Production of chemotactic factor for lymphocytes by neutral SH-dependent protease of rabbit PMN leukocytes from immunoglobulins,especially IgM

In inflammation, PMN leukocyte emigration is followed by lymphocyte emigration. Two neutral proteases were isolated from lysosomal fraction of rabbit PMN leukocytes and purified by chromatography. The SH-dependent protease converted in vitro a naturally occurring IgM and specific antibody IgM to a chemotactic factor for lymphocytes; its molecular size was assumed to be around 14,000. Lymphocytes were collected from the thoracic duct lymph of rats. The chemotactic generation was induced by a short treatment with small amount of the enzyme, but the chemotactic factor produced was inactivated by a prolonged digestion with the enzyme. The chemotactic generation by the enzyme of rabbit IgG was apparently less marked. On the other hand, the SH-independent protease was ineffective for such chemotactic generation, suggesting different enzymatic characteristics of these proteases.     

Spectrophotometric analysis of RNA and DNA using cetyltrimethylammonium bromide

A versatile procedure is described for the analysis of RNA and DNA in brain using cetyltrimethylammonium bromide as the initial precipitant. Optimal conditions are described for the precipitation, hydrolysis, and effective separation of the RNA and DNA fractions from contaminating protein. The RNA and DNA fraction can now be accurately estimated by uv absorbance without a two wavelength correction. This method has also been used for the analysis of other mammalian organs and for mammalian cells obtained from tissue culture. The method may also be used for the simultaneous determination of radioactivity in nucleic acids. The orcinol reaction is shown to give high values for brain RNA.     

Purification and characterization of kynurenine--2-oxoglutarate aminotransferase from the liver, brain and small intestine of rats.

1. Kynurenine-2-oxoglutarate aminotransferase (isoenzyme 1) was purified to homogeneity from the liver, brain and small intestine of rats by the same procedure. The three enzyme preparations had nearly identical pH optima, substrate specificities and molecular weights. Isoenzyme 1 was active with 2-oxoglutarate but not with pyruvate as amino acceptor, and utilized a wide range of amino acids as amino donors. Amino acids were effective in the following order to activity: L-aspartate greater than L-tyrosine greater than L-phenylalanine greater than L-tryptophan greater than 5-hydroxy-L-tryptophan greater than L-kynurenine. The molecular weight was approximately 88 000 as determined by sucrose-density-gradient centrifugation. The pH optimum was between 8.0 and 8.5. On the basis of substrate specificity, substrate inhibition, subcellular distribution and polyacrylamide-disc-gel electrophoresis, it is suggested that liver, brain and small intestinal kynurenine-2-oxoglutarate aminotransferase (isoenzyme 1) is identical with mitochondrial tyrosine-2-oxoglutarate aminotransferase and also with mitochondrial aspartate-2-oxoglutarate aminotransferase. 2. An additional kynurenine-2-oxoglutarate aminotransferase (isoenzyme 2) was purified from the liver. This enzyme was specific for 2-oxoglutarate and L-kynurenine. Sucrose-density-gradient centrifugation gave a molecular weight of approximately 100 000. The pH optimum was between 6.0 and 6.5. This enzyme was not detected in the brain or small intestine.     

Subunit interactions of lobster hemocyanin. I. Ultracentrifuge studies

Subunit interactions in the hemocyanin of New England lobster, Homarus americanus, were investigated by means of the ultracentrifuge, using sedimentation velocity and Archibald molecular weight methods. It was verified that a 17S species dimerizes rapidly and reversibly to form a 25S species in the pH range 9.4–9.7 in the presence of calcium ion. From the Ca²⁺ and pH dependence of the equilibrium constant for this process, the absorption of approximately five calcium ions and three protons accompany the formation of one molecule of the 25S species. The sedimentation velocity patterns were also found to shift in favor of the 17S species with the imposition of excess hydrostatic pressure.     

Restriction cleavage map of mitochonrial DNA from the yeast Saccharomyces cerevisiae.

