Biomedical subjects

H Degani

Publications and source records attributed to H Degani.

At least 55 records · Page 3Linked to original sources

PubMed1991

In Vivo pH Regulation by a Na/H Antiporter in the Halotolerant Alga Dunaliella salina.

Na(+)/H(+) exchange activity in whole cells of the halotolerant alga Dunaliella salina can be elicited by intracellular acidification due to addition of weak acids at appropriate external pH. The changes in both intracellular pH and Na(+) were followed. Following a mild intracellular acidification, intracellular Na(+) content increased dramatically and then decreased. We interpret the phase of Na(+) influx as due to the activation of the plasma membrane Na(+)/H(+) antiporter and the phase of Na(+) efflux as due to an active Na(+) extrusion process. The following observations are in agreement with this interpretation: (a) the Na(+) influx phase was sensitive to Li(+), which is an inhibitor of the Na(+)/H(+) antiporter, did not require energy, and was insensitive to vanadate; (b) the Na(+) efflux phase is energy-dependent and sensitive to the plasma membrane ATPase inhibitor, vanadate. Following intracellular acidification, a drastic decrease in the intracellular ATP content is observed that is reversed when the cells regain their neutral pH value. We suggest that the intracellular acidification-induced change in the internal Na(+) concentration is due to a combination of Na(+) uptake via the Na(+)/H(+) antiporter and an active, ATPase-dependent, Na(+) extrusion. The Na(+)/H(+) antiporter seems, therefore, to play a principal role in internal pH regulation in Dunaliella.

Journal Article↗

PubMed1991

In vivo studies by magnetic resonance imaging and spectroscopy of the response to tamoxifen of MCF7 human breast cancer implanted in nude mice.

Magnetic resonance 1H-imaging and 31P-localized spectroscopy were utilized to monitor, noninvasively, MCF7 human breast cancer tumors implanted in immunodeficient mice. The tumors were followed during estrogen dependent growth and tamoxifen induced remission. Early after tamoxifen administration enhanced necrosis developed, extending to most of the tumor volume. This was followed by growth of repair tissue along with tumor regression. The short-term tamoxifen treatment also modified the content of the phosphate metabolites, increasing the nucleoside triphosphate to inorganic phosphate ratio from 0.41 +/- 0.15 (n = 14) before treatment to 1.10 +/- 0.70 (n = 8, P less than 0.025) and to 1.75 +/- 0.66 (n = 9, P less than 0.0002) 4-7 days and 9-19 days, respectively, after treatment. This change was attributed to the growth of reparative tissue. The results provide new information regarding the response and the mechanism of action of tamoxifen.

Animals↗

PubMed1990

31P and 23Na nuclear magnetic resonance studies of resting and stimulated mast cells.

Exocytosis induced by crosslinking the type I receptor for Fc epsilon domains present on rat mucosal mast cells (RBL-2H3-line) requires the influx of Ca2+ ions and is markedly influenced by the concentration of monovalent cations (K+, Na+ and protons) in their medium. We investigated the role of these ions in coupling the immunological stimulus to secretion using NMR spectroscopy to monitor simultaneously intracellular pH, ATP and Na+ concentrations and the secretory response of living adherent mast cells. Using this methodology we observed that: (i) ATP concentration and intracellular pH are highly regulated and no changes could be resolved in them upon stimulation and during exocytosis. (ii) In the absence of potassium ions in the cells' medium, a decrease is observed in the intracellular pH and ATP concentration and an increase in the Na+ concentration. (iii) From the influx of extracellular Na+ following inhibition of the Na+, K(+)-ATPase by ouabain, we estimated the inward Na+ current of resting cells to 5 x 10(7) ions/(cell.s). This value does not vary by more than 10% during exocytosis.

Animals↗

PubMed1990

NMR studies of the lipid metabolism of T47D human breast cancer spheroids.

The in vivo 31P NMR spectrum of T47D human breast cancer cells grown as spheroids shows changes in the phosphomonoester lipid precursors as a function of spheroid size. The ratio of phosphorylethanolamine (PE) to phosphorylcholine (PC) was 1.0 +/- 0.3 for 3-day-old, 150 microns spheroids. This ratio increased to 2.4 +/- 0.4 for spheroids 7 days and older and which were at least 300 microns in diameter. To investigate the phosphatidylethanolamine to phosphatidylcholine (PdylE/PdylC) ratio in the membranes, chloroform/methanol extracts of spheroids were performed. The 31P spectrum of these extracts showed no change with spheroid size, namely the PdylE/PdylC ratio was 0.5 +/- 0.06 for spheroids of all ages suggesting that membrane composition is strongly regulated at the precursor level.

