Induction of autolysis in starved Escherichia coli cells by seminalplasmin, an antimicrobial protein from bovine seminal plasma

Abstract A pair of relA ⁺ and relA E. coli strains, otherwise isogenic, were studied with regard to the susceptibility of starved cells to lysis induced by the natural peptide seminalplasmin. Starved relA cells were more sensitive to seminalplasmin-induced lysis when compared to starved relA ⁺ cells. Nevertheless, pronounced lysis of starved relA ⁺ cells was observed with increase in the concentration of seminalplasmin. In conctrast, ampicillin could not lyse starved relA ⁺ cells even at very high concentrations. Further, seminalplasmin could cause loss of viability and degradation of peptidoglycan in starved relA ⁺ cells. These observations suggest that, unlike many other antibiotics, seminalplasmin can induce autolysis under the conditions of a stringent response.     

The Anton blood group antigen is the erythrocyte receptor for Haemophilus influenzae

Abstract The protein synthesis pattern was investigated in Bacillus subtilis relA ⁺ and relA after heat shock using the highly sensitive 2-dimensional O'Farrell technique [1]. The synthesis of several proteins is markedly enhanced upon temperature shift-up in both strains. At 52°C the growth rate is drastically diminished because the synthesis of cellular proteins is inhibited. However, the production of heat-shock proteins is maintained. The synthesis of some of these presumptive heat-shock proteins is stimulated at 37°C in cells treated with H₂O₂ as well as with norvaline, which induces a guanosine tetraphosphate (ppGpp)-dependent stringent response.     

Properties of the Sodium Extrusion Mechanism Controlled by Nerve Growth Factor in Chick Embryo Dorsal Root Ganglionic Cells

Abstract: Cell dissociates from embryonic chick dorsal root ganglia, incubated for 6 h with ²²Na⁺, accumulated four to six times more radioactivity in the absence than in the presence of Nerve Growth Factor (NGF). The accumulation of radioactivity paralleled the external Na⁺ concentration, indicating that the cells may have been reaching equilibrium with the medium. Delayed presentation of NGF to ²²Na⁺-loaded cells caused a rapid loss of radioactivity, even with extracellular ²²Na⁺ still present, demonstrating that NGF caused an overall efflux of Na⁺ rather than an accelerated equilibration. The Na⁺ exclusion from ²²Na⁺-loaded cells was dependent upon NGF concentration. Use of nutrient-rich medium, serum, and certain hormones and other proteins did not prevent the Na+ accumulation in the absence of NGF or its reversion by delayed NGF administration. Incubation of the ganglionic cells with ouabain or dinitrophenol during the ²²Na⁺ loading period (no NGF) increased the rate, but not the magnitude, of loading. The same incubation carried out in a Na+-free medium and followed by ²²Na⁺ presentation resulted in fast radioactive loading that was identical to that occurring in drug-free, NGF-deprived cells and was not prevented by presentation of NGF together with the ²²Na⁺. These data are consistent with a model in which NGF acts through a Na⁺ pump rather than by restricting Na+ influxes.     

Stringent response in Vibrio cholerae: genetic analysis of spoT gene function and identification of a novel (p)ppGpp synthetase gene

RelA and SpoT of Gram-negative organisms critically regulate cellular levels of (p)ppGpp. Here, we have dissected the spoT gene function of the cholera pathogen Vibrio cholerae by extensive genetic analysis. Unlike Escherichia coli , V. cholerae Δ relA Δ spoT cells accumulated (p)ppGpp upon fatty acid or glucose starvation. The result strongly suggests RelA-SpoT-independent (p)ppGpp synthesis in V. cholerae . By repeated subculturing of a V. cholerae Δ relA Δ spoT mutant, a suppressor strain with (p)ppGpp⁰ phenotype was isolated. Bioinformatics analysis of V. cholerae whole genome sequence allowed identification of a hypothetical gene ( VC1224 ), which codes for a small protein (∼29 kDa) with a (p)ppGpp synthetase domain and the gene is highly conserved in vibrios; hence it has been named relV . Using E. coli Δ relA or Δ relA Δ spoT mutant we showed that relV indeed codes for a novel (p)ppGpp synthetase. Further analysis indicated that relV gene of the suppressor strain carries a point mutation at nucleotide position 676 of its coding region (Δ relA Δ spoT relV676 ), which seems to be responsible for the (p)ppGpp⁰ phenotype. Analysis of a V. cholerae Δ relA Δ spoT Δ relV triple mutant confirmed that apart from canonical relA and spoT genes, relV is a novel gene in V. cholerae responsible for (p)ppGpp synthesis.     

