Anaerobic transport of amino acids coupled to the glycerol-3-phosphate-fumarate oxidoreductase system in a cytochrome-deficient mutant of Escherichia coli

The uptake of proline and glutamine by cytochrome-deficient cells of Escherichia coli SASX76 grown aerobically on glucose or anaerobically on pyruvate was stimulated by these two substrates. Pyruvate could not stimulate transport in the glucose-grown cells. Uptake of these amino acids energized by glucose was inhibited by inhibitors of the Ca²⁺, Mg²⁺-stimulated ATPase such as DCCD, pyrophosphate, and azide, and by the uncouplers CCCP and 2,4-dinitrophenol. Glycerol (or glycerol 3-phosphate) in the presence of fumarate stimulated the transport of proline and glutamine under anaerobic conditions in cytochrome-deficient cells but not in membrane vesicles prepared from these cells although glycerol 3-phosphate-fumarate oxidoreductase activity could be demonstrated in the vesicle preparation. In contrast, in vesicles prepared from cytochrome-containing cells of E. coli SASX76 amino acid transport was energized under anaerobic conditions by this system. Inhibitors of the Ca²⁺, Mg²⁺-activated ATPase and uncoupling agents inhibited the uptake of proline and glutamine in cytochrome-deficient cells dependent on the glycerol-fumarate oxidoreductase system. Ferricyanide could replace fumarate as an electron acceptor to permit transport of phenylalanine in cytochrome-deficient or cytochrome- containing cells under anaerobic conditions. It is concluded that in cytochrome-deficient cells using glucose, pyruvate, or glycerol in the presence of fumarate, transport of both proline and glutamine under anaerobic conditions is energized by ATP through the Ca²⁺, Mg²⁺-activated ATPase. In cytochrome-containing cells under anaerobic conditions electron transfer between glycerol and fumarate can also drive transport of these amino acids.     

Dramatic effect of glycolysis inhibition on the cerebellar granule cells bioenergetics

Cultured cerebellar granule cells were co-loaded with Ca²⁺-sensitive dye fura-2FF and rhodamine-123 sensitive to changes in the mitochondrial potential (????_m). A 60-min incubation of cells in glucose-free solution containing 2-deoxy-D-glucose (DG) induced a slow developing mitochondrial depolarization (sMD) without appreciable changes in basal [Ca²⁺]ⁱ. This sMD was insensitive to a removal of external Ca²⁺ or to the NMDA channels blocker memantine but could be readily suppressed by oligomycin due to inhibition of the inward proton current through the Fo channel of mitochondrial ATP synthase. In resting cells glucose deprivation caused a progressive decrease in mitochondrial NADH content ([NADH]), which strikingly enhanced the ability of glutamate to induce a delayed Ca²⁺ deregulation (DCD) associated with a profound mitochondrial depolarization. In glucose-containing medium this DCD appeared in young cells (usually 6?C8 days in vitro) after a prolonged latent period (lag phase). Substitution of glucose by DG led to a dramatic shortening of this lag phase, associated with a critical decrease in [NADH] in most neurons. Addition of pyruvate or lactate to DG-containing solution prevented the sMD and [NADH] decrease in resting cells and greatly diminished the number of cells exhibiting glutamate-induced DCD in glucose-free medium. Measurement of intracellular ATP level ([ATP]) in experiments on sister cells showed that glucose deprivation decreased [ATP] in resting cells and considerably deepened the fall of [ATP] caused by glutamate. This decrease in [ATP] was only slightly attenuated by pyruvate and lactate, despite their ability to prevent the shortening of lag phase preceding the DCD appearance under these conditions. Simultaneous monitoring of cytosolic ATP concentration ([ATP]_c) and ????_m changes in individual CGC expressing fluorescent ATP sensor (AT1.03) revealed that inhibition of either mitochondrial respiration or glycolysis caused a relatively small decrease in [ATP]_c and ????_m. Complete blockade of ATP synthesis in resting CGC with oligomycin in glucose-free DG-containing buffer caused fast ATP depletion and mitochondrial repolarization, indicating that mitochondrial respiratory chain still possess a reserve fuel to support ????_m despite inhibition of glycolysis. The data obtained suggest that the extraordinary enhancement of glutamate-induced deterioration in Ca²⁺ homeostasis caused by glucose deprivation in brain neurons is mainly determined by NADH depletion.     

