DNA as the Intracellular Secondary Target for Antibacterial Action of Human Neutrophil Peptide-I Against Mycobacterium tuberculosis H37Ra

The secondary intracellular target of human neutrophil peptide-1 has been examined in M. tuberculosis H₃₇Ra. Binding studies with radioiodinated HNP-1 revealed biphasic equilibrium binding kinetics with respect to time. The major site of HNP-1 binding was found to be plasma membrane/cell wall whereas the cytosol appears to be a secondary site. Among the different macromolecules examined, maximum inhibition (75%) was observed in DNA biosynthesis during treatment with HNP-1. The interaction of HNP-1 with mycobacterial genomic DNA on the basis of gel retardation assay revealed HNP-1 binding to DNA. These results indicate that HNP-1 has DNA as the secondary intracellular target for antibacterial action against mycobacteria. Received: 25 October 2000/Accepted: 10 January 2001     

Isocitrate dehydrogenase and glyoxylate cycle enzyme activities in Bradyrhizobium japonicum under various growth conditions

Bradyrhizobium japonicum, the nitrogen-fixing symbiotic partner of soybean, was grown on various carbon substrates and assayed for the presence of the glyoxylate cycle enzymes, isocitrate lyase and malate synthase. The highest levels of isocitrate lyase [165–170 nmol min^–1 (mg protein)^–1] were found in cells grown on acetate or β-hydroxybutyrate, intermediate activity was found after growth on pyruvate or galactose, and very little activity was found in cells grown on arabinose, malate, or glycerol. Malate synthase activity was present in arabinose- and malate-grown cultures and increased by only 50–80% when cells were grown on acetate. B. japonicum bacteroids, harvested at four different nodule ages, showed very little isocitrate lyase activity, implying that a complete glyoxylate cycle is not functional during symbiosis. The apparent K _m of isocitrate lyase for d,l-isocitrate was fourfold higher than that of isocitrate dehydrogenase (61.5 and 15.5 μM, respectively) in desalted crude extracts from acetate-grown B. japonicum. When isocitrate lyase was induced, neither the V _max nor the d,l-isocitrate K _m of isocitrate dehydrogenase changed, implying that isocitrate dehydrogenase is not inhibited by covalent modification to facilitate operation of the glyoxylate cycle in B. japonicum. Received: 10 October 1997 / Accepted: 16 January 1998     

Nitrate Uptake by the Halotolerant Cyanobacterium <Emphasis Type="Italic">Aphanothece halophytica</Emphasis> Grown Under Non-Stress and Salt-Stress Conditions

We have compared the characteristics of nitrate uptake by Aphanothece halophytica grown under non-stress and salt-stress conditions. Both cell types showed essentially similar patterns of nitrate uptake toward ammonium, nitrite, and DL-glyceraldehyde. Although the affinities of nitrate to non-stress cells and salt-stress cells were not significantly different, i.e., K_s = 416 and 450 µM, respectively, the V_max value for non-stress cells was about twofold of that for salt-stress cells (9.1 vs 5.3 µmol min^–1 mg^–1 Chl). Nitrate uptake by A. halophytica was found to be dependent on Na⁺. Ammonium inhibited nitrate uptake, and the presence of methionine sulfoximine could not release the inhibition by ammonium. Nitrite appeared to competitively inhibit nitrate uptake with a K_i value of 84 µM. Both chloride and phosphate anions did not affect nitrate uptake. DL-Glyceraldehyde, an inhibitor of CO₂ fixation, caused a reduction in the uptake of nitrate.Received: 22 October 2002 / Accepted: 6 December 2002     

Ca2+ Uptake Through Voltage-gated L-type Ca2+ Channels by Polarized Enterocytes from Atlantic Cod Gadus morhua

