Acidification Of Extracellular pH Research Articles

Expression Dynamics of CA IX Epitope in Cancer Cells under Intermittent Hypoxia Correlates with Extracellular pH Drop and Cell Killing by Ureido-Sulfonamide CA IX Inhibitors.

Carbonic anhydrase IX (CA IX) is a membrane-bound CA isozyme over-expressed in many hypoxic tumor cells, where it ensures pH homeostasis and has been implicated in tumor survival, metastasis and resistance to chemotherapy and radiotherapy. Given the functional importance of CA IX in tumor biochemistry, we investigated the expression dynamics of CA IX in normoxia, hypoxia and intermittent hypoxia, which are typical conditions experienced by tumor cells in aggressive carcinomas. We correlated the CA IX epitope expression dynamics with extracellular pH acidification and with viability of CA IX-expressing cancer cells upon treatment with CA IX inhibitors (CAIs) in colon HT-29, breast MDA-MB-231 and ovarian SKOV-3 tumor cell models. We observed that the CA IX epitope expressed under hypoxia by these cancer cells is retained in a significant amount upon reoxygenation, probably to preserve their proliferation ability. The extracellular pH drop correlated well with the level of CA IX expression, with the intermittent hypoxic cells showing a similar pH drop to fully hypoxic ones. All cancer cells showed higher sensitivity to CA IX inhibitors (CAIs) under hypoxia as compared to normoxia. The tumor cell sensitivity to CAIs under hypoxia and intermittent hypoxia were similar and higher than in normoxia and appeared to be correlated with the lipophilicity of the CAI.

Impact of tumor microenvironment on adoptive T cell transfer activity.

Recent advances in immunotherapy have revolutionized the treatment of cancer. The use of adoptive cell therapies (ACT) such as those based on tumor infiltrating lymphocytes (TILs) or genetically modified cells (transgenic TCR lymphocytes or CAR-T cells), has shown impressive results in the treatment of several types of cancers. However, cancer cells can exploit mechanisms to escape from immunosurveillance resulting in many patients not responding to these therapies or respond only transiently. The failure of immunotherapy to achieve long-term tumor control is multifactorial. On the one hand, only a limited percentage of the transferred lymphocytes is capable of circulating through the bloodstream, interacting and crossing the tumor endothelium to infiltrate the tumor. Metabolic competition, excessive glucose consumption, the high level of lactic acid secretion and the extracellular pH acidification, the shortage of essential amino acids, the hypoxic conditions or the accumulation of fatty acids in the tumor microenvironment (TME), greatly hinder the anti-tumor activity of the immune cells in ACT therapy strategies. Therefore, there is a new trend in immunotherapy research that seeks to unravel the fundamental biology that underpins the response to therapy and identifies new approaches to better amplify the efficacy of immunotherapies. In this review we address important aspects that may significantly affect the efficacy of ACT, indicating also the therapeutic alternatives that are currently being implemented to overcome these drawbacks.

Abstract 4987: ASS1 downregulation facilitates cancer cell survival in acidic environment

