Developmental regulation and properties of the cGMP-specific phosphodiesterase in Dictyostelium discoideum

A simple assay has been developed to measure cGMP-specific phosphodiesterase (cGPD) activity in crude soluble extracts of amoebae of Dictyostelium discoideum. When amoebae of different wild-type strains were starved on buffered agar, all strains exhibited an 8- to 12-fold increase in cGMP-specific hydrolyzing activity during development, with the major increase occurring at aggregation. cGMP-specific activity was found in both prestalk and prespore cells. To determine if the elevated cGMP-specific hydrolyzing activity observed during late development was associated with the same enzyme present in vegetative cells, cGMP-specific activities were partially purified from cells at different developmental stages and characterized. Activity in vegetative cells was fractionated by gel filtration into three components with molecular weights of approximately 172,000, 115,000 and 56,000. In contrast, cells starved 4 hr in suspension or 18 hr on agar possessed only the 172,000 or 115,000 Mr forms, respectively. The low-molecular-weight enzyme differed from the two larger forms in kinetic properties and in sensitivity to sulfhydryl reagents. Nevertheless, the three activities probably represent different forms of the same enzyme because mutants defective at the stmF locus lacked appreciable cGMP-specific hydrolyzing activity throughout development. These results indicate that D. discoideum produces a single cGPD which is strongly developmentally regulated. These findings further suggest that intracellular cGMP might be involved in regulating postaggregative as well as preaggregative development.     

Selection and proliferation of rapid growing cell lines from embryo derived cell cultures of yew tree (Taxus cuspidata Sieb. et Zucc)

Calli were induced from 300,000 embryos isolated from immature to mature stage of seeds collected on late September from 14 elite trees. When the embryos were cultured onto plastic Petri-dish containing 20 mL of modified B5 basal medium supplemented with 3% (w/v) sucrose, 500 mg/L casein hydrolysate, 250 mg/L myo-inositol, 0.5% (w/v) polyvinyl polypyrrolidon (PVPP), 2×MS vitamins, 0.5 mg/L gibberellic acid, and 10 mg/L 2,4-D after 2 weeks of culture, yellowish-white calli were immediately formed on the surfaces of embryos, and subcultured for 4 weeks in same culture medium. Because most of calli maintained for more than 3 months were revealed differences in their colors, surface texture, and growth rate, visual selection was made for first round screening. When the size of visually selected calli larger than 19 mm in their diameter were inoculated, persistent proliferation was observed. Among the plating methods tested for the selection of rapid growing cell lines at single cell and/or small cell aggregate level, 2-layer spread plating revealed as the best for single cell cloning. To enhance cell growth and maintain high rate of viability for long-term culture of yew cells in bioreactor, final cell volume less than 50% in SCV seemed to be the best. Time course study revealed that 30% of inoculum density was suitable for fed batch culture. Among the tested conditional media, the rate of 1∶2 (old medium: fresh medium) was recorded at the best for cell growth.     

Pattern formation in fragmented eggs of the short germ insect Schistocerca gregaria

Summary The effect of transverse fragmentation on the segment pattern of the short germ embryo of the locust Schistocerca gregaria has been investigated at two stages subsequent to the formation of the germ anlage. Following fragmentation both anterior and posterior partial embryos were observed, although rarely in a single egg. Anterior partial patterns usually terminated with a segment visible at the time of fragmentation or with the next segment due to appear. Posterior partial patterns began with a wide range of segments depending on the level of fragmentation.Anterior and posterior partial patterns developing in a single egg were usually not complementary and the segments missing sometimes included some segments visible when the embryo was fragmented. Non-complementary patterns resulted following fragmentation in all regions, while complementary patterns only occurred after fragmentation in the visibly-segmented region.The results suggest that following fragmentation isolated posterior portions of the embryo continue to form segments, while isolated anterior regions usually do not. This effect could result from variable damage to an existing pattern of unequally-sized segment primordia, or from the disruption of a process of sequential segmentation in the elongating posterior region of the embryo. The results are broadly compatible with the progress zone model proposed by Summerbell et al. (1973).     

