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S Grinstein

Publications and source records attributed to S Grinstein.

At least 289 records · Page 16Linked to original sources
PubMed1986

Epidemiology of human rotaviruses in Argentina as determined by RNA genome electrophoresis.

Rotavirus gastroenteritis was studied by enzyme-linked immunosorbent assay (ELISA) and by PAGE from April 1983 to April 1985 at the Buenos Aires Ricardo Gutierrez Children's Hospital and at the San Justo Children's Hospital. Based on examination of 576 cases, rotavirus was identified in 109 (18.9%) cases by ELISA and in 99 (17.2%) cases by PAGE. As a diagnostic tool PAGE presented a sensitivity of 90.8% compared with ELISA. Compared with the control SA-11 genome, 84 samples (84.9%) presented a long electropherotype and 15 isolates (15.1%) presented a short electropherotype. We detected 35 different long electropherotypes and report a short electropherotype that did not show genome variation and was responsible for an outbreak in the San Justo Children's Hospital population from September 1983 to July 1984.

Argentina↗
PubMed1985

Interleukin 2 induces a rapid increase in intracellular pH through activation of a Na+/H+ antiport. Cytoplasmic alkalinization is not required for lymphocyte proliferation.

In several cell types, proliferation initiated by growth factors is associated with a rapid increase in cytoplasmic pH (pHi). This cytoplasmic alkalinization is due to the activation of an amiloride-sensitive Na+/H+ antiport. It is unclear whether growth factor-induced activation of the antiport or the resultant increase in pHi is the trigger for proliferation, an obligatory requirement for proliferation, or simply an associated phenomenon. Interleukin 2 (IL 2) acts as a growth factor for mitogen or antigen-stimulated thymus-derived (T) lymphocytes. In this study, we established that IL 2 produces an increase in pHi and determined whether this increase in pHi plays a role in the proliferative response to IL 2. Monitoring pHi with an intracellularly trapped, pH-sensitive, fluorescent dye, 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein, we demonstrated that IL 2 rapidly (less than 90 s) initiates an increase in pHi in IL 2-sensitive human and murine T cells. Because intracellular alkalinization requires extracellular Na+ and is amiloride-sensitive, it likely occurs through activation of the Na+/H+ antiport. Using partitioning of a weak acid, 5,5-dimethyl-2,4-oxazolidinedione, we confirmed that the IL 2-dependent increase in pHi is sustained for several hours and returns to near base-line levels by 18 h. We also investigated the consequence of preventing Na+/H+ exchange on the proliferative response induced by IL 2. IL 2-driven proliferation occurred in nominally bicarbonate-free medium in the presence of concentrations of amiloride analogs sufficient to inhibit the Na+/H+ antiport and prevent intracellular alkalinization. These data suggest that although the antiport is activated by binding of IL 2 to its receptor, intracellular alkalinization is not essential for IL 2-dependent proliferation. It seems unlikely that either cytoplasmic alkalinization or activation of the Na+/H+ antiport are triggers for T cell proliferation.

Amiloride↗
PubMed1985

Control of free cytoplasmic calcium by intracellular pH in rat lymphocytes.

Activation of Na+-H+ exchange in rat thymocytes was found to be followed by an increase in free cytoplasmic Ca2+ concentration [( Ca2+]i). We determined whether the change in [Ca2+]i was secondary to the uptake of Na+, or to the cytoplasmic alkalinization that result from activation of the antiport. Increasing intracellular [Na+] by treating the cells with ouabain or gramicidin failed to affect [Ca2+]i. In contrast, procedures that increased the cytoplasmic pH, such as addition of monensin or NH3, significantly elevated [Ca2+]i. These results suggest an important role of cytoplasmic pH in the control of [Ca2+]i in lymphocytes.

Aminoquinolines↗
PubMed1985

Bromo-A23187: a nonfluorescent calcium ionophore for use with fluorescent probes.