Abstract ”pH gradient reversal”, namely the alkalinization of intracellular pH (pHi) and acidification of extracellular pH (pHe), is a unique phenotype of cancer cells which enable cancer survival, migration and invasion. Hence, not surprisingly, pH gradient reversal is emerging as a new hallmark of cancer. Argininosuccinate synthase, ASS1, is a urea cycle enzyme found to be downregulated by methylation in multiple different types of cancers in correlation with increased metastasis and poor prognosis. We recently showed that ASS1 downregulation increases proliferation by channeling aspartate towards pyrimidine synthesis by CAD. We now show that under hypoxia and acidic environments, ASS1 is downregulated together with CAD by HIF1α as part of an essential network necessary for cancer cells’ survival, invasion and migration. Similar to germline deficiency of ASS1 which increases the levels of glutamine and ammonia, ASS1 downregulation by HIF1αenables the cancer cells to increase glutamine levels allowing its utilization for glutathione and importantly, for preservation of alkali pHi by elevating ammonia levels and buffering the acidity caused by cancers’ enhanced glycolysis and the consequent increase in lactic acid. Moreover, cancer cells which express ASS1 when grown in culture, downregulate its expression in hypoxic regions when grown in vivo, suggesting that these cancers might also be sensitive to drugs previously utilized only in ASS1 deficient cancers. Finally, we show that treating cancers with ASS1 deficiency with ammonia scavengers as given to ASS1 deficient patients, increases apoptosis and restricts migration, and is hence beneficial therapeutically. In summary, ASS1 downregulation is regulated differently at altered carcinogenic states, promoting proliferation as well as cancer survival and metastasis. Citation Format: Ayelet Erez, Alon Silberman, Adi Jacob, Shiran Limanovich, Odeya Boukobza1, Erez Persi, Rom Keshet, Alona Sarver, Joo Sang Lee, Alexander Brandis, Keren Bahar Halpern, Raya Eilam, Ben Pode, Yair Anikster, Igor Ulitsky, Sandesh Nagamani, Eytan Ruppin. ASS1 downregulation facilitates cancer cell survival in acidic environment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4987. doi:10.1158/1538-7445.AM2017-4987

In vivo evaluation of tumour acidosis for assessing the early metabolic response and onset of resistance to dichloroacetate by using magnetic resonance pH imaging.

Dichloroacetate (DCA) can reverse the glycolytic phenotype that is responsible of increased lactate production and extracellular pH acidification in cancer cells. Magnetic resonance imaging-chemical exchange saturation transfer (MRI-CEST) pH mapping is a novel non-invasive imaging approach that can measure invivo extracellular tumour pH. We examined whether MRI-CEST pH mapping can monitor invivo changes in tumour acidosis for assessing treatment response to DCA. Cell viability and extracellular pH were assessed in TS/A breast cancer cells treated with 1-10mM DCA for 24h in normoxia or hypoxia (1% O2) conditions. Extracellular tumour pH values were measured invivo by MRI-CEST pH mapping of TS/A tumour-bearing mice before, three days and fifteen days after DCA or saline treatment. Reduced extracellular acidification and vitality were observed in DCA-treated TS/A cells. Tumour-bearing mice showed a marked and significant increase of tumour extracellular pH at 3 days post-DCA treatment, reflecting DCA-induced glycolysis inhibition, as confirmed by reduced lactate production. After 15 days of DCA treatment, the onset of resistance to DCA was observed, with recover of tumour extracellular acidification and lactate levels that returned to baseline values. A significant correlation was observed between tumour extracellular pH values and lactate levels (r=-0.97, P<0.05). These results suggest that MRI-CEST pH imaging is a promising tool to monitor the early response and efficacy of cancer metabolic targeting drugs.

ASIC1a Deficient Mice Show Unaltered Neurodegeneration in the Subacute MPTP Model of Parkinson Disease.

Inflammation contributes to the death of dopaminergic neurons in Parkinson disease and can be accompanied by acidification of extracellular pH, which may activate acid-sensing ion channels (ASIC). Accordingly, amiloride, a non-selective inhibitor of ASIC, was protective in an acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson disease. To complement these findings we determined MPTP toxicity in mice deficient for ASIC1a, the most common ASIC isoform in neurons. MPTP was applied i.p. in doses of 30 mg per kg on five consecutive days. We determined the number of dopaminergic neurons in the substantia nigra, assayed by stereological counting 14 days after the last MPTP injection, the number of Nissl positive neurons in the substantia nigra, and the concentration of catecholamines in the striatum. There was no difference between ASIC1a-deficient mice and wildtype controls. We are therefore not able to confirm that ASIC1a are involved in MPTP toxicity. The difference might relate to the subacute MPTP model we used, which more closely resembles the pathogenesis of Parkinson disease, or to further targets of amiloride.

Presence of an H+/Quinidine Antiport System in Madin-Darby Canine Kidney Cells.