Mitochondrial DNA (mtDNA) from the yeast Saccharomyces cerevisiae was cleaved by restriction endonucleases Eco RI, Hpa I, Bam HI, Hind III, Pst I, and Sal I, yielding 10, 7, 5, 6, 1, and 1 fragments, respectively. A physical ordering of the restriction sites on yeast mtDNA has been derived. Yeast mtDNA cannot be isolated as intact molecules, and it contains nicks and gaps which complicate the use of conventional fragment mapping procedures. Nevertheless, the position of each of the restriction sites was obtained primarily by reciprocal redigestion of isolated restriction fragments. This procedure was supplemented by co-digestion of mtDNA with a multisite enzyme and a single-site enzyme (i.e., Sal I or Pst I) which provided a unique orientation for overlapping fragments cleaved by Sal I or Pst I. The data obtained from these approaches were confirmed by analysis of double and triple enzyme digests. Analysis of partial digest fragments was used for positioning of the smallest Eco RI fragment. A comparison of mtDNA from four grande strains (MH41-7B, 19d, TR3-15A, and MH32-12D) revealed similar, but slightly varying restriction patterns, with an identical genome size for each of approximately 5 X 10(-7) d or 75 kb. A fifth grande strain, D273-10B from S. cerevisiae, revealed restriction patterns different from those of the above strains, with a smaller genome size of 70 kb.     

RIBOSOME CRYSTALLIZATION IN CHICKEN EMBRYOS : II. Conditions for the Formation of Ribosome Tetramers In Vivo

Slow cooling of fertilized chicken eggs permits the elongation and termination of nascen^t polypeptides in the polysomes but prevents the initiation of new protein chains. This leads to polysome disaggregation during the first 30 min of cooling, and to the formation, of a pool of inactive ribosomes prone to crystallization. After 2 hr these ribosomes began to form tetramers, which do not contain any labeled proteins synthesized during cooling. If protein synthesis is inhibited by cycloheximide, added to eggs before cooling, tetramer formation in the embryos is prevented. Puromycin, on the other hand, leads to polysome disassembly and does not prevent tetramer formation. Rapid cooling of explanted embryos after short incubation at 37°C, with or without cycloheximide, largely prevents polysome disaggregation and the formation of tetramers. On the other hand, the addition of puromycin to explanted embryos promotes tetramer formation after rapid cooling. When cooled eggs are rewarmed, tetramers are disassembled into monomers, even if protein synthesis is inhibited. When those embryos were rapidly recooled tetramers reformed spontaneously from tetramer-derived monomers, even in the presence of cycloheximide. We conclude that the formation of tetramers at low temperature is an inherent property of the normal ribosomes.     

Sister-chromatid exchanges and cell-cycle kinetics in human lymphocyte cultures exposed to alkylating mutagens: apparent deformity in dose-response relationships

Experiments have been carried out using human whole-blood cultures to determine the effects of sampling times and of the duration of 5-bromodeoxyuridine (BrdUrd) treatment before fixation on sister-chromatid exchange (SCE) frequencies following exposure to mitomycin C (MMC). Cells were pulse treated for 1 h with 3 X 10(-6) M MMC at G1, and then sampled at 4-h intervals up to 88 h after stimulation of cultures with phytohemagglutinin (PHA). Results showed that this MMC treatment induced a 5-6 h proliferation delay per cell cycle, and that SCE frequencies first increased with time of fixation, peaking at 68 h, and then decreased. When cells were similarly treated with MMC, but subsequently exposed to BrdUrd for various times before fixation of cultures at 72 h, the SCE frequencies markedly increased with increasing durations of BrdUrd incubation times. These data indicate that, in mutagen-treated cultures, lymphocytes having relatively longer cell-cycle times show a higher mean frequency of SCEs. In a subsequent experiment, cells were treated for 1 h with increasing doses of MMC or 4-nitroquinoline 1-oxide (4NQO) at 0, 24, or 48 h, and then fixed at 72 h after PHA stimulation. Results showed that the optimal treatment times at which the agents could most efficiently produce SCEs were different for MMC and 4NQO, and that the dose-response curves tended to 'bend down' at very high doses; that is, treatments with very high doses induced smaller than expected numbers of SCEs. However, cells similarly treated with very high doses showed a higher, expected frequency of SCEs when sampled at 84 h, but again had a lower than expected SCE frequency when fixed at 96 h. The results indicate that there is an optimal time for sampling at which one can observe the maximum increase in SCE frequencies following mutagen exposure, and strongly suggest that the higher the dose, the later the optimal sampling time. Because of the apparent deformity of dose-response curves obtained after various treatments and sampling times, it seems necessary that extra fixation-time points be included in test protocols so as to avoid false negatives or confirm possible positives.