Differential effects of sodium on hydrogen- and glucose-dependent growth of the acetogenic bacterium Acetogenium kivui.

Acetogenium kivui could not be revived or maintained in a sodium-deficient medium (0.2 mM sodium) under H2-dependent conditions, and neither lithium nor potassium replaced the sodium requirement of H2-cultivated cells. Conversely, the revival and maintenance of glucose-cultivated cells did not display a dependency on supplemental sodium. In the absence of growth, formate became a major end product in both sodium-deficient and metabolically impaired H2-grown cultures of A. kivui. Harmaline, a putative inhibitor of Na+/H+ antiporters, uncoupled acetogenesis from H2-dependent growth but was less effective when growth was at the expense of glucose. Significantly, carbon monoxide (CO) stimulated H2-dependent growth of A. kivui but inhibited glucose-dependent growth. Collectively, these findings demonstrate that sodium plays a critical role in the H2-dependent bioenergetics of A. kivui and indicate that autotrophic and heterotrophic cells may utilize dissimilar mechanisms of energy conservation. In contrast to the growth of A. kivui, supplemental sodium was not required for the glucose-, H2-, and CO-dependent growth of Clostridium thermoaceticum.     

Characterization of the H2- and CO-dependent chemolithotrophic potentials of the acetogens Clostridium thermoaceticum and Acetogenium kivui.

Strains of Clostridium thermoaceticum were tested for H2- and CO-dependent growth in a defined medium containing metals, minerals, vitamins, cysteine-sulfide, CO2-bicarbonate, and H2 or CO. Ten of the thirteen strains tested grew at the expense of H2 and CO, and C. thermoaceticum ATCC 39073 was chosen for further study. The doubling times for H2- and CO-dependent growth under chemolithotrophic conditions (the defined medium with nicotinic acid as sole essential vitamin and sulfide as sole reducer) were 25 and 10 h, respectively. Product stiochiometries for chemolithotrophic cultures approximated: 4.1H2 + 2.4CO2----CH3COOH + 0.1 cell C + 0.3 unrecovered C and 6.8CO----CH3COOH + 3.5CO2 + 0.4 cell C + 0.9 unrecovered C. H2-dependent growth produced significantly higher acetate concentrations per unit of biomass synthesized than did CO- or glucose-dependent growth. In contrast, the doubling time for H2-dependent growth under chemolithotrophic conditions (the defined medium without vitamins and sulfide as sole reducer) by Acetogenium kivui ATCC 33488 was 2.7 h; as a sole energy source, CO was not growth supportive for A. kivui. The YH2 values for A. kivui and C. thermoaceticum were 0.91 and 0.46 g of cell dry weight per mol of H2 consumed, respectively; the YCO value for C. thermoaceticum was 1.28 g of cell dry weight per mol of CO consumed. The specific activities of hydrogenase and CO dehydrogenase in both acetogens were influenced by the energy source utilized for growth and were significantly lower in C. thermoaceticum than in A. kivui. With extracts of H2-cultivated cells and benzyl viologen as electron acceptor, the Vmax values for hydrogenase from C. thermoaceticum and A. kivui were 155.7 and 1,670 micromoles of H2 oxidized per min mg of protein, respectively; the Vmax values for CO dehydrogenase from C. thermoaceticum and A. kivui were 90.6 and 2,973 micromoles of CO oxidized per min per mg of protein, respectively.     

Nickel transport by the thermophilic acetogen Acetogenium kivui

Exogenous 63Ni was incorporated into carbon monoxide dehydrogenase when Acetogenium kivui ATCC 33488 was cultivated in the presence of 63NiCl2. The capacity for nickel (63NiCl2) transport was greatest with cells harvested from the mid- to late exponential phases of growth. Nickel transport was linear during the transport assay period and displayed saturation kinetics. The apparent Km and Vmax for nickel transport by H2-cultivated cells approximated 2.3 microM Ni and 670 pmol of Ni transported per min per mg (dry weight) of cells, respectively. The nickel transport system was not appreciably affected by the other divalent cations that were tested, and transported nickel was not readily exchangeable with exogenous nickel. Nickel transport was stimulated by glucose or H2 and was decreased by various metabolic inhibitors; however, nickel uptake by glucose- and H2-cultivated cells displayed differential sensitivities to ATPase inhibitors.     