We have recently found an H+/quinidine antiport system in human kidney HEK 293 cells. The aim of the present study was to evaluate whether the H+/quinidine antiport system is expressed in Madin-Darby canine kidney (MDCK) cells. We investigated the uptake and efflux of quinidine in MDCK cells. The uptake of 100µM quinidine into MDCK cells was decreased by acidification of extracellular pH or alkalization of intracellular pH. In addition, the uptake of quinidine was highly temperature sensitive, but was extracellular Na+ and membrane potential independent. Furthermore, tetraethylammonium, a typical substrate of renal organic cation transporters, did not inhibit the uptake of quinidine in MDCK cells. On the other hand, lipophilic cationic drugs, such as clonidine, bisoprolol, diphenhydramine, pyrilamine, and imipramine, significantly decreased the uptake of quinidine in MDCK cells. The uptake of quinidine was saturable, and the Michaelis-Menten constant was estimated to be approximately 0.5mM. In addition, the efflux of quinidine from MDCK cells was increased by the acidification of extracellular pH, suggesting that the transport system mediates not only the uptake, but also secretion of quinidine. The present findings suggested that the renal new antiport system is involved in the bidirectional membrane transport of quinidine in MDCK cells.

PH Gradient Reversal: An Emerging Hallmark of Cancers.

Several tumors exhibit pH gradient reversal, with acidification of extracellular pH (pHe) and alkalinization of intracellular pH (pHi). The pH gradient reversal is evident even during the preliminary stages of tumorigenesis and is crucial for survival and propagation of tumors, irrespective of their pathology, genetics and origins. Moreover, this hallmark seems to be present ubiquitously in all malignant tumors. Based on these facts, we propose a new emerging hallmark of cancer "pH gradient reversal". Normalizing pH gradient reversal through inhibition of various proton transporters such as Na(+)-H(+) exchanger (NHE), Vacuolar-type H(+)-ATPase (V-ATPase), H(+)/K(+)-ATPases and carbonic anhydrases (CAs) has demonstrated substantial therapeutic benefits. Indeed, inhibition of NHE1 is now being regarded as the latest concept in cancer treatment. A recent patent deals with the utilization of cis-Urocanic acid to acidify the pHi and induce apoptosis in tumors. Another patent reports therapeutic benefit by inhibiting Lactate Dehydrogenase - 5 (LDH-5) in various cancers. Several patents have been formulated by designing drugs activated through acidic pHe providing a cancer specific action. The purpose of this review is to analyze the available literature and help design selective therapies that could be a valuable adjunct to the conventional therapies or even replace them.

Evaluating tumor metastatic potential by imaging intratumoral acidosis via pH-activatable near-infrared fluorescent probe.

Metastasis accounts for the vast majority of cancer deaths. To minimize metastasis-associated mortality, it is crucially important to evaluate the metastatic potential (M.P.), that is, defined as a tendency of a primary tumor to colonize a distant organ. Dysregulated pH in solid tumors, especially the acidification of extracellular pH (pHe ) promotes dormant metastasis by driving protease-mediated digestion, disrupting cell-matrix interaction and increasing migration of cancer cells. Therefore, imaging intratumoral acidosis creates a unique opportunity to evaluate the M.P. In this work, a novel pH activatable probe was developed, in which two near-infrared (NIR) fluorophores were conjugated via a flexible and acid liable linkage. While the fluorescence of this probe is quenched due to intramolecular dimeric aggregate under neutral environment, the cleavage of pH liable linkage with the concomitant disruption of aggregates in acidic tumor microenvironment results in a remarkable fluorescence enhancement. This probe not only visualized the primary tumors with high target to background (T/B) signal ratio in vivo, but also revealed the correlation between the M.P. and acidosis distribution pattern in tumor. While the acidosis locate dispersedly at tumor periphery in highly metastatic tumor, it distribute more widely in lowly metastatic tumor and the acidification degree increases substantially from the margin to core areas. This pH activatable NIR fluorescent probe holds the potential to evaluate the M.P., monitor the therapeutic response and predict the prognosis by delineating acidosis in tumors.

