热带假丝酵母细胞内pH的测定及其与生长代谢活性的关系

应用荧光探针5(6)-双醋酸羧基荧光素 (Carboxyfluorescein diacetate) 测定了产长链二元酸热带假丝酵母 (Candida tropicalis) 细胞内pH (pHi) 值,确定了该探针载入C. tropicalis细胞的适宜条件。用摇瓶培养C. tropicalis细胞,考察了细胞外pH和生长碳源对pH_I的影响,实验结果表明：细胞外pH对pH_I略有影响,而生长碳源对pH_I的影响略为明显。利用5L发酵罐进一步研究了细胞生长代谢活性与pHi的关系,结果表明：细胞比生长速率、CO₂比生产速率和葡萄糖比消耗速率与pHi变化密切相关,pH_I的增加伴随着细胞生长活力的增加,反之亦然。在pH6.0条件下用葡萄糖和醋酸钠共作碳源培养C. tropicalis细胞时,测得的pH_I值维持在5.72～6.15范围内。     

Regulation of intracellular pH in the myocardium; relevance to pathology

Intracellular pH affects the contractile function of the heart, metabolic reactions, ion exchange and calcium homoeostasis. Numerous studies have concluded that a fall of extracellular pH, by whatever mechanism, causes a fall of contractility by alteration of intracellular pH. Measurement of cytosolic intracellular pH using microelectrodes has confirmed that earlier deduction. Acidosis reduces the slow calcium current and the release of calcium from the sarcoplasmic reticumul but, because the cytosolic calcium does not fall, the major site of action of hydrogen ions appears to be on the calcium sensitivity of the contractile proteins. In man acidosis can be detected 15 s after the occlusion of a coronary artery and is a major mechanism for the simultaneous loss of contractility in ischaemia. A transient alkalosis is not detected in man but has been reported in isolated heart preparations where ATP consumption is low.An imposed mild respiratory acidosis during hypoxia increases the subsequent recovery of mechanical function on reoxygenation whereas a severe acidosis can be harmful. Acidosis in ischaemic may be advantageous due to a cardioplegic effect, inhibition of transsarcolemmal calcium fluxes or a reduction of mitochondrial calcium overload. Calcium uptake on reperfusion or reoxygenation has been linked to an inward movement of sodium in exchange for hydrogen ions on reperfusion and subsequent sodium-calcium exchange. Such a mechanism in its simplest form cannot account for the similar uptake of calcium on reoxygenation and reperfusion. Acidosis is a cause of early contractile failure in ischaemia but the role of acidosis in causing cell necrosis is not established.     

Effects of extracellular pH on the metabolic pathways in sulfur-deprived,H2-producing Chlamydomonas reinhardtii cultures

Sustained photoproduction of H(2) by the green alga, Chlamydomonas reinhardtii, can be obtained by incubating cells in sulfur-deprived medium [Ghirardi et al. (2000b) Trends Biotechnol. 18: 506; Melis et al. (2000) Plant Physiol. 122: 127]. The current work focuses on (a) the effects of different initial extracellular pHs on the inactivation of photosystem II (PSII) and O(2)-sensitive H(2)-production activity in sulfur-deprived algal cells and (b) the relationships among H(2)-production, photosynthetic, aerobic and anaerobic metabolisms under different pH regimens. The maximum rate and yield of H(2) production occur when the pH at the start of the sulfur deprivation period is 7.7 and decrease when the initial pH is lowered to 6.5 or increased to 8.2. The pH profile of hydrogen photoproduction correlates with that of the residual PSII activity (optimum pH 7.3-7.9), but not with the pH profiles of photosynthetic electron transport through photosystem I or of starch and protein degradation. In vitro hydrogenase activity over this pH range is much higher than the actual in situ rates of H(2) production, indicating that hydrogenase activity per se is not limiting. Starch and protein catabolisms generate formate, acetate and ethanol; contribute some reductant for H(2) photoproduction, as indicated by 3-(3,4-dichlorophenyl)-1,1-dimethylurea and 2,5-dibromo-6-isopropyl-3-methyl-1,4-benzoquinone inhibition results; and are the primary sources of reductant for respiratory processes that remove photosynthetically generated O(2). Carbon balances demonstrate that alternative metabolic pathways predominate at different pHs, and these depend on whether residual photosynthetic activity is present or not.     

