Search PubMed⌕ Search

Biomedical subjects

S Treves

Publications and source records attributed to S Treves.

At least 37 records · Page 2Linked to original sources
PubMed1995

A ryanodine receptor-like Ca2+ channel is expressed in nonexcitable cells.

In this study we investigated the effects of 4-chloro-3-ethylphenol on Ca2+ homeostasis, and we report that this compound induces Ca2+ release from a ruthenium red-sensitive Ca2+ release channel present in skeletal muscle terminal cisternae. When tested with bovine cerebellar microsomes, the Ca(2+)-releasing activity of 4-chloro-3-ethylphenol was reduced by ruthenium red and unaffected by heparin. In PC-12 cells, HL-60 cells, human fibroblasts, the human hepatoma cell line PLC/PRF/5, and Jurkat cells, 4-chloro-3-ethylphenol released Ca2+ from intracellular thapsigargin-sensitive stores. Although decreased, its effect was retained after treatment of intact/permeabilized cells with inositol-1,4,5-trisphosphate (IP3)-mobilizing agonists/IP3, whereas pretreatment of permeabilized cells with ruthenium red reduced the Ca(2+)-releasing activity of 4-chloro-3-ethylphenol. These results provide functional evidence for the presence of a Ca2+ channel distinct from the IP3 receptor, having pharmacological similarities to the ryanodine receptor, in the intracellular Ca2+ stores of a variety of nonexcitable cells. We also demonstrate that a monoclonal anti-ryanodine receptor antibody recognizes a protein in human fibroblasts with similar apparent molecular mass the ryanodine receptor. Thus, the intracellular Ca2+ stores of mammalian cells appear to be endowed with two distinct intracellular Ca2+ channels.

Animals↗
PubMed1994

Identification and characterization of three calmodulin binding sites of the skeletal muscle ryanodine receptor.

In the present study, we have identified calmodulin binding sequences in the skeletal muscle ryanodine receptor Ca2+ release channel. Ligand overlays on RYR fusion proteins indicate that the skeletal muscle RYR contains three calmodulin binding regions defined by residues 2937-3225, 3546-3655, and 4425-4621. The RYR fusion protein PC28 (residues 2937-3225) bound calmodulin in the presence of EGTA and Ca2+, while RYR fusion protein PC26 (residues 3546-3655) exhibited strong calmodulin binding at 10 microM Ca2+. The RYR fusion protein PC15 (residues 4425-4621) did not bind calmodulin in the presence of either EGTA or 10-50 microM Ca2+. In the presence of 100-500 microM Ca2+, the RYR fusion protein PC15 exhibited an affinity for calmodulin of approximately 50 nM. Peptides RYR1 PM2 (residues 3610-3629) and RYR1 PM3 (4534-4552) encompassing putative RYR-calmodulin binding sites were synthesized. The synthetic peptides interacted directly with dansylcalmodulin as demonstrated by their capacity to affect the fluorescence emission of dansylcalmodulin. Missense mutation analysis indicates that the Lys and Arg residues are essential for calmodulin binding to the synthetic peptide RYR1 PM3. The RYR calmodulin binding site defined by peptide PM3 lies in the myoplasmic loop 2, a few residues upstream of the putative transmembrane segment M5; the other two calmodulin binding sites are next to the putative transmembrane segments M' and M''. Thus, the effect of calmodulin on Ca2+ release might involve the regulation of the putative transmembrane segments M5, M', and M''.

Amino Acid Sequence↗
PubMed1994

Alteration of intracellular Ca2+ transients in COS-7 cells transfected with the cDNA encoding skeletal-muscle ryanodine receptor carrying a mutation associated with malignant hyperthermia.