The presence and localization of voltage-gated Ca²⁺ channels of L-type were investigated in intestinal cells of the Atlantic cod. Enterocytes were loaded with the fluorescent Ca²⁺ probe, fure-2/AM and changes in intracellular Ca²⁺ concentrations ([Ca²⁺] _i ) were measured, in cell suspensions, in the presence of high potassium levels (100 mm), BAY K-8644 (5 μm), nifedipine (5 μm) or ω-conotoxin (1 μm). L-type Ca²⁺ channels were visualized on intestinal sections using the fluorescent dihydropyridine (-)-STBodipy. Depolarization of the plasma membrane produced a rapid (within 5 sec) and transient (at basal levels after 21 sec) increase in [Ca²⁺] _i . BAY K-8644 increased the [Ca²⁺] _i by 7.2%. Cells in a Ca²⁺-free buffer increased [Ca²⁺] _i after addition of 10 mm Ca²⁺, and this increase was abolished by nifedipine in both depolarizing and normal medium but not by ω-conotoxin. Single cell experiments using video microscopy revealed that enterocytes remained polarized several hours after preparation and that the Ca²⁺ entry and extrusion occurred at specific and different regions of the enterocyte outer membrane. Fluorescent staining of L-type Ca²⁺ channels in the intestinal mucosa showed the most intense staining at the brushborder membrane. These results demonstrate the presence of voltage gated L-type Ca²⁺ channels in enterocytes from the Atlantic cod. The channels are mainly located at the apical side of the cells, and there is a polarized uptake of Ca²⁺ into the enterocytes. This suggests that the L-type Ca²⁺ channels are involved in the transcellular Ca²⁺ entry into the enterocytes. Received: 21 August 1997/Revised: 15 April 1998     

Identification of a sheath-associated protein involved in phosphate transport in Sphaerotilus natans

Summary Sphaerotilus natans was shown to have a fourfold lower K _mof phosphate transport when grown in medium containing 0.1 mm phosphate, compared to cells grown in 10.0 mm phosphate. Analysis of sheath proteins from cells grown at these two phosphate levels revealed a protein of 53 kDa present in the sheath of cells grown at a phosphate concentration of 0.1 mm. This sheath-associated, phosphate-regulated protein, designated SapP, was gel purified and used to raise a polyclonal antibody. Enzyme-linked immunosorbent assay was used to localize this protein to the surface of the sheathed cells. Phosphate uptake assays done in the presence of the antibody also showed a rise in the K _mof phosphate transport in cells grown in 0.1 mm phosphate, indicating that this protein is involved in high-affinity phosphate transport.Offprint requests to: C. F. Kulpa Jr     

Inhibitory effect of ethanol on growth and solute accumulation by Saccharomyces cerevisiae as affected by plasma-membrane lipid composition

Incorporation of ethanol (1.0 or 1.25 M) into exponential-phase cultures of Saccharomyces cerevisiae NCYC 366 growing anaerobically in a medium supplemented with ergosterol and an unsaturated fatty acid caused a retardation in growth rate, which was greater when the medium contained oleic rather than linoleic acid. Ethanol incorporation led to an immediate drop in growth rate, and ethanol-containing cultures grew at the slower rate for at least 10 h. Incorporation of ethanol (0.5 M) into buffered (pH 4.5) cell suspensions containing d-[6-³H] glucose, d-[1-¹⁴C] glucosamine, l-[U-¹⁴C] lysine or arginine, or KH₂ ³²PO₄ lowered the rate of solute accumulation by cells. Rates of accumulation of glucose, lysine and arginine were retarded to a greater extent when cells had been grown in the presence of oleic rather than linoleic acid. This difference was not observed with accumulation of phosphate. Ethanol was extracted from exponential-phase cells by four different methods. Cells grown in the presence of linoleic acid contained a slightly, but consistently, lower concentration of ethanol than cells grown in oleic acid-containing medium. The ethanol concentration in cells was 5–7 times greater than that in the cell-free medium.     