The influences of calcium and magnesium ions on the exchange of monovalent cations in Neurospora crassa

Low-K⁺, high-Na⁺ cells of strain RL21a of Neurospora crassa , in steady state with 25 m M Na⁺, were used to study K⁺/Na⁺ exchanges in the presence or absence of Ca²⁺ and Mg²⁺. In the presence of Ca²⁺ and Mg²⁺, a low concentration of K⁺ (0.3 m M ) triggered a rapid exchange, but in the absence of the divalents, a high K⁺ concentration (30 m M ) was required to initiate the exchange at a rapid rate. In the absence of Ca²⁺ and Mg²⁺, K⁺ uptake did not occur at low K⁺ concentration, internal K⁺ did not regulate Na⁺ influx in the presence of external K⁺, and the efflux of Na⁺ proceeded at maximum activity at very low-K⁺ contents.     

Rates of sulfate reduction and thiosulfate consumption in a marine microbial mat

Abstract The sulfur cycle in a microbial mat was studied by determining viable counts of sulfate-reducing bacteria, chemolithoautotrophic sulfur bacteria and anoxygenic phototrophic bacteria. All three functional groups of sulfur bacteria revealed a maximum population density in the uppermost 5 mm of the mat: 1.1 × 10⁸ cells of sulfate reducers cm⁻³ sediment, 2.0 × 10⁹ cells of chemolithoautotrophs cm⁻³ sediment, and 4.0 × 10⁷ cells of anoxygenic phototrophs cm⁻³ sediment. Bacterial dynamics were studied by sulfate reduction rate measurements, both under anoxic conditions (dark incubation) and oxic conditions (incubation in the light), and determination of the vertical distribution of the potential rate of thiosulfate consumption under oxic conditions. Sulfate reduction rates in the top 5 mm of the sediment were 566 nmol cm⁻³ d⁻¹ in the absence of oxygen, and 123 nmol cm⁻³ d⁻¹ in the presence of oxygen. In the latter case, the maximum rate was found in the 5–10-mm depth horizon (361 nmol cm⁻³ d⁻¹). Biological consumption of amended thiosulfate was rapid and decreased with depth, while in the presence of molybdate, thiosulfate consumption decreased to 10–30% of the original rate.     

Investigation of the Role of Conserved Serine Residues in the Long Form of the Rat D2 Dopamine Receptor Using Site-Directed Mutagenesis

Abstract: Three serine residues (Ser¹⁹³, Ser¹⁹⁴, Ser¹⁹⁷) in the fifth transmembrane-spanning region of the D₂ dopamine receptor have been mutated separately to alanine and the effects of the mutations determined in ligand-binding experiments with [³H]spiperone. For many antagonists the mutations had little effect, showing that the overall conformation of the mutant receptors was similar to that of the native, although there were effects on the binding of certain antagonists. The effect of the mutations on agonist binding to the free receptor (uncoupled from G proteins) was determined in the presence of GTP (100 µ M ). This showed that there was no single mode of binding of catecholamine agonists to the receptor and that all three serine residues can participate in the binding of some agonists, possibly through hydrogen bonds to the catechol hydroxyl groups. Coupling of the mutant receptors to G proteins was assessed from agonist-binding curves in the absence of GTP, when higher and lower affinity agonist-binding sites were seen. Receptor/G protein coupling was generally unaffected by the Ala¹⁹³ and Ala¹⁹⁴ mutations, but the Ala¹⁹⁷ mutation eliminated receptor/G protein coupling for some agonists. These data show that the interactions of agonists with the free and coupled forms of the receptor are different.     

Proteolysis and nitrogen fixation in faba-bean (Vicia faba) nodules under water stress

Nodulated faba-beans ( Vicia faba L. var. minor) exhibiting high rates of N₂ fixation (133 μmol C₂H₄ g⁻¹ dry weight h⁻¹), were subjected to water restriction. A loss of C₂H₂ reduction due to water stress was always associated with a decline of the leghemoglobin content for each of the 4 decreasing values of Ψ_mod. Electron micrographs showed ultrastructural alterations of the fixing tissue, which affected both partners and increased with the severity of water stress. In the nodule cytosol, the alkaline proteolysis approximately doubled when Ψ_mod decreased from −0.55 MPa to −1.55 MPa. Concomitantly, an increase of the nodule intracellular pH from 6.3 to 7.0 was observed. Proteolysis was due to serine proteases, exhibiting a pH-optimum of 8 and which actively degraded purified leghemoglobin in vitro (K_m=100 μ M ). The degradation of leghemoglobin during water stress may contribute to the loss of C₂H₂ reduction and may affect the pattern of recovery upon rewatering.     

