Arginine utilization by Mycoplasma fermentans is not regulated by glucose metabolism: A 13C-NMR study

Abstract Electrofusion of protoplasts of two mutant strains of Hansenula polymorpha resulted in high fusion and hybrid yields when the calcium ions present in the conventional fusion medium replaced by zinc ions. The optimal fusion conditions were an alignment field of 0.4 kV cm⁻¹ strength and 2 MHz frequency for 30 s, followed by two consecutive pulses of 12 kV cm⁻¹ strength and 15 μs duration. With 0.05–0.1 mM zinc ions in the fusion medium an average clone number of 10⁴–10⁵ clones per 10⁸ input cells was reached. The presence of about 0.6 mM magnesium ions in the zinc fusion medium was essential.     

Transformation of Streptococcus bovis protoplasts by plasmid DNA

M. MAREKOVÁ, V. KMET' AND P. JAVORSKÝ. 1996. The transformation and subsequent regeneration of ruminal strain Streptococcus bovis AO24/85 protoplasts by plasmid DNA was studied. The best stabilizer for regeneration of protoplasted cells was 5% sucrose in the regeneration medium and in the agar plates. Optimal concentration of polyethylene glycol 6000 in the transformation medium was 25% for both plasmids tested. Addition of Ca²⁺ and Mg²⁺ ions (2.5 mmol l^-1) to the transformation medium increased the proportion of regenerated cells. Transformation frequencies were 3 times 10³ transformants per μg of pNZ12 and 2.4 times 10² per μg of pJK108, respectively.     

Electrotransfection of Propionibacterium freudenreichii TL 110

The first highly efficient protocol is described for the electrotransfection of Propionibacterium freudenreichii with DNA phage. The transfection efficiency is 7 times 10⁵ transfectants per μg of DNA under optimal conditions. Optimized parameters included the field strength (12.5 kV, 200 Ohms, 25 μF), phage DNA concentration (1 μg ml^-1) and cell density (1.5 times 10¹⁰ cells ml^-1). Growth in the presence of glycine and harvesting of cells during the early exponential growth phase increased the transfection efficiency. This electrotransfection protocol is of importance for the genetic improvement of dairy propionibacteria.     

45Ca2+ Uptake into Rat Whole Brain Synaptosomes Unaltered by Dihydropyridine Calcium Antagonists

Abstract: Voltage-dependent ⁴⁵Ca²⁺ uptake into rat whole brain synaptosomes was measured after 3-s KCl-induced depolarization to investigate possible inhibitory effects of calcium antagonists, nitrendipine, nimodipine, and nisoldipine. At a Ca²⁺ concentration of 1.2 m M , nitrendipine, in concentrations ranging from 0.1 n M to 10 μ M , had no effect on ⁴⁵Ca²⁺ uptake. When the Ca²⁺ concentration was lowered to 0.06 and 0.12 m M , nitrendipine, 10 μ M , inhibited ⁴⁵Ca²⁺ uptake in response to 109 m M KCl depolarization. However, in a separate concentration response study, nitrendipine, nimodipine, and nisoldipine, 0.1 n M to 10 μ M , failed to alter the uptake of ⁴⁵Ca²⁺ (0.06 m M Ca²⁺) into 30 m M KCl-depolarized synaptosomes. The high concentrations of these agents required to depress ⁴⁵Ca²⁺ uptake indicate that the dihydropyridine calcium antagonists are considerably less potent in brain tissue than in peripheral tissue.     

An efficient transformation system for Bifidobacterium spp.

This study describes a broad host transformation protocol that enables the uptake of plasmid DNA into 10 different species of Bifidobacterium , some of which have never been transformed before. The vector pNC7 (4·9 kb) was used to optimize the electroporation protocol. Transformation efficiencies ranged from 3·6×10⁻¹ to 1·2×10⁵ transformations per μg DNA. The impact of growth medium composition and electric field strength on transformation efficiency were independently optimized. Electrocompetent cells were grown in Iwata medium broth enriched with Actilight^RP 16%, harvested during the early exponential growth phase, and pulsed at 12·5 kV cm⁻¹, 100 Ω and 25 μF.     