Nickel transport by the thermophilic acetogen Acetogenium kivui.

Exogenous 63Ni was incorporated into carbon monoxide dehydrogenase when Acetogenium kivui ATCC 33488 was cultivated in the presence of 63NiCl2. The capacity for nickel (63NiCl2) transport was greatest with cells harvested from the mid- to late exponential phases of growth. Nickel transport was linear during the transport assay period and displayed saturation kinetics. The apparent Km and Vmax for nickel transport by H2-cultivated cells approximated 2.3 microM Ni and 670 pmol of Ni transported per min per mg (dry weight) of cells, respectively. The nickel transport system was not appreciably affected by the other divalent cations that were tested, and transported nickel was not readily exchangeable with exogenous nickel. Nickel transport was stimulated by glucose or H2 and was decreased by various metabolic inhibitors; however, nickel uptake by glucose- and H2-cultivated cells displayed differential sensitivities to ATPase inhibitors.     

Non-proton-motive-force-dependent sodium efflux from the ruminal bacterium Streptococcus bovis: bound versus free pools

Growing cells of Streptococcus bovis JB1 had a sodium content of 1,125 nmol/mg of protein and, based on a ratio of cell volume to protein of 4.3 microliters/mg, the apparent intracellular sodium concentration was more than 240 mM. Much of this sodium could not be removed by water washing even if cells were boiled or treated with the pore-forming ionophore, gramicidin, but it could be exchanged for potassium. Stationary cultures had a 2.6-microliters volume per milligram of protein and a total sodium content of 410 mM. When stationary cultures were energized with glucose at pH 6 to 8, sodium (more than 200 mM) was expelled within 2 min, and it appeared that growing cells had a very small pool of free intracellular sodium. Sodium-proton antiport activity could not be demonstrated with a sodium pulse, and the protonophore SF6847, valinomycin, and the H+-ATPase inhibitor dicyclohexylcarbodiimide (DCCD) had little effect on sodium efflux, even though these inhibitors greatly reduced the proton-motive force. SF6847, valinomycin, and DCCD had little effect on intracellular ATP, but iodoacetate, an inhibitor of glycolysis, decreased ATP as well as sodium efflux. Stationary cells from sodium-deficient medium expelled little sodium after glucose addition and had 35% more ATP than stationary cells which were grown in sodium medium and expelled sodium. An artificial electrochemical gradient of sodium was able to drive ATP synthesis in stationary cells, and this ATP formation was not sensitive to DCCD. These results indicated that bacteria could have a significant pool of bound sodium and that sodium expulsion from S. bovis was directly coupled to ATP hydrolysis.     

Effect of Sodium Deficiency on Ammonium-Grown Cells of Anabaena PCC 7119

This study examined how the absence of sodium affects growth,photosynthesis, photosynthetic pigments and superoxide dismutase(SOD) activity of ammonium-grown cells of AnabaenaPCC 7119 culturedunder low CO₂ levels (air) and alkaline pH. The growth rateswereseverely inhibited. After 24-h culture, photoreduction ofdioxygen was observed which, however, did not induce any photooxidationsuch as that found in sodium-deficient cells grown on nitratesor dinitrogen [Sanchez Maeso et al. (1987) Plant Physiol. 85:585–587]. In fact, SOD increasedsignificantly with sodiumdeficiency. No change was observed in the contents of photosyntheticpigments. (Received February 8, 1988; Accepted September 16, 1988)     

Non-proton-motive-force-dependent sodium efflux from the ruminal bacterium Streptococcus bovis: bound versus free pools.

