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S Grinstein

Publications and source records attributed to S Grinstein.

At least 109 records · Page 6Linked to original sources
PubMed1996

Chemotactic peptide-induced activation of MEK-2, the predominant isoform in human neutrophils. Inhibition by wortmannin.

Exposure of neutrophils to a variety of agonists including chemoattractant peptides and cytokines induces degranulation and activation of the oxidative burst which are required for bacterial killing. The signaling pathways regulating these important functions are incompletely characterized. Mitogen-activated protein (MAP) kinases, which include the extracellular signal-regulated kinases (ERKs), are activated rapidly in neutrophils, suggesting that they may regulate cell activation. We found that neutrophils express two isoforms of MAP/ERK kinase (MEK), mixed-function kinases that are responsible for phosphorylation and activation of ERK. Like MEK-1, MEK-2 was found to reside in the cytosol both before and after stimulation. Studies were undertaken to define the relative abundance and functional contribution of MEK-1 and MEK-2 in neutrophils and to characterize the signaling pathways leading to their activation. Although the abundance of the two isoforms was similar, the activity of MEK-2 was at least 3-fold greater than that of MEK-1. A rise in cytosolic [Ca2+] was insufficient for MEK stimulation, and blunting the [Ca2+] change with intracellular chelators failed to prevent receptor-mediated activation of either isoform, implying that cytosolic Ca2+ transients are not necessary. In contrast, both MEK-1 and MEK-2 were activated by exposure of cells to protein kinase C (PKC) agonists. Conversely, PKC antagonists inhibited the chemotactic stimulation of both isoforms, suggesting that PKC was required for their activation. Despite these similarities, clear differences were also found in the pathways leading to activation of the MEK isoforms. In particular, MEK-2 was considerably more sensitive than MEK-1 to the phosphatidylinositol 3-kinase inhibitor wortmannin. Phosphorylation and activation of ERK-1 and ERK-2 were also reduced by this inhibitor. In summary, MEK-2 is stimulated in formyl-methionyl-leucyl-phenylalanine-treated neutrophils, where it appears to be functionally the predominant isoform. The time course and inhibitor sensitivity of MEK-2 activation parallel those of several components of the microbicidal response, suggesting a signaling role of the MEK-ERK pathway.

Amino Acid Sequence↗
PubMed1996

Insulin activates a p21-activated kinase in muscle cells via phosphatidylinositol 3-kinase.

Insulin activates rapidly a complex cascade of lipid and protein kinases leading to stimulation of mitogenic and metabolic events. Here we describe a renaturable kinase of 65 kDa (PK65) that becomes rapidly activated by insulin in differentiated L6 muscle cells (myotubes) and can phosphorylate histones immobilized in polyacrylamide gels. Insulin activation of PK65 was abolished by the tyrosine kinase inhibitor erbstatin and by the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor wortmannin, but was unaffected by inhibitors of protein kinase C or of the activation of p70(S6K). Recently, a number of protein kinases have been described which become activated through interaction with the small GTP-binding proteins Rac and Cdc42 (21-ctivated inases, or PAKs) and lead to activation of the stress-induced mitogen-activated protein kinase (MAPK) p38 MAPK. Two different polyclonal antibodies recognizing the carboxyl-terminal or the Rac-binding domain of a 65-kDa PAK (PAK65) immunoprecipitated the myotube PK65. The insulin-induced activation of PK65 in myotubes was detectable following immunoprecipitation of the kinase. Furthermore, PK65 associated with and became activated by glutathione S-transferase-Cdc42Hs in the presence of GTPgammaS (guanosine 5'-3-O-(thio)triphosphate). In myotubes insulin also induced tyrosine phosphorylation of p38 MAPK. However, this phosphorylation was insensitive to wortmannin, indicating that p38 MAPK is not activated by PK65 in insulin-stimulated cells. The results suggest that insulin activates in muscle cells a renaturable kinase (PK65) closely related to PAK65. Tyrosine kinases and PI 3-kinase act upstream of PK65 in the insulin signaling cascade. Insulin activates p38 MAPK in myotubes, but this occurs by a pathway independent of PI 3-kinase and PK65.

Animals↗
PubMed1996

Activation of proton pumping in human neutrophils occurs by exocytosis of vesicles bearing vacuolar-type H+-ATPases.