Breast Neoplasms↗

PubMed1990

Chemotherapy-induced changes in the energetics of human breast cancer cells; 31P- and 13C-NMR studies.

The early changes in the energetics of T47D-clone 11 human breast cancer cells, following treatment with adriamycin and several other anti-cancer drugs were characterized by 31P- and 13C-NMR spectroscopy. Treatment of the cells with cytotoxic doses of either adriamycin (10(-5) M), daunomycin (10(-5) M) or actinomycin-D (2 x 10(-6) M) induced an immediate increase in the content of the nucleoside triphosphate (NTP) pool. A maximum increase of 30 to 50% was reached 6 to 8 h after treatment, and was followed by a gradual decrease, in accord with the decline in cell number due to cell death. High-performance liquid chromatography measurements indicated that the adriamycin-induced build-up of the NTP pool was mainly due to a specific increase in ATP and GTP. Treatment with cytotoxic doses of cytosine arabinofuranoside (10(-4) M) and cis-platin (10(-4) M) and with the antiestrogen tamoxifen at a dose which inhibited growth (2 x 10(-6) M) did not induce an early increase in the NTP content. Adriamycin and actinomycin-D did not alter significantly the rates of glucose consumption and lactate production via glycolysis during the first 4 to 8 h of treatment. Both drug, however, caused during this time interval a 50% inhibition in the rate of glutamate synthesis via the Krebs cycle. Complementary flow cytometry studies have indicated that within 4 h of treatment with either adriamycin or actinomycin-D there is no detectable change in cell cycle distribution. Treatment for longer time periods indicated that each drug affects the cell cycle distribution in a different manner. Thus, the early increase in NTP can not be associated with a specific cell cycle distribution. The results suggest therefore that drugs of the anthracycline and actinomycin type exert a similar specific and early metabolic induction which may affect the energy state of the cells. This induction may relate to the cytotoxic mechanism and could potentially serve as an early marker for response to treatment.

Antineoplastic Agents↗

PubMed1990

Metabolic studies with NMR spectroscopy of the alga Dunaliella salina trapped within agarose beads.

A technique for the entrapment of the unicellular algae Dunaliella salina in agarose beads and their perfusion during NMR measurements is presented. The trapped cells maintained their ability to proliferate under normal growth conditions, and remained viable and stable under steady-state conditions for long periods during NMR measurements. Following osmotic shock in the dark, prominent changes were observed in the intracellular level of ATP and polyphosphates, but little to no changes in the intracellular pH or orthoposphate content. When cells were subjected to hyperosmotic shock, the ATP level decreased. The content of NMR-visible polyphosphates decreased as well, presumably due to the production of longer, NMR-invisible structures. Following hypoosmotic shock, the ATP content increased and longer polyphosphates were broken down to shorter, more mobile polymers.

Adenosine Triphosphate↗

PubMed1990

The role of intracellular orthophosphate in triggering osmoregulation in the alga Dunaliella salina.

A new hypothesis is presented for the mechanism of metabolic response during osmoregulation in the alga Dunaliella salina. We propose that the osmotic response is initiated by differential volume changes of the cytoplasm and the chloroplast (observed using the electron microscope) which alter the cytoplasmic orthophosphate concentration. This triggers a flow through the Pi/triose-phosphate shuttle, activating chloroplast enzymes in the direction of either starch or glycerol synthesis. The Pi-dependent response was investigated in vivo using NMR. The rates of glycerol synthesis or elimination following osmotic shocks were modulated by the intracellular Pi level as predicted by the hypothesis.

Chemical Phenomena↗

PubMed1989

Metabolic studies of estrogen- and tamoxifen-treated human breast cancer cells by nuclear magnetic resonance spectroscopy.