Malignant hyperthermia (MH), an inherited neuromuscular disease triggered by halogenated inhalational anaesthetics and skeletal-muscle relaxants, appears to be due to an alteration of intracellular Ca2+ homoeostasis. MH occurs in 1 out of 20,000 anaesthetized adults and is characterized by hypermetabolism, skeletal-muscle rigidity and elevation in body temperature, which is frequently fatal [MacLennan and Phillips (1992) Science 256, 789-794]. The defect responsible for the disease may lie within the mechanism controlling the release of Ca2+ from sarcoplasmic reticulum via the ryanodine-receptor (RYR) Ca2+ channel; in fact a point mutation in the RYR has been associated with MH in some human families, as well as in the MH-susceptible pig. To date, however, no direct evidence has been obtained demonstrating that the point mutation is both necessary and sufficient to cause functional alterations in RYR-mediated Ca2+ release. In the present report we show that the presence of the Arg-to-Cys point mutation in the recombinant RYR expressed in COS-7 transfected cells causes abnormal cytosolic Ca2+ transients in response to 4-chloro-m-cresol, an agent capable of eliciting in vitro contracture of MH-susceptible muscles.

Amino Acid Sequence↗
PubMed1994

Apoptosis is dependent on intracellular zinc and independent of intracellular calcium in lymphocytes.

Changes in intracellular Ca2+ concentration ([Ca2+]i) are believed to have a crucial role in triggering apoptosis, the process whereby cells are eliminated during tissue turnover, hormone-mediated tissue reabsorption, clonal selection in the thymus, T cell-mediated cytotoxic reactions, and many other physiological and pathological processes. In the present report we have explored the role of intracellular Ca2+ and Zn2+ in the regulation of apoptosis in peripheral blood lymphocytes (PBL). Unless they were stimulated by a mitogen, e.g., phytohemagglutinin (PHA), PBL showed typical apoptotic changes (nuclear condensation and DNA fragmentation) after a 48- to 72-h culture. Neither a sustained nor a transient increase in [Ca2+]i induced by the Ca2+ ionophore ionomycin in the presence or absence of extracellular Ca2+, respectively, was able to trigger DNA fragmentation. On the contrary, [Ca2+]i chelation to a level 10- to 20-fold lower than physiological resting levels induced DNA cleavage into nucleosome-size DNA fragments. DNA cleavage was also induced by incubation in the presence of the intracellular, membrane-permeant Zn2+ chelator N,N,N',N'-tetrakis(2-pyridyl-methyl)ethylenediamine. Total intracellular Zn2+ content was increased by mitogenic stimulation with PHA and decreased by treatment of PBL cultures with dexamethasone or extracellular ATP, stimuli known to trigger apoptosis in lymphocytes. Our results suggest that Zn2+ rather than Ca2+ could be a key intracellular regulator of apoptosis.

Apoptosis↗
PubMed1993

Identification of the domain recognized by anti-(ryanodine receptor) antibodies which affect Ca(2+)-induced Ca2+ release.

In the present paper we have defined putative functional domains of the ryanodine receptor Ca2+ channel. cDNA fragments of the skeletal muscle ryanodine receptor were fused in-frame with the Escherichia coli trpe protein and the resulting fusion proteins were evaluated for their ability to react with anti-(ryanodine receptor) antibodies, which are known to block Ca(2+)-dependent activation of the Ca(2+)-release channel. Anti-(ryanodine receptor) antibodies react with epitopes lying within a 245-amino-acid-long polypeptide which is located in a region (residues 4380-4625) encompassing most of myoplasmic loop 2, the predicted transmembrane segment M5 and part of the next lumenal loop (45 residues). Purification of the anti-(ryanodine receptor) antibodies by affinity chromatography led to the isolation of a population of antibodies which was capable of decreasing (by > 30%) the doxorubicin-induced Ca2+ release from isolated terminal cisternae. Polyclonal antibodies raised against a ryanodine receptor fusion encompassing part (198 out of 245 residues) of the immunopositive polypeptide decreased by 2-fold the first-order rate constant of Ca(2+)-induced 45Ca2+ efflux from isolated terminal cisternae. These results suggest strongly that the Ca(2+)-activating domain of the skeletal muscle Ca(2+)-release channel is close to, or associated with, myoplasmic loop 2.