Spatially resolved monitoring of cellular metabolic activity with a semiconductor-based biosensor

Metabolic activity of cultured cells can be monitored by measuring changes in the pH of the surrounding medium caused by metabolic products such as protons, carbon dioxide or lactic acid. Although many systems designed for this purpose have been reported, almost all of them are based on bulk measurements, where the average metabolic activity of all cells in contact with the device is recorded. Here, we report on a novel biosensor, based on a modified light-addressable potentiometric sensor (LAPS) device, which enables the metabolic activity of cultured cells to be measured with spatial resolution. This is demonstrated here by detecting the differential sensitivity to a cholinergic receptor agonist of two different co-cultured cellular populations. By making simultaneous measurements of the metabolic activity of different cell types seeded on different segments of one sensor, this device not only provides a rapid means of assessing cellular specificity of pharmaceutical compounds but also has the potential of being used to non-invasively monitor humoral as well as synaptic communication between different cell populations in co-culture. The temporal and spatial resolution of the device were investigated and are discussed.     

Interrelationship between sodium cyanate and pH in the regulation of tumor cell division

Previous studies have suggested that the selective inhibitory effects of sodium cyanate on tumor metabolism in vivo may be related to a lower interstitial pH in tumors. In the present work, the influence of extracellular pH on the actions of sodium cyanate was studied with one rat hepatoma cell line (HTC) and two human colon tumor cell lines (HT29 and LS174T) and with rat hepatocytes to determine if the effects are accompanied by changes in intracellular pH. With some tumor cells, an inhibition of cell proliferation was observed when the cells were exposed to an acidic medium (pH 6.6). However, the LS174T line of human tumor cells divided at pH 6.6 essentially as fast as at pH 7.4. In the concentration range of 0.02-0.1 mg/ml, a greater inhibitory effect of cyanate on cell proliferation was observed at the lower pH. Intracellular pH was found to be influenced by the sodium ion concentration of the medium to a similar degree in the three tumor lines that were examined. The intracellular pH was found to be significantly affected by cyanate in rat hepatocytes and in two of the tumor cell lines (HT29 and LS174T). The data suggested that not only does extracellular pH influence the inhibitory effect of cyanate on tumor cell proliferation but also that cyanate can affect the regulation of intracellular pH in normal and neoplastic cells.     

A 31P-NMR study on the recovery of intracellular pH in LLC-PK1/Cl4 cells from intracellular alkalinization

The regulation of intracellular pH (pHi) in a renal epithelial cell line, LLC-PK1/Cl4, during re-acidification from an alkaline load was studied by 31P-NMR. Intracellular alkalinization was induced by 10 mM ammonium glucuronate or by preloading with and subsequent removal of 20% CO2; the rate of re-acidification was found to be 0.047 pH units/min and 0.053 pH units/min, respectively. This rate of re-acidification was inhibited by 83% if Cl- was removed from the extracellular medium. A similar inhibition was found in the presence of 1 mM 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid (SITS) (76% inhibition) and 1 mM bumetanide (81% inhibition). No change in recovery was found after removing sodium from the extracellular medium, indicating that LLC-PK1/Cl4 cells recover from an intracellular alkaline load by a Cl-/HCO3- exchanger, which is SITS- and bumetanide-sensitive and has no requirement for sodium. In addition, the steady-state pHi in Cl4 cells was monitored by 31P-NMR. Removal of Cl- from the extracellular medium introduced an increase in pHi by 0.33 pH units, whereas 1 mM SITS and 1 mM bumetanide caused an increase in pHi by 0.14 or 0.13 pH units. In the presence of 1 mM amiloride, an inhibitor of the Na+/H+ exchanger, the steady-state pHi did not change significantly. These results indicate that at pHo 7.4 the steady-state intracellular pH of LLC-PK1/Cl4 cells strongly depends on the activity of the Cl-/HCO3- exchanger. Under the same conditions the activity of the Na+/H+ exchanger seems to be negligible.     