The effects of 17 beta-estradiol treatment versus tamoxifen on the metabolism of human breast cancer T47D-clone 11 cells were studied by noninvasive 31P and 13C nuclear magnetic resonance techniques. 31P nuclear magnetic resonance spectra revealed differences between estrogen and tamoxifen treated cells. The steady state content of phosphorylcholine and of the nucleoside diphosphates was higher in the tamoxifen treated cells by 33 and 140%, respectively, relative to estrogen treated cells. The intracellular pH of 7.2 and the content of the nucleoside triphosphates, Pi, phosphocreatine, glycerolphosphorylcholine, and glycerolphosphorylethanolamine and uridine diphosphoglucose remained the same in both treatments. Glucose utilization and subsequent lactate, glutamate, alanine, and glycerol 3-phosphate synthesis were monitored on line following administration of specifically labeled [13C]glucose. In estrogen treated cells the rate of lactate production via glycolysis was 560 fmol/cell/h and the initial rate of 13C labeling of the glutamate pool via the Krebs cycle was 6.8 fmol/cell/h. In the tamoxifen treated cells these rates were 2-fold lower, at 250 and 2.9 fmol/cell/h for lactate and glutamate labeling, respectively. In estrogen treated cells, the calculated content of glutamate (19 fmol/cell), alanine (11 fmol/cell), and glycerol 3-phosphate (8 fmol/cell) was higher than in tamoxifen treated cells, where only glutamate labeling was detected (13 fmol/cell). The observed differences in the in vivo kinetics of glucose metabolism may provide a sensitive measure for detecting the response of human breast cancer cells to estrogen versus tamoxifen treatments.

Breast Neoplasms↗

PubMed1989

31P NMR studies of the thermodynamics and kinetics of the creatine kinase reaction.

The thermodynamic and kinetic parameters of the reaction catalyzed by creatine kinase (CK) were measured in vitro in the temperature range 13 to 35 degrees C, using 31P NMR spectroscopy, including magnetization transfer methods. The apparent equilibrium constant of the reaction and the associated enthalpy for the formation of ATP at 35 degrees C, pH 8.2, and excess [Mg2+] were 3.5 x 10(9) M-1 and -2.4 +/- 0.5 kcal/mol, respectively. The rates at equilibrium at 35 degrees C catalyzed by 1 unit/ml CK were 12.4 and 10.7 microM/s at pH 7 and 8, respectively. The rate constants per 1 unit CK/1 ml at 35 degrees C, pH 7, were 1.3 x 10(8) s-1 M-2 and 9.9 x 10(-3) s-1 M-1 in the direction of ATP and PCr formation, respectively. The activation energies in both directions were similar and corresponded to 15 +/- 2 kcal/mol at pH 7 and 17.5 +/- 1.5 kcal/mol at pH 8. Comparison of in vivo results with the above in vitro data may provide information regarding the activity and kinetics of the CK reaction.

Creatine Kinase↗

PubMed1989

Studies of the metabolism of human breast cancer spheroids by NMR.

Spheroids present a model for tumor behavior in vivo. In NMR studies, a problem of spheroid adhesion occurs. Here, we present a method which overcomes this problem and show the variations in phosphate metabolite concentrations as well as the rate of glucose metabolism as a function of spheroid age.

Adenosine Triphosphate↗

PubMed1989

Estradiol induction of accelerated energy metabolism in prepuberal rat uteri in vitro: mRNA hybridization and [13C]NMR studies.

In vitro treatment with 30 nM 17 beta-estradiol stimulated the induction of mRNA for the brain type isozyme of creatine kinase BB (CKBB) and stimulated glucose metabolism in perifused uteri from 27-29-day-old rats. The perifusion conditions maintained the normal NMR spectrum of high energy phosphates for at least 24 h. This technique permitted the demonstration that perifused rat uteri stimulated by 17 beta-estradiol show increased mRNA for creatine kinase BB, 1 h after estrogen addition. The time-course of increase, measured by Northern blot hybridization, parallels that seen in mRNA extracted from uteri after in vivo induction by i.p. injection of 5 micrograms 17 beta-estradiol; the maximal increase is seen at 2-4 h. Experiments utilizing actinomycin D (4 micrograms/ml) for inhibition of RNA synthesis showed that CKB mRNA from both untreated and estradiol stimulated uteri had a similar half-life, of approximately 2 h, indicating that CKB mRNA is transcriptionally regulated. In the same system, the rate of glycolysis was measured by NMR spectroscopy using [1-13C]glucose. Following in vitro stimulation with 30 nM estradiol, glycolysis increased within 3 h, in parallel to increases previously found in uteri from in vivo stimulated rats.

