Biomedical subjects

Y Eilam

Publications and source records attributed to Y Eilam.

At least 19 recordsLinked to original sources

PubMed1992

An intracellular ATP-dependent calcium pump within the yeast Schizosaccharomyces pombe, encoded by the gene cta3.

We have permeabilized the plasma membranes of Schizosaccharomyces pombe cell with nystatin and measured ATP-dependent Ca2+ uptake in the presence of KNO3 and a protonophore in order to inhibit Ca2+ uptake into the vacuole. ATP-dependent Ca2+ accumulation into non-vacuolar Ca(2+)-storing organelles was detected. This Ca2+ uptake activity was maximal at pH 6 and inhibited by vanadate, the inhibitor of P-type ATPases. The null mutation of cta3, a putative Ca2+ gene, [Ghislain, M., Goffeau, A., Halachmi, D. and Eilam, Y. (1990) J. Biol. Chem. 265, 18400-18407] strongly reduced the level of ATP-dependent Ca2+ uptake into non-vacuolar intracellular storing organelles. This result suggests that cta3 encodes an intracellular ATP-dependent Ca2+ pump. The residual ATP-dependent Ca2+ uptake in the mutant strain indicated the presence of a second nonvacuolar, intracellular Ca(2+)-ATPase encoded by a different gene.

Adenosine Triphosphate↗

PubMed1991

Binding properties and biological effects of oxidized-ouabain on cultured neonatal-rat cardiac myocytes. Implications on the mechanism of action of the digitalis-glycosides.

Mild oxidation of ouabain with NaIO4, causes the cleavage of the bond between C2' and C3' of the rhamnose ring, leaving the steroid moiety intact. The oxidized ouabain (ox-ouabain) was examined on spontaneously contracting cultured rat-cardiac myocytes. Two classes of binding sites, with high and low affinities, were detected for both ox-ouabain and unmodified ouabain. The dissociation constants (KD) were found to be similar for both compounds, but the rate constants of association (ka) and dissociation (kd) of the low affinity sites were higher for ox-ouabain as compared with ouabain. Displacement experiments showed that ox-ouabain and ouabain bind to the same sites. The effects of ox-ouabain and ouabain on the activity of Na+, K(+)-ATPase were determined in microsomal preparations. Similar dose-response curves for the inhibition of the enzyme activity were determined for both drugs. Inhibition was observed only at concentrations above 10(-6) M. The biological effects of the drugs were examined by their capacity to induce positive inotropic or toxic effects. Concentrations of ox-ouabain which induced positive inotropic effects (increase in amplitude of systolic cell motion), ranged from 5 x 10(-8) M to 5 x 10(-6) M, as compared with 10(-7) M to 5 x 10(-7) M with ouabain. "Toxic" effects (decrease in the amplitude of systolic motion, increased beating frequencies and elevation in the position of maximal relaxation) was observed only with 10(-5) M ox-ouabain as compared with 10(-6) M ouabain. The mechanism of the inotropic action of ox-ouabain at the lower concentration range was investigated by measuring the effect of the drugs on 86Rb+ (analogue of K+) influx. Dose-response curves of effects of ouabain and ox-ouabain on 86Rb+ influx were bi-phasic. At low concentrations stimulation was observed, whereas at high concentrations 86Rb+ influx was inhibited. Ox-ouabain stimulated 86Rb+ influx by lower concentrations and to a greater extent than ouabain. A part of 86Rb+ influx into cardiac myocytes is mediated by the K+/Na+/Cl- cotransporter, which can be inhibited by loop diuretic drugs such as bumetanide. We have previously shown that ouabain, at low concentrations, stimulates the activity of the cotransporter. It is shown in the present work that ox-ouabain stimulates the activity of the cotransporter by lower concentrations and to a greater extent than ouabain.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1991

Antitumor effects of ketoconazole and trifluoperazine in murine T-cell lymphomas.

