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Biomedical subjects

K C Robbins

Publications and source records attributed to K C Robbins.

At least 145 records · Page 8Linked to original sources
PubMed1979

Molecular cloning of Moloney murine sarcoma virus: arrangement of virus-related sequences within the normal mouse genome.

The unintegrated circular DNA form of Moloney murine sarcoma virus (MSV) has been cloned in bacteriophage lambda. Discrete deletions in the viral genome were shown to occur during propagation of recombinant phage in Escherichia coli. Heteroduplex and restriction enzyme analyses indicated the deletion of tandemly repeated sequences within certain of the cloned MSV DNA inserts. Cloned MSV DNA was used to prepare a probe composed of its acquired cellular (src) sequences, shown previously to be necessary for MSV transformation. Analysis of EcoRI digests of normal mouse cellular DNA revealed the presence of a single 14-kilobase-pair fragment containing these sequences which lacked contiguity with endogenous type C helper viral information of the same cells. Thus, the sarcoma virus-specific sequences of MSV are represented within the normal mouse genome in a manner analogous to that of a cellular gene.

Animals↗
PubMed1978

The human plasma fibrinolytic system: regulation and control.

The factors involved in the regulation and control of the human plasma fibrinolytic system at the cellular level are unknown at this time. The physiological regulation of plasmin formation in plasma depends primarily on the nature of the circulating zymogen, plasminogen, the physiological activators formed both in the blood and in the vascular endothelium, and the specific plasmin inhibitors found both in plasma and in certain of the cellular elements of the blood. The biosynthesis of the zymogen must be under genetic control, and the activators are probably released, after thrombus and clot formation, from components involved in the surface-mediated initiation of the coagulation system, and from the vascular endothelium. Activation of plasminogen can occur both in the fluid phase surrounding the thrombus and probably at thrombus surfaces, involving both the fibrin clot and the platelet membrane. The plasmin inhibitors act to control the system in order to prevent proteolytic degradation of important physiologic trace proteins of the coagulation, complement and kallikrein-kinin systems by the enzyme.

Chemical Phenomena↗
PubMed1978

Genetic recombination between mouse type C RNA viruses: a mechanism for endogenous viral gene amplification in mammalian cells.

A strategy based on the identification of type-specific antigenic determinants in the transitional products of gag (p15, p12, and p30 proteins), pol (reverse transcriptase), and env (gp70 glycoproteins) genes of mammalian type C viruses has been used to study genetic recombination between these RNA viruses. By this approach, recombinants involving exogenous and endogenous mouse type C viruses have been identified and genetically mapped. Analogous techniques have been applied to investigate the genetic relationships between different classes of endogenous virus that exist within the same mouse cells. Proteins of the inducible class of xenotropic virus were shown to exhibit extensive antigenic homology with the gag but not the env gene products of the ecotropic virus class. Instead, the env gene-coded glycoproteins of the inducible and noninducible xenotropic virus classes possessed striking antigenic relatedness. These results, as well as supporting findings from molecular hybridization, favor the concept that the inducible xenotropic virus of mouse cells arose by a recombinational mechanism involving the progenitors of the other two endogenous virus classes.

Animals↗
PubMed1978

Molecular mechanisms involved in the differential expression of gag gene products by clonal isolates of a primate sarcoma virus.

Clonal isolates of an early passage stock of woolly monkey sarcoma virus (WSV) have been shown to code for different numbers of woolly monkey helper leukemia virus gag gene products. In the present report, the molecular mechanisms responsible for their differential expression of gag gene products have been analyzed. Three WSV RNA genomes were shown to possess sedimentation coefficients consistent with the differences demonstrated in their allotments of helper viral sequences. The WSV variant (WSV clone 9) that expressed no detectable proteins was shown to contain the largest amount of helper viral information. Moreover, there was no additive hybridization of the WLV complementary DNA probe by RNA of this WSV clone and that of a WSV clone coding for several gag gene products. These results suggest that the lack of expression of gag gene products by WSV clone 9 is not due to a major deletion of helper viral gag gene sequences. Similar levels of WLV-specific RNA were demonstrated in cells nonproductively transformed by each WSV clone, arguing that the ability to express gag gene proteins was not related to the magnitude of viral RNA transcription. Taken together, the results are most consistent with a mechanism by which small deletions or point mutations in the genomes of some WSV variants result in premature termination of translation or synthesis of immunologically nonreactive gag gene proteins. The present findings have implications concerning the effects of evolutionary selective pressures on helper viral genetic information in mammalian transforming viruses.