Inhibitory effect of osmotic concentration, potassium and pH on motility of the sperm of the North American burbot Lota lota maculosa

Seminal plasma factors maintaining North American (NA) burbot Lota lota maculosa sperm quiescent were examined. Sperm were diluted into buffered saline solutions of various compositions and motility assessed. After 1 h in these solutions at 10° C, aliquots of the suspension were diluted with tap water and motility again assessed. Dilution of sperm in an incubation solution containing Ca²⁺ in the absence of K⁺ initiated sperm motility resulting in low motility when sperm were subsequently diluted in tap water. Incubation solutions with osmolalities >200 mOsm kg⁻¹ and containing 12·5 mM K⁺ prevented the onset of sperm motility and were associated with maximal sperm motility upon dilution in tap water. Sperm maintained at lower osmolalities exhibited limited motility upon dilution in tap water indicating interdependence between K⁺ and osmolality in maintaining sperm quiescent in the presence of Ca²⁺. Sperm kept in incubation solution at pH values < c. 7·5 for 1 h demonstrated reduced motility when subsequently diluted in tap water. That motility of sperm was pH sensitive was further indicated by CO₂ inhibition of motility. Therefore, NA burbot sperm are probably maintained in an immotile state, yet with potential for motility, by combination of high K⁺, osmolality and possibly pH. The results from this study differ from published information on sperm quiescence in the temporally and geographically distinct Eurasian burbot Lota lota lota .     

Cation-stimulated H+ efflux by intact roots of barley

Abstract. Rates of proton extrusion and potassium (⁸⁶Rb) influx by intact roots of barley ( Hordeum vulgare cvs . Fergus, Conquest and Betzes) plants were simultaneously measured in short-term (15min) experiments. The nature and extent of apparent coupling between these ion fluxes was explored by manipulating conditions of temperature, pH and cation composition and concentration during flux determinations. In addition, the influence of salt status upon these fluxes was examined. At low K⁺ concentrations (0.01 to 1 mol m⁻³), H⁺ efflux and K⁺ influx were strongly correlated in both low- and high-K⁺ roots, although K⁺: H⁺ exchange stoichiometries were almost consistently greater than 2:1. At higher concentrations (1 to 5 mol m⁻³), H⁺ efflux was either reduced or remained unchanged while K⁺ influxes increased. In the presence of Na₂SO₄, rates of H⁺ extrusion demonstrated similar cation dependence, although below 10 mol m⁻³ Na₂SO₄, H⁺ fluxes were generally 50% lower than in equivalent concentrations of K₂SO₄. These observations are considered in the context of current hypotheses regarding the mechanisms of k⁺/H⁺ exchange.     

Compartments of Labeled and Endogenous γ-Aminobutyric Acid Giving Rise to Release Evoked by Potassium or Veratridine in Rat Cortical Slices

Abstract— To establish compartments involved in depolarization-induced release of γ-aminobutyric acid (GABA) in rat brain slices, the amount of exogenous labeled and endogenous GABA released and retained was followed during 48 min exposure to 50 m m -K⁺ or to 50 μ m -veratridine. Endogenous GABA was measured with high performance liquid chromatography. The presence of 10 μ m -aminooxyacetic acid throughout prevented both the metabolism of GABA and the formation of endogenous GABA due to depolarization. During super-fusion with 50 m m -K⁺ and 2.6 m m -Ca²⁺ the efflux of labeled and endogenous GABA after an initial large increase declined to 10% of the highest value with constant and identical rates. Kinetic analysis of efflux showed that 10% of endogenous and 25% of labeled GABA present is available for release by high K⁺ and Ca²⁺. In the absence of Ca²⁺, release by high K⁺ of both labeled and endogenous GABA was nearly suppressed. Veratridine, unlike high K⁺, caused an efflux which declined with an initial fast and late very slow phase. The slow efflux by veratridine was doubled in the absence of Ca²⁺. Exposure to veratridine in the absence of Ca²⁺ during 120 min released nearly 70% of labeled and endogenous GABA present. Results suggest that only about 0.25 μmol g⁻¹ endogenous GABA is the source of physiological Ca²⁺-dependent release, while much of the remaining GABA present is released only under unphysiological conditions.     