In vitro and in vivo antitumor effects of ketoconazole (KTZ), trifluoperazine (TFP), and combinations of both drugs were examined in cell lines established from radiation leukemia virus (RadLV)-induced T-cell lymphomas. KTZ inhibited [3H]-thymidine incorporation in the tumor cells in vitro; 50% inhibition of DNA synthesis was observed at concentrations of 4-7 micrograms/ml. [3H]-thymidine uptake in bone-marrow and spleen cells prepared from healthy mice was also inhibited by KTZ, but 50% inhibition was observed only at a concentration of 50 micrograms/ml. Stimulation of spleen cells with concanavalin A led to an increase in their sensitivity to the inhibition of DNA synthesis by KTZ. The tumor-cell lines varied in their sensitivity to the inhibition of DNA synthesis by TFP, and the effects of TFP on DNA synthesis in bone-marrow and spleen cells were similar to those observed in the tumor cells. Synergistic, additive, or less than additive effects of the drug combinations on the inhibition of DNA synthesis in vitro were observed both in tumor cells and in bone-marrow cells. In vivo experiments were conducted on groups of C57BL/6 (B6) mice that were inoculated s.c. with tumor cells and then treated with i.p. injections of KTZ, TFP or both. Control groups were injected with phosphate-buffered saline (PBS). Each of the drugs alone as well as their combinations caused a significant delay in the appearance of palpable tumors, a decrease in tumor size, and a marked prolongation of survival. The concentrations of the drugs used in in vivo experiments did not affect the WBC counts in the peripheral blood of healthy mice. KTZ is currently used for the treatment of prostatic cancer because of its inhibitory effect on testosterone biosynthesis. The results of the present study indicate the hormone-independent chemotherapeutic potential of KTZ, TFP, and combinations of the two drugs.

Animals↗

PubMed1990

Calcium homeostasis and transport are affected by disruption of cta3, a novel gene encoding Ca2(+)-ATPase in Schizosaccharomyces pombe.

A new P-type ATPase gene, cta3, has been identified in Schizosaccharomyces pombe. The deduced amino acid sequence presents a 45% identity with the Saccharomyces cerevisiae putative Ca2(+)-ATPase encoded by the PMR2 gene. The cta3 protein contains 7 out of the 8 amino acid residues involved in high affinity Ca2+ binding in the sarcoplasmic reticulum Ca2(+)-ATPase from muscles. It also contains a region similar to the phospholamban-binding domain that characterizes this Ca2+ pump. A null mutation of cta3 leads to higher levels of cytosolic free Ca2+ and to lower amounts of sequestered and bound Ca2+. Cellular Ca2+ efflux and rates of uptake into intracellular compartments are reduced by the loss of cta3 function. The sequence analysis and the physiological results strongly support the conclusion that the cta3 gene encodes a Ca2(+)-ATPase, probably located in intracellular membranes.

Amino Acid Sequence↗

PubMed1990

Decrease in the basal levels of cytosolic free calcium in chondrocytes during aging in culture: possible role as differentiation-signal.

Cell- and matrix-related parameters, which characterize the aging and differentiation process of cartilage in vivo, were measured in cultured chick epiphyseal chondrocytes during maintenance in a suspension culture for 34 days. A gradual decrease in the rates of proliferation and an increase in the size of the cells were observed. Ultrastructural examination revealed increased vacuolization and appearance of glycogen-storing pools. The rate of proteoglycan synthesis gradually increased. Age-related changes in the composition of the proteoglycan consisted of an increase in the ratio of keratan sulfate/chondroitin sulfate. The results indicate that the process of aging in culture resembles maturation and differentiation of cartilage tissue in vivo. The levels of cytosolic free calcium ions ([Ca2+]i) were measured in fura-2-loaded cells during the course of aging in culture. A gradual decrease in [Ca2+]i was observed. In 5-day cultures, a value of 184 nM [Ca2+]i was measured; this value decreased to 61 nM in 34-day cultures. On the basis of the present data and the previous results, which showed that cartilage-derived growth factors caused a decrease in [Ca2+]i, concomitantly with enhancing differentiation, whereas factors which elevated [Ca2+]i caused an increase in proliferation and a decrease in proteoglycan synthesis, we suggest a model for control of chondrocyte differentiation and aging. The model suggests that the rate of differentiation may be paced by changes in steady-state levels of [Ca2+]i.

Animals↗

PubMed1990

Role of the Na+/K+/Cl- transporter in the positive inotropic effect of ouabain in cardiac myocytes.