Calcium-dependent transferrin receptor recycling in bovine chromaffin cells

The release of regulated secretory granules is known to be calcium dependent. To examine the Ca²⁺-dependence of other exocytic fusion events, transferrin recycling in bovine chromaffin cells was examined. Internalised ¹²⁵I-transferrin was released constitutively from cells with a half-time of about 7 min. Secretagogues that triggered catecholamine secretion doubled the rate of ¹²⁵I-transferrin release, the time courses of the two triggered secretory responses being similar. The triggered ¹²⁵I-transferrin release came from recycling endosomes rather than from sorting endosomes or a triggered secretory vesicle pool. Triggered ¹²⁵I-transferrin release, like catecholamine secretion from the same cells, was calcium dependent but the affinities for calcium were very different. The extracellular calcium concentrations that gave rise to half-maximal evoked secretion were 0.1 m m for ¹²⁵I-transferrin and 1.0 m m for catecholamine, and the intracellular concentrations were 0.1 μ m and 1 μ m , respectively. There was significant ¹²⁵I-transferrin recycling in the virtual absence of intracellular Ca²⁺, but the rate increased when Ca²⁺ was raised above 1 n m , and peaked at 1 μ m when the rate had doubled. Botulinum toxin type D blocked both transferrin recycling and catecholamine secretion. These results indicate that a major component of the vesicular transport required for the constitutive recycling of transferrin in quiescent cells is calcium dependent and thus under physiological control, and also that some of the molecular machinery involved in transferrin recycling/fusion processes is shared with that for triggered neurosecretion.     

The Use of Sulphamezathine for Inhibiting Proteus Spp. on Baird-Parker's Isolation Medium for Staphylococcus aureus

S ummary : The addition of 50 μg of sulphamezathine/ml to egg-tellurite-glycine-pyruvate agar was effective in suppressing the growth and swarming of Proteus spp. Small numbers of Staphylococcus aureus (10³/g) could be recovered quantitatively on the modified medium in the presence of up to 10⁶/g of mixed Proteus vulgaris and Proteus mirabilis strains.     

The Effects of Electroshock Convulsions on Calcium Transport within Synaptic Terminals

Abstract: Calcium transport was assessed within synaptic terminals isolated from cerebral cortices of rats which experienced one maximal electroshock (ES) convulsion daily. No significant change in calcium content [(Ca₁)] of synaptosomes was present after 2 consecutive days of maximal convulsions. After 4 and 6 days of maximal seizures, (Ca₁) rose 20% and 37%, respectively. ¹⁵Ca²⁺ influx within synaptosomes in vitro increased after 6 days of ES convulsions (1.94 ± 0.4 μmol/g protein/min in ES convulsions versus 1.54 ± .03 μmol/g protein/min in controls). The higher rate of ⁴⁵Ca²⁺ influx in convulsed animals was accounted for by elevated internal sodium [(Na₁)] values. Maximal ⁴⁵Ca²⁺ efflux decreased after ES convulsions (0.48 μmol/g protei/min in ES convulsions versus 0.8 μmol/g protei/min in controls). The slower rate of ⁴⁵Ca²⁺ efflux after convulsions was also accounted for by elevated (Na₁). Our results suggest that (Ca₁) increased within synapses after in vivo ES convulsions secondary to a primary ionic event, namely, elevated (Na₁).     

The inhibition of photosynthesis and photorespiration in isolated mesophyll cells of Phaseolus and Lycopersicon by reduced osmotic potentials

Mesophyll cells isolated from Phaseolus vulgaris and Lycopersicon esculentum show decreasing photosynthetic rates when suspended in media containing increasing concentrations of osmoticum. The photosynthetic activity was sensitive to small changes in osmotic potential over a range of sorbitol concentrations from 0.44 M (−1.08 MPa) to 0.77 M (−1.88 MPa). Photorespiration assayed by ¹⁴CO₂ release in CO₂-free air and by ¹⁴CO₂ release from the oxidation of [1–¹⁴C] glycolate also decreased as the osmotic potential of the incubation medium was reduced. The CO₂ compensation points of the cells increased with increasing concentration of osmoticum from approximately 60 μ I⁻¹1 at −1.08 MPa to 130 μl 1⁻¹ for cells stressed at −1.88 MPa. Changes in photosynthetic and photorespiratory activities occurred at moderate osmotic potentials in these cells suggesting that in whole leaves during a reduction in water potential, non- stomatal inhibition of CO₂ assimilation and glycolate pathway metabolism occurs simultaneously with stomatal closure.     