Growing cells of Streptococcus bovis JB1 had a sodium content of 1,125 nmol/mg of protein and, based on a ratio of cell volume to protein of 4.3 microliters/mg, the apparent intracellular sodium concentration was more than 240 mM. Much of this sodium could not be removed by water washing even if cells were boiled or treated with the pore-forming ionophore, gramicidin, but it could be exchanged for potassium. Stationary cultures had a 2.6-microliters volume per milligram of protein and a total sodium content of 410 mM. When stationary cultures were energized with glucose at pH 6 to 8, sodium (more than 200 mM) was expelled within 2 min, and it appeared that growing cells had a very small pool of free intracellular sodium. Sodium-proton antiport activity could not be demonstrated with a sodium pulse, and the protonophore SF6847, valinomycin, and the H+-ATPase inhibitor dicyclohexylcarbodiimide (DCCD) had little effect on sodium efflux, even though these inhibitors greatly reduced the proton-motive force. SF6847, valinomycin, and DCCD had little effect on intracellular ATP, but iodoacetate, an inhibitor of glycolysis, decreased ATP as well as sodium efflux. Stationary cells from sodium-deficient medium expelled little sodium after glucose addition and had 35% more ATP than stationary cells which were grown in sodium medium and expelled sodium. An artificial electrochemical gradient of sodium was able to drive ATP synthesis in stationary cells, and this ATP formation was not sensitive to DCCD. These results indicated that bacteria could have a significant pool of bound sodium and that sodium expulsion from S. bovis was directly coupled to ATP hydrolysis.     

Arrest of cell division but not protein synthesis in sodium-deficient cells of the marine diatom Phaeodactylum tricornutum Bohlin

Cell division in the marine diatom Phaeodactylum tricornutum was prevented when cultures were maintained in the absence of sodium, regardless of the nitrogen status of the cells or medium. Addition of 10 mM ammonium and 50 mM sodium to cultures preconditioned in nitrogen and sodium-deficient medium for 5 d led to a recovery in cell division and chlorophyll a, and net protein synthesis. Sodium added in the absence of ammonium led to a recovery in cell division, but not net protein synthesis. Ammonium added in the absence of sodium was partially assimilated (as NH₃) and resulted in a small amount of protein synthesis, but without cell division. This effect was enhanced if the cells had lower protein quotas prior to ammonium addition, with total consumption of the added 1 mM ammonium and appreciable net protein synthesis. Respiration was enhanced by 1 or 10 mM ammonium or 10 mM methylammonium addition to nitrogen-deficient cultures maintained in the presence or absence of sodium. In contrast to respiration, photosynthesis was inhibited by these additions in sodium-replete cultures, but was enhanced in sodium-deficient cultures.This research was supported by the Auckland University Research Committee and University Grants Committee of New Zealand.     

Arsenite induces premature senescence via p53/p21 pathway as a result of DNA damage in human malignant glioblastoma cells

In this study, we investigate whether arsenite-induced DNA damage leads to p53-dependent premature senescence using human glioblastoma cells with p53-wild type (U87MG-neo) and p53 deficient (U87MG-E6). A dose dependent relationship between arsenite and reduced cell growth is demonstrated, as well as induced γH2AX foci formation in both U87MG-neo and U87MG-E6 cells at low concentrations of arsenite. Senescence was induced by arsenite with senescence-associated β-galactosidase staining. Dimethyl- and trimethyl-lysine 9 of histone H3 (H3DMK9 and H3TMK9) foci formation was accompanied by p21 accumulation only in U87MG-neo but not in U87MG-E6 cells. This suggests that arsenite induces premature senescence as a result of DNA damage with heterochromatin forming through a p53/p21 dependent pathway. p21 and p53 siRNA consistently decreased H3TMK9 foci formation in U87M G-neo but not in U87MG-E6 cells after arsenite treatment. Taken together, arsenite reduces cell growth independently of p53 and induces premature senescence via p53/p21-dependent pathway following DNA damage. [BMB Reports 2014; 47(10): 575-580]     