The effect of calcium ions on the glycolytic activity of Ehrlich ascites-tumour cells

1. Added Ca²⁺ inhibited lactate formation from sugar phosphates by intact Ehrlich ascites-tumour cells. Lactate formation from glucose by these cells was unaffected by added Ca²⁺. 2. The Ca²⁺ inhibition of lactate formation by intact cells occurred in the extracellular medium. 3. Intact ascites-tumour cells did not take up Ca²⁺ in vitro. 4. Glycolysis of sugar phosphates by cell extracts as well as pyruvate formation from 3-phosphoglycerate and phosphoenolpyruvate was inhibited by Ca²⁺. 5. It was concluded that Ca²⁺ inhibited the pyruvate-kinase (EC 2.7.1.40) reaction. Further, Ca²⁺ inhibition of pyruvate kinase could be correlated with the overall inhibition of glycolysis. 6. Concentrations of Ca²⁺ usually present in Krebs–Ringer buffers, inhibited glycolysis and pyruvate-kinase activity by approx. 50%. 7. The inhibition of glycolysis by added Ca²⁺ could be partially reversed by K⁺ and completely reversed by Mg²⁺ or by stoicheiometric amounts of EDTA. 8. The hypothesis is advanced that the inability of tumour cells to take up Ca²⁺ is a factor contributing towards their high rate of glycolysis.     

Fed-batch culture optimization of a growth-associated hybridoma cell line in chemically defined protein-free media

An investigation was made to study the processes of fed-batch cultures of a hybridoma cell line in chemically defined protein-free media. First of all, a strong growth-associated pattern was correlated between the production of MAb and growth of cells through the kinetic studies of batch cultures, suggesting the potential effectiveness of extending the duration of exponential growth in the improvement of MAb titers. Second, compositions of amino acids in the feeding solution were balanced stepwisely according to their stoichiometrical correlations with glucose uptake in batch and fed-batch cultures. Moreover, a limiting factor screening revealed the constitutive nature of Ca²⁺ and Mg²⁺ for cell growth, and the importance of their feeding in fed-batch cultures. Finally, a fed-batch process was executed with a glucose uptake coupled feeding of balanced amino acids together with groups of nutrients and a feeding of CaCl₂ and MgCl₂ concentrate. The duration of exponential cell growth was extended from 70 h in batch culture and 98 h in fed-batch culture without Ca²⁺/Mg²⁺ feeding to 117 h with Ca²⁺/Mg²⁺ feeding. As a result of the prolonged exponential cell growth, the viable and total cell densities reached 7.04 × 10⁶ and 9.12 × 10⁶ cells ml⁻¹, respectively. The maximal MAb concentration achieved was increased to approximately eight times of that in serum supplemented batch culture.     

Increased intracellular calcium level and impaired nutrient absorption are important pathogenicity traits in the chicken intestinal epithelium during <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> colonization