The mechanism of pH-dependent hydrogen peroxide cytotoxicity in vitro

The present paper is concerned with the influence of hydrogen ion concentration and composition of the medium on clonogenic survival of epithelial cells exposed to hydrogen peroxide in vitro. The survival of cells incubated with H2O2 in phosphate-buffered saline at pH 6.5 was 1 x 10(-2) and increased abruptly to 9 x 10(-2) at pH 7.0. The pH dependence of the cytocidal effect was particularly conspicuous when Eagle's minimum essential medium (SFMEM) was used for cell exposure to H2O2: the survival was characterized by exponential pH dependence and varied from 1 x 10(-1) to 9 x 10(-1) for pH 6.5 and 7.5, respectively, with a superimposed sharp peak value of 9 x 10(-1) at pH 7.0. The enhanced pH dependence of the H2O2 cytotoxicity in SFMEM was found to result from the additive action of glucose and histidine present in this medium. Glucose alone protected the cells with the efficiency decreasing with increasing hydrogen ion concentration. Histidine was responsible for the intermediate maximum in the pH-dependent survival spectrum. In addition, the changes in cell survival were accompanied by pH-dependent release of GSSG from the exposed cells. The GSSG efflux was inhibited by glucose in the medium. The influence of glucose on both the pattern of cell survival and the associated GSSG release indicate that the glutathione peroxidase activity supported by the pentose phosphate pathway is crucial in cell protection against extracellular H2O2 toxicity.     

INFLUENCE OF TEMPERATURE AND HYDROGEN ION CONCENTRATION UPON THE SPORE CYCLE OF BACILLUS SUBTILIS

1. At 5°C. no germination took place. 2. At 25°C. and at 37°C. germination occurs if the hydrogen ion concentration of the broth is kept between pH 5 and pH 10, but not at higher or lower pH values. 3. The completion of the spore cycle likewise requires a hydrogen ion concentration between pH 5 and pH 10. 4. The spores can germinate when the pH value is 10, although after germination the vegetative cells multiply only to a very slight extent and soon pass into spores. 5. The slight growth and multiplication of vegetative cells in broth of pH 10 suggest that the formation of endospores in this medium must be caused largely by the unfavorable reaction of the medium rather than by the accumulation of metabolic products. 6. Automatic adjustment of the medium seems to play a rôle in the completion of the spore cycle. 7. The results are not only of theoretical importance but they have a practical application to the preservation of food by canning and by other methods.     

Role of the Na/H antiport in pH-dependent cell death in pulmonary artery endothelial cells

We investigated the role of intracellular pH (pH(i)) and Na/H exchange in cell death in human pulmonary artery endothelial cells (HPAEC) following a metabolic insult (inhibition-oxidative phosphorylation, glycolysis). Metabolic inhibition in medium at pH 7. 4 decreased viability (0-15% live cells) over 6 h. Cell death was attenuated by maneuvers that decreased pH(i) and inhibited Na/H exchange (acidosis, Na/H antiport inhibitors). In contrast, cell death was potentiated by maneuvers that elevated pH(i) or increased Na/H exchange (monensin, phorbol ester treatment) before the insult. HPAEC demonstrated a biphasic pH(i) response following a metabolic insult. An initial decrease in pH(i) was followed by a return to baseline over 60 min. Maneuvers that protected HPAEC and inhibited Na/H exchange (acidosis, Na(+)-free medium, antiport inhibitors) altered this pattern. pH(i) decreased, but no recovery was observed, suggesting that the return of pH(i) to normal was mediated by antiport activation. Although Na/H antiport activity was reduced (55-60% of control) following a metabolic insult, the cells still demonstrated active Na/H exchange despite significant ATP depletion. Phorbol ester pretreatment, which potentiated cell death, increased Na/H antiport activity above the level observed in monolayers subjected to a metabolic insult alone. These results demonstrate that HPAEC undergo a pH-dependent loss of viability linked to active Na/H exchange following a metabolic insult. Potentiation of cell death with phorbol ester treatment suggests that this cell death pathway involves protein kinase C-mediated phosphorylation events.     

Growth, metabolic, and antibody production kinetics of hybridoma cell culture: 1. Analysis of data from controlled batch reactors.

