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.     

Angiotensin III and IV activation of the brain AT1 receptor subtype in cardiovascular function

The present investigation determined that native angiotensins II and III (ANG II and III) were equipotent as pressor agents when ICV infused in alert rats, whereas native angiotensin IV (ANG IV) was less potent. An analogue of each of these angiotensins was prepared with a hydroxyethylamine (HEA) amide bond replacement at the N-terminus, yielding additional resistance to degradation. These three angiotensin analogues, HEA-ANG II, HEA-ANG III, and HEA-ANG IV, were equivalent with respect to maximum elevation in pressor responses when ICV infused; and each evidenced significantly extended durations of effect compared with their respective native angiotensin. Comparing analogues, HEA-ANG II had a significantly longer effect compared with HEA-ANG III, and HEA-ANG IV, whereas the latter were equivalent. Pretreatment with the AT₁ receptor subtype antagonist, Losartan (DuP753), blocked subsequent pressor responses to each of these analogues, suggesting that these responses were mediated by the AT₁ receptor subtype. Pretreatment with the specific AT₄ receptor subtype antagonist, Divalinal (HED 1291), failed to influence pressor responses induced by the subsequent infusion of these analogues. These results suggest an important role for Ang III, and perhaps ANG IV, in brain angiotensin pressor responses mediated by the AT₁ receptor subtype.     

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.     

Testosterone, and winning and losing in human competition

Testosterone and cortisol were measured in six university tennis players across six matches during their varsity season. Testosterone rose just before most matches, and players with the highest prematch testosterone had the most positive improvement in mood before their matches. After matches, mean testosterone rose for winners relative to losers, especially for winners with very positive moods after their victories and who evaluated their own performance highly. Winners with rising testosterone had higher testosterone before their next match, in contrast to losers with falling testosterone, who had lower testosterone before their next match. Cortisol was not related to winning or losing, but it was related to seed (top players having low cortisol), and cortisol generally declined as the season progressed. These results are consistent with a biosocial theory of status.     

Growth of lipolytic psychrotrophic pseudomonads in raw and ultra-heat-treated milk

The lipolytic floras of 36 raw milk samples showing lipolytic defects were dominated by pseudomonads. Representative lipolytic isolates were selected and tested for growth, lipase activity and lipolysis in ultra-heat-treated milk at temperatures ranging from 5 degrees to 30 degrees C. Pseudomonas fluorescens was the most frequently encountered species but Ps. fragi was found to cause more severe lipolytic defects in both single and mixed strain milk cultures. A representative strain of Ps. fragi multiplied faster in cold-stored milk than did three representative strains of Ps. fluorescens. The lipases produced by Ps. fragi strains were more heat-stable than those produced by Ps. fluorescens strains.     

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).     

Enzymic Dehalogenation of 4-Chlorobenzoyl Coenzyme A in Acinetobacter sp. Strain 4-CB1

4-Chlorobenzoate degradation in cell extracts of Acinetobacter sp. strain 4-CB1 occurs by initial synthesis of 4-chlorobenzoyl coenzyme A (4-chlorobenzoyl CoA) from 4-chlorobenzoate, CoA, and ATP. 4-Chlorobenzoyl CoA is dehalogenated to 4-hydroxybenzoyl CoA. Following the dehalogenation reaction, 4-hydroxybenzoyl CoA is hydrolyzed to 4-hydroxybenzoate and CoA. Possible roles for the CoA moiety in the dehalogenation reaction are discussed.     

Thermodynamic analysis of trinitrotoluene biodegradation and mineralization pathways

Biodegradation of 2,4,6-trinitrotoluene (TNT) proceeds through several different metabolic pathways. However, the reaction steps which are considered rate-controlling have not been fully determined. Glycolysis and other biological pathways contain biochemical reactions which are acutely rate-limiting due to enzyme control. These rate-limiting steps also have large negative Gibbs free energy changes. Because xenobiotic compounds such as TNT can be used by biological systems as nitrogen, carbon, and energy sources, it is likely that their degradation pathways also contain acutely rate-limiting steps. Identification of these rate-controlling reactions will enhance and better direct genetic engineering techniques to increase specific enzyme levels.This article identifies likely rate-controlling steps (or sets of steps) in reported TNT biodegradation pathways by estimating the Gibbs free energy change for each step and for the overall pathways. The biological standard Gibbs free energy change of reaction was calculated for each pathway step using a group contribution method specifically tailored for biomolecules. The method was also applied to hypothetical "pathways" constructed to mineralize TNT using several different microorganisms. Pathways steps that have large negative Gibbs free energy changes are postulated to be potentially rate-controlling. The microorganisms which utilize degradation pathways with the largest overall (from TNT to citrate) negatiave Gibbs free energy changes were also determined. Such microorganisms can extract more energy from the starting substrate and are thus assumed to have a competitive advantage over other microorganisms. Results from this modeling-based research are consistent with much experimental work available in the literature. (c) 1996 John Wiley & Sons, Inc.