Animals↗

PubMed1989

Early estrogen-induced metabolic changes and their inhibition by actinomycin D and cycloheximide in human breast cancer cells: 31P and 13C NMR studies.

Metabolic changes following estrogen stimulation and the inhibition of these changes in the presence of actinomycin D and cycloheximide were monitored continuously in perfused human breast cancer T47D clone 11 cells with 31P and 13C NMR techniques. The experiments were performed by estrogen rescue of tamoxifen-treated cells. Immediately after perfusion with estrogen-containing medium, a continuous enhancement in the rates of glucose consumption, lactate production by glycolysis, and glutamate synthesis by the Krebs cycle occurred with a persistent 2-fold increase at 4 hr. The content of phosphocholine had increased by 10% to 30% within the first hour of estrogen stimulation, but the content of the other observed phosphate metabolites as well as the pH remained unchanged. Pretreatment with either actinomycin D or cycloheximide, at concentrations known to inhibit mRNA and protein synthesis, respectively, and simultaneous treatment with estrogen and each inhibitor prevented the estrogen-induced changes in glucose metabolism. This suggested that the observed estrogen stimulation required synthesis of mRNA and protein. These inhibitors also modulated several metabolic activities that were not related to estrogen stimulation. The observed changes in the in vivo kinetics of glucose metabolism may provide a means for the early detection of the response of human breast cancer cells to estrogen versus tamoxifen treatment.

Breast Neoplasms↗

PubMed1988

Adaptation of culture methods for NMR studies of anchorage-dependent cells.

Two methods for growing anchorage-dependent cells were adapted for nuclear magnetic resonance (NMR) measurements: growing cells on agarose polyacrolein microsphere beads and on "filters" made of nonwoven polyester fabric. Both were found to be convenient and most suitable for NMR studies in any conventional spectrometer without probe modification. These methods were employed in studies of human breast cancer T47D-A11 cells, using scanning electron microscopy and 31P NMR spectroscopy. The results show that the contents per cell of phosphorylcholine, phosphorylethanolamine, and their glycerol derivatives depend on the mode of cell assembly and decrease gradually with the increase in cell-cell interaction along the growth curve.

Cell Adhesion↗

PubMed1988

P and C-NMR Studies of the Phosphorus and Carbon Metabolites in the Halotolerant Alga, Dunaliella salina.

The intracellular phosphorus and carbon metabolites in the halotolerant alga Dunaliella salina adapted to different salinities were monitored in living cells by (31)P- and (13)C-nuclear magnetic resonance (NMR) spectroscopy. The (13)C-NMR studies showed that the composition of the visible intracellular carbon metabolites other than glycerol is not significantly affected by the salinity of the growth medium. The T(1) relaxation rates of the (13)C-glycerol signals in intact cells were enhanced with increasing salinity of the growth medium, in parallel to the expected increase in the intracellular viscosity due to the increase in intracellular glycerol. The (31)P-NMR studies showed that cells adapted to the various salinities contained inorganic phosphate, phosphomonoesters, high energy phosphate compounds, and long chain polyphosphates. In addition, cells grown in media containing up to 1 molar NaCl contained tripolyphosphates. The tripolyphosphate content was also controlled by the availability of inorganic phosphate during cell growth. Phosphate-depleted D. salina contained no detectable tripolyphosphate signal. Excess phosphate, however, did not result in the appearance of tripolyphosphate in (31)P-NMR spectra of cells adapted to high (>1.5 molar NaCl) salinites.

Journal Article↗

PubMed1988

Na-NMR Studies of the Intracellular Sodium Ion Concentration in the Halotolerant Alga Dunaliella salina.

The Intracellular Na(+) concentration in the halotolerant alga Dunaliella salina was measured in intact cells by (23)Na-NMR spectroscopy, utilizing the dysprosium tripolyphosphate complex as a sodium shift reagent, and was found to be 88 +/- 28 millimolar. Intracellular sodium ion content and intracellular volume were the same, within the experimental error, in cells adapted to grow in media containing between 0.1 and 4.0 molar NaCl. These values assume extracellular and intracellular NMR visibilities of the (23)Na nuclei of 100 and 40%, respectively. The relaxation rate of intracellular sodium was enhanced with increasing salinity of the growth medium, in parallel to the intracellular osmosity due to the presence of glycerol, indicating that Na(+) ions and glycerol are codistribbuted within the cell volume.