Although a high number of chickens carry Campylobacter jejuni, the mechanistic action of colonization in the intestine is still poorly understood. The current study was therefore designed to investigate the effects of C. jejuni on glucose uptake, amino acids availability in digesta, and intracellular calcium [Ca²⁺]_i signaling in the intestines of broiler chickens. For this, we compared: control birds (n?=?60) and C. jejuni-infected birds (n?=?60; infected orally with 1?×?10⁸ CFU of C. jejuni NCTC 12744 at 14 days of age). Our results showed that glucose uptake was reduced due to C. jejuni infection in isolated jejunal, but not in cecal mucosa at 14 days postinfection (dpi). The decrease in intestinal glucose absorption coincided with a decrease in body weight gain during the 2-week post-infectious period. A reduction in the amount of the amino acids (serine, proline, valine, leucine, phenylalanine, arginine, histidine, and lysine) in ileal digesta of the infected birds at 2 and/or 7 dpi was found, indicating that Campylobacter utilizes amino acids as a carbon source for their multiplication. Applying the cell-permeable Ca²⁺ indicator Fluo-4 and two-photon microscopy, we revealed that [Ca²⁺]_i was increased in the jejunal and cecal mucosa of infected birds. The muscarinic agonist carbachol induced an increase in [Ca²⁺]_i in jejunum and cecum mucosa of control chickens, a response absent in the mucosa of infected chickens, demonstrating that the modulation of [Ca²⁺]_i by Campylobacter might be involved in facilitating the necessary cytoskeletal rearrangements that occur during the bacterial invasion of epithelial cells. In conclusion, this study demonstrates the multifaceted interactions of C. jejuni with the gastrointestinal mucosa of broiler chickens. For the first time, it could be shown that a Campylobacter infection could interfere with intracellular Ca²⁺ signaling and nutrient absorption in the small intestine with consequences on intestinal function, performance, and Campylobacter colonization. Altogether, these findings indicate that Campylobacter is not entirely a commensal and can be recognized as an important factor contributing to an impaired chicken gut health.     

Lanthanum is transported by the sodium/calcium exchanger and regulates its activity

La³⁺ uptake was measured in fura 2-loaded Chinese hamster ovary cells expressing the bovine cardiac Na⁺/Ca²⁺ exchanger (NCX1.1). La³⁺ was taken up by the cells after an initial lag phase of 50-60 s and achieved a steady state within 5-6 min. Neonatal cardiac myocytes accumulated La³⁺ in a similar manner. La³⁺ uptake was due to the activity of the exchanger, because no uptake was seen in nontransfected cells or in transfected cells that had been treated with gramicidin to remove cytosolic Na⁺. The low rate of La³⁺ uptake during the lag period resulted from insufficient cytosolic Ca²⁺ to activate the exchanger at its regulatory sites, as shown by the following observations. La³⁺ uptake occurred without a lag period in cells expressing a mutant of NCX1.1 that does not exhibit regulatory activation by cytosolic Ca²⁺. The rate of La³⁺ uptake by wild-type cells was increased, and the lag phase was reduced or eliminated, when the cytosolic Ca²⁺ concentration was increased before initiating La³⁺ uptake. La³⁺ could substitute for Ca²⁺ at very low concentrations to activate exchange activity. Thus preloading cells expressing NCX1.1 with a small quantity of La³⁺ increased the rate of exchange-mediated Ca²⁺ influx by 20-fold; in contrast, cytosolic La³⁺ partially inhibited Ca²⁺ uptake by the regulation-deficient mutant. With an estimated K_D of 30 pM for the binding of La³⁺ to fura 2, we conclude that cytosolic La³⁺ activates exchange activity at picomolar concentrations. We speculatively suggest that endogenous trace metals might activate exchange activity under physiological conditions. fura 2; NCX1.1; myocyte     

Characterization of the Human Intestinal Calcium Transporter, CaT1, Stably Expressed in CHO Cells

The human calcium transporter, hCaT1, was cloned and analyzed. The obtained amino acid sequence was slightly different from the ortholog of hCaT1 which had been identified by Peng et al. (2000. Biochem. Biophys. Res. Commun 278: 326-332). An mRNA analysis of human gastrointestinal segments demonstrates that hCaT1 was expressed in the stomach, duodenum, jejunum, ileum, ileocecum, cecum, ascending colon, transverse colon, descending colon, and, at very low levels, in the esophagus and rectum. hCaT1 was transiently expressed by transfecting COS-1 cells and was stably expressed by the transfected CHO cells. The transfected cells expressed hCaT1 with a molecular mass of 75 kDa. Stable expression of hCaT1 in the CHO cells increased the cellular uptake of Ca²⁺. hCaT1 was inhibited by La³⁺, Gd³⁺ and Cd²⁺, whereas Co²⁺, Fe²⁺, Mn²⁺ and Mg²⁺ showed no significant effects on the activity. Acidification of the extracellular solution to pH 5.5 reduced the ⁴⁵Ca²⁺uptake by hCaT1 in the CHO cells. The addition of lactose and raffinose had no effect on the ⁴⁵Ca²⁺ uptake, whereas galactose and glucose increased the ⁴⁵Ca²⁺ uptake. CHO cells stably expressing hCaT1 will be useful to detect and analyze food substances that could modulate the hCaT1 activity.     