In this study we have characterized the bumetanide-sensitive K+/Na+/Cl- cotransport in cultured rat cardiac myocytes. 1) It carries about 10% of the total K+ influx. 2) It is sensitive to furosemide (Ki0.5 = 10(-6)M) and bumetanide (Ki0.5 = 10(-7)M). 3) It is strongly dependent on the extracellular concentrations of Na+ and Cl-. 4) It carries out influx of both ions, K+ and Na+. A therapeutic concentration of ouabain (10(-7) M) stimulated the bumetanide-sensitive K+ influx (as measured by 86Rb+), in the cultured myocytes, with no effect on the bumetanide-resistant K+ influx, which was mediated mostly by the Na+/K+ pump. Stimulation of the bumetanide-sensitive Rb+ influx by a low ouabain concentration was strongly dependent on Na+ and Cl- in the extracellular medium. A low concentration of ouabain (10(-7) M) was found to increase the steady-state level of cytosolic Na+ by 15%. This increase was abolished by the addition of bumetanide or furosemide. These findings suggest that ouabain, at a low (10(-7) M) concentration, induced its positive inotropic effect in rat cardiac myocytes by increasing Na+ influx into the cells through the bumetanide-sensitive Na+/K+/Cl- cotransporter. In order to examine this hypothesis, we measured the effect of bumetanide on the increased amplitude of systolic cell motion induced by ouabain. Bumetanide or furosemide, added to cultured cardiac myocytes, inhibited the increased amplitude of systolic cell motion induced by ouabain. Neither bumetanide nor furosemide alone has any significant effect on the basal amplitude of systolic cell motion. We propose that stimulation of bumetanide-sensitive Na+ influx plays an essential role in the positive inotropic effect in rat cardiac myocytes induced by low concentration of ouabain.

Animals↗

PubMed1990

Transient increase in Ca2+ influx in Saccharomyces cerevisiae in response to glucose: effects of intracellular acidification and cAMP levels.

Influx of 45Ca2+ into Saccharomyces cerevisiae was measured under experimental conditions which enabled measurements of initial rate of transport across the plasma membrane, without interference by the vacuolar Ca2+ transport system. Addition of glucose or glycerol to the cells, after pre-incubation in glucose-free medium for 5 min, caused a rapid, transient increase in 45Ca2+ influx, reaching a peak at 3-5 min after addition of substrate. Ethanol, or glycerol added with antimycin A, had no effect on 45Ca2+ influx. We have shown previously that this increase is not mediated by an effect of the substrates on intracellular ATP levels. Changes in membrane potential accounted for only a part of the glucose-stimulated 45Ca2+ influx. The roles of intracellular acidification and changes in cellular cAMP in mediating the effects of glucose on 45Ca2+ influx were examined. After a short preincubation in glucose-free medium addition of glucose caused a decrease in the intracellular pH, [pH]i, which reached a minimum value after 3 min. A transient increase in the cellular cAMP level was also observed. Addition of glycerol also caused intracellular acidification, but ethanol or glycerol added with antimycin A had no effect on [pH]i. Artificial intracellular acidification induced by exposure to isobutyric acid or to CCCP caused a transient rise in Ca2+ influx but the extent of the increase was smaller than that caused by glucose, and the time-course was different. We conclude that intracellular acidification may be responsible for part of the glucose stimulation of Ca2+ influx.(ABSTRACT TRUNCATED AT 250 WORDS)

Antimycin A↗

PubMed1990

Activation of Ca2+ influx by metabolic substrates in Saccharomyces cerevisiae: role of membrane potential and cellular ATP levels.

Influx of Ca2+ into cells of Saccharomyces cerevisiae was measured under non-steady-state conditions, which enable measurements of the initial rate of transport across plasma membranes without interference by the vacuolar Ca2+ transport system. Removal of glucose from the incubation medium led to inactivation of Ca2+ influx within 5 min. Readdition of glucose led to a transient increase in the rate of Ca2+ transport, reaching a peak after 3-5 min. A second increase was observed 60-80 min later. To examine whether the first transient activation of Ca2+ influx by glucose was mediated by membrane hyperpolarization, influx of 45Ca2+ was measured in the presence and absence of metabolic substrates (glucose, glycerol, and glucose plus antimycin A) in cells hyperpolarized to different values of membrane potential (delta psi). Logarithms of the rate of Ca2+ influx were plotted against values of delta psi. Two different slopes were obtained, depending upon whether the metabolic substrate was present or absent. Ca2+ influx in the presence of the metabolic substrates was always higher than expected by their effect on delta psi. Glycerol plus antimycin A did not affect Ca2+ influx. It was concluded that metabolized substrates activate Ca2+ influx not only by effects on delta psi but also by additional mechanism(s). Since no simple correlation between Ca2+ influx and intracellular ATP levels was observed, it was concluded that ATP levels do not affect the initial rates of Ca2+ transport across the plasma membrane of S. cerevisiae.