Maintenance of two genetic entities by habitat selection

Summary In the laboratory, the two species of copepodsLepeophtheirus thompsoni andLepeophtheirus europaensis, ectoparasites of flatfishes, can meet and mate on at least one host species. In the wild however, these two species are found isolated on their sympatric hosts. Habitat selection theoretically represents a powerful enough mechanism to explain the maintenance of genetic heterogeneity in the wide sense. In this paper, the host colonization process is studied for both parasite species. It is shown that each parasite can develop and reach adult age on each host species. However,L. thompsoni is highly selective; it almost totally refuses to colonize hosts other than its natural one.Lepeophtheirus europaensis, on the contrary, readily infests turbot and brill in single-host experiments, but strongly prefers the brill when it has a choice. It appears that these two genetic entities are sympatrically maintained due to strong habitat selection. Such a pattern could theoretically only occur in a soft-selection context (density dependence). This point is discussed with respect to the different patterns in host use found in the geographical distribution of these parasites.     

Proposals for optimization of the international phage typing system for Listeria monocytogenes: combined analysis of phage lytic spectrum and variability of typing results.

Combined analysis of 5,179 serial phage reactions of 20 Listeria monocytogenes propagating strains over 14 years and phage typing results from 2,659 further L. monocytogenes strains allowed us to estimate lytic spectrum specificity and the variability of the lytic reactions of 35 phages. These included the 26 phages recommended for the international method for phage typing defined in 1985 by Rocourt et al. (J. Rocourt, A. Audurier, A. L. Courtieu, J. Durst, S. Ortel, A. Schrettenbrunner, and A. G. Taylor, Zentralbl. Bakteriol. Abt. 1 Orig. A 259:489-497, 1985). The results are discussed individually for each phage. Proposals for modifying the present system are made with the aim of producing an optimal bacteriophage set for routine use.     

Ginkgolide B production in cultured cells derived from Ginkgo biloba L. leaves

Callus cultures and cell suspension cultures derived from Ginkgo biloba L. leaves produced ginkgolidc B. In cell suspension cultures, the production reached a maximum by the 13th day of subculture and followed by a sharp decrease. The medium of Murashige and Skoog induced the highest ginkgolide B content in cultures while the medium of Schenk and Hildebrandt promoted cell growth. For the maximal production of ginkgolide B, cells were cultured in Murashige and Skoog medium modified to contain 1.0 mg/l of -naphthaleneacetic acid, 0.1 mg/1 of kinetin, 30 g/1 sucrose and 1.25 mM potassium phosphate with a molar ratio of ammonium to nitrate ions of 1 3.Abbreviations B5 Gamborg et al (1968) medium - GKB Ginkgolide B - MS Murashige and Skoog (1962) medium - NAA -naphthaleneacetic aicd - SH Schenk and Hildebrandt (1972) medium     

Mitochondrial DNA sequence evolution in sharks: rates, patterns, and phylogenetic inferences

Abundant representation of sharks in the fossil record makes this group a superb system in which to investigate rates and patterns of molecular evolution and to explore the strengths and weaknesses of phylogenetic inferences from molecular data. In this report, the molecular evolution of the cytochrome b gene in sharks is described and the information related to results from phylogenetic analysis of the data evaluated in the light of a phylogeny derived independently of the molecular data. Across divergent lineages of sharks there is evidence for significant substitution rate variation, departure from compositional equilibrium, and substantial homoplasy; nevertheless, the signal of evolutionary history is evident in patterns of shared transversions and amino acid replacements.      

Metabolic rate and directional nucleotide substitution in animal mitochondrial DNA

There is marked heterogeneity of nucleotide composition in mitochondrial DNA across divergent animals. Differences in nucleotide composition presumably reflect differences in directional nucleotide substitution for A+T or G+C nucleotides. In mitochondrial DNA, there is A+T directional nucleotide substitution in most (if not all) animals surveyed, and the magnitude of directional A+T nucleotide substitution differs greatly within and among groups. Differences in directional nucleotide substitution among lineages of mammals can be explained by changes in metabolic physiology. This relationship is thought to be mediated by the effect of oxygen radicals because these toxic compounds are by-products of aerobic metabolism and are known mutagens. Association between metabolism and nucleotide composition provides additional evidence in favor of the hypothesis that rates and patterns of nucleotide substitution in mitochondrial DNA can be influenced by factors that impinge on rates of endogenous DNA damage.      

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.     

Metronidazole in prevention and treatment of bacteroides infections in elective colonic surgery.

A double-blind randomised trial was carried out among 46 patients undergoing elective colonic surgery; 27 patients received prophylactic metronidazole and 19 received placebo. Anaerobic infections did not develop in any of the metronidazole-treated patients, but did develop in 11 (58%) of 19 controls who were subsequently successfully treated with metronidazole.