MAGNESIUM REQUIREMENT IN FERTILIZATION PROCESS OF SEA URCHINS

The Mg²⁺ requirement in fertilization was investigated in sea urchins. It was found that when sea urchin eggs were inseminated in sea water free of Mg²⁺, little fertilization took place. Even when spermatozoa pre-treated with dissolved egg-jelly to induce the acrosome reaction, which needs Ca²⁺, were used, the fertilization rate remained quite low in the absence of Mg²⁺. In Strongylo-centrotus intermedius , the lowest concentration of Mg²⁺ required for 50% fertilization was 0.05 mM in the presence of 10 mM Ca²⁺, whereas that of calcium was 3 mM in the presence of 49 mM Mg²⁺. These critical concentrations increased when the concentration of the other ion decreased. Removal of Mg²⁺ or Ca²⁺ or both from the suspending medium had little adverse effect on sperm motility. The elevation of the fertilization membrane was also induced by butyric acid independent of the presence or absence of Mg²⁺ and/or Ca²⁺. These results indicate that Mg²⁺ are required at least in some process(es) between acrosome reaction and fertilization membrane elevation, such as sperm penetration or membrane fusion.     

On the participation of chloride in bicarbonate-induced reversal of anion inhibition of photosystem II electron transport in spinach thylakoids

Inhibition of electron transport through photosystem II (PS II) by formate (HCO⁻₂) or nitrite (NO⁻₂) in the presence or absence of chloride ions was studied. The inhibition induced by HCO⁻₂ or NO⁻₂ is overcome by HCO⁻₃ more in the presence, than in the absence of Cl⁻. The data on electron transport are supported by chlorophyll a fluorescence measurements. In experiments. In experiments in which water oxidation was blocked. Cl⁻ was found to facilitate electron transport between bound quinone A (Q⁻_A) and the plastoquinone (PQ) pool. It can thus be concluded that in addition to the well known site of action of Cl⁻ on water oxidation, another site of Cl⁻ action is between Q⁻_A and the PQ pool.     

Role of benthic insect feces in a Rocky Mountain stream: fecal production and support of consumer growth

The feces of stream insects may be a major component of fine particulate organic matter (FPOM) available to collector organisms. In Mink Creek, Idaho, winter defecation rates for 9 species ranged from 86 mg dry feces (g dry body wt)⁻¹ d⁻¹ (Ephemerella spinifera) to 154 mg g⁻¹ d⁻¹ (Paraleptophlebia heteronea) . Detailed studies of 3 species in summer revealed that rates were much greater than in winter and exhibited high between-individual variation (e.g., 695 ± 184.8 mg (g body wt)⁻¹ d⁻¹ for Baetis tricaudatus ). By combining measured and literature values, it is estimated that the benthic insect community egests approximately 3.4 kg feces m⁻² yr⁻¹. This value lies within the range of FPOM estimated to be derived from degradation of leaf litter input (0.7–9.9 kg m⁻² yr⁻¹, depending upon assimilation efficiency of the organisms).
Short-term winter growth experiments showed that, with two exceptions, collectors fed on fecal detritus will grow as well as on other food resources; relative growth rates depend upon the species. Because fecal detritus is abundant throughout the year, and is treated by many species as a source of food, it could be a major mechanism by which seasonal fluctuation in availability of allochthonous litter input is minimized and homeostasis of structure and function maintained.     

PHOTORESPIRATION IN CONTINUOUS CULTURE OF DUNALIELLA TERTIOLECTA (CHLOROPHYTA): RELATIONSHIPS BETWEEN SERINE, GLYCINE, AND EXTRACELLULAR GLYCOLATE

The concentrations of extracellular glycolate and intracellular free pools of serine and glycine were monitored in nitrogen-limited continuous cultures of Dunaliella tertiolecta (Butcher) UTEX LB999, grown at two different irradiances on a light:dark cycle. Under steady-state conditions, this microalga excreted into the medium a large amount of glycolate during the light phase, up to 100 nmol·(10⁶ cells)⁻¹ for a cell concentration of around 1.5 10⁸ cells·L⁻¹, but glycolate disappeared from the dissolved phase in the dark. Cells grown at 70 and those grown at 430 μmol photons·m⁻²·s⁻¹ differed in maximal glycolate concentration, intracellular serine and glycine concentrations, and serine:glycine ratio. Reversal of these photon flux densities to which the cultures were exposed caused rapid modification of the extracellular glycolate and intracellular serine and glycine pools. These results suggest that photorespiratory metabolism in D. tertiolecta could be approximately quantified by measuring the changes in dissolved glycolate and intracellular serine and glycine concentrations, extending previous results from cultured phytoplankton and suggesting methods for field studies.     