Regulation of Ca2+ homeostasis by glucose metabolism in rat brain

In a previous communication we reported that glucose deprivation from KHRB medium resulted in a marked stimulation of Ca²⁺ uptake by brain tissue, suggesting a relationship between glucose and Ca²⁺ homeostasis in brain tissue [17]. Experiments were carried out to investigate the significance of glucose in Ca²⁺ transport in brain cells. The replacement of glucose with either D-methylglucoside or 2-deoxyglucose, non-metabolizable analogues of glucose, resulted in stimulation of Ca²⁺ uptake just as by glucose deprivation. These data show that glucose metabolism rather than glucose transfer was necessary to stimulate Ca²⁺ uptake in brain tissue. Inhibition of glucose metabolism with either NaF, NaCN, or iodoacetate resulted in stimulation of Ca²⁺ uptake similar to that produced by glucose deprivation. These results lend further support for the concept that glucose metabolism is essential for Ca²⁺ homeostasis in brain. Anoxia promotes glucose metabolism through glycolytic pathway to keep up with the demand for ATP by cellular processes (the Pasteur effect). Incubation of brain slices under nitrogen gas did not alter Ca²⁺ uptake by brain tissue, as did glucose deprivation and the inhibitors of glucose metabolism. We conclude that glucose metabolism resulting in the synthesis of ATP is essential for Ca²⁺ homeostasis in brain. Verapamil and nifedipine which block voltage-gated Ca²⁺ channels, did not alter Ca²⁺ uptake stimulated by glucose deprivation, indicating that glucose deprivation-enhanced Ca²⁺ uptake was not mediated by Ca²⁺ channels. Tetrodotoxin which specifically blocks Na⁺ channels, abolished Ca²⁺ uptake enhanced by glucose deprivation, but had no effect on Ca²⁺ uptake in presence of glucose (controls). These results suggest that stimulation of Ca²⁺ uptake by glucose deprivation may be related to Na⁺ transfer via Na-Ca exchange in brain.     

Effects of Calcium on Sugar Transport in Azotobacter vinelandii

A fast and environmentally safe procedure was used to study sugar uptake by Azotobacter vinelandii. Transport experiments were performed in a 24-well plate and aerated by rapid oscillatory vibration. Samples were washed by centrifugation and dissolved in biodegradable scintillation cocktail for counting. At cell concentrations up to 6 × 10⁸ cells per ml, the uptake of sucrose was a function of time and was proportional to the cell concentration. This modified uptake assay was used to test the effect of cations on sugar uptake in A. vinelandii. Results showed that Ca²⁺ at 1 to 2 mM stimulated sucrose uptake by decreasing the apparent K_m of sucrose transport. Higher Ca²⁺ concentrations inhibited sucrose uptake in this organism.     