Involvement of colchicine-sensitive cytoplasmic element in premitotic nuclear positioning ofAdiantum protonemata

Summary When the red-light grown protonema ofAdiantum capillus-veneris was transferred to the dark, the nucleus ceased its migration ca. 5 hours before cell plate formation (Mineyuki andFuruya 1980). To see whether the nucleus was held by some cytoplasmic structure during nuclear positioning, protonemata were treated with various centrifugal forces at different stages of the cell cycle. Nuclei of G₁ phase were easily displaced by centrifugation at 360×g for 15 minutes, but those of G₂ or M phase were not displaced by it, suggesting that the nuclei were held by some cytoplasmic elements in G₂ or M phase. This nuclear anchoring was not detectable in protonemata that were treated with 5mM colchicine. With this treatment, the nucleus did not stop its migration at late G₂ and moved even in prophase. And the retardation of organelle movement which was observed in cytoplasm on the lateral side of the nucleus after the cessation of premitotic nuclear migration (Mineyuki andFuruya 1984) was not observed in the presence of colchicine. Thus the nuclei appear to be held by colchicine-sensitive structure in cytoplasm between the lateral surface of the nucleus and cell wall during the premitotic nuclear positioning. Electron micrographs showing cytoplasmic microtubules were consistent with the idea.Abbreviations PPN Premitotic positioning of the nucleus - L region Cytoplasm between the lateral surface of the nucleus and cell wall (seeMineyuki et al. 1984)     

Die Aminosäuresequenz des DAP-haltigen Mureins von Lactobacillus plantarum und Lactobacillus inulinus

Zusammenfassung Von L. plantarum und L. inulinus wurden die Zellwände isoliert und durch Inkubation mit Trypsin gereinigt. Durch Extraktion mit TES und Formamid konnte das Murein (Peptidoglycan) bis zu rund 85% der Trockenmasse angereichert werden. Die Zellwände von L. plantarum enthielten rund 30% Teichonsäure des Ribit-Typs, die von L. inulinus waren frei von Teichonsäure.Im Hydrolysat der teichonsäurefreien Zellwände ergaben sich folgende aufbzw. abgerundete Molverhältnisse Mur: GlNH₂:Glu:DAPl-Alad-Ala=1:1:1:1:1:0,5. Außerdem waren 2 Mole Ammoniak enthalten, was das Vorliegen von Glu und DAP als Amide anzeigt. Die durch Hemmung mit d-Cycloserin angereicherte unvollständige Mureinvorstufe hatte ein Molverhältnis von UDP:Murl-Ala:Glu:DAP=1:1:1:1:1.Nach Dinitrophenylierung der Zellwand ließen sich rund 50% der gesamten DAP als mono-DNP-DAP nachweisen. Die Hydrazinolyse der Zellwand zum Nachweis C-terminaler Aminosäuren ergab 4% freies DAP und 0,8% freies Alanin.Durch die Analyse der in Partialhydrolysaten der Zellwand auftretenden Peptide konnte die folgende Aminosäuresequenz des an die Muraminsäure gebundenen Tetrapeptides bestimmt werden: l-Ala-d-Glu-l-Lys-d-Ala. Im Murein ist vermutlich nur etwa die Hälfte der Muraminsäure mit einem Tetrapeptid, die andere Hälfte mit einem Tripeptid, dessen d-Alanin fehlt, substituiert.Die Quervernetzung erfolgt zwischen der 2. Aminogruppe der DAP und der Carboxylgruppe des d-Alanins eines benachbarten Tetrapeptids.

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The amino acid sequence of the DAP-containing murein of Lactobacillus plantarum and Lactobacillus inulinus

Summary Cell walls of L. plantarum and L. inulinus were isolated and purified by incubation with trypsin. After extraction with TCA and formamide, 85% of the dry weight consists of murein (peptidoglycan).The cell walls of L. plantarum contained about 30% teichoic acid (ribit-type), whereas no teichoic acid was present in the cell walls of L. inulinus.The quantitative determination of amino sugars and amino acids in the hydrolysate of the cell walls showed the following molar ratios: Mur: Gl-NH₂:Glu:DAP l-Alad-Ala=1:1:1:1:1:0.5. In addition, 2 mols of NH₃ were found per mol of glutamic acid, indicating, that DAP as well as glutamic acid are present as amides.The UDP-activated cell wall precursor which was accumulated by inhibiting the cells by d-cycloserine showed the following molar ratios: UDP:Murl-Ala: Glu:DAP=1:1:1:1:1.After dinitrophenylation and hydrolysation of the cell wall 50% of the DAP were present as mono-DNP-DAP. Hydrozinolysis of the cell wall yielded 4% free DAP and 0.8% free alanine. This shows that only a very small amount of these amino acids are C-terminal in the whole murein.The analysis of various peptides from acid partial hydrolysates of the cell wall indicates the following amino acid sequence of the tetrapeptides attached to muramic acid: l-Ala-d-Glu-meso-DAP-d-Ala. Only half of the muramic acid molecules are substituted by tetrapeptides, while the other half carries a tripeptide in which the terminal d-alanine is missing.The cross-linking of the muropeptides is achieved by a peptide-bond between the second amino group of DAP and the carboxylgroup of the d-alanine of an adjacent muropeptide.

