Induction of spherule formation in Physarum polycephalum by polyols

A method has been developed for inducing spherule formation (spherulation) in the myxomycete Physarum polycephalum by transferring the culture to synthetic medium containing 0.5 m mannitol or other polyols. This morphogenetic process occurred within 12 to 35 hr after the inducer was added. The mature spherules existed as distinct morphogenetic units, in contrast to the clusters of spherules formed during starvation. Ninety per cent of the spherules germinated by 24 hr in synthetic medium. The changes in the synthesis of ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and protein during plasmodial growth, spherulation, and germination of spherules are described. When spherule formation was completed, RNA, protein, and DNA decreased, compared with the values at the beginning of the conversion. The incorporation of (3)H-uridine into trichloroacetic acid-insoluble material was different in each of these periods, and this incorporation was sensitive to actinomycin D. The amount of glycogen increased during growth, whereas it decreased during spherulation. (14)C-glucose could be taken up by the cells in the presence of the inducer, and mannitol could not replace glucose as a source of energy. The mode of action of mannitol and its mechanism of induction are discussed.     

Activity of some enzymes in Physarum polycephalum

Summary The activity levels of seven enzymes were studied in growing plasmodia of Physarum polycephalum. The enzymes were isocitrate dehydrogenase, glucose-6-phosphate dehydrogenase, glutamate dehydrogenase, acid phosphatase, phosphodiesterase, -glucosidase, and histidase. Six of the enzymes showed a continuous increase in activity during the mitotic cycle; glutamate dehydrogenase exhibited a stepwise increase about 5 h after mitosis. Cycloheximide immediately inhibited the activity of all enzymes. Actinomycin D was ineffective in inhibiting enzyme activity until after one mitotic cycle had been completed; this indicates that mRNA was stable for all of these enzymes during the G2 period. Attempts to induce enzyme activity were unsuccessful.

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Zusammenfassung Der Aktivitätsverlauf von sieben Enzymen wurde in wachsenden Plasmodien von Physarum polycephalum untersucht. Es handelte sich um die Enzyme: Isocitrat-Dehydrogenase, Glucose-6-Phosphat-Dehydrogenase, Glutamat-Dehydrogenase, saure Phosphatase, Phosphodiesterase, -Glucosidase und Histidase. Sechs dieser Enzyme wiesen einen kontinuierlichen Aktivitätsanstieg während des Mitosecyclus auf; Glutamat-Dehydrogenase zeigte einen stufenförmigen Anstieg etwa 5 Std nach der Kernteilung. Cycloheximid hemmte sofort die Aktivität der Enzyme, während Actinomycin D erst nach Ablauf eines halben Teilungscyclus inhibierend wirkte. Dies deutet auf eine relativ stabile mRNA hin. Versuche, die Aktivität zu induzieren, schlugen fehl.

     

8-Bromo-cyclic GMP inhibits the calcium channel current in embryonic chick ventricular myocytes

Superfusion with 8-bromo-cyclic GMP or intracellular injection of cyclic GMP inhibits calcium-dependent slow action potentials in embryonic chick or guinea pig ventricular cells, suggesting that cyclic GMP inhibits calcium currents. Recently, cyclic GMP has been shown to reduce cyclic AMP-stimulated calcium currents in voltage-clamped ventricular myocytes. Since earlier results in intact cells had suggested that cyclic GMP might inhibit basal (i.e., unstimulated by cyclic AMP) calcium currents, we directly investigated the effect of 8-bromo-cyclic GMP on basal calcium channel currents (using barium as the charge carrier) in voltage-clamped ventricular myocytes isolated from embryonic chick hearts. Superfusion with 1 mM 8-bromo-cyclic GMP (without prior cyclic AMP elevation) progressively decreased peak calcium channel currents (-68% at 15 min after the onset of drug exposure). In contrast, the currents were unchanged during 15 min superfusion with control solution, or 1 mM 8-bromo-GMP (the noncyclic inactive analog of 8-bromo-cyclic GMP). The present results in voltage-clamped embryonic chick heart cells indicate that cyclic GMP can inhibit basal calcium channel currents, apparently through a cyclic AMP-independent mechanism.     

Characterization of a Serine Hydrolase Targeted by Acyl-protein Thioesterase Inhibitors in Toxoplasma gondii

In eukaryotic organisms, cysteine palmitoylation is an important reversible modification that impacts protein targeting, folding, stability, and interactions with partners. Evidence suggests that protein palmitoylation contributes to key biological processes in Apicomplexa with the recent palmitome of the malaria parasite Plasmodium falciparum reporting over 400 substrates that are modified with palmitate by a broad range of protein S-acyl transferases. Dynamic palmitoylation cycles require the action of an acyl-protein thioesterase (APT) that cleaves palmitate from substrates and conveys reversibility to this posttranslational modification. In this work, we identified candidates for APT activity in Toxoplasma gondii. Treatment of parasites with low micromolar concentrations of β-lactone- or triazole urea-based inhibitors that target human APT1 showed varied detrimental effects at multiple steps of the parasite lytic cycle. The use of an activity-based probe in combination with these inhibitors revealed the existence of several serine hydrolases that are targeted by APT1 inhibitors. The active serine hydrolase, TgASH1, identified as the homologue closest to human APT1 and APT2, was characterized further. Biochemical analysis of TgASH1 indicated that this enzyme cleaves substrates with a specificity similar to APTs, and homology modeling points toward an APT-like enzyme. TgASH1 is dispensable for parasite survival, which indicates that the severe effects observed with the β-lactone inhibitors are caused by the inhibition of non-TgASH1 targets. Other ASH candidates for APT activity were functionally characterized, and one of them was found to be resistant to gene disruption due to the potential essential nature of the protein.     