Animals↗
PubMed1993

Purification of calreticulin-like protein(s) from spinach leaves.

In a search for the plant equivalent of calsequestrin or calreticulin, the high capacity, low affinity Ca2+ binding proteins of muscle and non-muscle cells thought to play important roles in Ca2+ storage, we purified two Ca(2+)-binding proteins from spinach leaves. The proteins had apparent molecular weights of 55 and 53 kDa. On Western blot, they did not react either with anti-rabbit skeletal muscle, anti-dog cardiac muscle calsequestrin or anti-rabbit or anti-rat liver calreticulin antibodies, indicating that they were antigenically distinct. Periodic acid Schiff staining (PAS) revealed that the larger protein was glycosylated while the 53 kDa one was PAS-negative. When the proteins were subjected to NH2-terminus amino acid sequencing, the 55 and 53 kDa proteins turned out to be identical, thus probably representing different isoforms of the same protein. Comparison with published amino acid sequences of calreticulin reveals regions of similarity indicating that the plant Ca(2+)-binding proteins probably belong to the calreticulin family.

Amino Acid Sequence↗
PubMed1993

Chlorocresol: an activator of ryanodine receptor-mediated Ca2+ release.

In the present study we investigated the effect of the compound chlorocresol on intracellular Ca2+ homeostasis. Three different systems that have been shown to express the ryanodine receptor Ca2+ channel were chosen, i.e., skeletal muscle sarcoplasmic reticulum, cerebellar microsomes, and PC12 cells. In skeletal muscle sarcoplasmic reticulum, 4-chloro-m-cresol was found to be a potent activator of Ca2+ release mediated by a ruthenium red/caffeine-sensitive Ca2+ release channel. In cerebellar microsomes, this compound released Ca2+ from an inositol-1,4,5-trisphosphate-insensitive store, suggesting that there too it was acting at the ryanodine receptor level. When tested on PC12 cells, chlorocresol released Ca2+ from a caffeine- and thapsigargin-sensitive intracellular store. In addition, the compound was capable of releasing Ca2+ after pretreatment of PC12 cells with bradykinin, suggesting that it acts on a channel contained within an intracellular Ca2+ store that is distinct from that sensitive to inositol-1,4,5-trisphosphate. Structure-activity relationship analyses suggest that the chloro and methyl groups in chlorocresols are important for the activation of the ryanodine receptor Ca2+ release channel.

Animals↗
PubMed1992

Identification of calreticulin isoforms in the central nervous system.

In the present paper we report the cloning and sequencing of the cDNA encoding two calreticulin isoforms from Xenopus laevis central nervous system. The two isoforms display 93% identity at the amino acid level. The predicted amino acid sequences of the amphibian calreticulins are very similar (76%) to those of mammalian liver and skeletal muscle. Xenopus laevis calreticulins are characterized by a very acidic c-terminal domain endowed with the endoplasmic-reticulum retention signal KDEL. The cDNAs of both clones encode an N-glycosylation consensus sequence. A third clone of calreticulin was also identified. The restriction map of this clone was clearly distinct from that of the two sequenced clones. These results indicate the existence of multiple calreticulin isoforms in the central nervous system and open questions about their functional role in different cells and/or subcellular compartments.

Amino Acid Sequence↗
PubMed1992

Molecular cloning, functional expression and tissue distribution of the cDNA encoding frog skeletal muscle calsequestrin.

