Purfication and molecular weight of an RNA-dependant RNA polymerase from Brassicae oleracea var. Botrytis]

An RNA-dependent RNA polymerase has been completely purified from Cauliflower inflorescences. Analysis of the purified enzyme on SDS-polyacrylamide gels showed one polypeptide chain with a molecular weight of 140,000 dalton. The enzyme is monomeric in its native state. The in vitro activity was completely dependent on added RNA, the most efficient templates being poly (U), poly (U, C), poly (I) and viral RNA.     

A quick and efficient method to generate mammalian stable cell lines based on a novel inducible alphavirus DNA/RNA layered system

We report a new method to generate high-expressing mammalian cell lines in a quick and efficient way. For that purpose, we developed a master cell line (MCL) containing an inducible alphavirus vector expressing GFP integrated into the genome. In the MCL, recombinant RNA levels increased >4,600-fold after induction, due to a doxycycline-dependent RNA amplification loop. The MCL maintained inducibility and expression during 50 passages, being more efficient for protein expression than a conventional cell line. To generate new cell lines, mutant LoxP sites were inserted into the MCL, allowing transgene and selection gene exchange by Cre-directed recombination, leading to quick generation of inducible cell lines expressing proteins of therapeutic interest, like human cardiotrophin-1 and oncostatin-M at several mg/l/24 h. These proteins contained posttranslational modifications, showed bioactivity, and were efficiently purified. Remarkably, this system allowed production of toxic proteins, like oncostatin-M, since cells able to express it could be grown to the desired amount before induction. These cell lines were easily adapted to growth in suspension, making this methodology very attractive for therapeutic protein production.     

Degradation of ribosomal RNA from Escherichia coli by ribonuclease U2 and in vitro transcription of "RNA-fragments" into complementary DNA]

Under well defined conditions ribosomal RNAs purified from Escherichia coli can be degraded by ribonuclease U2 giving rise to RNA fragments of 60--70 nucleotides. In vitro, these fragments are efficiently transcribed into a complementary DNA by DNA polymerase RNA dependent, partially purified from extracts of E. coli. In vivo, "RNA-fragments-U2" inhibit the development of plant tumors.     

Adenovirus type 12 infection of defined mouse-human hybrid cell clones

Summary Human adenovirus type 12 does not multiply in mouse cells; only viral T-antigen is detected. Mouse-human cell hybrid clones containing human chromosomes A3, B5, C7, C11, C12, D14, E17, F19 and F20, support synthesis of adenovirus DNA and capsid antigens.The hybrid clones used in these experiments were kindly supplied by Drs.Carlo Croce andHilary Koprowski of the Wistar Institute, Philadelphia, Pennsylvania. Supported by Grants from National Cancer Institute.     

Isolation and properties of cell walls fromAgrobacterium tumefaciens B 6 1

Summary Purified cell walls were prepared fromAgrobacterium tumefaciens B ₆ by extraction of intact cells with hot sodium dodecyl sulfate and digestion with proteases. Such preparations contained peptidoglycan that accounted for about 40% of their dry weight. Electron micrographs of the purified walls showed that they conserved their characteristic shape despite the drastic extraction procedure.This work was supported in part by a contribution from a friend of the Weizmann Institute of Science in Buenos Aires, Argentine.I wish to thank Professor Nathan Sharon for his constant interest and critical discussion during this work.     

Presence of autocomplementary RNA with viral specificity in cells infected with herpes virus]

RNA from cells infected with Herpes simplex virus contain a higher percentage of double-stranded RNA than non-infected cells. This percentage increases three-fold upon self-annealing. The complementary RNA sequences were shown to be virus-specific by the following criteria: (1) high melting temperature than double-stranded RNA from non infected cells; (2) higher density in caesium sulphate; (3) specific hybridization with viral DNA.     

High salt effect on RNA synthesis in Krebs ascites tumor cells

Summary The incubation of Krebs ascites tumor cells in medium with a high salt concentration resulted in a partial inhibition of nuclear RNA synthesis. The residual RNA polymerase activity in such nuclei was only slightly inhibited by low concentrations (50 nM) of -amanitin. This finding suggested an inhibition of RNA polymerase II activity under conditions of medium hypertonicity. Indeed, RNA polymerase II, isolated from the nuclei of cells exposed to hypertonicity, revealed only half of the control activity. On the other hand, RNA polymerase I was not affected by hypertonicity. Moreover, chromatin fractions isolated from cells incubated in hypertonic or isotonic medium were equally template-active in RNA synthesis.This investigation was supported by the Turkish Scientific and Technical Research Council (TÜBITAK), Project No. TAG 339.     

Hemagglutination by simian adenovirus 7]

Simian adenovirus 7, either complete virus or its capsid subunits, agglutinates Rat (Sprague-Dowley) red blood cells in the presence of heterotypical antiserum. Haemagglutination takes place at 4 degrees C and room temperature. The antigen could not be eluted and its haemagglutinin properties are heat-stable. The reaction is specific. It is inhibited by homologous antiserum only. This property and its characteristics permit a camparison of this strongly oncogenic adenovirus to the human adenovirus of subgroup III of Rosen.     

The mode of action of bacteriocin N5 purified from Clostridium perfringens]

The purified bacteriocin N5 produced by Clostridium perfringens BP6K inhibited simultaneously the syntheses of DNA, RNA and protein in sensitive cells, without DNA degradation. Bacteriocin N5 inhibited the accumulation of leucin and caused the exit of the previously accumulated amino acid. The effects of bacteriocin N5 are very similar to those observed for colicins E1, K, A and I.     