Calcium signaling in insulin action on striated muscle

Striated muscles (skeletal and cardiac) are major physiological targets of insulin and this hormone triggers complex signaling pathways regulating cell growth and energy metabolism. Insulin increases glucose uptake into muscle cells by stimulating glucose transporter (GLUT4) translocation from intracellular compartments to the cell surface. The canonical insulin-triggered signaling cascade controlling this process is constituted by well-mapped tyrosine, lipid and serine/threonine phosphorylation reactions. In parallel to these signals, recent findings reveal insulin-dependent Ca²⁺ mobilization in skeletal muscle cells and cardiomyocytes. Specifically, insulin activates the sarco-endoplasmic reticulum (SER) channels that release Ca²⁺ into the cytosol i.e., the Ryanodine Receptor (RyR) and the inositol 1,4,5-triphosphate receptor (IP₃R). In skeletal muscle cells, a rapid, insulin-triggered Ca²⁺ release occurs through RyR, that is brought about upon S-glutathionylation of cysteine residues in the channel by reactive oxygen species (ROS) produced by the early activation of the NADPH oxidase (NOX2). In cardiomyocytes insulin induces a fast and transient increase in cytoplasmic [Ca²⁺]_i trough L-type Ca²⁺ channels activation. In both cell types, a relatively slower Ca²⁺ release also occurs through IP₃R activation, and is required for GLUT4 translocation and glucose uptake. The insulin-dependent Ca²⁺ released from IP₃R of skeletal muscle also promotes mitochondrial Ca²⁺ uptake. We review here these actions of insulin on intracellular Ca²⁺ channel activation and their impact on GLUT4 traffic in muscle cells, as well as other implications of insulin-dependent Ca²⁺ release from the SER.     

Characterization of Calumenin-SERCA2 Interaction in Mouse Cardiac Sarcoplasmic Reticulum

Calumenin is a multiple EF-hand Ca²⁺-binding protein localized in the sarcoplasmic reticulum (SR) with C-terminal SR retention signal HDEF. Recently, we showed evidence that calumenin interacts with SERCA2 in rat cardiac SR (Sahoo, S. K., and Kim, D. H. (2008) Mol. Cells 26, 265–269). The present study was undertaken to further characterize the association of calumenin with SERCA2 in mouse heart by various gene manipulation approaches. Immunocytochemical analysis showed that calumenin and SERCA2 were partially co-localized in HL-1 cells. Knockdown (KD) of calumenin was conducted in HL-1 cells and 80% reduction of calumenin did not induce any expressional changes of other Ca²⁺-cycling proteins. But it enhanced Ca²⁺ transient amplitude and showed shortened time to reach peak and decreased time to reach 50% of baseline. Oxalate-supported Ca²⁺ uptake showed increased Ca²⁺ sensitivity of SERCA2 in calumenin KD HL-1 cells. Calumenin and SERCA2 interaction was significantly lower in the presence of thapsigargin, vanadate, or ATP, as compared with 1.3 μm Ca²⁺, suggesting that the interaction is favored in the E1 state of SERCA2. A glutathione S-transferase-pulldown assay of calumenin deletion fragments and SERCA2 luminal domains suggested that regions of 132–222 amino acids of calumenin and 853–892 amino acids of SERCA2-L4 are the major binding partners. On the basis of our in vitro binding data and available information on three-dimensional structure of Ca²⁺-ATPases, a molecular model was proposed for the interaction between calumenin and SERCA2. Taken together, the present results suggest that calumenin is a novel regulator of SERCA2, and its expressional changes are tightly coupled with Ca²⁺-cycling of cardiomyocytes.     

Participation of the microtubular-microfilamentous system on intracellular Ca transport and acid secretion in dispersed parietal cells