Base Sequence↗
PubMed1977

Comparison of the esterase and human plasminogen activator activities of various activated forms of human plasminogen and their equimolar streptokinase complexes.

A comparison was made of the esterase and activator activities of the various activated forms of human plasminogen and their streptokinase complexes with Nalpha-Cbz-L-lysine-p-nitrophenyl ester as the substrate. The steady state kinetic properties of Glu- and Lys-plasmins, and Glu- and Lys-plasminogen-streptokinase complexes were identical, while the Lys-plasmin-streptokinase complex showed a 2-fold increase in Km with the same kcat and a 3-fold increase in Ki for the competitive inhibitor leupeptin. Lys-plasminogen (zymogen with an active site) was prepared which incorporated 0.7 mol of [3H]idisopropyl phosphorofluoridate and 0.43 mol of p-nitrophenyl-p'-guanidinobenzoate/mol of protein. The Km for Lys-plasminogen was 3-fold higher than that of Lys-plasmin, and its maximum velocity 10-fold lower. The steady state kinetic parameters of a plasmin-derived light (B) chain (CmCys)3, and a derived equimolar light (B) chain-streptokinase complex (CmCys)3, isolated from human plasmin and equimolar plasmin-streptokinase, or plasminogen-streptokinase, complexes, respectively, were determined. When the light (B) chain-streptokinase complex is isolated from its parent complexes, there is a complete retention of the original parent's esterase activities, with respect to Km and kcat, and interaction with the competitive inhibitors benzamidine and leupeptin. The plasmin-derived light (B) chain does not retain its parent esterase activities. This chain has very similar kinetic properties to Lys-plasminogen except that streptokinase, in an equal molar amount, does not impart full esterase activity to the light (B) chain whereas the zymogen can be completely activated by streptokinase. The kcat of the plasmin-derived light (B) chain, and its streptokinase complex can be enhanced by 50 and 30%, respectively, in the presence of 10(-4) M leupeptin, a competitive inhibitor of plasmin, attesting to the increased structural flexibility within the active site of this enzyme species. Urokinase hydrolyzes Nalpha-Cbz-L-lysine p-nitrophenyl ester efficiently with a kcat/Km of one-third that of plasmin. The human plasminogen activator activities of various activated forms of human plasminogen and their equimolar streptokinase complexes were compared in a kinetic assay. The Lys-plasmin-streptokinase complex, and streptokinase were the least active of the activator species and were approximately equal in their activator activities. Glu- and Lys-plasminogen-streptokinase complexes had approximately 1.5 times the activity of streptokinase, whereas the equimolar light (B) chain-streptokinase complexes had approximately 2- to 3-times the activator activity of streptokinase. Since the esterase activity remained unchanged, this indicates a greater degree of specificity in the active site of the equimolar light (B) chain-streptokinase activator complex. Urokinase proved to be a poor activator species...

Enzyme Activation↗
PubMed1977

Segregation of genetic information for a B-tropic leukemia virus with the structural locus for BALB:virus-1.

A B-tropic type-C RNA virus isolatable from lymphoreticular tumors of the inbred BALB/c mouse strain has previously been shown to be leukemogenic in its natural host. This virus is not chemically inducible from BALB/c embryo cells or from embryo lines containing segregating inducibility loci for two known endogenous type-C viruses of BALB/c cells. Molecular hybridization and type-specific immunologic assays demonstrate a high degree of genetic homology between the B-tropic leukemia virus and BALB:virus-1, an N-tropic endogenous virus of BALB/c cells. Genetic sequences specific for BALB:virus-1 are shown to segregate with the locus for BALB:virus-1 induction in genetic crosses between BALB/c and the noninducible NIH Swiss strain. Thus, if the information of the B-tropic virus is encoded in the genome of the animal, it must be closely linked to the structural locus for BALB:virus-1. The evidence is consistent with a mechanism by which a small genetic alteration in BALB:virus-1 leads to a virus, whose growth is unrestricted, and subsequently to the development of neoplasia.

