Importance of lysine-286 at the NADP site of glutamate dehydrogenase from Salmonella typhimurium.

Affinity labeling studies of NADP(+)-glutamate dehydrogenase from Salmonella typhimurium have shown that the peptide Leu-282-Lys-286 is located near the coenzyme site [Haeffner-Gormley et al. (1991) J. Biol. Chem. 266, 5388-5394]. The present study was undertaken to evaluate the role of lysine-286. The mutant enzymes K286R, K286Q, and K286E were prepared by site-directed mutagenesis, expressed in Escherichia coli, and purified. The Vmax values (micromoles of NADPH per minute per milligram of protein) were similar for WT (270), K286R (529), K296Q (409), and K286E (382) enzymes. As measured at pH 7.9, the Km value for NADPH was much greater for K286E (280 microM) than for WT (9.8 microM), K286R (30 microM), or K286Q (66 microM) enzymes. The efficiencies (kcat/Km) of the WT and K286R mutant were similar (1.2 x 10(3) min-1 microM-1 and 1.0 x 10(3) min-1 microM-1, respectively) while those of K286Q (0.30 x 10(3) min-1 microM-1) and K286E (0.07 x 10(3) min-1 microM-1) were greatly reduced. The decreased efficiency of the K286E mutant results from the increase in Km-NADPH, consistent with a role for a basic residue at position 286 which enhances the binding of NADPH. Plots of Vmax vs pH showed the pH optima to be 8.1-8.3 for all enzymes at saturating NADPH concentrations. A 40-fold increase in Km-NADPH for K286E was observed as the pH increased from 5.98 to 8.08, from which a unique pKe of 6.5 was calculated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Production of multimeric forms of CD4 through a sugar-based cross-linking strategy

We have developed a three-step cross-linking procedure that is specifically targeted at the carbohydrate on a protein and applied it to CD4 as a model system for studying the role of multivalent interactions in function. In the first step CD4 was oxidized with periodate, creating aldehydes that served as targets for the subsequent chemistry. Next the aldehydes were modified with cystamine, converting the reactive group into a thiol. Finally cross-linking through the thiol moiety was generated with the homobifunctional cross-linker bismaleimidohexane. With this procedure, approximately 60% of the CD4 was converted into higher molecular weight complexes that were soluble and retained function as assessed by glycoprotein gp120 binding activity. CD4 dimers and tetramers by mass were 4 and 15 times as active as CD4 monomer in blocking virus infection with HTLV-IIIB in an in vitro cellular assay. The cross-linking chemistry provides an efficient method for producing homomultimers of a glycoprotein.     

Colonization and extinction of land planarians (Platyhelminthes,Tricladida) in a Brazilian Atlantic Forest regrowth remnant

Long-term assessments of species assemblages are valuable tools for detecting species ecological preferences and their dispersal tracks, as well as for assessing the possible effects of alien species on native communities. Here we report a 50-year-long study on population dynamics of the four species of land flatworms (Platyhelminthes, Tricladida, Terricola) that have colonized or become extinct in a 70-year-old Atlantic Forest regrowth remnant through the period 1955–2006. On the one hand, the two initially most abundant species, which are native to the study site, Notogynaphallia ernesti and Geoplana multicolor have declined over decades and at present do not exist in the forest remnant. The extinction of these species is most likely related with their preference for open vegetation areas, which presently do not exist in the forest remnant. On the other hand, the neotropical Geoplaninae 1 and the exotic Endeavouria septemlineata were detected in the forest only very recently. The long-term study allowed us to conclude that Geoplaninae 1 was introduced into the study area, although it is only known from the study site. Endeavouria septemlineata, an active predator of the exotic giant African snail, is originally known from Hawaii. This land flatworm species was observed repeatedly in Brazilian anthropogenic areas, and this is the first report of the species in relatively well preserved native forest, which may be evidence of an ongoing adaptive process. Monitoring of its geographic spread and its ecological role would be a good practice for preventing potential damaging effects, since it also feeds on native mollusk fauna, as we observed in lab conditions. Júlio Pedroni: Granted by CNPQ–Brazil.     

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.     

Population dynamics of two small pelagic fish in the central-south area off Chile: delayed density-dependence and biological interaction

It is widely believed that environmental variability is the main cause for fluctuations in commercially exploited small pelagic fish populations around the world. Nevertheless, density-dependent factors also can drive population dynamics. In this paper, we analyzed thirteen years of a relative abundance index of two clupeoids fish populations coexisting in the central-south area off Chile, namely the common sardine, Strangomera bentincki, and anchovy, Engraulis ringens. We applied the classical diagnostic tools of time series analysis to the observed time-series. Also, the realized per capita population growth rate was studied with the aim of detecting the feedback structure that is characterizing the population dynamics of the two species. The analysis suggests that population fluctuations of the two species have an important density-dependent component, displaying first-order (direct density-dependent) and second-order (delayed density-dependent) simultaneously. The density-dependent component explained 70.5 and 55.6 % of the realized per capita population growth rate of common sardine and anchovy, respectively. The deterministic skeleton model showed an asymptotic convergence to equilibrium density. In presence of a stochastic environment, fluctuations were reproduced for the species showing a component of fluctuation with a period of 4 year. The intrinsic dynamics of each species is typical of interacting species resulting from trophic interactions. It is postulated that the second-order dynamics of S. bentincki and E. ringens in central-south Chile, may be the result from interactions with a specialist predator (the fishing fleet), interacting with exogenous environmental factors.     

Ethylene and IAA interactions in the inhibition of photoperiodic flower induction of <Emphasis Type="Italic">Pharbitis nil</Emphasis>

It has been shown that both IAA and ethylene application inhibit flower induction in the short-day plant Pharbitis nil. However application of IAA has elevated ethylene production in this plant, as well. Strong enhancement of ethylene production is also correlated with the night-break effect, which completely inhibits flowering. In order to determine what the role of IAA and ethylene is in the photoperiodic flower induction in Pharbitis nil, we measured changes in their levels during inductive and non-inductive photoperiods, and the effects of ethylene biosynthesis and action inhibitors on inhibition of flowering by IAA. Our results have shown that the inhibitory effect of IAA on Pharbitis nil flowering is not physiological but is connected with its effect on ethylene biosynthesis.     

A strategy for high cell density culture of heterotrophic microalgae with inhibitory substrates

Substrate inhibition is one of the major problems preventing high cell densities of microalgae in heterotrophic culture, so the possibility of overcoming the problem by various culture techniques was examined. It was found that perfusion culture may be the most appropriate technique for high cell densities in heterotrophic culture using inhibitory substrates. An experimental example in which a hollow fibre cell recycle system (HFCRS) was employed to achieve high cell densities of Chlamydomonas reinhardtii on acetate under heterotrophic conditions of growth was demonstrated. The cell density in the HFCRS was much higher than that reported in the literature for this species.