Interactions of amiloride and small monovalent cations with the epithelial sodium channel. Inferences about the nature of the channel pore.

The voltage dependence of amiloride-induced inhibition of current flow through apical membrane sodium channels in toad urinary bladder was studied at different ionic conditions. The "inert" salt N-methyl-D-glucamine HCl (NMDG HCl) affected neither the apparent inhibition constant (Kl) for the amiloride-induced current inhibition nor the apparent fraction of the transmembrane voltage that falls between the mucosal solution and the amiloride-binding site (delta). When NMDG+ was replaced with Na+, Kl increased, reflecting amiloride-Na+ competition, whereas delta was unchanged. Similar results were obtained with another permeant cation, Li+. When NMDG+ was replaced by K+, an impermeant but channel-blocking cation, Kl increased whereas delta decreased. Similar results were obtained using another impermeant, channel-blocking cation guanidinium. The results are interpreted on the premise that Na+ and K+ compete with amiloride by binding to cation binding sites within the channel lumen such that ion occupancy of these sites vary with voltage. Occupancy by K+, which cannot traverse the channel, will increase as the mucosal solution becomes positive, relative to the serosal solution. Occupancy by Na+, which can traverse the channel, is comparatively voltage independent. Ion movement through the channels was simulated using discrete-state kinetic models. Two types of models could describe the shape of the current-voltage relationship and the voltage dependence of the amiloride-induced channel block. One model had a single ion-binding site with a broad energy barrier at the inner (cytoplasmic) side of the site. The other model had two binding sites separated from each other and from the aqueous solutions by sharp energy barriers.     

Field Study Comparing Growth and Viability of a Population of Phototrophic Bacteria

The growth and viability of an anoxygenic, phototrophic bacterial community in the hypolimnion of Zaca Lake, Calif., were compared throughout the summer. The community is dominated by a single species, “Thiopedia rosea,” that inhabits the entire hypolimnion (6 to 8 m) for approximately 11 months. Suboptimal conditions in the hypolimnion (extremely low light intensity, high or low H₂S levels) result in zero or extremely low growth rates (doubling times > 1 month) for most of the population, most of the time, yet cells remain viable and capable of high specific growth rates (doubling times of 1 to 10 days) when placed under favorable conditions (higher light intensities and temperatures). We first conclude that phototrophic bacterial populations in situ may frequently exist in a viable yet nongrowing state. Second, the viability of cells is likely to be reduced with depth owing to higher concentrations of potentially toxic chemicals and to changes in the physiological state associated with the prolonged periods of darkness commonly found at the bottom of bacterial plates.     

Nitric oxide from L-arginine stimulates the soluble guanylate cyclase in adrenal glands

The formation of nitric oxide (NO) by an L-arginine:NO synthase and its stimulation of the soluble guanylate cyclase was studied in rat whole adrenal and bovine cortex and medulla cytosol. In the presence of L-arginine, the stimulation of soluble guanylate cyclase was accompanied by the formation of citrulline and NO2-, formed from NO. The NO synthase was NADPH- and Ca(2+)-dependent and was inhibited by several L-arginine analogues. These results indicate that rat and bovine adrenal cytosol contains an L-arginine:NO synthase.     

Mass measurement of inositol 1,4,5-trisphosphate and sn-1,2-diacylglycerol in bombesin-stimulated Swiss 3T3 mouse fibroblasts.

Two specific and selective assays were used to measure changes in the mass of Ins(1,4,5)P3 and sn-1,2-diacylglycerol in bombesin-stimulated Swiss 3T3 cells. The results demonstrate that the increase in Ins(1,4,5)P3 was extremely rapid, but transient, returning to basal levels by 30 s. In contrast, the increase in sn-1,2-diacylglycerol was biphasic: the first phase mirrored the transient Ins(1,4,5)P3 response, whereas the second phase was sustained and occurred in the absence of elevated Ins(1,4,5)P3. The possible source of the second phase of diacylglycerol is discussed.     

The chemical modification of cysteine-69 of rat liver fatty acid-binding protein (FABP): a fluorescence approach to FABP structure and function

Summary Hepatic-FABP was labelled at cysteine-69 with the fluorescent environmentally sensitive reporter group AEDANS. The labelled protein had an emission maximum at 465 nm indicating that cysteine-69 was buried in a non-polar environment. The modified protein was still able to bind ligands such as oleic acid, oleoyl CoA and haem. The affinity of AEDANS-FABP for haem was unaltered as compared with the native protein indicating that cysteine-69 must be remote from the ligand binding site. The binding of oleic acid did not significantly perturb the fluorescence emission spectrum of the fluorescent reporter group suggesting that there are not large conformational changes in the region of cysteine-69 on fatty acid binding. The binding of stoichiometric amounts of oleoyl CoA was accompanied by a small fluorescence enhancement which suggests that fatty acyl CoAs may interact with other regions of the FABP molecule not involved in fatty acid binding.Abbreviations FABP Fatty Acid-Binding Protein - AEDANS 5-[2-(Acetyl)aminoEthyl]Aminonaphthalene-1-Sulphonic Acid - AAEDANS 5-[2-(Iodoacetyl-AminolEthyl] aminonaphthalene-1-Sulphonic Acid - DTNB 5,5Dithiobis-(2-Nitrobenzoic acid)     

