody binding was both competitive with 125I-labeled alpha-bungarotoxin and irreversible on a time scale of hours. (ii) 125I-Labeled alpha-bungarotoxin was not displaced by antibody from preformed complexes and, conversely, antibody was not displaced by toxin. (iii) Antibody binding was competitive with some, but not all, nicotinic agents. Thus, acetylcholine, carbamoylcholine, and dimethyltubocurarine competed effectively whereas decamethonium and hexamethonium did not, suggesting that the two classes of nicotinic ligands probably interact at different, nonoverlapping receptor subsites. (iv) There was no competitive binding between these antibodies and the muscarinic antagonist atropine. (v) Both this class of myasthenic immunoglobulins and rabbit antibodies raised against Torpedo acetylcholine receptors increased the rate of receptor degradation. However, synthesis and degradation remained coupled and there was a compensating increase in receptor synthesis. We propose that immunoglobulins directed against the ligand-binding site of acetylcholine receptors may account for the characteristic curare-like symptoms of early myasthenia and their response to cholinesterase inhibition, for the apparent decrease in receptors measurable by 125I-labeled alpha-bungarotoxin binding, and for initiating localized complement activation at the postsynaptic membrane.

Animals↗
PubMed1978

Metabolism of acetylcholine receptor in chick embryo muscle cells: effects of RSV and PMA.

We have investigated some aspects of the metabolism of the integral membrane protein acetylcholine receptor (AChR) in normal and transformed cultures of chick embryo muscle cells. Turnover of AChR in control muscle cell cultures was compared with turnover in cultures infected and transformed by a temperature-sensitive mutant of Rous sarcoma virus (RSV) and with cultures treated with the tumor promoter phorbol myristate acetate (PMA). The parameters of AChR metabolism were estimated using 125I-alpha-bungarotoxin as a stoichiometric high affinity ligand for the AChR. We found that both RSV transformation and PMA increased the rate of degradation and decreased the rate of synthesis of AChR. The consequent reduction in steady state receptor levels suggests that oncogenic transformation and tumor promoter significantly alter the metabolism of cell surface membranes. We also observed that parameters of AChR metabolism in control cultures changed systematically in a pattern which depended upon the age of the culture as well as the use of embryo extract or fetal bovine serum as medium supplements. The muscle cell system allows quantitative measurement of an integral membrane protein and its metabolism, and may serve as a more general model for alterations in membrane and surface receptor metabolism associated with the transformed state.

Animals↗
PubMed1978

Plasminogen activator in chick embryo muscle cells: induction of enzyme by RSV, PMA and retinoic acid.

To explore the generality of the effects of sarcoma viruses, tumor-promoting phorbol esters and retinoic acid, we have studied plasminogen activator production in differentiating chick myogenic cultures. Although slightly higher than in chick fibroblast cultures, the level of spontaneously synthesized enzyme is low; it reaches a peak shortly after maximum cell fusion has been completed and then declines. Rous sarcoma virus (RSV) transformation of differentiating myotubes was accomplished by infecting myoblasts with a temperature-sensitive mutant, maintaining cultures at the nonpermissive temperature until completion of fusion and shifting to permissive temperatures at selected times thereafter. RSV transformation, phorbol myristate acetate (PMA) and retinoic acid all induced high levels of plasminogen activator production by differentiating myotubes in the absence of DNA synthesis. In comparison with fibroblasts, virus-induced enzyme synthesis by myogenic cultures proceeded more slowly but ultimately reached comparably high levels. Whereas cAMP strongly repressed RSV- and PMA-induced plasminogen activator production by chick fibroblasts, it weakly stimulated enzyme synthesis by myotubes. This suggests that enzyme induction by RSV and PMA is not mediated primarily through effects on cAMP metabolism.

Animals↗
PubMed1976

A protein factor enhancing activation of inactive 30-S ribosomal subunits of Escherichia coli.

A protein factor partially purified from the S-100 fraction (supernatant from 100 000 x g spin) of Escherichia coli stimulates the conversion of 30-S ribosomal subunits from an inactive to an active form. The subunits are normally inactivated at low concentrations of Mg2+ and reactivated when heated under appropriate ionic conditions. The factor, which differs from the known soluble factors required for protein biosynthesis as well as from proteins of the 30-S subunit, enhances activation under conditions which by themselves allow for only a low level of activity to reappear. Fully active ribosomes and the assay reactions are not affected by the factor, indicating that its effect must be restricted to the activation process. The factor is likely to function by facilitating a conformational transition in the ribosome, and might act in vivo in a similar manner.

Bacterial Proteins↗