Chemical rescue of ΔF508-CFTR in C127 epithelial cells reverses aberrant extracellular pH acidification to wild-type alkalization as monitored by microphysiometry

Cystic fibrosis (CF) is caused by mutations in the gene for CFTR, a cAMP-activated anion channel expressed in apical membranes of wet epithelia. Since CFTR is permeable to HCO3−, and may regulate bicarbonate exchangers, it is not surprising evidence of changes in extracellular pH (pHo) have been found in CF. Previously we have shown that tracking pHo can be used to differentiate cells expressing wild-type CFTR from controls in mouse mammary epithelial (C127) and fibroblast (NIH/3T3) cell lines. In this study we characterized forskolin-stimulated extracellular acidification rates in epithelia where chemical correction of mutant ΔF508-CFTR converted an aberrant response in acidification (10%+ increase) to wild-type (25%+ decrease). Thus treatment with corrector (10% glycerol) and the resulting increased expression of ΔF508-CFTR at the surface was detected by microphysiometry as a significant reversal from acidification to alkalization of pHo. These results suggest that CFTR activation as well as correction can be detected by carefully monitoring pHo and support findings in the field that extracellular pH acidification may impact the function of airway surface liquid in CF.

Acute Effects of β-Amyloid (1-42) Oligomers on Rat Pyramidal Entorhinal Neurons

Deposition of beta-amyloid peptide (Aβ) in senile plaques is the hallmark of Alzheimer disease neuropathology. Soluble Aβ oligomers block voltage-gated ion channels as P/Q type calcium channels (Nimmrich et al., 2008) as well as the transient potassium current (Liudyno et al., 2012) and calcium dependent potassium channels, BK type (Yamamoto et al., 2011). Exposure to Aβ may cause a loss of cellular calcium homeostasis, but the mechanism by which this occurs is uncertain. In this work we evaluated the acute response of rat pyramidal entorhinal neurons to oligomers formed from purified Aβ1-42 in both entorhinal cortex slice preparations and isolated pyramidal neurons in culture using current- and voltage-clamp conditions. Exposure to Aβ oligomers but not monomers increased the input resistance and enlarged the action potential in the slice preparation, whereas in culture neurons produced a reversible inhibition of the inward potassium current generated by voltage ramps from −70 to 70 mV in symmetric potassium conditions. This current is generated by inward rectifier potassium channels as well as the leak potassium channels, it was blocked by barium, arachidonic acid, bupivacaine and extracellular pH acidification, suggesting that TASK type K2P channels are targets of the toxic effects of Aβ. Blockage of potassium channels by Aβ could lead to prolonged cell depolarization, thereby increasing calcium influx.Supported by CONACyT (324341) Mexico.

The effects of CA IX catalysis products within tumor microenvironment

Solid tumors are composed of both cancer cells and various types of accessory cells, mainly fibroblasts, that collectively compose the so called tumor-microenvironment. Cancer-associated fibroblasts have been described to actively participate in cancer progression by establishing a cytokine-mediated as well as metabolic crosstalk with cancer cells. In the present paper we show that activated human fibroblasts are able to boost tumor cells proliferation and that this effect is greatly dependent on stromal carbonic anhydrase IX (CA IX) activity. In fact fibroblasts show a strong upregulation of CA IX expression upon activation by cancer cells, while CA IX products, protons and bicarbonate, exert differential effects on cancer cells proliferation. While acidification of extracellular pH, a typical condition of rapidly growing solid tumors, is detrimental for tumor cells proliferation, bicarbonate, through its organication, supplies cancer cells with intermediates useful to sustain their high proliferation rate. Here we propose a new kind of fibroblasts/tumor cells crosstalk within tumor microenvironment, mediated by stromal CA IX products, aimed to favor cancer cells growth, opening new perspectives on CA IX role in tumor microenvironment.