Interactive effects of pests increase seed yield

Loss in seed yield and therefore decrease in plant fitness due to simultaneous attacks by multiple herbivores is not necessarily additive, as demonstrated in evolutionary studies on wild plants. However, it is not clear how this transfers to crop plants that grow in very different conditions compared to wild plants. Nevertheless, loss in crop seed yield caused by any single pest is most often studied in isolation although crop plants are attacked by many pests that can cause substantial yield losses. This is especially important for crops able to compensate and even overcompensate for the damage. We investigated the interactive impacts on crop yield of four insect pests attacking different plant parts at different times during the cropping season. In 15 oilseed rape fields in Sweden, we estimated the damage caused by seed and stem weevils, pollen beetles, and pod midges. Pest pressure varied drastically among fields with very low correlation among pests, allowing us to explore interactive impacts on yield from attacks by multiple species. The plant damage caused by each pest species individually had, as expected, either no, or a negative impact on seed yield and the strongest negative effect was caused by pollen beetles. However, seed yield increased when plant damage caused by both seed and stem weevils was high, presumably due to the joint plant compensatory reaction to insect attack leading to overcompensation. Hence, attacks by several pests can change the impact on yield of individual pest species. Economic thresholds based on single species, on which pest management decisions currently rely, may therefore result in economically suboptimal choices being made and unnecessary excessive use of insecticides.     

The microtubule plus end tracking protein Orbit/MAST/CLASP acts downstream of the tyrosine kinase Abl in mediating axon guidance

Axon guidance requires coordinated remodeling of actin and microtubule polymers. Using a genetic screen, we identified the microtubule-associated protein Orbit/MAST as a partner of the Abelson (Abl) tyrosine kinase. We find identical axon guidance phenotypes in orbit/MAST and Abl mutants at the midline, where the repellent Slit restricts axon crossing. Genetic interaction and epistasis assays indicate that Orbit/MAST mediates the action of Slit and its receptors, acting downstream of Abl. We find that Orbit/MAST protein localizes to Drosophila growth cones. Higher-resolution imaging of the Orbit/MAST ortholog CLASP in Xenopus growth cones suggests that this family of microtubule plus end tracking proteins identifies a subset of microtubules that probe the actin-rich peripheral growth cone domain, where guidance signals exert their initial influence on cytoskeletal organization. These and other data suggest a model where Abl acts as a central signaling node to coordinate actin and microtubule dynamics downstream of guidance receptors.     

Short-term responses of ecosystem carbon fluxes to experimental soil warming at the Swiss alpine treeline

Climatic warming will probably have particularly large impacts on carbon fluxes in high altitude and latitude ecosystems due to their great stocks of labile soil C and high temperature sensitivity. At the alpine treeline, we experimentally warmed undisturbed soils by 4 K for one growing season with heating cables at the soil surface and measured the response of net C uptake by plants, of soil respiration, and of leaching of dissolved organic carbon (DOC). Soil warming increased soil CO₂ effluxes instantaneously and throughout the whole vegetation period (+45%; +120 g C m y^?1). In contrast, DOC leaching showed a negligible response of a 5% increase (NS). Annual C uptake of new shoots was not significantly affected by elevated soil temperatures, with a 17, 12, and 14% increase for larch, pine, and dwarf shrubs, respectively, resulting in an overall increase in net C uptake by plants of 20–40 g C m^?2y^?1. The Q ₁₀ of 3.0 measured for soil respiration did not change compared to a 3-year period before the warming treatment started, suggesting little impact of warming-induced lower soil moisture (?15% relative decrease) or increased soil C losses. The fraction of recent plant-derived C in soil respired CO₂ from warmed soils was smaller than that from control soils (25 vs. 40% of total C respired), which implies that the warming-induced increase in soil CO₂ efflux resulted mainly from mineralization of older SOM rather than from stimulated root respiration. In summary, one season of 4 K soil warming, representative of hot years, led to C losses from the studied alpine treeline ecosystem by increasing SOM decomposition more than C gains through plant growth.     

CBOD5 treatment using the marshland upwelling system

Five-day carbonaceous biochemical oxygen demand (CBOD₅) removal efficiency was evaluated for the marshland upwelling system (MUS) under both intermediate and saltwater conditions. The MUS treated decentralized wastewater from two private camps and a public restroom in the Grand Bay National Estuarine Research Reserve, Moss Point, Mississippi, and one private camp in the Barataria Terrebonne National Estuary, along Bayou Segnette, Louisiana. Raw wastewater was injected into the surrounding subsurface at a depth of 3.8 or 4.3 m. Various injection flow rates and frequencies were tested in addition to a synthetic wastewater trial. All trials followed a first-order background corrected removal equation, resulting in removal constants ranging from 0.49 to 3.32 m^?1 and predicted surface concentrations from 5.7 to 33.0 mg L^?1. CBOD₅ (unfiltered) influent concentrations of 282 ± 173 mg L^?1 were reduced to an overall effluent mean of 13 ± 13 mg L^?1 by a vector distance of 7 m at Moss Point and from 365 ± 151 mg L^?1 to 3.6 ± 7.6 mg L^?1 by a vector distance of 6 m for Bayou Segnette. Of seven trials, only one failed to achieve effluent CBOD₅ levels below a National Pollutant Discharge Elimination System (NPDES) standard level of 25 mg L^?1.