Biphasic responses of amino[¹⁴C]pyrine accumulation and oxygen consumption were registered by gastrin stimulation in dispersed parietal cells from guinea pig gastric mucosa, and this was mimicked with the calcium ionophore A23187. The characteristics of these phases (first phase and second phase) were distinguished by the differences in the requirements of extracellular Ca²⁺. The first phase evoked by gastrin or ionophore A23187 was independent of extracellular Ca²⁺, whereas the second phase was not. In the first phase, fluorescence of a cytosolic Ca²⁺ indicator (quin2-AM) increased with the stimulation of ionophore A23187 and carbamylcholine chloride in the presence of extracellular Ca²⁺. In addition, an increase in cytosolic Ca²⁺ induced by ionophore A23187, but not by carbamylcholine chloride was also observed in the absence of extracellular Ca²⁺, suggesting that Ca²⁺ pool(s) in parietal cells might be present in the intracellular organelle. Cytochalasin B and colchicine, but not oligomycin, could eliminate this cytosolic Ca²⁺ increase induced by A23187 in a Ca²⁺-free medium. On the other hand, in a Ca²⁺-free medium, addition of ATP after pretreatment with digitonin could diminish the cytosolic Ca²⁺ increase brought about by A23187. This was also observed with oligomycin-treated cells, but not with cytochalasin B-treated cells. Similarly, subcellular fractionation of a parietal cell which had been pretreated with cytochalasin B or colchicine in an intact cell system reduced the rate of ATP-dependent Ca²⁺ uptake. These observations indicate that intracellular Ca²⁺ transport in dispersed parietal cells may be regulated by the microtubular-microfilamentous system. In conclusion, this study demonstrates the possibility of the existence of intracellular Ca²⁺ transport mediated by gastrin or ionophore A23187 and regulated by the microtubular-microfilamentous system in parietal cells.     

Electrophysiological Studies into the Safety of the Anti-diarrheal Drug Clotrimazole during Oral Rehydration Therapy

ResultsTreatment of small intestinal tissue with clotrimazole inhibited the Cl^- secretory currents that resulted from challenge with the cAMP-agonist vasoactive intestinal peptide (VIP) or Ca²⁺-agonist carbachol in a dose-dependent fashion. A dose of 30 μM was effective in significantly reducing the Isc response to VIP and carbachol by 50% and 72%, respectively. At this dose, uptake of glucose was only marginally affected (decreased by 14%, p = 0.37). There was no measurable effect on SGLT1-mediated sugar transport, as uptake of SGLT1-restricted 3-O-methyl glucose was equivalent between clotrimazole-treated and untreated tissue (98% vs. 100%, p = 0.90).ConclusionTreatment of intestinal tissue with clotrimazole significantly reduced secretory responses caused by both cAMP- and Ca²⁺-dependent agonists as expected, but did not affect Na⁺-coupled glucose absorption. Clotrimazole could thus be used in conjunction with oral rehydration solution as a low-cost, auxiliary treatment of acute secretory diarrheas.     

Physiological and Biochemical Responses Reveal the Drought Tolerance Efficacy of the Halophyte Salicornia brachiata

The drought tolerance of Salicornia brachiata seedlings was assessed by monitoring growth, nutrient uptake, electrolyte leakage, lipid peroxidation, and biochemical responses under drought conditions simulated with 0, 10, 20, and 30 % polyethylene glycol (PEG 6000). After 7 days of drought induction, plants were harvested for measurement of various parameters. The biomass decreased and the plant height remained unchanged with PEG treatment. The total plant water content (TWC%) decreased by 11 % at the highest concentration of PEG (30 %). The electrolyte leakage and lipid peroxidation of shoots increased by 17 and 5 %, respectively, in 30 % PEG-treated plants. K⁺ and Ca²⁺ contents of shoots increased in a dose-dependent manner. However, in roots K⁺ content decreased and Ca²⁺ content remained unaffected by PEG treatment. Mg²⁺ content increased at high concentrations of PEG (20–30 %) in shoots and decreased at the highest concentration of PEG (30 %) in roots. Total free amino acids, proline, and polyphenol contents increased progressively with increase in severity of the drought stress. Total sugar content and reducing sugar content increased in 10 and 20 % PEG-treated plants and decreased in 30 % PEG-treated plants. Our results suggest that proline and other free amino acids, sugars, and polyphenols are the main compatible solutes in S. brachiata for maintenance of osmotic balance, protection of cellular macromolecules, detoxification of the cells, and scavenging of free radicals under drought stress. A greater accumulation of compatible solutes also facilitates the maintenance of nutrient uptake and adequate tissue water status and protection of membranes under drought conditions in S. brachiata. The results from the present study suggest that S. brachiata can be used for restoration of arid and semiarid lands of coastal ecosystems.     

