Stabilization and protection of an enzymatic system, participating in the transformation of beta-carotene into retinal, during its isolation]

The authors studied the effect of dithiothreitol (DTT), carotenoids and protease inhibitors on stabilization and protection of the enzyme catalysing the conversion of beta-carotene into retinal during the enzyme isolation from the rabbit small intestine. The addition of 1 mM DTT into the homogenization mixture increased the activity of the enzyme 5 times compared with control. The additional introduction of 0.7 mg/ml soybean trypsin inhibitor or 2.10(-4) M phenylmethylsulfonyl fluoride increased the enzyme activity 2.1 and 1.2 times, respectively. Lutein, beta-carotene and lycopene at a concentration of 10 mg/ml increased the enzyme activity 2.1, 1.9 and 1.6 times respectively. The effects of DTT, lutein and the protease inhibitor depended on their concentrations and was of an independent additive character. The maximum activity of the isolated enzyme exceeded the control without DTT 15 times.     

Effect of calcium chloride on interferon production and the biosynthesis of cellular macromolecules

The influence of calcium chloride in production of interferon and biosynthesis of cell proteins, RNA and DNA in the cultures of chick embryo fibroblast cells and murine cells L-929 in response to induction of the cells by poly(I).poly(C) was studied. It was shown that calcium ions in concentrations of 10 to 30 mM markedly increased formation of interferon in the cell cultures.     

The state of hyporeactivity in interferon production: localization and partial purification of the factor repressing the interferon production

The hyporeactivity factor in interferon production by L-929 cells designated IRP (interferon repressing protein) has been studied. In particular, its localization and methods of its purification have been studied. The kinetics of IRP accumulation by producing cells correlate with the development of hyporeactivity condition. Most of IRP is localized in cell sap and in ribosomal fraction in evidence to regulatory role of repressor at the level of interferon mRNA translation. A 100-fold increase in repressor activity was achieved by IRP concentration by ammonium sulfate precipitation. IRP as well as interferon have been shown to possess high affinity to polyU sepharose. The preparations of IRP and interferon concentrated by ammonium sulfate precipitation were subsequently purified by fractioning in a polyI sepharose column. A 10,000-fold (6 x 10(4) U/mg) purification was achieved for IRP and 250-fold (10(4) U/mg) for interferon.     

Recovery of activity of the gene coding for tetracycline resistance in the plasmin pBRS188

The spontaneous recovery of activity of tet gene deleted of the promoter region was studied. Plasmid pBRS188 was used as a model for studying this problem. The plasmid has the fragment of tet gene of pBR322, from which it originates, between the sites of restriction endonucleases EcoRI and HindIII cleavage resulting in inactivation of tet promoter. E. coli cells harbouring the plasmid were shown to revert the TcR phenotype with the frequency 10(-9). The gene activation coincided with intraplasmid recombination revealed by restriction analysis. In some cases the recovery of tet gene activity coincided with the formation of multimeric plasmids.     

Role of macrophages in interferon production]

The role of macrophages in production of interferons (IF) is manysided. They are able to synthesize IFs after any induction. However, the function of macrophages as producers of IFs is not, probably, basic. The levels of IFs produced by them are mainly low. When they are stimulated by inducers of "early" IF the synthesis is performed by intact macrophages whereas with the use of inducers of "late" IF there is always observed joint activity of macrophages and other immunocompetent cells. The main role of macrophages in production of IFs is in regulation of synthesis of these proteins in the host. In addition, they are able to serve as stimulating cells in inducing IF production by the majority of drugs by transmitting information on IF synthesis to lymphocytes. This function of macrophages is not species-specific.     