     

Membrane Potassium Channels and Human Bladder Tumor Cells: II. Growth Properties

These experiments were done to determine the effect of glibenclamide and diazoxide on the growth of human bladder carcinoma (HTB-9) cells in vitro. Cell growth was assayed by cell counts, protein accumulation, and ³H-thymidine uptake. Glibenclamide added at 75 and 150 μm for 48 hr reduced cell proliferation. Dose-inhibition curves showed that glibenclamide added for 48 hr reduced cell growth at concentrations as low as 1 μm (IC₅₀= 73 μm) when growth was assayed in the absence of added serum. This μM-effect on cell growth was in agreement with the dose range in which glibenclamide decreased open probability of membrane K_ATP channels. Addition of glibenclamide for 48 hr also altered the distribution of cells within stages of the cell cycle as determined by flow cytometry using 10⁻⁵ m bromodeoxyuridine. Glibenclamide (100 μm) increased the percentage of cells in G₀/G₁ from 33.6% (vehicle control) to 38.3% (P < 0.05), and it reduced the percentage of cells in S phase from 38.3% to 30.6%. On the other hand, diazoxide, which opens membrane K_ATP channels in HTB-9 cells, stimulated growth measured by protein accumulation, but it did not increase the cell number. We conclude that the sulfonylurea receptor and the corresponding membrane K_ATP channel are involved in mechanisms controlling HTB-9 cell growth. However, K_ATP is not rate-limiting among the signaling mechanisms or molecular switches that regulate the cell cycle. Received: 12 June 1997/Revised: 21 October 1997     

A method to increase silver biosorption by an industrial strain of Saccharomyces cerevisiae

 Ag⁺ biosorption by an industrial strain of Saccharomyces cerevisiae was investigated. Older (96 h old) biomass had half the biosorption capacity of younger (24 h old) biomass (0.187 and 0.387 mmol Ag⁺/g dry mass respectively). Comparisons of cell walls isolated from biomass of either age indicated that chemical composition and Ag⁺ biosorption capacity varied little over the time span examined and that cell walls from either age of culture had small Ag⁺ biosorption capacities compared to whole cells of a similar age. Silver-containing precipitates were observed both on the cell wall and within the cell, indicating that intracellular components sorbed Ag⁺. The concentration of these precipitates within the cell appeared visually to decrease with age in Ag⁺-exposed cells. Incorporation of L-cysteine into the growth medium resulted in biomass with increased silver biosorption capacities, protein and sulphydryl group content. Increasing the concentration of L-cysteine in the growth medium from 0 to 5.0 mM increased silver biosorption from 0.389 to 0.556 mmol Ag⁺/g dry mass Isolated cell walls of biomass grown in supplemented media also showed a possible link between silver biosorption capacities, protein and sulphydryl group content. No precipitates were observed in silver-exposed biomass that had been grown in the presence of 5.0 mM L-cysteine. Received: 12 April 1995 / Received revision: 7 August 1995 / Accepted: 22 August 1995     

``Frustrated Exocytosis' — A Novel Phenomenon: Membrane Fusion without Contents Release, Followed by Detachment and Reattachment of Dense Core Vesicles in Paramecium Cells