Role of calcium in the regulation of sugar transport in the avian erythrocyte: Effects of the calcium ionophore,A23187

The tissue/medium distribution of the nonmetabolized glucose analog [¹⁴C]-3-0-methyl-D-glucose was measured in pigeon erythrocytes and related to changes in ⁴⁵Ca uptake and efflux, total calcium content and ATP levels. Sugar transport was not affected by changes in external Ca²⁺. However, both sugar and ⁴⁵Ca influx were increased by the Ca-ionophore A23187. In the absence of external Ca²⁺, the ionophore caused a delayed increase in sugar transport and net loss of calcium, probably through releasing Ca²⁺ from internal storage sites into the cytoplasm. Increasing internal Na⁺ through Na⁺ pump inhibition or using the sodium ionophore monensin did not alter influx of sugar or ⁴⁵Ca, indicating Na⁺-Ca²⁺ exchange was absent in these cells. The results are consistent with A23187 causing increased Ca²⁺ influx or release from mitochondrial storage and the resulting rise in cytoplasmic Ca²⁺ stimulating hexose transport. Experiments with low Mg⁺⁺ and high K⁺ media and measurements of ATP levels exclude alternative explanations for the action of A23187. We conclude that sugar transport regulation in avian erythrocytes is Ca²⁺-dependent and resembles that in muscle in its basic mechanism. It differs in the response to some modulating agents, largely because of a different pattern of Ca²⁺ fluxes in these cells.     

Metabolism of Separated Leaf Cells: III. Effects of Calcium and Ammonium on Product Distribution During Photosynthesis with Cotton Cells

Separated mesophyll cells from cotton (Gossypium hirsutum var. Stoneville 1613 Glandless) were isolated with pectinase and mechanical agitation. The separated cells had rates of light-dependent CO₂ fixation between 50 to 100 μmoles CO₂ per mg chlorophyll per hour. The presence of Ca²⁺ in the incubation medium did not significantly affect the type of photosynthetic products formed, but 2 mm Ca²⁺ did cause a 50% decrease in the appearance of photosynthetic products in the incubation medium. The movement of all types of products (sugars, organic, and amino acids) out of the cells was reduced similarly by the Ca²⁺. Light had no affect on the movement of products out of the cells, whereas 1 mm ethylenediaminetetra-acetate greatly increased the movement. The addition of 1.6 mm NH₄Cl to the cell suspensions caused a large increase in the amount of fixed ¹⁴C appearing in the amino acid fraction and a decrease in the sugar fraction. These metabolic changes in the cells were reflected in the movement of products out of the cells so that the incubation medium also contained a larger amount of label in amino acids and a smaller amount in sucrose. Although the cell plasma membrane restricted the movement of soluble products, it did not discriminate significantly between the types of products moved.     

The regulation of ionic nickel uptake and cytotoxicity by specific amino acids and serum components

The effects of serum components and amino acids on the uptake and cytotoxicity of NiCl₂ were examined in cultured Chinese hamster ovary (CHO) cells. CHO cells maintained in a minimal salts/glucose medium accumulated 10-fold more⁶³Ni than did cells maintained in complete medium supplemented with 10% fetal bovine serum. Cell-surface binding of⁶³Ni appeared to account for the majority of this increased accumulation of cell-associated nickel observed in the simple maintenance medium since such increases were reduced 70% by trypsin treatment. The addition of the Ni²⁺-binding amino acids cysteine or histidine to the salts/glucose medium markedly decreased⁶³Ni accumulations, an effect not observed following addition of any of several amino acids that do not bind Ni²⁺. Supplementation of the salts/glucose medium with fetal bovine serum decreased in a concentration dependent fashion both the⁶³Ni²⁺ uptake and cell detachment caused by Ni²⁺, while dialyzed (amino acid-free) serum was 3–5-fold less effective than undialyzed serum at reducing⁶³Ni²⁺ uptake and similarly exhibited only a slight protective effect against nickel-induced cytotoxicity. Supplementation of dialyzed serum with cysteine at levels approximating those in whole serum partially restored its inhibitory activity toward nickel uptake by cells and restored completely its inhibition of nickel's cytotoxicity, indicating the predominant role of specific amino acids over serum proteins in regulating the uptake and subsequent cytotoxicity of Ni²⁺. Addition of cysteine to the salts/glucose medium during a 2 h exposure of cells to either 100 μM HgCl₂ or 1 mM NiCl₂ masked the cytotoxic effects of these metal ions. These results demonstrate the importance of extracellular small molecular weight metal ion chelators in altering the biological effects of metal ions at the level of metal uptake.     