Elemental distribution in striated muscle and the effects of hypertonicity: Electron probe analysis of cryo sections

A method of rapid freezing in supercooled Freon 22 (monochlorodifluoromethane) followed by cryoultramicrotomy is described and shown to yield ultrathin sections in which both the cellular ultrastructure and the distribution of diffusible ions across the cell membrane are preserved and intracellular compartmentalization of diffusabler ions can be quantitated. Quantitative electron probe analysis (Shuman, H., A.V. Somlyo, and A.P. Somlyo. 1976. Ultramicros. 1:317-339.) of freeze-dried ultrathin cryto sections was found to provide a valid measure of the composition of cells and cellular organelles and was used to determine the ionic composition of the in situ terminal cisternae of the sarcoplasmic reticulum (SR), the distribution of CI in skeletal muscle, and the effects of hypertonic solutions on the subcellular composition if striated muscle. There was no evidence of sequestered CI in the terminal cisternae of resting muscles, although calcium (66mmol/kg dry wt +/- 4.6 SE) was detected. The values of [C1](i) determined with small (50-100 nm) diameter probes over cytoplasm excluding organelles over nuclei or terminal cisternae were not significantly different. Mitochondria partially excluded C1, with a cytoplasmic/ mitochondrial Ci ratio of 2.4 +/- 0.88 SD. The elemental concentrations (mmol/kg dry wt +/- SD) of muscle fibers measured with 0.5-9-μm diameter electron probes in normal frog striated muscle were: P, 302 +/- 4.3; S, 189 +/- 2.9;C1, 24 +/- 1.1;K, 404 +/- 4.3, and Mg, 39 +/- 2.1. It is concluded that: (a) in normal muscle the "excess CI" measured with previous bulk chemical analyses and flux studies is not compartmentalized in the SR or in other cellular organelles, and (b) the cytoplasmic C1 in low [K](0) solutions exceeds that predicted by a passive electrochemical distribution. Hypertonic 2.2 X NaCl, 2.5 X sucrose, or 2.2 X Na isethionate produced: (a) swollen vacuoles, frequently paired, adjacent to the Z lines and containing significantly higher than cytoplasmic concentrations of Na and Cl or S (isethionate), but no detectable Ca, and (b) granules of Ca, Mg, and P = approximately (6 Ca + 1 Mg)/6P in the longitudinal SR. It is concluded that hypertonicity produces compartmentalized domains of extracellular solutes within the muscle fibers and translocates Ca into the longitudinal tubules.     

Stimulation of interferon production]

Data on the effect of some factors on interferon production in vitro are presented The kinetics of interferon synthesis in response to superinduction was similar to the respective curve of the effect of UV-radiation on the cell. Possible similarity in the effect of these factors on the mechanisms controlling interferon production is noted. An increase in interferon synthesis under the effect of ascorbic acid in cells of chick embryo fibroblast and L-929 was found. Combined use of the inductors provided an increase in the tests of interferon.     

Translation of chicken interferon messenger in a cell-free protein synthesis system and in a cell culture

A highly effective cell-free system for protein synthesis was obtained from rabbit reticulocytes and for the first time used for synthesis of biologically active chicken interferon. The optimal conditions for translation of its mRNA were developed. The translation efficacy in the cell-free system was 10-50 times higher than that in the culture of heterologous cells. The higher the purity level of RNA, the higher the translation level. With respect to poly (A+) RNA sedimenting in the sucrose gradient 9S the efficacy reached 2560 units per 1 microgram of RNA. By the content of poly (A), sequences and rate of the sedimentation, mRNA of the chicken interferon was similar to that of the human fibroblast cell interferon. The possible translation of mRNA of the chicken interferon at low concentrations of exogenic potassium ions in the cell-free system is explained by production of interferon in infected cells where the concentration of the intracellular potassium significantly decreases which is indicative of the mRNA interferon similarity with virus templates. It was found that only albino New Zealand rabbits, but also chinchilla may be used for preparation of the cell-free protein synthesizing system. Various exogenic templates in the mRNA-dependent cell-free system prepared from reticulocyte nonfractionated lysate by treatment with micrococcal nuclease stimulated the protein synthesis by 7-15 times.