Animals↗
PubMed1976

Isolation of a human plasmin-derived, functionally active, light (B) chain capable of forming with streptokinase an equimolar light (B) chain-streptokinase complex with plasminogen activator activity.

A functionally active human plasmin light (B) chain derivative, stabilized by the streptomyces plasmin inhibitor leupeptin, was isolated from a partially reduced and alkylated enzyme preparation by an affinity chromatography method with a L-lysine-substituted Sepharose column. This light (B) chain derivative was found to be relatively homogeneous by electrophoretic analysis in both an acrylamide gel/dodecyl sulfate system and on cellulose acetate. It possessed approximately 3% of the proteolytic activity (casein substrate) of the original enzyme, and it incorporated 0.09 mol of [3H]diisopropyl phosphorofluoridate per mol of protein. It contained 3.1 +/- 0.3 carboxymethylated cysteines per mol of protein and can be designated as a CmCys5-light (B) chain (CmCys)3. When this isolated light (B) chain derivative was mixed in equal molar amounts with streptokinase, the mixture developed both human and bovine plasminogen activator activities; the bovine activator activity was approximately 66% of the bovine activator activity of the equimolar human plasmin-streptokinase complex. Although this complex now incorporated 0.50 mol of [3H]diisopropyl phosphorofluoridate per mol of protein, its proteolytic activity, on a molar basis, was the same as the proteolytic activity of the isolated light (B) chain derivative. It was shown by electrophoretic analysis in both an acrylamide gel/epsilon-aminocaproic acid system and on cellulose acetate that the light (B) chain derivative and streptokinase forms an equimolar light (B) chain-streptokinase complex, indicating that the binding site for streptokinase is located on the light (B) chain of the enzyme. A functionally active equimolar light (B) chain-streptokinase complex was also isolated from a partially reduced and alkylated equimolar human plasmin-streptokinase complex by the affinity chromatography method. The plasminogen activator activities (human and bovine) of this light (B) chain-streptokinase complex were similar to those of the plasmin-streptokinase complex from which it was derived. Although this complex incorporated 0.70 mol of [3H]diisopropyl phosphorofluoridate per mol of protein, its proteolytic activity, on a molar basis, was only 14% of proteolytic activity of the plasmin-streptokinase complex.

Amino Acids↗
PubMed1976

Isolation and characterization of the affinity chromatography forms of human Glu- and Lys-plasminogens and plasmins.