Adenosine Triphosphate↗

PubMed1989

Cytosolic and vacuolar Ca2+ concentrations in yeast cells measured with the Ca2+-sensitive fluorescence dye indo-1.

Cells of Saccharomyces cerevisiae were loaded with indo-1, by incubation in a medium of pH 4.5, which contained penta-potassium indo-1. Cells were then washed and resuspended in a buffer of pH 4.0. The emission fluorescence spectra were recorded between 390 and 500 nm (excitation at 355 nm) and the autofluorescent spectra of the matched controls were subtracted. A 19-fold cellular accumulation of indo-1 was achieved. By permeabilization of plasma membranes, leaving the vacuolar membrane intact, it was proved that indo-1 was accumulated in the cytosol. It was also shown that intracellular indo-1 did not leak out of the cells and was not modified by cellular metabolism. Using the emission fluorescence ratio at 410/480 nm, the concentration of a free cytosolic Ca2+ was found to be 346 nM. Vacuolar Ca2+ concentration, calculated from indo-1 fluorescence after lysis of vacuolar and cellular membranes, was found to be 1.3 mM.

Calcium↗

PubMed1989

Rapid intracellular alkalinization of Saccharomyces cerevisiae MATa cells in response to alpha-factor requires the CDC25 gene product.

The alpha-factor mating pheromone induces a transient intracellular alkalinization of MATa cells within minutes after exposure to the pheromone, and is the earliest biochemical event that can be identified subsequent to the exposure. Dissipation of the pheromone induced pH gradient, using 2,4-dinitrophenol or sodium orthovanadate, does not inhibit the biological response of the yeast to the pheromone such as mating and 'schmoo' formation. These findings suggest that the pheromone mediated pH change per se is not a part of the transmembrane signalling but rather the consequence of a biochemical reaction triggered by the alpha-pheromone interaction with its receptor and may have a permissive effect on the pheromonal response. The cdc25ts mutation causes MATa cells to become nonresponsive to alpha-factor subsequent to a shift to the restrictive temperature, suggesting that the CDC25 gene product participates in the pheromone response pathway.

Cell Cycle Proteins↗

PubMed1989

Effect of ouabain on the concentration of free cytosolic Ca++ and on contractility in cultured rat cardiac myocytes.

Effects of ouabain on [Ca++]i and on contractility was measured in quin2 and fura2 loaded cultured neonatal rat cardiac myocytes. Addition of ouabain (5 x 10(-8) to 5 x 10(-6) M) to cultured myocytes exposed to balanced buffered salt solution (BSS) caused a transient increase in [Ca++]i, followed by slow oscillations for about 10 min, and by an elevated steady state level of [Ca++]i thereafter. Concentrations of ouabain between 10(-7) and 5 x 10(-7) M caused an increase in the amplitude of systolic motion (ASM) whereas concentrations above 10(-6) caused a decrease in the ASM, an increase in the beating frequency and an upward shift of the base line, indicating impaired relaxation. When ouabain was added to cardiac myocytes exposed to Ca++-free BSS the increase in [Ca++]i was not observed, but only a transient decrease. To investigate the effect of [K+]o on the ouabain-induced changes in [Ca++]i, ouabain was added to cells exposed to BSS containing low K+ concentration (1 mM instead of 5 mM in balanced BSS). In this medium the increase in ASM by ouabain was similar to that in balanced BSS. Addition of ouabain caused a transient decrease in [Ca++]i. There was no initial increase in [Ca++]i and the steady state level of [Ca++]i was not elevated as compared with the same cells before the addition of ouabain. Similar results were observed in cells loaded with quin2 or with fura2. In view of these results the mechanism of action of ouabain on cardiac myocytes is discussed.