The lipophilic fluorescent dye, FM1-43, as now frequently used to stain cell membranes and to monitor exo-endocytosis and membrane recycling, induces a cortical [Ca²⁺] _i transient and exocytosis of dense core vesicles (``trichocysts') in Paramecium cells, when applied at usual concentrations (≤10 μm) in presence of extracellular Ca<sup>2+</sup> ([Ca<sup>2+</sup>] <sub>o</sub> = 50 μm). When [Ca<sup>2+</sup>] <sub>o</sub> is kept at 30 nm (<[Ca<sup>2+</sup>]<sup>rest</sup> <sub>i</sub> ), in about one third of the population of extrudable trichocysts docked at the cell membrane, FM1-43 induces membrane fusion, visible by FM1-43 fluorescence of the vesicle membrane. However, in this system extrusion of secretory contents cannot occur in absence of any sufficient Ca<sup>2+</sup> <sub>o</sub> . Upon readdition of Ca<sup>2+</sup> <sub>o</sub> or some other appropriate Me<sup>2+</sup> <sub>o</sub> at 90 μm, secretory contents can be released (complete exocytosis). Resulting ghosts formed in presence of Ca<sup>2+</sup>, Sr<sup>2+</sup> or Mn<sup>2+</sup> are vesicular, but when formed in presence of Mg<sup>2+</sup>, for reasons to be elucidated, they are tubular, though both types are endocytosed and lose their FM1-43 stain. In contrast, in presence of [Mg<sup>2+</sup>] <sub>o</sub> = 3 mm (which inhibits contents release), the exocytotic openings reseal and intact trichocysts with labeled membranes and with still condensed contents are detached from the cell surface (``frustrated exocytosis') within ∼15 min. They undergo cytoplasmic streaming and saltatory redocking, with a half-time of ∼35 min. During this time, the population of redocked trichocysts amenable to exocytosis upon a second stimulus increases with a half-time of ∼35 min. Therefore, acquirement of competence for exocytotic membrane fusion may occur with only a small delay after docking, and this maturation process may last only a short time. A similar number of trichocysts can be detached by merely increasing [Mg²⁺] _o to 3 mm, or by application of the anti-calmodulin drug, R21547 (calmidazolium). Essentially we show (i) requirement of calmodulin and appropriate [Me²⁺] to maintain docking sites in a functional state, (ii) requirement of Ca²⁺ _o or of some other Me²⁺ _o to drive membrane resealing during exo-endocytosis, (iii) requirement of an ``empty' signal to go to the regular endocytotic pathway (with fading fluorescence), and (iv) occurrence of a ``filled' signal for trichocysts to undergo detachment and redocking (with fluorescence) after ``frustrated exocytosis'. Received: 20 January 2000/Revised: 5 May 2000     

Nitric Oxide Activates or Inhibits Skeletal Muscle Ryanodine Receptors Depending on Its Concentration, Membrane Potential and Ligand Binding

We show that rabbit skeletal RyR channels in lipid bilayers can be activated or inhibited by NO, in a manner that depends on donor concentration, membrane potential and the presence of channel agonists. 10 μm S-nitroso-N-acetyl-penicillamine (SNAP) increased RyR activity at −40 mV within 15 sec of addition to the cis chamber, with a 2-fold increase in frequency of channel opening (F _o ). 10 μm SNAP did not alter activity at +40 mV and did not further activate RyRs previously activated by 2 mm cis ATP at +40 or −40 mV. In contrast to the increase in F _o with 10 μm SNAP, 1 mm SNAP caused a 2-fold reduction in F _o but a 1.5-fold increase in mean open time (T _o ) at −40 mV in the absence of ATP. 1 mm SNAP or 0.5 mm sodium nitroprusside (SNP) induced ∼3-fold reductions in F _o and T _o at +40 or −40 mV when channels were activated by 2 mm cis ATP or in channels activated by 6.5 μm peptide A at −40 mV (peptide A corresponds to part of the II–III loop of the skeletal dihydropyridine receptor). Both SNAP-induced activation and SNAP/SNP-induced inhibition were reversed by 2 mm dithiothreitol. The results suggest that S-Nitrosylation or oxidation of at least three classes of protein thiols by NO each produced characteristic changes in RyR activity. We propose that, in vivo, initial release of NO activates RyRs, but stronger release increases [NO] and inhibits RyR activity and contraction. Received: 27 August 1999/Revised: 25 October 1999     

