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Biomedical subjects

J Renart

Publications and source records attributed to J Renart.

31 records · Page 2Linked to original sources
PubMed1984

Purification and subunit structure of RNA polymerase II from different stages of Artemia development.

RNA polymerase II has been purified from different stages of Artemia development: cryptobiotic gastrulae, developing embryos and nauplii. RNA polymerase from dormant and developing embryos consists of 12 subunits of 205 000, 140 000, 32 500, 25 600, 22 000, 19 400, 18 000, 16 800, 147 000, 14 000, 12 700 and 11 500 daltons, as determined by dodecyl sulphate polyacrylamide gel electrophoresis. The enzyme from larvae contains the same polypeptides except for the higher-molecular-mass component: the larval enzyme has a subunit of 172 kDa instead of the 205-kDa subunit present in the enzyme purified from embryos. Incubation of embryonic RNA polymerase II with larval extracts results in the conversion of the 205-kDa subunit into the 172-kDa one. This conversion is inhibited by soybean trypsin inhibitor, a compound which inhibits three proteases induced during Artemia early larval development. RNA polymerase II purified from a mixture of embryos and larvae contain only the 172-kDa subunit. Our results indicate that the 172-kDa subunit present in RNA polymerase II purified from larvae is produced by proteolysis of the 205-kDa subunit during the extraction and purification of the enzyme and therefore that Artemia has only one class of RNA polymerase II.

Animals↗
PubMed1984

Antibodies to vimentin intermediate filaments in sera from patients with systemic lupus erythematosus.

Sera from patients with systemic lupus erythematosus and from healthy subjects were tested, using immunofluorescence and blotting techniques, for the presence of antibodies to intermediate filaments of the cytoskeleton of human skin fibroblasts. Both techniques showed that antibodies to intermediate filaments were found in a higher proportion of sera from patients (53%) than healthy subjects (9%). The antigen target was found to be the protein band that corresponded to vimentin (Mr 57,000).

Adolescent↗
PubMed1984

Western blots.

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Electrophoresis, Polyacrylamide Gel↗
PubMed1982

Purification and characterization of a novel UpN-specific endoribonuclease VI from Artemia larvae.

Artemia larval ribonuclease (Sebastián, J., and Heredia, C. F., (1978) Eur. J. Bichem. 90, 405-411) has been purified near homogeneity and its properties were studied. It consists of a single polypeptide chain of 38,000 daltons. It requires a divalent cation for activity. Ca2+ is the most effective among the metals tested. The metal dependence of the activity is biphasic. Maximal activity is obtained at 5-10 mM. In the absence of metals and chelating agents in the assay, 30-40% of the activity is observed. However, if chelating agents are added, the activity is abolished. At low concentrations of free metal (1-20 microM), 30-40% of maximal activity is obtained with Ca2+ or Mn2+, but not with Mg2+, Ca2+, but not Mn2+ or Mg2+, protects the enzyme from thermal inactivation. The best substrates for Artemia ribonuclease are poly(U) and poly(A), although with the latter it has only 10% the activity shown with the former. Using poly(U) as substrate, the products of a terminal digestion are P-2':3'-Urd and 3'-UMP. Using dinucleoside monophosphates as substrates, the enzyme is highly specific for a U residue at the 3' side of the phosphodiester bond (UpN), especially UpA, being inactive if the U residue is at the 5' side (NpU). Although some of its properties are similar to other eukaryotic or prokaryotic ribonucleases, its high specificity for UpN bonds suggest that this is a new type of ribonuclease. Moreover, it is a potentially useful enzyme for RNA analysis and/or sequencing.

Animals↗
PubMed1980

Specific association of simian virus 40 tumor antigen with simian virus 40 chromatin.

Simian virus 40 tumor antigen (SV40 T antigen) was bound to both replicating and fully replicated SV40 chromatin extracted with a low-salt buffer from the nuclei of infected cells, and at least a part of the association was tight specific. T antigen cosedimented on sucrose gradients with SV40 chromatin, and T antigen-chromatin complexes could be precipitated from the nuclear extract specifically with anti-T serum. From 10 to 20% of viral DNA labeled to steady state with [3H]thymidine for 12 h late in infection or 40 to 50% of replicating viral DNA pulse-labeled for 5 min was associated with T antigen in such immunoprecipitates. After reaction with antibody, most of the T antigen-chromatin complex was stable to washing with 0.5 M NaCl, but only about 20% of the DNA label remained in the precipitate after washing with 0.5 M NaCl-0.4% Sarkosyl. This tightly bound class of T antigen was associated preferentially with a subfraction of pulse-labeled replicating DNA which comigrated with an SV40 form I marker. A tight binding site for T antigen was identified tentatively by removing the histones with dextran sulfate and heparin from immunoprecipitated chromatin labeled with [32P]phosphate to steady state and then digesting the DNA with restriction endonucleases HinfI and HpaII. The site was within the fragment spanning the origin of replication, 0.641 to 0.725 on the SV40 map.