Aminoquinolines↗

PubMed1988

Slowing down aging of cultured embryonal chick chondrocytes by maintenance under lowered oxygen tension.

Cultured epiphyseal-chondrocytes from embryonic chick may serve as a useful in vitro model to study aging processes in cartilage. The accelerated aging process in cultured chondrocytes is completed within a month and is manifested by typical changes in both cellular and extracellular compartments. Under common maintenance conditions, cells show a gradual loss of replicative capacity, increase in the rate of proteoglycan synthesis and age-dependent changes in the structure and composition of proteoglycan. An environmental factor--reduced oxygen tension--was found to slow down aging processes and preserve the young features of chondrocytes for a longer duration in culture. Cultures maintained under lower oxygen tension had higher proliferation rate, smaller cell size, lower rate of proteoglycan synthesis, and lower content of keratan sulfate side chains in the proteoglycan. In addition higher concentrations of free cytosolic calcium [Ca2+]in as compared to control cultures, was found. It is suggested that the increased proliferation rate and the decrease in proteoglycan synthesis caused by low oxygen tension may be signalled by the higher [Ca2+]in in these cells.

Animals↗

PubMed1988

Low concentrations of trifluoperazine arrest the cell division cycle of Saccharomyces cerevisiae at two specific stages.

Low concentrations of trifluoperazine (TFP) reversibly inhibited vegetative growth of Saccharomyces cerevisiae. The cell cycle was analysed by flow cytometry using haploid a cells synchronized by alpha-factor arrest and several temperature-sensitive cell division cycle mutants (cdc). Cells were pulse-labelled with fluorescein-labelled concanavalin A (ConA-FITC) to determine cell division or stained with propidium iodide to determine the stage of cell cycle arrest by TFP. Cell growth was estimated from the changes in the relative intensity of scattered light, and budding was determined microscopically. When TFP was added before Start on release from alpha-factor arrest, after release of cdc28-arrested cells, and at transition from stationary phase to vegetative growth, cell growth, budding and DNA synthesis were inhibited. When TFP was added after execution of spindle pole body duplication, cell growth, bud emergence and DNA synthesis were not inhibited but cell division was inhibited and the cells arrested with buds at G2M Using cdc mutants, the second stage of arrest by TFP was determined to be just before medial-nuclear division.

Cell Division↗

PubMed1988

In vitro synergistic activity of ketoconazole with trifluoperazine and with chlorpromazine against medically important yeasts.

Combination of ketoconazole and trifluoperazine or chlorpromazine yielded an in vitro synergistic effect on growth inhibition of Candida albicans, Torulopsis glabrata, Cryptococcus neoformans, Candida parapsilosis and Candida tropicalis. The optimal pH range for the synergistic effects was 7.0-7.6. At pH 5.0 the drug combination was antagonistic. (Application for patent protection has been filed).

Chlorpromazine↗

PubMed1987

Poly L-histidine. A potent stimulator of superoxide generation in human blood leukocytes.