Adaptational changes in cellular phospholipids and fatty acid composition of the food pathogen Listeria monocytogenes as a stress response to disinfectant sanitizer benzalkonium chloride

Aims: This study provides a first approach to observing the alterations of the cell membrane lipids in the adaptation response of Listeria monocytogenes to the sanitizer benzalkonium chloride. Methods and Results: A thorough investigation of the composition of polar and neutral lipids from L. monocytogenes grown when exposed to benzalkonium chloride is compared to cells optimally grown. The adaptation mechanism of L. monocytogenes in the presence of benzalkonium chloride caused (i) an increase in saturated‐chain fatty acids (mainly C_16:0 and C_18:0) and unsaturated fatty acids (mainly C_16:1 and C_18:1) at the expense of branched‐chain fatty acids (mainly C_a‐15:0 and C_a‐17:0) mainly because of neutral fatty acids; (ii) no alteration in the percentage of neutral and polar lipid content among total lipids; (iii) a decrease in lipid phosphorus and (iv) an obvious increase in the anionic phospholipids and a decrease in the amphiphilic phosphoaminolipid. Conclusions: These lipid changes could lead to decreased membrane fluidity and also to modifications of physicochemical properties of cell surface and thus changes in bacterial adhesion to abiotic surfaces. Significance and Impact of the Study: The adaptation and resistance of L. monocytogenes to disinfectants is able to change its physiology to allow growth in food‐processing plants. Understanding microbial stress response mechanisms would improve the effective use of disinfectants.     

Expression of recombinant antibody and secreted alkaline phosphatase in mammalian cells. Influence of cell line and culture system upon production kinetics

The growth and productivity of an Sp2/0 cell line, F3b10, expressing a recombinant antibody (rAb) and BHK21 cells expressing either the same rAb from the same plasmids (BHK.IgG) or secreted alkaline phosphatase (SEAP) (BHK.SEAP) were investigated. The F3b10 line was grown as a single cell suspension. The BHK lines were grown either as suspended natural aggregates or on Cytodex 3 microcarriers. The data for F3b10 showed that the cell-specific rAb production rate (Qs _rAb) increased in parallel with increases in the specific growth rate (). A similar result was obtained for suspended aggregate cultures of both recombinant BHK cell lines. In contrast, for microcarrier cultures of both BHK cell lines, Qs _product increased as decreased. This report shows that the relationship between cell growth and Qs _product for the cell lines and products studied is dependent upon the culture process. In systems where recombinant cells are growing as a single cell suspension or within a natural suspension aggregate, Qs _product increased with increases in . In such systems, the cells have a rounded morphology. When cells were grown on microcarriers, Qs _product decreased as increased. Cells growing attached to a surface are flat and elongated. The observed differences in the relationship of Qs _product to are correlated with changes in cell morphology. The relationship between Qs _product and is also affected by the choice of cell line. Correspondence to: A. J. Racher     

Pathways of d-fructose and d-glucose catabolism in marine species of Alcaligenes,Pseudomonas marina,and Alteromonas communis