杏仁中央核损毁对缺钠大鼠钠欲行为启动和表达的影响

目的:探讨杏仁中央核(CeA)损毁对缺钠大鼠钠欲行为启动和表达的影响。方法:将18只成年雄性SD大鼠随机分为3组(n=6):双侧Ce A损毁组、假损毁组和不损毁组,手术恢复后给予大鼠14 d低钠饲料摄食以建立缺钠大鼠模型,运用单笼双瓶选择测试方法观察缺钠大鼠在24 h内5个不同时间段对0.3 mol/L NaCl和自由饮水的摄入情况。应用免疫荧光化学染色方法观察杏仁中央核损毁与否对缺钠或正常大鼠孤束核内醛固酮敏感神经元活动的影响。结果:低钠饮食14 d后,大鼠对0.3 mol/L NaCl 24 h内饮用量和偏爱率比低钠饮食前明显增加(P<0.01);杏仁中央核损毁后缺钠大鼠对0.3 mol/L Na Cl溶液的摄入量和偏爱率显著下降(P<0.01)。杏仁中央核损毁对低钠饮食诱发的大鼠孤束核内醛固酮敏感神经元活动增加没有影响。结论:低钠饮食诱导大鼠钠欲行为表达增加;杏仁中央核损毁压抑缺钠大鼠钠欲行为的表达,而对缺钠大鼠的钠欲行为的启动没有影响。     

Tolerance and metabolic response of acetogenic bacteria toward oxygen

The acetogens Sporomusa silvacetica, Moorella thermoacetica, Clostridium magnum, Acetobacterium woodii, and Thermoanaerobacter kivui (i) grew in both semisolid and liquid cultivation media containing O(2) and (ii) consumed small amounts of O(2). Low concentrations of O(2) caused a lag phase in growth but did not alter the ability of these acetogens to synthesize acetate via the acetyl coenzyme A pathway. Cell extracts of S. silvacetica, M. thermoacetica, and C. magnum contained peroxidase and NADH oxidase activities; catalase and superoxide dismutase activities were not detected.     

Bicarbonate determines cytoplasmic pH and suppresses mitogen-induced alkalinization in fibroblastic cells

Addition of growth factors to responsive cells in HCO3- -free media results in a rapid rise in cytoplasmic pH (pHi) caused by activation of Na+/H+ exchange. In this paper, we have examined how pHi regulation and growth factor responsiveness are affected by HCO3(-)using quiescent mouse MES-1 fibroblastic cells as a model. When cells are exposed to 25 mM HCO3-, 5% CO2, steady-state pHi reaches a new more alkaline level (by 0.25 unit) within 10 min. This rise in pHi is both Na+- and HCO3- -dependent, does not occur in Cl(-)-depleted cells, and is inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, but not by 5-(n,n-dimethyl)-amiloride, indicating the involvement of Na+-dependent HCO3-/Cl- exchange. Furthermore, the recovery of pHi from acute acid loads is accelerated by HCO3- in a Na+-dependent and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-sensitive manner and is blocked in Cl(-) -depleted cells. Similar results were obtained for mouse 3T3 cells and human fibroblasts. In the presence of HCO3-/CO2 (pH 7.35), mitogens and phorbol esters fail to induce a detectable rise in pHi. However, when steady-state pHi is artificially lowered by approximately 0.4 unit, growth factors evoke significant increases in pHi due to activation of Na+/H+ exchange. In the absence of HCO3-, mitogen-induced alkalinizations are readily detectable but not when pHi is artificially elevated to the value normally observed in HCO3- media. From these results we conclude that: 1) Na+-dependent HCO3-/Cl- exchange determines steady-state pHi and acts in parallel with Na+/H+ exchange to stimulate pHi recovery from acid loading; 2) Na+-dependent HCO3-/Cl- exchange raises steady-state pHi to a level beyond the operating range of the Na+/H+ exchanger and thereby prevents growth factors from alkalinizing the cytoplasm any further. The results also imply that, unlike Na+/H+ exchange, Na+-dependent HCO3-/Cl- exchange is not activated by mitogens.     