Nitroprusside and Cyclic GMP Stimulate Na+-Ca2+ Exchange Activity in Neuronal Preparations and Cultured Rat Astrocytes

Abstract: The effects of nitric oxide (NO)-generating agents on ⁴⁵Ca²⁺ uptake in rat brain slices and cultured rat astrocytes were studied in the presence of monensin, which is considered to drive the Na⁺-Ca²⁺ exchanger in the reverse mode. Sodium nitroprusside (SNP) at >10 µ M increased monensin-stimulated Ca²⁺ uptake in the slices, although it did not affect high K⁺-stimulated Ca²⁺ uptake. Another NO donor, 3-morpholinosydnonimine, was effective. The effect of SNP was antagonized by hemoglobin (50 µ M ), a NO scavenger, and mimicked by 8-bromo-cyclic GMP (100 µ M ). In rat brain synaptosomes, SNP increased monensin-stimulated Ca²⁺ uptake, but it did not affect high K⁺-stimulated Ca²⁺ uptake. 8-Bromocyclic GMP, but not SNP, increased Na⁺-dependent Ca²⁺ uptake significantly in synaptic membrane vesicles in the absence of monensin. In cultured rat astrocytes, SNP and 8-bromo-cyclic GMP increased Ca²⁺ uptake in the presence of ouabain and monensin, which were required for the Ca²⁺ uptake in the cells. These findings suggest that NO stimulates the Na⁺-Ca²⁺ exchanger in neuronal preparations and astrocytes in a cyclic GMP-dependent mechanism.     

Nitrate reductase activity in nodules of pea inoculated with hydrogenase positive and hydrogenase negative strains of Rhizobium leguminosarum

The nitrate reductase (NR, EC 1.6.6.1) activity in root nodules formed by hydrogenase positive (Hup⁺) and hydrogenase negative (Hup⁻) Rhizobium leguminosarum strains was examined in symbioses with the pea cultivar Alaska ( Pisum sativum L.), Rates of activity were determined by the in vivo assay in nodules from plants that were only N₂-dependent or grown in the presence of 2 m M KNO₃. The rates varied widely among strains, regardless of the Hup phenotype of the R. leguminosarum strain used for inoculation, but the overall results indicated that nodules formed by Hup⁻ strains accumulated more nitrite in the incubation medium than did those with Hup⁻ phenotypes. Total plant dry weight and reduced nitrogen content of pea plants grown in the presence of 2 m M KNO₃ and inoculated with single Hup⁺ and Hup⁻ R. leguminosarum strains were statistically different among some strains. These observations suggest that the possible advantages derived from the presence of the Hup system on whole plant growth may be counteracted by the higher rates of NR activity in the Hup⁻ strains in the R. leguminosarum -pea symbiosis.     

Photosynthetic characteristics of the submerged macrophyte Ceratophyllum demersum

The photosynthetic and growth characteristics of Ceratophyllum demersum L. were investigated under laboratory conditions which simulated those encountered in the plants' normal environment. The carbon fixation rate of C. demersum measured with ¹⁴C at light and carbon saturation at pH 8.0 was 4.48 mg C (g ash-free dry weight)⁻¹ h⁻¹. It was lower at pH 6.5 than at pH 8.0. The light use efficiencies in quiescent plants and actively growing plants were 6.3 and 8.7 × 10⁻⁹ kg CO₂ J⁻¹, respectively, with corresponding maximum photosynthetic rates of 2.67 and 4.36 mg C (g ash-free dry weight)⁻¹ h⁻¹. Photorespiration in actively growing plants consumed 24% of the carbon fixed. Incubation with DCMU demonstrated that about one-third was refixed. The optimum temperature for carbon fixation was 25°C. The C₃-photosynthetic pathway was the main operational route as indicated by the early photosynthetic products (largely C₃-acids) and the absence of Krantz anatomy and the chlorophyll a:b ratio (2.7). The maximum relative growth rates ranged from 0.025 to 0.041 g ash-free dry weight (g ash-free dry weight)⁻¹ day⁻¹ in the field (Lake Vechten, 1 to 3 m depth classes).