A mouse-mouse hybridoma cell line (167.4G5.3) was cultivated in a 1.5-L stirred-tank bioreactor under constant pH and dissolved oxygen concentration. The transient kinetics of cell growth, metabolism, and antibody production were followed by biochemical and flow cytometric methods. The cell-specific kinetic parameters (growth and metabolic rates) as well as cell size were constant throughout the exponential phase. Intracellular protein and RNA content followed a similar trend. Cell growth stopped when the glutamine in the medium was depleted. Glucose could not substitute for glutamine, as glucose consumption ceased after glutamine depletion. Ammonia and lactate production followed closely glutamine and glucose consumption, respectively. Alanine, glutamate, serine, and glycine were produced but other amino acids were consumed. The cells are estimated to obtain about 45% of the total energy from glycolysis, with the balance of the metabolic energy provided by oxidative phosphorylation. The antibody was produced at a constant rate in both the exponential and decline phases of growth. The intracellular antibody content of the cells remained relatively constant during the exponential phase of growth and decreased slightly afterwards.     

Choice of DMEM, formulated with or without pyruvate, plays an important role in assessing the in vitro cytotoxicity of oxidants and prooxidant nutraceuticals

There is much interest in the positive health effects of nutraceuticals, in particular, polyphenols, which have both antioxidant and prooxidant characteristics. Pyruvate, a scavenger of hydrogen peroxide, is a component in some, but not in all, commercial formulations of cell culture media, Dulbecco’s modified Eagle’s medium in particular. This study showed that the cytotoxicities to human fibroblasts of hydrogen peroxide, tert-butyl hydroperoxide, and various prooxidant nutraceuticals were lessened in Dulbecco’s modified Eagle’s medium formulated with pyruvate, as compared to the same medium but formulated without pyruvate. Intracellular glutathione was unaffected in cells treated with hydrogen peroxide in Dulbecco’s modified Eagle’s medium formulated with pyruvate, as compared to medium formulated without pyruvate. In these studies, intracellular glutathione was analyzed in acid-soluble cell extracts by determining the oxidation of reduced glutathione by 5,5′-dithiobis(2-nitrobenzoic acid) to glutathione disulfide, with the formation of the yellow chromagen, 5-thio-2-nitrobenzoic acid, measured spectrophotometrically at 412 nm and by the visualization of reduced glutathione in cells stained with the fluorescent dye, Cell Tracker? Green 5-chloromethylfluorescein diacetate. A survey of various cell culture media, formulated with and without pyruvate, confirmed that the level of added hydrogen peroxide was greatly lessened in those media formulated with pyruvate. This study suggested that the pyruvate status of Dulbecco’s modified Eagle’s medium be specified in the experimental design, especially in studies involving oxidative stress.     

Some Aspects of Growth and Metabolism of Paul's Scarlet Rose Cell Suspensions

The growth pattern of suspension cultures of Paul’s Scarletrose cells has been examined. No distinct phases of cell divisionand cell expanison could be recognized. The 2-day lag phaseobserved after inoculation of stationary-phase cells into freshmedium was followed by a rapid entry into exponential growthduring which a mean cell-generation time of 36 h was recorded.Metabolic development assessed in terms of respiratory activityand RNA, DNA, and protein accumulation has been related to thephases of the growth cycle. A high metabolic activity developsimmediately after inoculation reaching a peak by early-exponentialphase. This activity sustains exponential growth until factorsin the medium become limiting. The pattern of DNA accumulationis closely correlated with increase in cell number and freshweight whereas changes in RNA levels are accompanied by similarchanges in protein levels and respiratory activity. The accumulationof phenolics follows a pattern unlike those of the other parametersexamined.     

Reduction of intracellular pH inhibits constitutive expression of cyclooxygenase-2 in human colon cancer cells

Cyclooxygenase-2 (COX-2) over-expression is critically involved in tumor formation. Intracellular pH (pHi) has been shown to be alkaline in cancer cells, and to be an important trigger for cell proliferation. This study therefore analyzed the relationship between pHi and COX-2 expression. HRT-18 and Caco-2 cells cultured in medium with bicarbonate maintained a pHi of approximately 7.6, which is higher than that of non-neoplastic cells. Cells grown in bicarbonate-free medium with a pH at 6.8 showed a reduction in pHi to approximately 7.0. Importantly, reduction of pHi resulted in a complete inhibition of COX-2 mRNA and protein expression. When cells were grown in bicarbonate-supplemented medium at pH 6.8, pHi maintained at approximately 7.6 and COX-2 expression was not inhibited. Additionally, analysis utilizing protein synthesis inhibitor cycloheximide demonstrated that pHi mediated inhibition of COX-2 mRNA expression requires de novo protein synthesis of regulatory protein(s). These data strongly suggest that an alkaline pHi is an important trigger for constitutive COX-2 expression. Defining pHi-mediated mechanisms that govern the constitutive COX-2 expression may help in developing new strategies to block COX-2 over-expression in cancer cells.     