4-Bromo-A23187, a halogenated analog of the widely studied divalent cation ionophore A23187, is a nonfluorescent Ca2+ ionophore suitable for use in the calibration of cytoplasmic free Ca2+ by fluorescent probes. Br-A23187 is shown to saturate Ca2+ sites in quin-2-loaded rat thymic lymphocytes in a manner essentially identical to ionomycin.

Aminoquinolines↗
PubMed1985

Chemical modification of the Na+/H+ exchanger of thymic lymphocytes. Inhibition by N-ethylmaleimide.

A Na+/H+ exchanger is involved in the regulation of cytoplasmic pH and cellular volume in a variety of cells. Little is known about the molecular nature of this exchanger. The purpose of this study was to survey a variety of group-specific covalent reagents as potential inhibitors of the exchanger. Na+/H+ countertransport activity was assayed as the amiloride-sensitive rate of Na+-induced alkalinization in acid-loaded lymphocytes, or as the rate of swelling in cells suspended in sodium propionate medium. Activity was not affected by proteinases or by carboxyl-group and amino-group specific reagents. A significant inhibition was produced by diethylpyrocarbonate, a histidine-specific reagent and by N-ethylmaleimide, a sulfhydryl group reagent. A similarly reactive but nonpermeating sulfhydryl agent, glutathione-maleimide, failed to inhibit Na+-H+ exchange. Moreover, the reaction with N-ethylmaleimide was sensitive to changes in the cytoplasmic pH. The data suggest that the chemically reactive groups of the Na+/H+ exchanger of lymphocytes have limited exposure to the extracellular medium but that an internally located sulfhydryl group is critical for the cation-exchange activity.

Animals↗
PubMed1985

The free cytoplasmic Ca2+ levels in Duchenne muscular dystrophy lymphocytes.

An increased cellular Ca2+ content has been associated with Duchenne muscular dystrophy (DMD). However, estimates of the free cytoplasmic Ca2+ concentration ([Ca2+]i) in cells of DMD patients were not available. We compared the [Ca2+]i levels of normal and DMD peripheral blood lymphocytes and Epstein-Barr virus-transformed lymphoblasts using the novel probe, quin 2, an internally trapped fluorescent indicator. The [Ca2+]i levels of normal and DMD cells were not significantly different.

Calcium↗
PubMed1985

Characterization of the activation of Na+/H+ exchange in lymphocytes by phorbol esters: change in cytoplasmic pH dependence of the antiport.

Phorbol 12-myristate 13-acetate and other phorbol esters induce an intracellular alkalinization in rat thymic lymphocytes. An extracellular acidification can be recorded concomitantly. This transmembrane H+ (equivalent) flux is dependent on external Na+ and is amiloride sensitive. Phorbol esters also stimulate an amiloride-sensitive uptake of 22Na+, suggesting activation of Na+o/H+i exchange. Only those phorbol derivatives that are tumor promoters and activators of protein kinase C stimulate the antiport. Activation of the Na+/H+ exchange is brought about by a change in the cytoplasmic pH sensitivity of the antiport. Activation of the Na+/H+ exchanger by phorbol esters results in membrane hyperpolarization, due to indirect stimulation of the electrogenic Na+/K+ pump by the increased intracellular Na+ concentration. Increased Na+/H+ exchange also produces cell swelling, which may be one of the earliest manifestations of the growth-promoting properties of the phorbol esters.

Amiloride↗
PubMed1985

Osmotic and phorbol ester-induced activation of Na+/H+ exchange: possible role of protein phosphorylation in lymphocyte volume regulation.

