Further studies on the genus Berkeleya Grev.

Detailed light and electron microscopical studies on the diatom genus Berkeleya Grev. clarify relationships between the four species and support the separtion of B. fragilis and B. micans as two distinct taxa.     

Further studies on the thymocyte stimulating factor.

The thymocyte-stimulating activity produced by spleen cells cultured in the presence of PHA (cell. Immunol.17, 495, 1975) was further investigated. It was found that this activity is caused by a factor having the chemical properties of a protein with a molecular weight of 30,000–32,000 that, at high concentration, probably dimerizes to a molecule of molecular weight about 55,000 as measured by Sephadex gel filtration. This factor is produced also in mixed lymphocyte cultures of CBA and C57 spleen cells. The observation that the factor produced in the presence of PHA and the factor produced in the mixed lymphocyte cultures have the same molecular weight and the same rate of denaturation at two different temperatures suggests that they are the same, or very similar, substance. The fact that spleen cells of nu/nu mice do not produce thymocyte-stimulating factor suggests that thymus-dependent cells are involved in the production of this factor. This assumption is consistent with the observation that spleen cells of mice less than two weeks old (which show a very small response to PHA) do not produce significant amounts of thymocyte-stimulating factor. On the other hand, results of experiments with hydrocortisone-injected mice suggest that the target cell for thymocyte-stimulating factor is the immature, cortisone-sensitive thymocyte. The properties of thymocyte-stimulating factor are discussed and compared to those of factors with similar actions reported in the literature.     

Further studies on endo-beta-N-acetylglucosaminidase D1.

The purification procedure for endo-beta-N-acetylglucosaminidase D was improved to yield an enzyme preparation which was homogeneous upon gel electrophoresis. The molecular weight of the enzyme as estimated by Sephadex G-200 column chromatography was 280,000, while SDS-gel electrophoresis after reduction with 2-mercaptoethanol gave a value of 150,000. The purified enzyme did not show any chitinase, hyaluronidase or lysozyme activity. In the presence of exoglycosidases removing peripheral sugars, the endoglycosidase acted on serum glycoproteins such as transferrin and fetuin. The enzyme also hydrolyzed an oligosaccharide, (Man)5(GlcNAc)2, indicating that the peptide portion of substrates does not have much effect on susceptibility to the enzyme.     

Further studies on the cardiolipin phosphodiesterase of Escherichia coli.

The cardiolipin phosphodiesterase of Escherichia coli was further characterized. This enzyme has a pH optimum of 7.0 and is Mg2+ dependent. Mn2+ and Co2+ could replace Mg2+ but other divalent cations were inhibitory or without effect. The enzyme is not periplasmic and does not appear to be associated with membrane fractions prepared by different methods. It is recovered as a soluble protein in the cytosol fraction but could not be readily purified because of its instability. With cell-free systems, a requirement for ATP or ADP could be shown under certain defined conditions. Other nucleotides were less effective or ineffective in stimulating the phosphodiesterase. The cells displayed the highest activity during the middle to late exponential stage but no marked requirement for ATP was apparent when the phosphodiesterase was obtained from such freshly grown cells. If, however, cells were starved for several hours in saline medium, the cardiolipin phosphodiesterase level fell and a requirement for added ATP could be shown. The cardiolipin phosphodiesterase is an enzyme distinct from cardiolipin synthase. The assay conditions are quite different from each of these enzymes as are their subcellular distributions.     

Further studies on the mechanism involved in differential staining of BUdR-substituted Vicia faba chromosomes

In a preceding publication we reported that photolysis of BUdR-substituted Vicia faba chromatids occurs during observation with a fluorescence microscope when chromosomes were mounted in a solution containing trypsin and a photosensitive dye (Hoechst 33258 or acridine orange). The present investigations support the hypothesis that the rapid dissolving of the double BUdR-substituted (BB) chromatids observed with our method is due to single-strand breaks induced by a photosensitive dye-visible light system. The agents cysteamine and potassium iodide which reduce BUdR radicals and in this way may inhibit single-strand breaks modify the rate of chromosomes showing differential staining. It was totally suppressed by high cysteamine concentrations and markedly reduced by potassium iodide. Several acridine dyes were tested concerning their ability to induce differential staining. Some of them, e.g. aurophosphine and coriphosphine O, yield good results, others, e.g. acriflavine and acridine yellow, give poor differential staining. In an experiment in which the trypsin concentration was varied to induce approximately optimum and non-optimum digestion conditions the necessity of trypsin treatment in our method was confirmed.     

Further studies on phytoferritin

Summary Crystalline and paracrystalline inclusions found in plastids of willow cambium, grown under dim-light conditions, are considered to be phytoferritin. Phytoferritin is completely unaffected following treatment of cambial cells with deoxyribonuclease or ribonuclease, although other components are, at least in part, denatured. This is considered to exclude the possibility that the inclusions are viral particles. While not normally arranged in crystalline form, phytoferritin from embryonic axes of pea appears identical in structure with the complex found in cambial plastids. It, also, is not affected by treatment with nucleases for periods of up to 18 hours. Phytoferritin has been found only rarely in willow grown under conditions of high illumination.Four, five, or six electron-opaque subunits are most often found in the individual particles of phytoferritin. The most satisfactory explanation of the arrangements seen seems to be that the micellar configuration is in the form of an octahedron with a subunit at each of the six vertices.     

Further studies on hypothermia and the blood-brain barrier system.

Rats subjected to five episodes of recurrent, progressively deeper hypothermia showed no difference in cerebral deposition of rubidium tracer at 16 °C and/or apnea from animals lowered to this temperature and/or condition only once. Rats allowed to rewarm from 16 °C showed persisting increased cerebral deposition of tracer at 20 °C with gradual diminution at higher temperatures; at 37 °C, deposition of rubidium tracer in brains of rewarmed rats was not different from that of euthermic rats which were not subjected to hypothermia.