Secretion of Sucrase by Leishmania donovani

ABSTRACT. Leishmania donovani promastigotes were collected, washed, resuspended in buffer, and assayed for sucrase activity. No activity was observed in the intact washed cells, but activity was measurable when the cells were permeabilized with Triton X-100. Intracellular sucrase activity was highest in promastigotes grown at pH 7.4, somewhat lower in promastigotes grown at pH 5.5, and significantly lower in "amastigotes" grown at pH 5.5. No trehalase, lactase, or maltase activities were observed. Assay of the medium in which the cells had grown showed that most the sucrase activity was extracellular, i.e. was secreted into the medium during growth.     

Buffer capacity of cotton cells and effects of extracellular pH on growth and somatic embryogenesis in cotton cell suspensions

Summary This research was designed to: a) characterize the normal pH changes that occur when cotton cell are grown in culture; b) determine if cotton cells can regulate the pH of their extracellular medium; and c) explore the effects of starting pH on cellular differentiation in culture, including formation of somatic embryos. When an aliquot of cotton cell suspension culture (Gossypium hirsutum L. cv Coker 312) was inoculated into fresh Murashige and Skoog (MS) medium at pH 4.5, the pH stabilized near 5.5 during the log phase of growth and then rose to pH 7.25. Cotton cells actively adjust medium with initial pH between 3 and 8 to near pH 5.5 in the early culture period. By acid/base titration, it has been shown that living cotton cells increase the buffer capacity of water and MS medium. Therefore, the metabolic activity of living cells accounts for the adjustment and stabilization of pH during the log phase of growth. The starting pH of the culture medium affects longterm viability, growth, and differentiation of the cells; pH 3 to 5 is best for cell viability, pH 3 to 4 enhances cell elongation; and pH 4, 7, or 8 stimulates somatic embryogenesis. Cultured cotton cells and the pH of their extracellular medium are in a complex, interactive relationship. This study was supported by the Texas Advanced Technology Program and the USDA-ARS. The journal no. for this paper is T-4-280, The College of Agriculture, Texas Tech University.     

Biodegradation of soil humic acids by Streptomyces viridosporus.

Biodegradation of soil humic acids by Streptomyces viridosporus ATCC 39115 growing in a mineral salts--glucose medium was demonstrated. This biodegradation accompanies bacterial growth and is, therefore, presumed to be a primary metabolic activity, but humic acids were not used as the sole source of carbon. This bacterial activity was enhanced when cells were shaken and within a pH range of 6.5-8.5. In further experiments, the relative abilities of S. viridosporus to mineralized [14C]melanoidin, used as synthetic humic acid, were also established. In contrast to the white rot fungus Phanerochaete chrysosporium, another microorganism exhibiting humic acid degrading activity at acidic pH, poor extracellular activities were found in culture medium of S. viridosporus, and veratryl alcohol does not result in increased humic acid degradation. In spite of some peroxidase activity measured in culture filtrates and analyzed by polyacrylamide gel electrophoresis, the humic acid degrading system of S. viridosporus, in these experimental conditions, seems to be cell associated.     

The landscape of metabolic pathway dependencies in cancer cell lines

The metabolic reprogramming of cancer cells creates metabolic vulnerabilities that can be therapeutically targeted. However, our understanding of metabolic dependencies and the pathway crosstalk that creates these vulnerabilities in cancer cells remains incomplete. Here, by integrating gene expression data with genetic loss-of-function and pharmacological screening data from hundreds of cancer cell lines, we identified metabolic vulnerabilities at the level of pathways rather than individual genes. This approach revealed that metabolic pathway dependencies are highly context-specific such that cancer cells are vulnerable to inhibition of one metabolic pathway only when activity of another metabolic pathway is altered. Notably, we also found that the no single metabolic pathway was universally essential, suggesting that cancer cells are not invariably dependent on any metabolic pathway. In addition, we confirmed that cell culture medium is a major confounding factor for the analysis of metabolic pathway vulnerabilities. Nevertheless, we found robust associations between metabolic pathway activity and sensitivity to clinically approved drugs that were independent of cell culture medium. Lastly, we used parallel integration of pharmacological and genetic dependency data to confidently identify metabolic pathway vulnerabilities. Taken together, this study serves as a comprehensive characterization of the landscape of metabolic pathway vulnerabilities in cancer cell lines.     