Carbonic Anhydrase IX Interacts with Bicarbonate Transporters in Lamellipodia and Increases Cell Migration via Its Catalytic Domain

Carbonic anhydrase IX (CA IX) is a hypoxia-induced cell surface enzyme expressed in solid tumors, and functionally involved in acidification of extracellular pH and destabilization of intercellular contacts. Since both extracellular acidosis and reduced cell adhesion facilitate invasion and metastasis, we investigated the role of CA IX in cell migration, which promotes the metastatic cascade. As demonstrated here, ectopically expressed CA IX increases scattering, wound healing and transwell migration of MDCK cells, while an inactive CA IX variant lacking the catalytic domain (ΔCA) fails to do so. Correspondingly, hypoxic HeLa cells exhibit diminished migration upon inactivation of the endogenous CA IX either by forced expression of the dominant-negative ΔCA variant or by treatment with CA inhibitor, implying that the catalytic activity is indispensable for the CA IX function. Interestingly, CA IX improves cell migration both in the absence and presence of hepatocyte growth factor (HGF), an established inducer of epithelial-mesenchymal transition. On the other hand, HGF up-regulates CA IX transcription and triggers CA IX protein accumulation at the leading edge of lamellipodia. In these membrane regions CA IX co-localizes with sodium bicarbonate co-transporter (NBCe1) and anion exchanger 2 (AE2) that are both components of the migration apparatus and form bicarbonate transport metabolon with CA IX. Moreover, CA IX physically interacts with AE2 and NBCe1 in situ, as shown here for the first time. Thus, our findings suggest that CA IX actively contributes to cell migration via its ability to facilitate ion transport and pH control at protruding fronts of moving cells.

Monoamine carboxylate transporters are involved in BI-1-associated cancer metastasis in HT1080 colon fibrosarcoma cells

The overexpression of BI-1 induces an acidic extracellular pH due to alterations of mitochondrial function, leading to cancer metastasis through anaerobic glucose metabolism. In this study, ion transporters such as sodium hydrogen exchangers (NHE) and monoamine carboxylate transporters (MCTs) were studied in BI-1-overexpressing HT1080 cells (BI-1 cells). The extracellular pH became acidic as culture time of BI-1 cells increased, while intracellular pH stayed relatively stable at pH 7.2. The expression of MCTs increased in BI-1 cells as culture time passed. 5-(N-ethyl-N-isopropyl) amiloride or dimethylamiloride, NHE inhibitor, abrogated the elevated MCT expression, indicating that MCT followed NHE activation. An MCT inhibitor, lonidamine, regulated the acidification of extracellular pH, also inhibiting both increased cancer cell migration and infiltration and MMP2/9 activity. The inhibition of either NHE or MCT affected the intracellular pH, leading to a severely acidic intracellular pH and cell death. In BI-1 cells, the activation of ion transporters such as NHE or MCT may offer a survival strategy against metabolism-associated acidic stresses. These findings suggest that BI-1 can lead cancer invasion and metastasis by inducing extracellular environment acidic.

Cytosolic pH and the inflammatory microenvironment modulate cell death in human neutrophils after phagocytosis

AbstractFollowing phagocytosis in vivo, acidification of extracellular pH (pHo) and intracellular metabolic acid generation contribute to cytosolic proton loading in neutrophils. Cytosolic pH (pHi) affects neutrophil function, although its regulation is incompletely understood. Its effect on mechanisms of neutrophil death is also uncertain. Thus, we investigated pHi regulation in Escherichia coli–exposed neutrophils, at various pathogen-to-phagocyte ratios (0:1-50:1), under conditions simulating the inflammatory milieu in vivo and correlated pHi changes with mechanisms of neutrophil death. Following phagocytosis, proton extrusion was dominated early by passive proton conductance channels, and later by Na+/H+ exchange (NHE). H+-translocating adenosine triphosphatase (V-ATPase) pHi regulation was evident primarily at lower bacterial densities. At physiologic pHo, lower pathogen-to-phagocyte ratios alkalinized pHi and inhibited apoptosis, whereas higher ratios acidified pHi (despite proton extrusive mechanisms) and promoted apoptosis. Necrosis was induced by high-density bacterial exposure at reduced pHo. Following phagocytosis, targeted inhibition of NHEs, proton conductance channels, or V-ATPases (amiloride, ZnCl2, or bafilomycin, respectively) moderately hyperacidified pHi and accelerated apoptosis. However, in combination they profoundly acidified pHi and induced necrosis. Proinflammatory mediators in vivo might affect both pHi regulation and cell death, so we tested the effects of bronchoalveolar lavage (BAL) fluid from patients with cystic fibrosis (CF) and healthy subjects. Only CF BAL fluid alkalinized pHi and suppressed apoptosis at physiologic pHo, but failed to prevent necrosis following phagocytosis at low pHo. Thus, a precarious balance between cytosolic proton loading and extrusion after phagocytosis dictates the mode of neutrophil cell death. pHi/pHo might be therapeutically targeted to limit neutrophil necrosis and protect host tissues during necrotizing infections.