Poly-L-histidine (PHSTD) of molecular weight 26,000 induced the generation of large amounts of superoxide (O2-) and hydrogen peroxide (H2O2) in human neutrophils (PMNs). Despite its low solubility at neutral pH, PHSTD was bound very rapidly to the PMN surfaces. Maximal generation of O2- took place with 4-5 X 10(-6) M of PHSTD, starting after a lag of about 25 sec and proceeding for 15-17 min at a rate of 150 nmol/10(7) PMNs/min, suggesting that this polycation is one of the most potent stimulators of O2- generation known, PHSTD was found to be non-toxic for PMNs even at millimolar concentrations. Generation of O2- by PHSTD depended on extracellular calcium; it was inhibited by calcium channel blockers and by trifluoperazine, and it triggered a sharp rise in intracellular calcium as determined by the Quin 2 fluorescence technique. The generation of both O2- and H2O2 by PHSTD was partially inhibited by cytochalasin B or (CYB, CYE). On the other hand, CYB markedly enhanced the generation of both O2- and H2O2 following stimulation of PMNs either by PHSTD, polyarginine, histone, or by antibody-opsonized group A streptococci. Electron microscopic analysis and NBT reduction tests revealed that both PHSTD and PHSTD-opsonized streptococci were avidly phagocytosed by PMNs. Since CYB totally inhibited internalization of both PHSTD and the PHSTD-opsonized streptococci, it was suggested that these agents stimulated oxygen radical generation mainly on the leukocyte surfaces. Complexes (CX) formed between PHSTD and polyanethole sulfonate (a strong polyanion) or between histone and the polyanion mimicked immune CX in their ability to trigger the generation of large amounts of O2- which were inhibited by CYB. Generation of O2- and chemiluminescence either by PHSTD or by PHSTD-opsonized streptococci were markedly inhibited by poly-L-glutamate, suggesting that PHSTD acted as a cationic agent which interacted via electrostatic forces with some negatively charged sites in the leukocyte membrane. Generation of H2O2 by PHSTD was also markedly inhibited by deoxyglucose, KCN, DASA, as well as by the lipoxygenase inhibitors nordihydroguaiaretic acid, phenidone, and propylgallate. On the other hand, cyclooxygenase inhibitors such as aspirin, indomethacin, and piroxicam were inactive, suggesting that arachidonic acid metabolism via lipoxygenase pathway might have been involved in the activation by PHSTD of the NADPH oxidase in PMNs.(ABSTRACT TRUNCATED AT 400 WORDS)

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed1987

Uptake of Ca2+ driven by the membrane potential in energy-depleted yeast cells.

The time-course of 45Ca2+ influx into yeast cells was measured under non-steady-state conditions obtained by preincubating the cells in a Ca2+-free medium containing glucose and buffer. Two components were distinguished: a saturable component which reached a steady-state after about 40 s of 45Ca2+ uptake and a linear increase in cellular 45Ca2+ starting after 60-90 s. Using differential extraction methods it was determined that after 20 s of uptake, 45Ca2+ was localized in the cytoplasmic pool and in bound form with no 45Ca2+ in the vacuole. After 3 min most of the cellular 45Ca2+ was concentrated in the vacuole and in bound form. The initial rate of 45Ca2+ uptake under non-steady-state conditions thus measured 45Ca2+ transport across the plasma membrane without interference by vacuolar uptake. The effect of membrane potential (delta psi) on this transport was investigated in cells depleted of ATP. A high delta psi was produced by preincubating the cells with trifluoperazine (TFP) and subsequently washing the cells free from TFP. Substantial 45Ca2+ influx was measured in the absence of metabolic energy in cells with a high delta psi. Below a threshold value of -69.5 mV the logarithms of the initial rate of 45Ca2+ influx and of the steady-state level of the first component were linear with respect to delta psi. It is suggested that 45Ca2+ influx across the plasma membrane is mediated by channels which open when delta psi is below a threshold value. The results indicated that Ca2+ influx across the plasma membrane was driven electrophoretically by delta psi.

Biological Transport↗

PubMed1987

Activity of phenothiazines against medically important yeasts.

Two phenothiazine compounds, trifluoperazine and chlorpromazine, inhibited growth in vitro of the five most common pathogenic yeasts, with MICs ranging from 10 to 40 micrograms/ml. Daily intraperitoneal injections of trifluoperazine (4 to 7 mg/kg of body weight) increased the survival of mice experimentally infected with Candida albicans or Cryptococcus neoformans. The potential use of these drugs against fungal meningitis is discussed.

Animals↗

PubMed1987

Age-related trend for osteopenia in femurs of female C57BL/6 mice.

Age-related changes in femoral cortical bone were quantified in female C57BL/6 mice. Variables included in this study were cortical thickness; number of osteocytes and periosteal preosteoblasts; bone DNA, protein, calcium, and phosphorus content; and uptake of 3H-thymidine, 3H-proline, and 45CaCl2. Also, the activity of bone alkaline and acid phosphatase was measured as well as the hydroxyproline content of the tissue. Femurs of old mice 28 months old, compared to those of young mice 7 months old, showed significant decreases in cortical thickness, number of bone cells, bone mineral content, and enzyme activity. The latter were accompanied by a marked reduction in ability to incorporate radiolabeled precursors of DNA and collagen synthesis as well as of the mineralization process. Hence, aging female C57BL/6 mice reveal structural and biochemical features indicative of developing osteopenia.

Aging↗