Journal Article↗

PubMed1988

Effects of 17 beta-estradiol on high energy phosphate concentrations and the flux catalyzed by creatine kinase in immature rat uteri: 31P nuclear magnetic resonance studies.

31P nuclear magnetic resonance (NMR) was used to study the effects of 17 beta-estradiol on the content of phosphates and on the flux catalyzed by creatine kinase in immature rat uteri. Perifusion with oxygenated medium at 36 C maintained the uteri in a viable state for at least 10 h in vitro during 31P NMR measurements. In vitro administration of 17 beta-estradiol to the perifused uteri induced changes in the concentration of the high energy phosphates similar to those found after in vivo stimulation: a rapid fall in the concentrations of ATP, phospho-creatine, and the phosphomonoesters during the first 2 h, followed by a slower return to initial concentrations by approximately 6 h. Analysis of the time course of this modulation indicated that after estrogen stimulation, the energy utilization rate was about twice the production rate. The flux through the creatine kinase (CK) reaction was measured independently using 31P magnetization transfer techniques; it was found to increase in uteri 24 h after estradiol injection by the same extent (65%) as the specific activity of CK measured by a spectrophotometric assay. The congruence between the results of these two techniques (in the absence of increased substrate concentrations) provides evidence that the early stimulation of brain-type CK synthesis by estrogen results in a net increase in the concentration of this enzyme.

Algorithms↗

PubMed1987

31P-NMR studies of phosphate transfer rates in T47D human breast cancer cells.

The concentration of phosphates and the kinetics of phosphate transfer reactions were measured in the human breast cancer cell line, T47D, using 31P-NMR spectroscopy. The cells were embedded in agarose filaments and perifused with oxygenated medium during the NMR measurements. The following phosphates were identified in spectra of perifused cells and of cell extracts: phosphorylcholine (PC), phosphorylethanolamine (PE), the glycerol derivatives of PC and PE, inorganic phosphate (Pi), phosphocreatine (PCr), nucleoside triphosphate (primarily ATP) and uridine diphosphate glucose. The rates of the transfers: PC----gamma ATP (0.2 mM/s), Pi----gamma ATP (0.2 mM/s) and the conversion beta ATP----beta ADP (1.3 mM/s) were determined from analysis of data obtained in steady-state saturation transfer and inversion recovery experiments. Data from spectrophotometric assays of the specific activity of creatine kinase (approx. 0.1 mumol/min per mg protein) and adenylate kinase (approx. 0.4 mumol/min per mg protein) suggest that the beta ATP----beta ADP rate is dominated by the latter reaction. The ratio between the rate of ATP synthesis from Pi and the rate of consumption of oxygen atoms (4 X 10(-3) mM/s) was approx. 50. This high value and preliminary measurements of the rate of lactate production from glucose, indicated that aerobic glycolysis is the main pathway of ATP synthesis.

Adenosine Diphosphate↗

PubMed1987

31P magnetization transfer studies of creatine kinase kinetics in living rabbit brain.

The kinetics of the CK reaction in the living rabbit brain was studied by three 31P magnetization transfer methods: inversion recovery (IR), inversion transfer (IT), and steady-state saturation transfer (SSST). Longitudinal relaxation rates (tau -1) were determined from IR and IT experiments. The values were 0.88 +/- 0.08 s-1 for PCr and 2.45 +/- 0.39 s-1 for gamma-ATP. Analysis of the results of SSST experiments in which gamma-ATP was saturated yielded a forward rate constant kF of 0.53 +/- 0.07 s-1. Upon saturation of PCr no change in the gamma-ATP signal could be detected in SSST. An average reverse rate constant (kR +/- S.D.) of 0.52 +/- 0.04 s-1 was estimated by analyzing IT data from three experiments with selective inversion of gamma-ATP. The standard error of kR was 50%. The average [PCr]/[ATP] of 1.21 +/- 0.16 together with the values of kF and kR yielded a forward-to-reverse flux ratio of 1.23. Within our limits of accuracy this ratio was not significantly different from 1.0, thus suggesting that in the brain the forward and reverse CK fluxes are equal.

Adenosine Triphosphate↗