We have cloned, sequenced and expressed the cDNA encoding frog skeletal muscle calsequestrin. The processed frog calsequestrin is 398 residues long, with an Mr of 45941 (unglycosylated form), and exhibits 77% sequence similarity with its rabbit counterpart. Consensus sequences for glycosylation and phosphorylation of the protein were conserved. Compared with rabbit calsequestrin, the mature amphibian protein has peculiar structural properties, which include (i) a higher content of negatively charged residues (142 versus 109), and (ii) a striking repeat sequence at the C-terminal region of 44 aspartic acid residues. Furthermore, this is the first report on the expression of calsequestrin cDNA in COS-1 cells; the expressed protein exhibited an Mr and antigenic properties which were indistinguishable from those of the native protein. In addition, it was capable of binding 45Ca in a ligand overlay. Northern blot analysis of frog skeletal muscle, liver, heart and brain RNA showed that the protein is mainly expressed in skeletal muscle. The high density of negative charges at the C-terminus might constitute high-capacity low-affinity Ca(2+)-binding sites, which may account for the higher Ca(2+)-binding capacity of frog calsequestrin compared with other members of the calsequestrin family (56 mol/mol versus 40-44 mol/mol of protein).

Amino Acid Sequence↗
PubMed1992

Efficacy of domperidone in infants and children with gastroesophageal reflux.

This study sought to define the therapeutic efficacy of domperidone in infants and children with gastroesophageal reflux. A double-blind, placebo-controlled trial was performed in seventeen children (ages 5 months to 11.3 years) with moderate to severe gastroesophageal reflux who had not responded to standard nonpharmacological therapy. Subjective and objective measures (weight gain, esophageal pH probe study, radionuclide gastric emptying scan) of gastroesophageal reflux were evaluated. Therapy with domperidone for 4 weeks was effective only in reducing the total number of reflux episodes in the two-hour postprandial period (p less than 0.01); however, it did not result in symptomatic improvement or significant improvement in other measures of gastroesophageal reflux or gastric emptying. After therapy for 8 weeks symptomatic improvement was reported in some patients who had denied improvement after 4 weeks of therapy, suggesting that more than 4 weeks of therapy may be required for some patients to obtain a clinical response. Mild self-limited diarrhea was reported by six patients (four domperidone, two placebo). We conclude that domperidone is tolerated by most infants and children with gastroesophageal reflux; however, 4 weeks of therapy was only minimally effective in producing objective improvement of gastroesophageal reflux and did not result in symptomatic improvement. Further studies of longer duration are needed to resolve the question raised by this study: that domperidone may be beneficial for patients with gastroesophageal reflux when given for more than four weeks.

Child↗
PubMed1991

Na+/H+ antiporter has different properties in human B lymphocytes according to CD5 expression and malignant phenotype.

In B chronic lymphocytic leukemia (B-CLL) cells, lipopolysaccharide (LPS) and phorbol esters fail to activate the plasma membrane-associated Na+/H+ antiporter and, subsequently, to elicit a rise in cytosolic pH. Since these events are thought to be a prerequisite for LPS-induced proliferation of B normal lymphocytes, we analyzed the kinetic properties of Na+/H+ antiporter in B-CLL cells as compared to both CD5- and CD5+ normal B lymphocytes. In the present work we report that Na+/H+ exchange rate after acid loading is drastically decreased in B-CLL cells, as compared to normal CD5- B lymphocytes, although the antiporter affinity for external Na+ and internal H+ is not significantly different in both cell populations. Kinetic data account for a reduction in the number of operating antiport units in B-CLL. The Na+/H+ antiporter of CD5+ normal B lymphocytes exhibits both an exchange rate and an ion affinity significantly higher than that observed in both CD5+ B-CLL cells and CD5- B normal lymphocytes, thus suggesting a possible explanation for their activated phenotype.

Antigens, CD↗
PubMed1990

Calreticulin is a candidate for a calsequestrin-like function in Ca2(+)-storage compartments (calciosomes) of liver and brain.