Affinity chromatography forms, 1 and 2, were each isolated from human Glu- and Lys-plasminogens by gradient elution from a L-lysine-substituted Sepharose column with a linear gradient of epsilon-aminocaproic acid. Although each of the two zymogen forms contains two affinity chromatography forms, the relative concentrattions of these forms in each of the zymogen preparations depended upon the plasma sample or enriched plasma fraction used for the preparation of the zymogen. Specific analytical acrylamide gel electrophoretic systems were used for the characterization of the zymogen and enzyme forms, and their component affinity chromatography forms, 1 and 2. The four zymogen affinity chromatography forms, Glu-1-plasminogen, Glu-2-plasminogen, Lys-1-plasminogen, and Lys-2-plasmingoen, show distinct stepwise differences in their molecular size and charge. The Glu-1-form is the largest in molecular size and the most acidic, and the Lys-2-form is the smallest in molecular size and the most basic. The proteolytically altered Lys-1- and Lys-2- forms appear to be specifically df the zymogen affinity chromatography forms showed a different distribution of isoelectric forms. The major isoelectric forms isolated from Glu-plasminogen with pI values of 6.2, 6.3, 6.4, and 6.6, and the major isoelectric forms isolated from Lys-plasminogen with pI values of 6.7, 7.2, 7.5, 7.8, and 8.1, (Summaria, L., Arzadon, L., Bernabe, P., Robbins, K. C., and Barlow, G. H. (1973) J. Biol. Chem. 248, 2984-2991) were shown to be mixtures of the Glu-1- and Glu-2- forms, or the Lys-1- and Lys-2- forms, respectively. Although the sialic acid contents of the Glu- and Lys- forms appear to be similar, the isolated affinity chromatography forms show distinct differences. The sialic acid contents of the Glu-1- and Lys-1- forms are identical, and are substantially higher than the sialic acid contents of the Glu-2- and Lys-2- forms which are also identical to each other. It is possible that the charge difference between the zymogen-1- and -2- forms may be related to the differences in their sialic acid content. Each of the four zymogen affinity chromatography forms, when activated by urokinase in the presence of the plasmin inhibitor, Trasylol, was converted to an apparently unique and different enzyme form. The four enzyme forms show distinct stepwise differences in molecular size; Glu-1-plasmin is the largest in size whereas Lys-2-plasmin is the smallest in size. Each plasmin-derived carboxymethyl heavy(A) chain was found to be different in molecular size, but the two carboxymethyl light(B) chains found in each of the four enzyme forms appeared to be identical and of the same molecular sizes. The four heavy(A) chains show a stepwise difference in molecular size; the Glu-1-heavy(A) chain is the largest in size whereas the Lys-2-heavy(A) chain is the smallest in size...

Chromatography, Affinity↗
PubMed1976

Induction of mouse type-C virus by translational inhibitors: evidence for transcriptional derepression of a specific class of endogenous virus.

Several biologically distinguishable type-C RNA viruses are genetically transmitted in mouse cells. In the present report, chemicals that inhibit several different steps in protein synthesis are shown to cause marked increases in the cellular concentration of virus-specific RNA and the subsequent induction of virus. Analysis of the effect of translational inhibitors on mouse embryo cells of different genotypes indicates that activation of viral RNA is specific for one endogenous virus class and is a dominant genetic characteristic. Two lines of evidence favor the hypothesis that the induction of viral RNA involves transcriptional derepression rather than an alteration in its post-transcriptional processing. First, nuclear and cytoplasmic fractions of induced cells are shown to demonstrate similar increases in their concentrations of virus-specific RNA. Second, the decay of induced viral RNA following inhibition of further RNA synthesis by actinomycin D is not prevented by continued exposure to the inducer. These findings weigh heavily against the possibility that translational inhibitors act to stabilize viral RNA post-transcriptionally. The results are consistent with a model in which the expression of one class of endogenous virus is regulated by a labile repressor protein acting at a transcriptional level.

Animals↗
PubMed1976

Rat sequences of the Kirsten and Harvey murine sarcoma virus genomes: nature, origin, and expression in rat tumor RNA.

Two murine sarcoma viruses, the Kirsten and the Harvey, were isolated by passage of mouse type C leukemia viruses through rats. These sarcoma viruses have genomes containing portions of their parental type C mouse leukemia virus genomes, in stable association with specific rat cellular sequences that we find to be quite likely not those of a rat type C leukemia virus. To determine if these murine sarcoma viruses provide a model relevant to the events occurring in spontaneous tumors, we have hybridized DNA and RNA prepared from rat tumors and normal rat tissues to [3H]DNA prepared from the Kirsten murine sarcoma virus. We have also hybridized these rat tissue nucleic acids to [3H]DNA prepared from a respresentative endogenous rat type C leukemia virus, the WFU (Wistar-Furth). Sarcoma-viral rat cellular sequences and endogenous rat leukemia viral sequences were detected in the DNA of both tumor and normal tissues, with no evidence of either gene amplification or additional sequences being present in tumor DNA. Sarcoma-viral rat cellular sequences and endogenous rat leukemia viral sequences were detected at elevated concentrations in the RNA of many rat tumors and in specific groups of normal tissues.

Animals↗