The Na+/H+ antiport is stimulated by 12-O-tetradecanoylphorbol-13, acetate (TPA) and other phorbol esters in rat thymic lymphocytes. Mediation by protein kinase C is suggested by three findings: (a) 1-oleoyl-2-acetylglycerol also activated the antiport; (b) trifluoperazine, an inhibitor of protein kinase C, blocked the stimulation of Na+/H+ exchange; and (c) activation of countertransport was accompanied by increased phosphorylation of specific membrane proteins. The Na+/H+ antiport is also activated by osmotic cell shrinking. The time course, extent, and reversibility of the osmotically induced and phorbol ester-induced responses are similar. Moreover, the responses are not additive and they are equally susceptible to inhibition by trifluoperazine, N-ethylmaleimide, and ATP depletion. The extensive analogies between the TPA and osmotically induced effects suggested a common underlying mechanism, possibly activation of a protein kinase. It is conceivable that osmotic shrinkage initiates the following sequence of events: stimulation of protein kinase(s) followed by activation of the Na+/H+ antiport, resulting in cytoplasmic alkalinization. The Na+ taken up through the antiport, together with the HCO3- and Cl- accumulated in the cells as a result of the cytoplasmic alkalinization, would be followed by osmotically obliged water. This series of events could underlie the phenomenon of regulatory volume increase.

Adenosine Triphosphate↗
PubMed1985

Mechanism of osmotic activation of Na+/H+ exchange in rat thymic lymphocytes.

The activity of the Na+/H+ exchange system of rat thymic lymphocytes was determined by means of intracellular (pHi) and extracellular pH (pH0) measurements. In isotonic media, the antiport is virtually quiescent at physiological pHi (7.0-7.1), but is greatly activated by cytoplasmic acidification. At normal pHi, the antiport can also be activated by osmotic shrinking. Osmotic activation occurs after a delay of 20-30 s and is reversed several minutes after iso-osmolarity is restored. The mechanism of activation was analyzed by comparing the kinetic parameters of transport in resting (isotonic) and hyperosmotically stressed cells. The affinities of the external substrate site for Na+ and H+ are not altered in shrunken cells. In contrast, the Hi+ sensitivity of the antiport (which is largely dictated by an allosteric modifier site) was increased, which accounted for the activation. The concentration of free cytoplasmic Ca2+ [( Ca2+]i) increased after osmotic shrinking. This increase was dependent on the presence of extracellular Ca2+ and Na+ and was blocked by inhibitors of Na+/H+ exchange, which suggests that it is a consequence, rather than the cause, of the activation of the antiport. It is concluded that the shift in the pHi dependence of the modifier site of the Na+/H+ antiport is the primary event underlying the regulatory volume increase that follows osmotic shrinkage.

Amiloride↗
PubMed1985

Phorbol ester-induced changes of cytoplasmic pH in neutrophils: role of exocytosis in Na+-H+ exchange.

Cytoplasmic pH homeostasis was studied in intact and granule-free porcine neutrophils following activation with 12-O-tetradecanoyl-phorbol-13-acetate (TPA). In intact cells, TPA activated at least two separate processes: a Na+-independent and amiloride-insensitive acidification, and a compensatory acid extrusion. The latter is Na+ dependent and blocked by amiloride and is likely to represent Na+-H+ exchange. Enucleated and degranulated neutrophils (cytoplasts) were prepared by sedimentation of cytochalasin B-treated neutrophils through a discontinuous density gradient. Cytoplasts responded to an artificially imposed acid load with activation of an amiloride-sensitive Na+-H+ exchange. TPA also activated both acid production and Na+-dependent acid extrusion in cytoplasts. The magnitude of the responses was comparable in intact neutrophils and in cytoplasts. These data suggest that 1) the nucleus and the secretory granules are not involved in the acidifying response to TPA, and 2) exocytosis of secretory vesicles is not required for activation of Na+-H+ exchange during acid loading or following treatment with the phorbol ester TPA.

Amiloride↗
PubMed1985

Cation and anion transport pathways in volume regulatory response of human lymphocytes to hyposmotic media.