Intracellular pH regulation in a nonmalignant and a derived malignant human breast cell line

Tumor cells in vivo often exist in an ischemic microenvironment that would compromise the growth of normal cells. To minimize intracellular acidification under these conditions, these cells are thought to upregulate H(+) transport mechanisms and/or slow the rate at which metabolic processes generate intracellular protons. Proton extrusion has been compared under identical conditions in two closely related human breast cell lines: nonmalignant but immortalized HMT-3522/S1 and malignant HMT-3522/T4-2 cells derived from them. Only the latter were capable of tumor formation in host animals or long-term growth in a low-pH medium designed to mimic conditions in many solid tumors. However, detailed study of the dynamics of proton extrusion in the two cell lines revealed no significant differences. Thus, even though the ability to upregulate proton extrusion in a low pH environment (pH(e)) may be important for cell survival in a tumor, this ability is not acquired along with the capacity to form solid tumors and is not unique to the transformed cell. This conclusion was based on fluorescence measurements of intracellular pH (pH(i)) on cells that were plated on extracellular matrix, allowing them to remain adherent to proteins to which they had become attached 24 to 48 h earlier. Proton translocation under conditions of low pH(e) was observed by monitoring pH(i) after exposing cells to an acute acidification of the surrounding medium. Proton translocation at normal pH(e) was measured by monitoring the recovery after introduction of an intracellular proton load by treatment with ammonium chloride. Even in the presence of inhibitors of the three major mechanisms of proton translocation (sodium-proton antiport, bicarbonate transport, and proton-lactate symport) together with acidification of their medium, cells showed only about 0.4 units of reduction in pH(i). This was attributed to a slowing of metabolic proton generation because the inhibitors were shown to be effective when the same cells were given an intracellular acidification.     

The pH dependence of disobutamide-induced clear cytoplasmic vacuoles in cultured cells

Cellular uptake of disobutamide (D), and clear cytoplasmic vacuoles (CCV) induction by D in cultured rat urinary bladder carcinoma cells were dependent on the culture medium pH. At pH 6.0-6.7, drug uptake was slow and no CCV formed in 24 hr. At pH 7.0-8.0, the rate of D uptake and early appearance of CCV were directly proportional to increased basicity. This was explained by the increasing fraction of un-ionized D molecules at increasing basicity of the culture medium. It is only these electrically neutral D molecules which can penetrate the lipoidal cell membrane to induce formation of CCV. Intracellular presence of D was demonstrated by mass spectrometry methods. The results indicate that D is incorporated intracellularly, that D and not its metabolite(s) is in cells, and suggest that CCV are a result of drug sequesteration.     

Effect of pH on the calcium metabolism of isolated rat kidney cells

Summary The effects of metabolic and respiratory acidosis and alkalosis on cellular calcium metabolism were studied in rat kidney cells dispersed with collagenase. In both types of acidosis, the intracellular pH, total cell calcium, and the cell relative radioactivity after 60 min of labeling are significantly depressed. Kinetic analysis of⁴⁵Ca desaturation curves shows that acidosis decreases all three cellular calcium pools and depresses calcium fluxes between the superficial and cytosolic pools and between the cytosolic and mitochondrial pools. In alkalosis the intracelluar pH, the total cell calcium, and the cell relative radioactivity are significantly increased. Kinetic studies show that in alkalosis, only the mitochondrial pool is consistently increased. Calcium exchange between the mitochondrial and cytosolic pool is increased in metabolic alkalosis only. These results suggest that hydrogen ion is an important modulator of calcium metabolism, and that the intracellular pH rather than extracellular pH is the critical factor in determining the calcium status of cells during altered acid-base conditions.