Sodium deficiency enhances the behavioral responses to centrally administered vasopressin in rats

The ability of sodium deficiency to stimulate vasopressin (VP) release was examined by determining if sodium deficiency sensitizes the animal to the behavioral disruption caused by intraventricular injections of VP. In sodium-replete rats, intraventricular injections of 50 ng VP on Day 1 had no effect on behavior, but this dose elicited abnormal behaviors (barrel rolls, hind-limb extensions) when administered on Day 2, indicating a sensitization phenomenon. In separate experiments, the first intraventricular injection of 50 ng VP in sodium-deficient but not in sodium-replete rats also elicited barrel rotations followed by hind-limb extension. Intraventricular injection of VP also disrupted motor behavior in sodium-replete rats that had multiple prior experiences with sodium deficiency but not in naive rats. These results show that sodium deficiency can mimic the effect of central injections of VP in sensitizing the brain to the behavioral effects of exogenous VP. This suggests that sodium deficiency induces the central release of VP.     

Evidence for a role of p38 kinase in hypoxia-inducible factor 1-independent induction of vascular endothelial growth factor expression by sodium arsenite

Recently we have demonstrated that sodium arsenite induces the expression of hypoxia-inducible factor 1alpha (HIF-1alpha) protein and vascular endothelial growth factor (VEGF) in OVCAR-3 human ovarian cancer cells. We now show that arsenic trioxide, an experimental anticancer drug, exerts the same effects. The involvement of phosphatidylinositol 3-kinase and mitogen-activated protein kinase (MAPK) pathways in the effects of sodium arsenite was investigated. By using kinase inhibitors in OVCAR-3 cells, both effects of sodium arsenite were found to be independent of phosphatidylinositol 3-kinase and p44/p42 MAPKS but were attenuated by inhibition of p38 MAPK. A role for p38 in the regulation of HIF-1alpha and VEGF expression was supported further by analysis of activation kinetics. Experiments in mouse fibroblast cell lines, lacking expression of c-Jun N-terminal kinases 1 and 2, suggested that these kinases are not required for induction of HIF-1alpha protein and VEGF mRNA. Unexpectedly, sodium arsenite did not activate a HIF-1-dependent reporter gene in OVCAR-3 cells, indicating that functional HIF-1 was not induced. In agreement with this hypothesis, up-regulation of VEGF mRNA was not reduced in HIF-1alpha(-/-) mouse fibroblast cell lines. Altogether, these data suggest that not HIF-1, but rather p38, mediates induction of VEGF mRNA expression by sodium arsenite.     

Phorbol esters can persistently replace interleukin-2 (IL-2) for the growth of a human IL-2-dependent T-cell line

A selected clone from an IL-2-dependent human T-cell line was persistently propagated in the presence of phorbol esters with the ability to activate protein kinase C (PKC), such as 12-O-tetradecanoylphorbol-13-acetate (TPA) or phorbol-12,13-dibutylate (PDBu). Thus, a TPA(PDBu)-dependent T-cell line, designated TPA-Mat, was established from IL-2-dependent T cells. The TPA-dependency of TPA-Mat was not lost during cultivation for more than a year in the presence of TPA, and TPA-Mat cells still showed IL-2-dependent growth. However, the TPA (PDBu)-dependent growth of TPA-Mat did not seem to be mediated by an autocrine mechanism of IL-2 or by any other growth factor production, because these factors were not detected in TPA-Mat cell supernatants. Therefore, the phorbol esters substituted for IL-2 and may be directly involved in transduction of growth signals in TPA-Mat cells. Although activity of PKC was down-regulated, messenger ribonucleic acid (mRNA) of the PKC beta-gene was detected in TPA-Mat cells cultured with PDBu. Furthermore, the growth of TPA-Mat cells was stimulated not only by phorbol esters but also by nonphorbol ester tumor promoters with the ability to activate PKC. These observations suggest that the sustained activation of PKC by the phorbol esters could induce continuous growth of the IL-2-dependent TPA-Mat cells.     