The regulatory volume decrease of osmotically swollen human peripheral blood lymphocytes can be inhibited by agents acting on volume-activated K+- or Cl--transport pathways. Quinine, cetiedil, and 3,3'-dipropylthiadicarbocyanine were found to block the volume-induced K+ transport by interaction with sites on the outside face of the membrane, perhaps by competition with external K+. Drugs known to influence calmodulin action inhibit both volume-induced K+ and Cl- transport to varying degrees. Those inhibitors, particularly of K+ transport, are correlated with their calmodulin-antagonist activity. Penetrating sulfhydryl (SH) reagents (in contrast to nonpenetrating ones) are potent inhibitors of both volume-induced K+ and Cl- movements, indicating the presence of functionally important SH groups located within the membrane or at the cytoplasmic face. A number of agents, such as dipyridamole and oligomycin C, are specific inhibitors of the volume-activated anion pathway. In all respects studied, the inhibition characteristics of the volume-activated K+ pathway of lymphocytes resemble those of the Ca2+-activated K+ channel of red cells. In contrast, the volume-induced anion permeability differs from the primary anion-transport pathway of red cells.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1985

Analysis of Ca2+-induced K+-transport of human erythrocytes in propionate media.

A method is described which allows studying specific cation transport pathways of the cell membranes by converting ion fluxes into volume changes. Lipophilic weak electrolytes, such as propionate, rapidly penetrate the cell membranes in their undissociated acid from but not as negatively charged ions. When human red cells are incubated in isoosmotic K-propionate media an intracellular acidification occurs with a limited propionate uptake and volume increase (corresponding to the buffering capacity of the cytoplasm). If both protons and alkali cations are rendered permeable, a rapid salt influx and volume increase is observed. The latter can be quantitatively followed by electronic sizing methods. A detailed characterization of the system is provided through studies with ionophores, inhibitors of the red cell anion exchange system and drugs which activate or inhibit the Ca2+-induced K+ transport. It is demonstrated that in K-propionate media the permeability of the Ca2+-induced K+ pathway can be directly estimated. The method is suitable to observe population (all-or-none) responses in the activation of the K+ pathway under certain experimental conditions. The application of the method in the search for cation-proton exchanger systems is discussed and its use for demonstration purposes by producing selective lysis of red cells is described.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1985

[Comparison of enzyme immunoassay, counterimmunoelectrophoresis and polyacrylamide gel electrophoresis for the diagnosis of rotaviruses].

Detection of human rotaviruses by enzyme-linked immunosorbent assay (ELISA), provided by the World Health Organization, in 100 stool specimens was compared with the sensitivity of detection by counterimmunoelectrophoresis (CIED) and polyacrylamide gel electrophoresis (PAGE). Table 1 shows that 34 samples were found to contain rotavirus antigens by ELISA and in other 4 specimens false negative results were observed in the ELISA confirmatory test because of high antigen concentration. The ELISA confirmatory test was repeated with the 4 samples diluted 1/40 and they became positives. It was found that, of the 38 ELISA positive samples, 35 were positive by PAGE (92%) and 24 by CIED (63%). Detection of human rotaviruses in stools by PAGE were almost as sensitive as ELISA, but the direct visualization of viral RNA fragments, provided a distinct advantage since no confirmatory test was necessary. Besides, the surveillance of rotavirus electrophoretypes constitutes a very useful tool in epidemiological studies, in order to the future possible use of a rotavirus vaccine in our country. By the other hand, it was found CIED less sensitive than ELISA and PAGE. This disadvantage might be tolerated in a diagnostic setting, only if no other test was available for rotavirus diagnosis.

Counterimmunoelectrophoresis↗
PubMed1985

Increase in cytosolic free calcium concentration is an intracellular messenger for the production of interleukin 2 but not for expression of the interleukin 2 receptor.

Activation of lymphocytes by mitogenic lectins results in the production of a group of soluble factors, the lymphokines. Proliferation of activated T cells requires interaction of one of these lymphokines, interleukin 2 (IL 2), with its receptor. The induction of IL 2 receptor expression and IL 2 production may involve different activation signals; some mitogens or antigens may activate both, whereas others may activate only one. An increase in cytosolic free calcium concentrations [( Ca++]i) is one of the signals involved in cellular activation by lectins. By using the fluorescent indicator quin-2, we have demonstrated that increases in [Ca++]i accompany phytohemagglutinin induced proliferative responses of human T lymphocytes. Preventing the increase in [Ca++]i also prevents proliferation. We demonstrate that an increase in [Ca++]i is not required for the expression of the IL 2 receptor, which is expressed even in the presence of extremely low external calcium concentrations. In contrast, IL 2 production requires an increase in [Ca++]i and does not occur in the absence of extracellular free calcium. IL 2 production appears to be the critical step requiring transmembrane calcium flux. In the absence of transmembrane calcium flux and subsequent IL 2 production, lectins are not able to trigger DNA synthesis and cell proliferation.

