Stretch-activated composite ion channels in Bacillus subtilis.

The presence of ion-conducting pores in the membrane of Bacillus subtilis giant protoplasts was discovered using the patch-clamp technique. Membrane stretch caused the activation of several conductances with values in the nS range. The observations indicate the presence of substate levels and of aggregates of channels behaving in a cooperative manner. Following repeated stretch cycles, the channels exhibited spontaneous activity. The characteristics of the electrical phenomena afterwards changed in time in a manner suggesting the decay of the giant channels into lower-conductance species, presumably corresponding to building blocks of the giant stretch-activated channels.     

Effects of exogenous fatty acids on calcium uptake by brush-border membrane vesicles from rabbit small intestine

The uptake of a variety of fatty acids by isolated brush-border membranes from rabbit small intestine was studied. This uptake increased with acyl chain-length and was not diminished by washing of the lipid-treated membranes with 0.25 M CsBr. The binding of fatty acid was not accompanied by a decrease in endogenous acyl groups or of cholesterol and therefore corresponded to a net uptake accountable qualitatively and quantitatively by the fatty acid added to the membranes. The uptake of Ca2+ was stimulated by treatment of the membranes with low concentrations of unsaturated fatty acids (0.05 mM) as well as with various concentrations of caprylic acid (0.10-3.00 mM) and inhibited by treatment with higher concentrations of unsaturated fatty acids (0.20-0.60 mM). Saturated fatty acids had no marked effects on Ca2+ uptake. The stimulatory concentrations of unsaturated fatty acids did not change the Ca2+-binding characteristics of the membranes, whereas the higher concentrations decreased equilibrium binding of Ca2+ and very probably the number of high-affinity binding sites. The results of this study are assessed in terms of the effects of normal fatty acids found in the diet on the absorptive properties of the brush-border membranes.     

Aneurysms after microvascular anastomoses. Incidence and pathogenesis in experimental animals.

This study has demonstrated that aneurysms occur in about 50 percent of the rat femoral arteries subjected to microvascular anastomosis. These aneurysms are consistent histologically--being characterized by medial necrosis, loss of elastic lamellae, and subintimal hyperplasia. Mechanical trauma is implicated as a factor in their pathogenesis, though further study in necessary to define the exact etiology.     

Dimorphism inYarrowia lipolytica: Filament formation is suppressed by nitrogen starvation and inhibition of respiration

In contrast toSaccharomyces cerevisiae, nitrogen starvation inhibited formation of hyphae in liquid cultures ofY. lipolytica, while carbon source did not seem to be important for filament formation. Inhibitors of mitochondrial respiration strongly suppressed the development of hyphae, indicating that energy conversion processes, and thus carbon metabolism, may be involved. pH of the medium also strongly affected the morphology, but only in the presence of a complex nitrogen source, implying that the cells respond to altered nutrition in media with different pH rather than to pH itself. The results suggest that theXPR2 gene encodingY. lipolytica alkaline extracellular proteinase is involved in the regulation of dimorphism in this species.     

Fluorescent diphenylphosphonate-based probes for detection of serine protease activity during inflammation

Activity-based probes are small molecules that covalently bind to the active site of a protease in an activity-dependent manner. We synthesized and characterized two fluorescent activity-based probes that target serine proteases with trypsin-like or elastase-like activity. We assessed the selectivity and potency of these probes against recombinant enzymes and demonstrated that while they are efficacious at labeling active proteases in complex protein mixtures in vitro, they are less valuable for in vivo studies. We used these probes to evaluate serine protease activity in two mouse models of acute inflammation, including pancreatitis and colitis. As anticipated, the activity of trypsin-like proteases was increased during pancreatitis. Levels of elastase-like proteases were low in pancreatic lysates and colonic luminal fluids, whether healthy or inflamed. Exogenously added recombinant neutrophil elastase was inhibited upon incubation with these samples, an effect that was augmented in inflamed samples compared to controls. These data suggest that endogenous inhibitors and elastase-degrading proteases are upregulated during inflammation.     

Glutathione peroxidase‐like enzymes cover five distinct cell compartments and membrane surfaces in Arabidopsis thaliana

Glutathione peroxidase‐like enzymes (GPXLs) constitute a family of eight peroxidases in Arabidopsis thaliana. In contrast to the eponymous selenocysteine glutathione peroxidases in mammalian cells that use glutathione as electron donor, GPXLs rely on cysteine instead of selenocysteine for activity and depend on the thioredoxin system for reduction. Although plant GPXLs have been implicated in important agronomic traits such as drought tolerance, photooxidative tolerance and immune responses, there remain major ambiguities regarding their subcellular localization. Because their site of action is a prerequisite for an understanding of their function, we investigated the localization of all eight GPXLs in stable Arabidopsis lines expressing N‐terminal and C‐terminal fusions with redox‐sensitive green fluorescent protein 2 (roGFP2) using confocal microscopy. GPXL1 and GPXL7 were found in plastids, while GPXL2 and GPXL8 are cytosolic nuclear. The N‐terminal target peptide of GPXL6 is sufficient to direct roGFP2 into mitochondria. Interestingly, GPXL3, GPXL4 and GPXL5 all appear to be membrane bound. GPXL3 was found exclusively in the secretory pathway where it is anchored by a single N‐terminal transmembrane domain. GPXL4 and GPXL5 are anchored to the plasma membrane. Presence of an N‐terminal myristoylation motif and genetic disruption of membrane association through targeted mutagenesis point to myristoylation as essential for membrane localization.     

A voltage-gated calcium channel is linked to the antigen receptor in Jurkat T lymphocytes.

Activation of T lymphocytes results in an increase in intracellular Ca2+ due in large part to influx of extracellular Ca2+. Using the patch clamp technique, an inward current in Jurkat T lymphocytes was observed upon depolarization from a holding potential of -90 mV but not from -60 mV. This whole-cell current was insensitive to tetrodotoxin, carried by Ba2+, and blocked by Ni2+. Occupancy of the T lymphocyte antigen receptor increased the current's magnitude. These data suggest that antigen receptor-induced Ca2+ entry in T lymphocytes may be mediated by a voltage-regulated Ca channel.