Early biochemical events in lymphocyte activation. I. Investigations on the nature and significance of early calcium fluxes observed in mitogen-induced T and B lymphocytes.

⁴⁵Ca²⁺ uptake was detected within minutes following addition of T- and B-cell² mitogens to mouse lymphocytes. The T-cell mitogens (Con A and PHA) gave an ~twofold increase in ⁴⁵Ca²⁺ uptake (representing an influx of ~ 130 amol per lymphocyte, corresponding to an increase in average cellular Ca²⁺ of ~0.95 mM). B-cell mitogens which gave the largest ⁴⁵Ca²⁺ uptake (~twofold) were purified LPS preparations from Salmonella minnesota R595 and Escherichia coli 0111:2125. The ⁴⁵Ca²⁺ uptake by rabbit splenocytes using specific anti-b4 allotype antiserum was comparable to that obtained with the two purified LPS preparations. A23187, in low nontoxic doses, gave an ~sixfold increase in ⁴⁵Ca²⁺ uptake with mouse T cells. The ⁴⁵Ca²⁺ uptake was modulated by cyclic nucleotides showing a “yin-yang” effect. The results suggest a possible entry of ⁴⁵Ca²⁺ from the extracellular medium through “gated Ca²⁺ channels” in the plasma membrane into the cytosol by passive diffusion. The Ca²⁺ may be sequestered in the mitochondria, and the excess Ca²⁺ is later effluxed into the extracellular medium. The fact that ⁴⁵Ca²⁺ uptake appears to be one of the earliest events occurring after ligand binding to the cell, together with the demonstration of a Ca²⁺-dependent glucose uptake and a requirement for extracellular Ca²⁺ for DNA synthesis, suggest that, as it is now known to function in many other cellular responses, Ca²⁺ may operate as a second messenger for lymphocyte activation.     

Molecular cloning of YlPMR1, a S. cerevisiae PMR1 homologue encoding a novel P-type secretory pathway Ca2+-ATPase,in the yeast Yarrowia lipolytica

A novel P-type ATPase gene, Saccharomyces cerevisiae PMR1 homologue (YlPMR1), has been cloned and sequenced in the yeast, Yarrowia lipolytica. The putative gene product has 928 amino acids with a calculated molecular mass of 100 050 Da and a pI of 5.15. The deduced amino-acid sequence analysis demonstrated that the cloned gene product contains all 10 of the conserved regions in P-type ATPases and exhibits 55% amino-acid identity to the S. cerevisiae PMR1 gene product; however, it shows a relatively lower homology to PMCA (24%) and SERCA (33%), confirming the presence of a third class of Ca²⁺-ATPase (secretory pathway Ca²⁺-ATPase, SPCA). The YlPMR1-disrupted strain shows defective growth in low Ca²⁺ or EGTA-containing medium. In fact, a longer lag time (60 h) was observed in YlPMR1-defective mutant cells during cultivation in EGTA-containing YPD medium. These growth defects were overcome by adding Ca²⁺ and Mn²⁺ into the medium. Interestingly, whereas Mn²⁺ inhibits growth of the control strain, it significantly improves the growth of YlPMR1-disrupted cells. These results suggest an involvement of the YlPMR1 gene product in Ca²⁺ and Mn²⁺ ion homeostasis in Y. lipolytica.