Animals↗
PubMed1985

Interleukin 2-induced lymphocyte proliferation is independent of increases in cytosolic-free calcium concentrations.

Activation of lymphocytes by mitogenic lectins initiates a sequence of events that culminates in DNA synthesis and cell proliferation. The mitogenic effects of lectins on T lymphocytes leads to the production of a group of lymphokines including the interleukins. The binding of interleukin 2 (IL 2) to its receptor results in activation of the cell leading to DNA synthesis. An increase in cytosolic-free Ca++ ([Ca++]i) is associated with activation of lymphocytes by mitogenic lectins and also appears to be a prerequisite for induction of DNA synthesis and cell proliferation. We have determined whether the proliferative response triggered by IL 2 binding to its receptor is associated with or requires an increase in [Ca++]i. Using human and murine IL 2-sensitive cell lines, we have demonstrated that the IL 2-induced proliferative response, in contrast to that induced by mitogens such as phytohemagglutinin or concanavalin A, is not accompanied by an increase in [Ca++]i as monitored by the fluorescent indicator quin-2. Furthermore, IL 2-dependent triggering of lymphoblasts occurs in the presence of extremely low extracellular calcium concentrations that prevent transmembrane calcium flux. Activation of IL 2 receptor-bearing T cells, therefore, does not appear to be associated with or to require an increase in [Ca++]i as part of the activation and signaling process. The critical step requiring calcium flux in cell signaling by mitogenic lectins must therefore occur elsewhere in the activation cascade.

Animals↗
PubMed1985

Na+/H+ exchange in volume regulation and cytoplasmic pH homeostasis in lymphocytes.

Osmotic shrinking activates an amiloride-sensitive Na+/H+ exchange in the membrane of blood and thymic lymphocytes. The exchange, which is virtually quiescent in isotonic conditions, can also be activated by lowering the cytoplasmic pH (pHi). Activation by pHi is largely caused by an allosteric interaction of H+ with a kinetic modifier site, different from the internal substrate site. The set point or threshold pHi for activation of the exchanger is dictated by the protonation of the modifier. Evidence is presented that indicates that cell shrinking alters the pHi sensitivity of the modifier, shifting the set point to more alkaline levels. In the presence of HCO3- and Cl- a volume increase will accompany the change in pHi. Volume changes can also be produced in isotonic solutions if the exchange is activated by acidification of the cytoplasm, e.g., by addition of propionate to the medium. The latter phenomenon provides a simple method for the detection of the Na+/H+ antiport by electronic cell sizing.

Amiloride↗
PubMed1984

Volume regulation in lymphoid leukemia cells and assignment of cell lineage.

Normal T lymphocytes readjust their volume after swelling in hypotonic medium, whereas B lymphocytes remain swollen. The difference between the two cell types appears to lie in the inability of B cells to increase their permeability to potassium in response to swelling. We have studied mononuclear cells from the bone marrow of 65 patients with lymphocytic leukemia, to determine whether the response to a hypotonic environment could be used to assign cell lineage. In contrast to leukemic cells of B-cell lineage (as in B-cell acute or chronic lymphocytic leukemia and non-T-cell, non-B-cell acute lymphocytic leukemia), T-cell-leukemia cells responded within five minutes to hypotonic swelling with an increase in potassium permeability and restoration toward isotonic volume. We conclude that the response to hypotonic shock may distinguish leukemic cells of T-cell origin from those derived from B-cell precursors and provides a simple, rapid, and reliable means for assigning cell lineage to lymphoid leukemia cells.

B-Lymphocytes↗