Cell-free extracts of d-fructose grown cells of marine species of Alcaligenes as well as Pseudomonas marina contained an activity which catalyzed a P-enolpyruvate-dependent phosphorylation of d-fructose in the 1-position as well as activities of the following enzymes: 1-P-fructokinase, fructose-1,6-P₂ aldolase, PPi-dependent 6-P-fructokinase, fructokinase, glucokinase, P-hexose isomerase, glucose-6-P dehydrogenase, 6-P-gluconate dehydrase, and 2-keto-3-deoxy-6-P-gluconate aldolase. The presence of these enzyme activities would allow d-fructose to be degraded by the Embden-Meyerhof pathway and/or the Entner-Doudoroff pathway. In cell-free extracts of d-glucose grown cells, the activity catalyzing a P-enolpyruvate-dependent phosphorylation of d-fructose as well as 1-P-fructokinase activity were reduced or absent while the remaining enzymes were present at levels similar to those found in d-fructose grown cells. Radiolabeling experiments suggested that both d-fructose and d-glucose were utilized primarily via the Entner-Doudoroff pathway. Alteromonas communis, a marine species lacking 1-P-fructokinase and the PPi-dependent 6-P-fructokinase, contained all the enzyme activities necessary for the catabolism of d-fructose and d-glucose by the Entner-Doudoroff pathway; the involvement of this pathway was also consistent with the results of the radiolabeling experiments.Non-Standard Abbreviations EDP Entner-Doudoroff pathway - EMP Embden-Meyerhof pathway - FDP fructose-1,6-P₂ - FDPase FDP phosphatase - F-1-P fructose-1-P - F-6-P fructose-6-P - FPTS PEP: d-fructose phosphotransferase system - PPi-6-PFK PPi dependent 6-PFK - G-6-P glucose-6-P - KDPG 2-keto-3-deoxy-6-P-gluconate - PEP P-enolpyruvate - 1-PFK 1-P-fructokinase - 6-PFK 6-P-fructokinase - 6-PGA 6-P-gluconate     

Changes in Purinoceptor Distribution and Intracellular Calcium Levels following Noise Exposure in the Outer Hair Cells of the Guinea Pig

Among the cells of the inner ear, the outer hair cells (OHCs) are the most important targets of noise-induced effects, being the most sensitive cell types. The aim of this study was to examine the effects of noise (50 Hz-20 kHz, 80 dB sound pressure level, 14 days) on intracellular calcium levels and on the expression pattern of purinoceptors in the membrane of the OHCs of the guinea pig and to measure the stiffness changes of the lateral membrane of these cells. In noise-exposed animals, the resting intracellular calcium concentration increased compared to nontreated animals and was slightly higher in the cells of the basal (219 ± 29 nM) than in the apical (181 ± 24 nM) turns of the cochlea. After application of 180 μM adenosine triphosphate, the intracellular calcium level rose by 60 ± 22 nM in cells from the apical and by 44 ± 10 nM in cells from the basal turns, significantly less than in nontreated animals. Expression of the P_2X1, P_2X2, P_2X4, P_2X7, P_2Y1 and P_2Y4 receptor subtypes was suppressed, while expression of the P_2Y2 subtype did not decrease in either of the two preparations. In parallel with the increase in intracellular calcium concentration, the stiffness of the lateral wall of the OHCs was increased. Noise-induced changes in intracellular calcium homeostasis and subsequently in the calcium-dependent regulatory mechanisms may modify OHC lateral wall stiffness and may lead to reduction of the efficacy of the cochlear amplifier.     

Chloramphenicol inhibition of Pythium ultimum and Rhodotorula glutinis

Summary The growth of Rhodotorula glutinis is inhibited by both D-threo chloramphenicol and an L-threo isomer of chloramphenicol (lacking the dichloroacetyl group), causing an increase in the mean generation time, in a variety of media, approximately proportional to the concentration of antibiotic. The antibiotic is not removed from the growth medium in any quantity during this inhibition of growth. The oxygen uptakes of normal and chloramphenicol-grown cells of R. glutinis are similar when expressed on a dry weight basis. The oxygen uptake of normal and L-threo isomer-grown cells is strongly inhibited by antimycin A, whereas D-threo chloramphenicol-grown cells are unaffected. There was no evidence to suggest that any uncoupling of phosphorylation occurred with either isomer. Pythium ultimum mycelium also showed similar oxygen uptakes per unit dry weight whether grown in the presence or absence of D-threo chloramphenicol. The D-threo chloramphenicol-grown mycelium was also insensitive to antimycin A in contrast to the normal mycelium which was strongly inhibited. P. ultimum grows slowly in the presence of 100 g/ml D-threo chloramphenicol in a glucose salts medium, but is completely inhibited by a similar concentration in a glycerol salts medium. The L-threo isomer does not inhibit the growth of P. ultimum.The mitochondria of Rhodotorula glutinis show a progressive disorganization when grown in the presence of increasing concentrations of D-threo chloramphenicol up to 1000 g/ml. There is an associated over synthesis of cell wall material in the higher concentrations of the antibiotic. The L-threo isomer produces no obvious fine structural abnormalities even at concentrations of 1000 g/ml.     