In a search for the non-muscle equivalent of calsequestrin (the low-affinity high-capacity Ca2(+)-binding protein responsible for Ca2+ storage within the terminal cisternae of the sarcoplasmic reticulum), acidic proteins were extracted from rat liver and brain microsomal preparations and purified by column chromatography. No calsequestrin was observed in these extracts, but the N-terminal amino acid sequence of the major Ca2(+)-binding protein of the liver microsomal fraction was determined and found to correspond to that of calreticulin. This protein was found to bind approx. 50 mol of Ca2+/mol of protein, with low affinity (average Kd approx. 1.0 mM). A monoclonal antibody, C6, raised against skeletal-muscle calsequestrin cross-reacted with calreticulin in SDS/PAGE immunoblots, but polyclonal antibodies reacted with native calreticulin only weakly, or not at all, after SDS denaturation. Immuno-gold decoration of liver ultrathin cryosections with affinity-purified antibodies against liver calreticulin revealed luminal labelling of vacuolar profiles indistinguishable from calciosomes, the subcellular structures previously identified by the use of anti-calsequestrin antibodies. We conclude that calreticulin is the Ca2(+)-binding protein segregated within the calciosome lumen, previously described as being calsequestrin-like. Because of its properties and intraluminal location, calreticulin might play a critical role in Ca2+ storage and release in non-muscle cells, similar to that played by calsequestrin in the muscle sarcoplasmic reticulum.

Amino Acid Sequence↗
PubMed1990

Role of Na+/H+ exchange in the granulocyte-macrophage colony-stimulating factor-dependent growth of a leukemic cell line.

The growth of the human leukemia cell line AML-193 in a serum-free medium is strictly dependent on the presence of the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF), which is one of the major regulators of the myelomonocytic lineage. At present, little is known about the mechanisms by which this growth factor transduces the signal intracellularly. The results of this study demonstrate that GM-CSF needs the operation of a Na+/H+ exchanger, which is located in the plasma membrane of almost every vertebrate cell. In fact, the GM-CSF-dependent proliferation of AML-193 cells is strongly reduced in the presence of the amiloride analog EIPA, a specific inhibitor of the Na+/H+ exchanger. When acidified, AML-193 cells are able to recover the original pHi in a Na(+)-dependent and EIPA-inhibitable way; this demonstrates for the first time the presence of the Na+/H+ exchanger in these cells. Finally, GM-CSF, at doses superimposable to those needed for triggering proliferation, induces in AML-193 cells a sustained alkalinization, which is dependent on a operating Na+/H+ exchange, as it is inhibited by EIPA. These results suggest that GM-CSF, like other growth factors in other cell systems, exerts its mitogenic activity in AML-193 cells by inducing a Na+/H+ exchanger-mediated rise in pHi.

Amiloride↗
PubMed1989

Sequence homology of a canine brain calcium-binding protein with calregulin and the human Ro/SS-A antigen.

A 60 kDa calcium-binding protein (CBP) was purified from canine brain and its N-terminal sequence determined to be: Glu-Pro-Ala-Ile-Tyr-Phe-Lys-Glu-Gln-Phe-Leu-Asp-Gly-Asp-Gly-X-Thr-Arg-X- Ile- Glu-Ser-Lys. This sequence is very similar to that of "Ccalregulin", a CBP of unknown function which is similar in size and appears to be present in most animal tissues. An unexpected and even more striking similarity was found with the N-terminal sequence of the human Ro/SS-A antigen, a 60 kDa protein which has long been implicated in the pathogenesis of autoimmune diseases such as systemic lupus erythematosus. These findings suggest that the Ro/SS-A antigen is probably also a CBP.

Amino Acid Sequence↗
PubMed1989

Interaction of lymphokine-activated killer cells with susceptible targets does not induce second messenger generation and cytolytic granule exocytosis.

CTL activation by specific targets leads to a rapid rise of inositol phosphates (InsPs) and of cytoplasmic-free Ca2+ concentration ([Ca2+]i). While these events are considered necessary to trigger granule secretion, Ca2+-independent cytolytic mechanisms have been recently proposed in addition or as an alternative to the classical Ca2+-dependent exocytosis model. We observed that lymphokine-activated killer (LAK) cells, obtained after stimulation with supraoptimal concentrations of IL-2 in short- or long-term cultures, kill susceptible targets in the absence of a [Ca2+]i rise and InsP3 formation. Moreover, LAK cell-mediated lysis was not associated with an increase in cytotoxic granule exocytosis, as evaluated by BLT-esterase release into the culture supernatant. Furthermore, using an antigen-specific CTL clone, which acquires LAK-like activity when cultured in medium containing high IL-2 doses, second messenger generation and cytolytic granule content secretion were not detected during lysis of unrelated target cells, while killing of specific targets triggered both these processes. These findings suggest that two lytic pathways may coexist in the same effector cells: a second messenger-dependent pathway involving degranulation, which is activated after TCR interaction with specific targets, and another pathway, independent of any known second messenger generation, responsible for unrelated target cell lysis.