Effects of long-term acidification of extracellular pH on ATP-induced calcium mobilization in rabbit lens epithelial cells.

ATP-induced calcium (Ca2+) mobilization was investigated in rabbit lens epithelial cells that had been cultured in a medium with pH of 7.4 (group 1), 7.2 (group 2), or 7.0 (group 3) for 10 to 21 d. Intracellular free Ca2+ ([Ca2+]i and pH (pHi) were measured by using fluorescent dyes, fura-2 and BCECF, respectively. The long-term acidification decreased the pHi to 7.15 +/- 0.01, from 7.22 +/- 0.01, in group 2 and to 7.09 +/- 0.01 in group 3. The administration of 10 micromol/l ATP produced an initial peak followed by a sustained increase in [Ca2+]i in the lens cells of group 1. Both the initial peak and the sustained increase in [Ca2+]i were enhanced in groups 2 and 3. The initial peak was abolished by pretreatment with 1 micromol/l thapsigargin, an ER Ca2+ pump inhibitor, but was not affected by the removal of extracellular Ca2+. On the other hand, the sustained increase was suppressed either by the thapsigargin treatment or by the Ca2+ removal. Treatment with only thapsigargin caused a sustained increase in [Ca2+]i that was greater in group 3 than in group 1. These results suggest that (1) the ATP-induced initial peak in [Ca2+]i is due to Ca2+ release from the intracellular stores, (2) the sustained increase in [Ca2+]i is mediated through either Ca2+ influx from the extracellular space or Ca2+ release from the store triggered by the Ca2+ influx, and (3) long-term, moderate acidification enhances both the initial peak and the sustained increase in [Ca2+)]i in rabbit lens epithelial cells. One possible mechanism of the ATP-induced Ca2+ influx seems to be a capacitative Ca2+ entry pathway.

Identification of an Aspartic Residue in the P-loop of the Vanilloid Receptor That Modulates Pore Properties

Vanilloid receptor subunit 1 (VR1) is a nonselective cation channel that integrates multiple pain-producing stimuli. VR1 channels are blocked with high efficacy by the well established noncompetitive antagonist ruthenium red and exhibit high permeability to divalent cations. The molecular determinants that define these functional properties remain elusive. We have addressed this question and evaluated by site-specific neutralization the contribution on pore properties of acidic residues located in the putative VR1 pore region. Mutant receptors expressed in Xenopus oocytes exhibited capsaicin-operated ionic currents akin to those of wild type channels. Incorporation of glutamine residues at Glu(648) and Glu(651) rendered minor effects on VR1 pore attributes, while Glu(636) slightly modulated pore blockade. In contrast, replacement of Asp(646) by asparagine decreased 10-fold ruthenium red blockade efficacy and reduced 4-fold the relative permeability of the divalent cation Mg(2+) with respect to Na(+) without changing the selectivity of monovalent cations. At variance with wild type channels and E636Q, E648Q, and E651Q mutant receptors, ruthenium red blockade of D646N mutants was weakly sensitive to extracellular pH acidification. Collectively, our results suggest that Asp(646) is a molecular determinant of VR1 pore properties and imply that this residue may form a ring of negative charges that structures a high affinity binding site for cationic molecules at the extracellular entryway.