Pressure-stress effects on the ultrastructure of cells of the dimorphic yeast Candida tropicalis

Abstract Starved cells of cadmium-sensitive Staphylococcus aureus 17810S accumulated ¹⁰⁹Cd via the Mn²⁺ porter energized by the membrane potential (ΔΨ) generated by l-lactate oxidation. However, Cd²⁺ accumulation did not result in inhibition of respiration and consequent generation of electrochemical proton gradient (Δμ_H+) via the respiratory chain. Thus, Δμ_H+-consuming processes, such as ATP synthesis and [¹⁴C]glutamate transport proceeded normally, despite the presence of Cd²⁺ in the cytoplasm. The mechanism of the intrinsic cadmium-insensitivity of the l-lactate oxidizing system is discussed.     

Improved PCR detection sensitivity of Erwinia carotovora subsp. atroseptica in potato tuber peel extract by prior enrichment on a selective medium

A simple and sensitive method was developed to replace the need for complex and laborious DNA extraction to remove inhibitory substances in potato tuber peel extract before detection of Erwinia carotovora subsp. atroseptica (Eca) by PCR. Eca was enriched by a factor of 10⁵ when peel extract was inoculated onto a selective medium, CVP, and incubated at 27°C for 24 h. Bacterial micro-colonies which developed were suspended in 500 μl of water and the bacteria diluted in water 100-fold, or 10-fold followed by washing by centrifugation, before PCR testing. The sensitivity of detection obtained with the former was ca 10¹–10² cells ml⁻¹ and with the latter ca 10¹ cells ml⁻¹, when different numbers of streptomycin-resistant Eca strain were added to peel extract from three Eca-free potato cultivars. The method was validated and the sensitivity confirmed relative to two different commonly used Eca detection methods using naturally contaminated tubers.     

The regulation of Mn2+ and Cu2+ uptake in cells of the yeast Candida utilis grown in continuous culture

Abstract Transport of Mn²⁺ was repressed in Candida utilis cells grown in continuous culture in high-Mn²⁺ (100 μM Mn²⁺) medium as compared to cells grown in basic (0.45 μM Mn²⁺) and low-Mn²⁺ (< 0.05 μM Mn²⁺) media. In contrast, no repression of Cu²⁺ uptake occurred in high-Cu²⁺-grown (25 μM Cu²⁺) cells as compared to cells grown in basic medium (0.54 μM Cu²⁺). Cu²⁺-limited cells did not hyperaccumulate Cu²⁺ and there was not significant difference in initial uptake rates for all 3 Cu²⁺ conditions. Mn²⁺ uptake appears to be regulated by a mechanism sensitive to the external Mn²⁺ concentration, whereas Cu²⁺ transport is not governed in this way by the external Cu²⁺.     

ON THE MECHANISM OF OUABAIN INHIBITION OF SYNAPTOSOME PROTEIN SYNTHESIS

Abstract— Ouabain (200μ m ) inhibited incorporation of radiolabelled leucine or glycine into the protein of neonatal synaptosome fractions but had minimal effect on preparations from adult rats. Leucine uptake into synaptosomes was rapid but not influenced by 200μ m -ouabain in contrast to ouabain inhibition of [¹⁴C]glycine and [¹⁴C]γ-aminobutyric acid uptake. Ouabain blocked the Na⁺ -dependent (stimulated) component of synaptosome fraction protein synthesis in the presence of 25m m -K⁺. Ouabain inhibition was not alleviated by addition of ADP or ATP. 100μ m -atractylate failed to influence [³H]leucine uptake or incorporation. Synergistic inhibition by ouabain was observed with the cycloheximide-sensitive component of protein synthesis and the chloramphenicol sensitive phase. Increasing the medium Ca²⁺ concentration stimulated protein synthesis and this stimulated component was inhibited by ouabain. Ouabain inhibition was associated with decreasing intraterminal K⁺ concentration and [K]_i was linearly related to the protein synthesis rate in control and ouabain treated preparations.     