Action de la ribonucléase sur les cellules du carcinome d'Ehrlich

Summary The reduced form of ribonuclease acts on the cells ofEhrlich's carcinoma by producing important changes in the RNA content of cells:(1) First, there is an important synthesis of intracellular RNA and an accumulation of free nucleotides (from the external medium). This phenomenon accompanies the necrosis of the cells.(2) After this first stage begins a rapid degradation of intracellular RNA.Simultaneously, the respiration of cells decreases whilst protein content remains unchanged.The oxidized form of ribonuclease has no action on the cells.

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Aspirant du Fonds national belge de la Recherche scientifique.     

Mesenchymal stem cells or cardiac progenitors for cardiac repair? A comparative study

In the past, clinical trials transplanting bone marrow–derived mononuclear cells reported a limited improvement in cardiac function. Therefore, the search for stem cells leading to more successful stem cell therapies continues. Good candidates are the so-called cardiac stem cells (CSCs). To date, there is no clear evidence to show if these cells are intrinsic stem cells from the heart or mobilized cells from bone marrow. In this study we performed a comparative study between human mesenchymal stem cells (hMSCs), purified c-kit⁺ CSCs, and cardiosphere-derived cells (CDCs). Our results showed that hMSCs can be discriminated from CSCs by their differentiation capacity towards adipocytes and osteocytes and the expression of CD140b. On the other hand, cardiac progenitors display a greater cardiomyogenic differentiation capacity. Despite a different isolation protocol, no distinction could be made between c-kit⁺ CSCs and CDCs, indicating that they probably derive from the same precursor or even are the same cells.     

Purification and properties of heat-resistant exotoxin produced by Macrophomina phaseolina (Tassi) Goid in culture.

A partially purified preparation of a water-soluble, heat-resistant, nonspecific exotoxin produced by a strain of Macrophomina phaseolina, isolated from Phaseolus mungo L. could reduce Cu++ ions and produced a red colour with 2,4-dinitrophenyl hydrazine reagent. It caused inhibition of seed germination, wilting of cult seedlings, stunted growth of young seedlings and loss of permeability of the cell membrane. Seedlings of P. mungo, grown in presence of the toxin showed a slight increase in the contents of protein and total RNA over control, but a significant increase in the specific activities of F-1, 6-BP aldolase and G-6-P isomerase.     

Adenovirus type 12 infection of defined mouse-human hybrid cell clones.

Human adenovirus type 12 does not multiply in mouse cells; only viral T-antigen is detected. Mouse-human cell hybrid clones containing human chromosomes A3, B5, C7, C11, C12, D14, E17, F19 and F20, support synthesis of adenovirus DNA and capsid antigens.     

Purification and properties of a heat-resistant exotoxin produced byMacrophomina phaseolina (Tassi) Goid in culture

Summary A partially purified preparation of a water-soluble, heat-resistant, nonspecific exotoxin produced by a strain ofMacrophomina phaseolina, isolated fromPhaseolus mungo L. could reduce Cu⁺⁺ ions and produced a red colour with 2,4-dinitrophenyl hydrazine reagent. It caused inhibition of seed germination, wilting of cut seedlings, stunted growth of young seedlings and loss of permeability of the cell membrane. Seedlings ofP. mungo, grown in presence of the toxin showed a slight increase in the contents of protein and total RNA over control, but a significant increase in the specific activities of F-1, 6-BP aldolase and G-6-P isomerase.     

Decrease in stimulation and allogeneic response following experimental infection of mice with human adenovirus 5]

15 days after experimental infection with human adenovirus 5, C57 Bl/6 murine spleen cells were found to have altered in vitro properties. Proliferative response to phytohemagglutinine was slightly increased while the allogeneic response was decreased and the capability to stimulate allogeneic cells was significantly diminished. The degree of expression of surface antigens responsible for allogeneic stimulation is influenced by many regulatory factors, still poorly defined; alteration of some factors of antigenicity may result, directly or indirectly, from certain viral infections.     

Aplysianin-A, an antibacterial and antineoplastic glycoprotein in the albumen gland of a sea hare, Aplysia kurodai

Aplysianin-A, an antibacterial and antineoplastic factor in the albumen gland of the sea hare Aplysia kurodai, was isolated. It had a molecular weight of approximately 320 kD and consisted of subunits with a molecular weight of 85 kD. It contained 9.8% neutral sugar. Aplysianin A showed 50% inhibition of Bacillus subtilis growth at a concentration of 4 microgram protein/ml and 50% lysis of murine MM46 tumor cells at 14 ng protein/ml. A partial identity of antigenic specificity of the purified specimen with an antineoplastic factor from Aplysia eggs was observed in immunodiffusion tests.     

Inclusion of Gross virus cell surface associated antigen in liposomes]

The Gross virus associated cell surface antigen GCSAa was extracted from (C58NT)D cells by solubilization of membranes with detergent and partially purified. This antigen was entraped in liposomes. Absorption experiments of the cytotoxic activity towards EmaleG2 cells of a W/Fu anti (C58NT)D serum showed the presence of the antigen at the surface of sensibilized liposomes.     

High salt effect on RNA synthesis in Krebs ascites tumor cells

The incubation of Krebs ascites tumor cells in medium with a high salt concentration resulted in a partial inhibition of nuclear RNA synthesis. The residual RNA polymerase activity in such nuclei was only slightly inhibited by low concentrations (50 nM) of alpha-amanitin. This finding suggested an inhibition of RNA polymerase II activity under conditions of medium hypertonicity. Indeed, RNA polymerase II, isolated from the nuclei of cells exposed to hypertonicity, revealed only half of the control activity. On the other hand, RNA polymerase I was not affected by hypertonicity. Moreover, chromatin fractions isolated from cells incubated in hypertonic or isotonic medium were equally template-active in RNA synthesis.