Effects of fusicoccin and gibberellic acid on the germination of embryos from dormant barley grains: roles of starch degradation and external pH

AbstractAbstract In isolated embryos from dormant barley grains, synergistic effects of fusicoccin (FC) and gibberellic acid (GA3) were observed on the induction of α-amylase mRNA expression. However, no α-amylase mRNA expression could be induced by both agents in embryos from non-dormant grains. Both light- and electron-microscopy studies demonstrated that there were large numbers of starch granules present in mature embryos (mainly in scutellum) from dormant barley grains but none or almost none in embryos from non-dormant grains. Furthermore, the content of reducing sugars in embryos from dormant grains was about half of that from non-dormant grains. In contrast to GA3, FC was able to induce a strong acidification of extracellular pH (pHe). Clamping the pHeto prevent FC-induced acidification, by using 50 mM MES buffer (pH 5.6), caused an inhibition of GA3- or FC-induced α-amylase mRNA expression but did not affect the germination of embryos from dormant grains. In addition, in MES buffer, addition of FC or a combination of FC and GA3increased the germination rate of embryos isolated from dormant grains, though large numbers of starch granules were still present in these embryos. Based on these observations, the presence of starch granules and a low reducing sugar level in embryos from dormant grains is not a factor for control of grain dormancy and germination.

Proteolytic stability of recombinant human serum albumin secreted in the yeast Saccharomyces cerevisiae

In order to direct the persistent expression of recombinant human serum albumin (HSA) from the GAL10 promoter in the yeast Saccharomyces cerevisiae, we carried out periodic feeding of galactose during shake-flask cultures. Unexpectedly, the recombinant protein secreted was observed to undergo rapid degradation, which was apparently accelerated by carbon-source feeding. The extracellular degradation of HSA occurred even in the strain deficient in the major vacuolar proteases PrA and PrB, and in the strain lacking the acidic protease Yap3p (involved in the generation of HSA-truncated fragments). Interestingly, the degradation correlated closely with the acidification of extracellular pH and thus was significantly overcome either by buffering the culture medium above pH 5.0 or by adding amino acid-rich supplements to the culture medium, which could prevent the acidification of medium pH during cultivation. Addition of arginine or ammonium salt also substantially minimized the degradation of HSA, even without buffering. The extracellular degradation activity was not detected in the cell-free culture supernatant but was found to be associated with intact cells. The results of the present study strongly suggest that the HSA secreted in S. cerevisiae is highly susceptible to the pH-dependent proteolysis mediated by cell-bound protease(s) whose activity and expression are greatly affected by the composition of the medium.

Involvement of the proton pump and proton conductance change in the wave of depolarization induced by wounding in Bidens pilosa

The role of protons in the slow wave of depolarization induced by wounding in Bidens pilosa L. was studied by means of voltage measurements performed under differing extracellular conditions. The degree of depolarization caused by an extracellular pH acidification from 7 to 4 was different when performed either in the resting conditions or 20 min after eliciting the slow wave. In the presence of cyanide, a heat stimulation still induced a slow wave, the amplitude of which was reduced to 42% of the control. While cyanide remained in the perfusing medium, the membrane did not repolarize. An extracellular pH change from 7 to 4 in the presence of cyanide induced a higher depolarization in the resting conditions than 20 min after eliciting a slow wave. Furthermore, when pumps were previously stimulated either by acid loading or by fusicoccin, the amplitude of the slow wave was increased. We thus propose that the slow wave was largely dominated by a transient "switch off” of the proton pump and we discuss the possible involvement of proton conductance change during the slow wave. Key words: Bidens pilosa L., membrane potential, proton conductance, slow wave of depolarization, wounding.

Ion Efflux from Pulvinar Cells during Slow Downward Movement of the Petiole of Mimosa pudica L. Induced by Photostimulation

Change of extracellular ion concentration in the main pulvinus of Mimosa pudica L. during a slow downward movement induced by white light (0.24 mW cm(-2)) was investigated using K(+)- and Cl(-)-selective electrodes and a pH electrode. The photostimulation induced a transient and concurrent efflux of K(+) and Cl(-) from the motor cells and acidification of extracellular pH in the main pulvinus.