ISOLATION, PURIFICATION, AND CHARACTERIZATION OF DMSP LYASE (DIMETHYLPROPIOTHETIN DETHIOMETHYLASE (4.4.1.3)) FROM THE RED ALGA POLYSIPHONIA PANICULATA1

Polysiphonia paniculata Montagne is an intertidal red alga known to produce large amounts of the compound dimethylsulfoniopropionate (DMSP). Conversion of this substrate into dimethylsulfide is accomplished in P, paniculata by an enzyme called DMSP lyase (dimethylpropiothetin dethiomethyla.se (4.4.1.3)). DMSP lyase has been purified and characterized from P. paniculata. Enzymie activity is found in two different proteins: the larger with a molecular weight of 9.26 ± 10⁴ daltons and the smaller with a molecular weight of 3.65 ± 10⁴ daltons. Specific activity of the enzyme is 526 μmols min⁻¹mg⁻¹ for the smaller protein a nd 263 μmols min ⁻¹ mg⁻¹ for the la rger protein. The Michaelis-Menten constant (K_m) is 72.8 μM ± 17.15 and the v_max is 1.62 μmols min⁻¹± 0.928 for the 92.6-kDa protein. The p1 of the larger protein is 5.8 and 5.9 for the smaller protein. Interaction with cysteine protease inhibitors L-trans-epoxysuccinyl-leucylamido (4-guanidino)-butane, dithiobis-(2-nitrobenzoate), or N -ethylmaleimide inactivated enzyme activity. The presence of either magnesium or calcium with DMSP lyase enhanced activity al concentrations between 20 and 40 μM but had little effect above these levels. Addition of the divalent chelators ethylenebis(oxyethylenenitrilo) tetraacetic acid and ethylenediaminetetraacetate decreased activity of the enzyme, but activity was restored when either chelator was removed and magnesium or calcium was added to the enzyme .     

SYNTHESIS AND RELEASE OF [14C]ACETYLCH0LINE IN SYNAPTOSOMES

Abstract— Synaptosomes took up [¹⁴C]choline, about half or more of which was converted to [^I4C]acetylcholine when incubated in an appropriate medium containing 1 to 5 μ M-[¹⁴C] choline and neostigmine. The amount of [¹⁴C]acetylcholine synthesized in synaptosomes increased in parallel with the increase of Na⁺ concentration in the incubation medium. The effect of Na⁺ on the uptake of [^I4C]choline into synaptosomes was dependent on the concentration of choline in the incubation medium.
About 25 per cent of [¹⁴C]acetylcholine synthesized in synaptosomes was released rapidly into the medium by increasing the K⁺ concentration in the medium from 5 m m to 35 m m . The change of Na⁺ concentration hardly affected the release of [¹⁴C]acetylcholine. The effect of K⁺ on the release of [¹⁴C]choline was rather small compared to that on [¹⁴C] acetylcholine. Ouabain promoted the release of [¹⁴C]acetylcholine.     

Key Residues Defining the μ-Opioid Receptor Binding Pocket: A Site-Directed Mutagenesis Study

Abstract: Structural elements of the rat μ-opioid receptor important in ligand receptor binding and selectivity were examined using a site-directed mutagenesis approach. Five single amino acid mutations were made, three that altered conserved residues in the μ, δ, and κ receptors (Asn¹⁵⁰ to Ala, His²⁹⁷ to Ala, and Tyr³²⁶ to Phe) and two designed to test for μ/δ selectivity (Ile¹⁹⁸ to Val and Val²⁰² to Ile). Mutation of His²⁹⁷ in transmembrane domain 6 (TM6) resulted in no detectable binding with [³H]DAMGO (³H-labeled d -Ala², N -Me-Phe⁴,Gly-ol⁵-enkephalin), [³H]bremazocine, or [³H]ethylketocyclazocine. Mutation of Asn¹⁵⁰ in TM3 produces a three- to 20-fold increase in affinity for the opioid agonists morphine, DAMGO, fentanyl, β-endorphin_1–31, JOM-13, deltorphin II, dynorphin_1–13, and U50,488, with no change in the binding of antagonists such as naloxone, naltrexone, naltrindole, and nor-binaltorphamine. In contrast, the Tyr³²⁶ mutation in TM7 resulted in a decreased affinity for a wide spectrum of μ, δ, and κ agonists and antagonists. Altering Val²⁰² to Ile in TM4 produced no change on ligand affinity, but Ile¹⁹⁸ to Val resulted in a four- to fivefold decreased affinity for the μ agonists morphine and DAMGO, with no change in the binding affinities of κ and δ ligands.     