Animals↗
PubMed1979

Transfer of proteins from gels to diazobenzyloxymethyl-paper and detection with antisera: a method for studying antibody specificity and antigen structure.

We describe a rapid and very sensitive method for detecting proteins as antigens after their separation in polyacrylamide/agarose composite gels, with or without sodium dodecyl sulfate. The polyacrylamide matrix is crosslinked with a reagent that can be cleaved with periodate or alkali to facilitate transfer of the protein bands to diazobenzyloxymethyl-paper, where they are coupled covalently. Specific proteins are detected by autoradiography after sequential incubation with unfractionated, unlabeled specific antiserum and 125I-labeled protein A from Staphylococcus aureus. Antibody and protein A can be removed with urea and 2-mercaptoethanol, and the same paper can be probed again with a different antiserum. An antiserum specific for the simian virus 40 virion proteins VP3 and VP2 has been prepared; it does not crossreact with VP1, as demonstrated by this method. An antiserum raised in rabbits against simian virus 40-transformed rabbit kidney cells is shown to be directed primarily against a periodate-sensitive moiety present in tumor (T) antigen from infected or transformed cells, whereas an antiserum raised in rabbits against large T antigen purified from lytically infected monkey kidney cells by electrophoresis in the presence of sodium dodecyl sulfate [Lane, D.P. & Robbins, A.K. (1978) Virology 87, 182-193] is directed primarily against determinants that are not sensitive to periodate.

Antibody Specificity↗
PubMed1976

Guanosine monophosphate reductase from Artemia salina: Inhibition by xanthosine monophosphate and activation by diguanosine tetraphosphate.

In the course of studies on the metabolic role of diguanosine tetraphosphate during development of Artemia salina, a guanosine monophosphate (GMP) reductase has been found in partially purified from the 150 000g Artemia cysts supernatant. From Lineweaver-Burk plots, two apparent Km values of 5 and 50 muM were obtained for GMP. Xanthosine monophosphate (XMP) is a very strong inhibitor of the reaction. In the presence of 1.5 muM XMP hyperbolic kinetics are found. Diguanosine tetraphosphate counteracts very effectively the inhibition of the activity by XMP, concomitantly changing to hyperbolic the kinetics of the enzyme, with a unique Km value of about 5 muM. The complex kinetic and the existence of allosteric e-fectors at physiological concentrations, together with our lack of success in resolving two isoenzymes, makes it very likely that GMP reductase presents negative cooperativity towards its substrate. The effect of diguanosine tetraphosphate on the enzyme is very specific; other structural analogues, diadenosine tetraphosphate and diguanosine triphosphate, tested a micromolar concentrations had no detectable effect on the enzyme. Guanosine triphosphate (GTP) (mM) was also able to counteract the inhibition of guanosine monophosphate (GMP) reductase by XMP. The properties of the Artemia GMP reductase are here compared with those of the similar enzyme from calf thymus and Escherchia coli. As a consequence, the regulation of eukaryotic GMP reductase is resulting to be quite different from that of the reductase from prokaryotes.

Animals↗
PubMed1976

Characterization and levels of the RNA polymerases during the embryogenesis of Artemia salina.

Dormant embryos at the gastrula stage of Artemia salina contain three DNA-dependent RNA polymerases: I, II, and III. The enzymes are solubilized from whole embryos and they are separated by chromatography on DEAE Sephadex. The ratio of activities with native and denatured DNA at the optimal salt concentrations is 3.5 for RNA polymerase I, 0.1 for RNA polymerase II and 1.0 for RNA polymerase III.Mn(i2+) is more efficient than Mg(2+) for the three enzymes. RNA polymerase II is 50% inhibited by 5 ng/ml of alpha-amanitin while RNA polymerases I and III are 10% and 30% inhibited by 1 mg/ml. During the embryonic development there is am independent variation of the levels of the RNA polymerases. RNA polymerase I increases its specific activity 4-5-fold, RNA polymerase III increases 2-fold, and RNA polymerase II less than 2-fold. The increase in RNA polymerase activity may represent a mechanism to control the rate of synthesis of RNA during the embryogenesis of A. salina.

Amanitins↗
PubMedDate not supplied

Oleoylanilide, a possible causative agent of toxic oil syndrome, interferes with the cytoskeleton in a neuronal cell line.

We have examined the effects of oleoylanilide, one of the main candidates in the etiology of the toxic oil syndrome, in the neuroblastoma cell line N2A. Oleoylanilide treatment causes two kinds of phenomena: alteration of the actin cytoskeleton, creating a brush-like protrusion of actin at the periphery of the cells, and reduction of the adhesiveness of these cells to laminin and fibronectin, two of the main components of the extracellular matrix in the central nervous system. These effects could be correlated with symptoms shown in the acute and chronic phases of the disease.

Actins↗