Nutritional Studies of Rickettsia quintana: Nature of the Hematin Requirement

Rickettsia quintana grew in a liquid medium consisting of a brain-heart infusion base supplemented with starch and hematin. The growth requirement for hematin could not be substituted by compounds of known catalytic activity for H(2)O(2), viz., catalase, potassium pyruvate, or charcoal, or by the reducing compounds sodium sulfite and sodium thioglycollate. R. quintana was catalase-negative, but no H(2)O(2) production could be demonstrated by the catalase-aminotriazole technique. A minimum inoculum giving 10(5) cells/ml was required to initiate growth. The generation time at 33 C was 10 hr. The temperature range for growth was 28 to 37 C. Growth was enhanced when succinate or glutamate was added as energy source.     

Autoimmune MRL/lpr mice sera contain IgG with interleukin 3-like activity

The spleen cells, thymocytes, and bone marrow cells of autoimmune MRL/MP-lpr/lpr (MRL/lpr) mice do not constitutively produce interleukin 3 (IL-3), but these mice had IL-3-like activity in their sera. MRL/lpr sera supported the growth of the IL-3-dependent cell lines FDC-P2 and DA-1 but not the growth of IL-2-dependent T-572 cells. This IL-3-like activity increased with age. Biochemical analysis of the MRL/lpr sera by anion-exchange chromatography, gel filtration on a Superose 12 column, the binding to protein-A and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the serum factor with the IL-3-like activity was not IL-3 itself but was associated with IgG. Flow cytometric analysis also showed that the serum level of the Ig capable of binding FDC-P2 cells was high in MRL/lpr but not in MRL/+ mice. We suggest that IL-3 is not responsible for lymphoid hyperplasia, contrary to a previous report; rather auto-antibodies directed toward the IL-3 receptor may act pathogenically in MRL/lpr mice.     

Thiol-sensitive mast cell lines derived from mouse bone marrow respond to a mast cell growth-enhancing activity different from both IL-3 and IL-4

A series of permanent IL-3-dependent cell lines have been established from normal BALB/c or C3H bone marrow using alpha-thioglycerol-supplemented culture medium and PWM-stimulated spleen cell-conditioned medium as a source of IL-3. The cell lines and derivatives cloned in agar resembled "mucosal type" mast cells with respect to phenotypic and functional properties. In this report we demonstrate that in vitro growth of these mast cell lines was not only dependent on IL-3 and synergistically enhanced by IL-4, but in addition regulated by alpha-thioglycerol which could be replaced by 2-ME or cysteamine. We show that these thiol-sensitive mast cell lines respond to a mast cell growth enhancing activity (MEA) present in spleen cell-conditioned medium and acting in concert with IL-3. Partially purified MEA was not able to stimulate the growth of IL-3-dependent 32Dcl.23 cells, IL-2-dependent CTLL-2 cells or the mouse T cell line F4/4K.6 (L3T4+) adapted to grow in purified IL-4. Moreover, 11B11 hybridoma-derived anti-IL-4 mAb specifically neutralizing mouse Il-4 were unable to abolish the bioactivity of MEA. PWM, CSF-1, GM-CSF, IL-1, IL-2, IL-5, IL-6, IL-7, IFN-gamma, TGF-alpha, TNF-alpha, NGF, or EPO did not substitute for MEA in our standard proliferation assay.