Endogenous cannabinoid anandamide inhibits nicotinic acetylcholine receptor function in mouse thalamic synaptosomes

The effects of the endogenous cannabinoid anandamide [arachidonylethanolamide (AEA)] on the function of nicotinic acetylcholine receptor (nAChR) were investigated using the ⁸⁶Rb⁺ efflux assay in thalamic synaptosomes. AEA reversibly inhibited ⁸⁶Rb⁺ efflux induced by 300 μM ACh with an IC₅₀ value of 0.9 ± 2 μM. Pre-treatment with the cannabinoid (CB₁) receptor antagonist SR141716A (1 μM), the CB₂ receptor antagonist SR144528 (1 μM), or pertussis toxin (0.2 mg/mL) did not alter the inhibitory effects of AEA, suggesting that known CB receptors are not involved in AEA inhibition of nAChRs. AEA inhibition of ⁸⁶Rb⁺ efflux was not reversed by increasing acetylcholine (ACh) concentrations. In radioligand binding studies, the specific binding of [³H]-nicotine was not altered in the presence of AEA, indicating that AEA inhibits the function of nAChR in a non-competitive manner. Neither the amidohydrolase inhibitor phenylmethylsulfonyl fluoride (0.2 mM) nor the cyclooxygenase inhibitor, indomethacin, (5 μM) affected AEA inhibition of nAChRs, suggesting that the effect of AEA is not mediated by its metabolic products. Importantly, the extent of AEA inhibition of ⁸⁶Rb⁺ efflux was significantly attenuated by the absence of 1% fatty acid free bovine serum albumin pre-treatment, supporting previous findings that fatty acid-like compounds modulate the activity of nAChRs. Collectively, the results indicate that AEA inhibits the function of nAChRs in thalamic synaptosomes via a CB-independent mechanism and that the background activity of these receptors is affected by fatty acids and AEA.     

Electrotransformation of meat lactobacilli. Effect of several parameters on their efficiency of transformation

Intact cells of several lactobacilli isolated from Spanish dry fermented sausages ( Lactobacillus curvatus, Lact. sake, Lact. plantarum and Lact. bavaricus ) were transformed by electroporation. With pNZ12 as a vector, transformation efficiencies of 2.4 times 10⁵, 3.8 times 10³ and 8.8 times 10² transformants μg^-1 DNA were observed for Lact. curvatus CTC435, Lact. sake CTC335 and Lact. bavaricus CTC232, respectively.
Effects of variation in experimental parameters on transformation efficiency were evaluated. Strains, vectors and buffers were the determinant parameters. The growth phase of the culture, cell concentration, voltage, use of cell wall weakening agents and the purity of the vector influenced the transformation efficiency in most strains.     

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.     

Transformation of Methanococcus maripaludis and identification of a Pst I-like restriction system

Abstract An optimized polyethylene glycol (PEG) method of transformation was developed for Methanococcus maripaludis using the pKAS102 integration vector. The frequency of transformation with 0.8 μg of plasmid and 3×10⁹ cells was 4.8×10⁻⁵ transformants cfu⁻¹, or 1.8×10⁵ transformants μg⁻¹, which was four orders of magnitude greater than with the natural transformation method. A Pst I restriction activity in M. maripaludis was also identified. Methylation of the plasmid with Pst I methylase increased the methanococcal transformation frequency at least four-fold. Also, chromosomal DNA from M. maripaludis was resistant to digestion by the Pst I endonuclease.