Phenol-induced membrane changes in free and immobilized Escherichia coli

Summary Membranes of Escherichia coli cells grown in the presence of phenol were examined after isolation of the cytoplasmic and outer membrane fractions. Both membrane types showed reduced lipid-to-protein ratios compared to cells grown without phenol. Phenol-induced differences in the expression of individual proteins of the outer membrane, probably involved in the uptake of iron, were expressed in smaller quantities after phenol addition. Growth in the presence of phenol increased the respiratory activity of the cytoplasmic membrane, whereas the direct inhibition of O₂ consumption by phenol was not affected by the presence of this compound in the growth medium. E. coli cells grown entrapped in calcium alginate showed low lipid-to-protein ratios even without phenol in the growth medium. Immobilization of cells also markedly changed the protein pattern of the outer membrane. Offprint requests to: H.-J. Rehm     

A novel mechanism involved in the metabolism of the tartaric acid stereoisomers in Rhodopseudomonas sphaeroides: enzymatic conversion of meso-tartaric acid to D(-)-glyceric acid and CO2

In cell extracts of Rhodopseudomonas sphaeroides grown on meso-tartrate the activities of the bifunctional L(+)-tartrate dehydrogenase-D(+)-malate dehydrogenase (decarboxylating) (EC 1.1.1.93 and 1.1.1.83, respectively) could be measured spectrophotometrically but not the activity of a meso-tartrate dehydrogenase or dehydratase. However, an enzyme activity was detected manometrically that catalyzed the stoichiometric release of CO₂ from mesotartrate in a molar ratio of 1:1. This reaction required catalytic amounts of NAD and the presence of both divalent (Mn²⁺ or Mg²⁺) and monovalent (NH ₄ ⁺ or K⁺) cations. Purification of the meso-tartrate decarboxylase showed that it was part of the bifunctional L(+)-tartrate dehydrogenase-D(+)-malate dehydrogenase (decarboxylating), which thus possessed a third catalytic function. The homogeneous enzyme catalyzed the stoichiometric conversion of incso-tartaric acid to D(-)-glyceric acid and CO₂. All interfering catalytic activities had been eliminated during the course of enzyme purification.     

Cell wall and sheath constituents of the cyanobacterium Gloeobacter violaceus

Sheaths isolated from Gloeobacter violaceus were found to be composed of a major polysaccharide moiety (glucose, galactose, rhamnose, mannose, arabinose), a protein moiety, and negatively charged components (glucuronic acids, phosphate, sulfate). Outer membrane polypeptide patterns were dominated by two major peptidoglycan-associated proteins (M_r 62,000 and 53,000). Lipopolysaccharide constituents were glucosamine, 3-hydroxy fatty acids (3-OH-14:0, anteiso-3-OH-15:0, 3-OH-16:0, 3-OH-18:0), carbohydrates, and phosphate. A1-type peptidoglycan and non-peptidoglycan components (mannosamine, glucose, mannose, and glucosamine) indicated the presence of a peptidoglycan-polysaccharide complex in the cell walls of Gloeobacter violaceus.Abbreviations A₂pm diaminopimelic acid - ATCC American Type Culture Collection - CE cell envelope - CM cytoplasmic membrane - CW cell wall - dOcla 3-deoxy-d-manno-2-octulosonic acid - GalN galactosamine - GlcN glucosamine - GlcUA glucuronic acid - HF hydrofluoric acid - LPS lipopolysaccharide - ManN mannosamine - M relative molecular mass - MurN muramic acid - MurN-6-P muramic acid-6-phosphate - OMe O-methyl - PAGE polyacrylamide gel electrophoresis - PCC Pasteur Culture Collection - SDS sodium dodecyl sulfate - SH sheath