Patterns of [13N]ammonium uptake and assimilation by Frankia HFPArl3

Nitrogen-starved cells of Frankia strain HFPArl3 incorporated [¹³N]-labeled ammonium into glutamine serine (glutamate, alanine, aspartate), after five-minute radioisotope exposures. High initial endogenous pools of glutamate were reduced, while total glutamine increased, during short term NH _inf4 ^sup+ incubation. Preincubation of cells in methionine sulfoximine (MSX) resulted in [¹³N]glutamine reduced by more than 80%, while [¹³N]glutamate and [¹³N]alanine levels increased. The results suggest that glutamine synthetase is the primary enzyme of ammonium assimilation, and that glutamate dehydrogenase and alanine dehydrogenase may also function in ammonium assimilation at low levels. Efflux of [¹³N]serine and lesser amounts of [¹³N]glutamine was detected from the Frankia cells. The identity of both Ser and Gln in the extracellular compartment was confirmed with gas chromatography/mass spectrometry. Serine efflux may be of significance in nitrogen transfer in Frankia.Abbreviations Pthr phosphothreonine - Aad -amino-adipate - MSX methionine sulfoximine     

Cloning and expression of the toxin B gene ofClostridium difficile

Results from our cloning studies on toxin A indicated that the gene for toxin B resided approximately 1 kb upstream of the toxin A gene. Clone pCD19, which contains the 5-end of the toxin A gene and a small open reading frame, was found to contain 1.2 kb of DNA which, when subcloned, expressed a nontoxic peptide that reacted with toxin B antibodies. The rest of the toxin B gene was located on the 6.8 kb cloned fragment of plasmid pCD19L. The two fragments overlapped 0.8 kb. Lysates containing protein expressed by the 6.8 fragment were cytotoxic and lethal, and were neutralized by toxin B antibody. The two fragments were ligated to give the complete toxin B gene. The protein expressed by the complete gene was cytotoxic and lethal, and showed complete immunological identity with toxin B. Further analysis of the expressed protein and the toxin B gene confirmed our earlier findings showing that toxin B has a molecular weight of 240,000 or greater.     

Seed Germination Biology of the Narrowly Endemic Species Lesquerella stonensis (Brassicaceae)

Abstract Lesquerella stonensis (Brassicaceae) is an obligate winter annual endemic to a small portion of Rutherford County in the Central Basin of Tennessee, where it grows in disturbed habitats. This species forms a persistent seed bank, and seeds remain viable in the soil for at least 6 years. Seeds are dormant at maturity in May and are dispersed as soon as they ripen. Some of the seeds produced in the current year, as well as some of those in the persistent seed bank, afterripen during late spring and summer; others do not afterripen and thus remain dormant. Seeds require actual or simulated spring/summer temperatures to come out of dormancy. Germination occurs in September and October. Fully afterripened seeds germinate over a wide range of thermoperiods (15/6–35/20°C) and to a much higher percentage in light (14 h photoperiod) than in darkness. The optimum daily thermoperiod for germination was 30/15°C. Nondormant seeds that do not germinate in autumn are induced back into dormancy (secondary dormancy) by low temperatures (e.g., 5°C) during winter, and those that are dormant do not afterripen; thus seeds cannot germinate in spring. These seed dormancy/ germination characteristics of L. stonensis do not differ from those reported for some geographically widespread, weedy species of winter annuals and thus do not help account for the narrow endemism of this species.     

In vitro stimulation and inhibition of steroid hormone release from postovulatory follicles of the tilapia,Oreochromis mossambicus

Summary Postovulatory follicles of the tilapia, Oreochromis mossambicus, were incubated with graded doses of salmon gonadotropin to identify the steroid hormones released by this tissue. In addition, the effects of either cytochalasin B or colchicine on steroid hormone release were studied. After the incubation, the tissue was examined by electron microscopy. Postovulatory follicles released testosterone and estradiol-17B in a dose-dependent manner with gonadotropin. There was no detectable release of progesterone or 17a-OH-progesterone. When stimulated with high doses of gonadotropin, the steroidogenic cells showed an increase in smooth endoplasmic reticulum, Golgi complexes, and lipid droplets. Also, microfilaments became arranged in orderly bundles and were found close to the numerous secretory vesicles and lipid droplets. Upon incubation with gonadotropin and either colchicine or cytochalasin B, the cells still appeared steroidogenic, but the filaments were not organized nor associated with vesicles or lipid droplets. Release of steroid hormone decreased significantly. Also in these tissues, vesicles were no longer numerous in the apical region of the granulosa cells, but were located primarily near smooth endoplasmic reticulum and Golgi complexes. This suggests that disruption of the cytoskeleton results in reduced steroid hormone synthesis or release.