Animals↗
PubMed1989

Na+/H+ exchange activation mediates the lipopolysaccharide-induced proliferation of human B lymphocytes and is impaired in malignant B-chronic lymphocytic leukemia lymphocytes.

In various mammalian cell types the stimulation of the plasma membrane amiloride-sensitive Na+/H+ exchange and the resulting increase of intracellular pH (pHi) play a key role in the initiation of cell proliferation. In the present work we have investigated whether Na+/H+ exchange is involved in normal human B cell proliferation and whether it is also operating in malignant B-chronic lymphocytic leukemia (B-CLL) lymphocytes. Our results show that: 1) normal human B cells contain an operating Na+/H+ exchanger, as inferred by their ability to recover pHi after acid-loading in a HCO3- -free medium and by evidences that LPS and phorbol ester PMA elicit a pHi rise inhibitable by either 5-(N-ethyl-N-isopropyl)amiloride (EIPA) or a Na+-free medium; 2) LPS-induced proliferation of normal human B cells is strongly inhibited when the amiloride analog EIPA (5 microM) is present in the culture medium (after 72 h the proportion of B cells incorporation bromodeoxyuridine falls from 13.9 +/- 3.9% to 2.8 +/- 1.1%); 3) EIPA does not affect BdR incorporation when B cells proliferation is induced by the co-mitogenic activity of IL-4 and low m.w. B cell growth factor (BCGF); 4) B-CLL cells, which proliferate in response to IL-4/BCGF but not to LPS, fail to increase pHi above their pHi resting levels when challenged with LPS or PMA and pHi recovery after acid-loading is highly impaired. These results lead to conclude that Na+/H+ exchange operation is necessary for LPS-(but not for IL-4/BCGF)-induced proliferation of human normal B lymphocytes and that Na+/H+ exchange activation is impaired in malignant B-CLL lymphocytes.

Aged↗
PubMed1988

Role of Na+/H+ exchange in thrombin- and arachidonic acid-induced Ca2+ influx in platelets.

Platelet activation is accompanied by an increase of cytosolic free Ca2+ concentration, [Ca2+]i, (due to both extracellular Ca2+ influx and Ca2+ movements from the dense tubular system) and an Na+ influx associated with H+ extrusion. The latter event is attributable to the activation of Na+/H+ exchange, which requires Na+ in the extracellular medium and is inhibited by amiloride and its analogs. The present study was carried out to determine whether a link exists between Ca2+ transients (measured by the quin2 method and the 45CaCl2 technique) and Na+/H+ exchange activation (studied with the pH-sensitive intracellular probe, 6-carboxyfluorescein) during platelet stimulation. Washed human platelets, stimulated with thrombin and arachidonic acid, showed: (1) a large and rapid [Ca2+]i rise, mostly due to a Ca2+ influx through the plasma membrane; (2) a marked intracellular alkalinization. Both phenomena were markedly inhibited in the absence of extracellular Na+ or in the presence of an amiloride analog (EIPA). Monensin, a cation exchanger which elicits Na+ influx and alkalinization, and NH4Cl, which induces alkalinization only, were able to evoke an increase in [Ca2+]i, mostly as an influx from the extracellular medium. Our results suggest that Ca2+ influx induced by thrombin and arachidonic acid in human platelets is strictly dependent on Na+/H+-exchange activation.

Ammonium Chloride↗