Proton pump activity is not measurable in the plasma membrane of unstimulated neutrophils but becomes readily detectable upon activation by soluble agonists. The mechanism of pump activation was investigated in this report. V-type H+ pump activity, estimated as a bafilomycin A1-sensitive elevation of the cytosolic pH, was stimulated in suspended neutrophils by chemotactic peptides and by phorbol esters. Stimulation of pump activity induced by the agonists was greatly enhanced by cytochalasin B, an agent known to potentiate granular secretion in neutrophils. We therefore compared the rate and extent of pump activation with the pattern of exocytosis of the four types of secretory organelles present in neutrophils, using flow cytometry and enzyme-linked immunosorbent assay. The kinetics of exocytosis of secretory vesicles and secondary and tertiary granules but not primary granules paralleled the appearance of pump activity. The subcellular localization of the pump was defined by cellular fractionation and immunoblotting using an antibody to the C subunit of the V-type ATPase. The pump was abundant in tertiary granules, with significant amounts present also in primary granules and secretory vesicles. The pump was scarce in secondary granules and not detectable in the cytosol. Finally, the agonists failed to stimulate pump activity in neutrophil cytoplasts, which are intact cell fragments devoid of acidic granules. Together, our results suggest that the V-type H+-ATPase is not constitutively present in the plasma membrane of neutrophils but is delivered to the surface membrane by exocytosis during cellular activation. Tertiary granules and secretory vesicles are the most likely source of V-ATPases. Following insertion in the plasma membrane, the pump is poised to effectively extrude the excess metabolic acid that is generated during chemotaxis and bacterial killing.

Enzyme Activation↗
PubMed1996

Characterization and subcellular localization of target membrane soluble NSF attachment protein receptors (t-SNAREs) in macrophages. Syntaxins 2, 3, and 4 are present on phagosomal membranes.

Phagosomes formed during ingestion of microorganisms by leukocytes undergo a rapid maturation, generating an acidic, microbicidal organelle. Maturation requires interactions with intracellular vesicles that dock and fuse preferentially with the phagosomal membrane. The basis of specificity of vesiculo-phagosomal interaction has not been elucidated. By contrast, the molecular basis of vesicular fusion in other systems is better understood. At neural synapses, vesicular docking and fusion to the plasma membrane are mediated by a protein complex including syntaxin 1. We explored whether macrophages contain syntaxins, and whether selective fusion of vesicles with the phagosome results from the accumulation of syntaxins in the phagosomal membrane. Isoform-specific Abs were utilized to demonstrate utilized to demonstrate that syntaxins 2, 3, and 4, but not syntaxin 1, are present in murine and human macrophages. Biochemical characterization demonstrated the presence of these syntaxins on microsomes, where they are integral membrane proteins. Subcellular localization using confocal immunofluorescence microscopy demonstrated that syntaxins 3 and 4 are present on the plasma membrane as well as on intracellular vesicles. Importantly, phagosomes isolated by fractionation were shown by immunoblotting to contain syntaxins 2, 3, and 4, suggesting that they may participate in phagosomal maturation. The density of the syntaxins on the phagosomal membrane was found to be comparable with that on the surface membrane. This suggests that preferential fusion of vesicles with the phagosomal membrane is not the result of segregation of the syntaxins to this organelle. Instead, local generation of second messengers in the vicinity of the phagosomal membrane may trigger focal fusion.

Animals↗
PubMed1996

Distinct structural domains confer cAMP sensitivity and ATP dependence to the Na+/H+ exchanger NHE3 isoform.

Agents known to increase cAMP levels in renal and intestinal epithelia decrease sodium absorption by inhibiting NHE3, an isoform of the Na+/H+ exchanger expressed at high levels in apical membranes of these cells. In contrast, the ubiquitous, housekeeping isoform of the exchanger (NHE1) is stimulated by cAMP in some cell types. Optimal activity of NHE3 as well as NHE1 requires the presence of ATP. To gain insight into the molecular mechanisms of ATP dependence and cAMP regulation of NHE3, a series of mutations were constructed by progressively truncating segments of the C-terminal cytoplasmic domain of the transporter at amino acid positions 684, 638, and 579 (named NHE3delta684, NHE3delta638, and NHE3delta579). In addition, chimeric antiporters were constructed with the N-terminal transmembrane domain of NHE3 linked to the entire cytoplasmic region of NHE1 (chimera NHE3/1) or vice versa (chimera NHE1/3). These constructs were heterologously expressed in antiport-deficient Chinese hamster ovary cells, and their activities were assessed by fluorimetric measurements of intracellular pH and by radioisotope determinations of Na+ influx. Forskolin, which directly stimulates adenylate cyclase, inhibited NHE3 as well as NHE1/3, but not NHE3/1, suggesting that the cytoplasmic domain of NHE3 was sufficient to confer sensitivity to inhibition by cAMP. Forskolin also inhibited the truncated mutant NHE3delta684 to an extent similar to that for wild type NHE3. However, the inhibitory effect was greatly reduced in NHE3delta638 and more profound truncations (NHE3delta579 obliterated the effect of forskolin. These findings suggest that a region found between amino acids 579 and 684 is essential for the cAMP response of NHE3. In contrast, comparable ATP dependence was observed in all exchanger constructs examined. These observations indicate that ATP dependence is conferred by a region of the molecule in or adjacent to the transmembrane domain, which is most conserved between isoforms. It is concluded that different sites, and therefore different mechanisms, underlie inhibition of NHE3 by cAMP and by depletion of ATP.

Adenosine Triphosphate↗
PubMed1996

Interleukin-1 increases vacuolar-type H+-ATPase activity in murine peritoneal macrophages.

Maintenance of cytoplasmic pH (pHi) within a narrow physiological range is crucial to normal cellular function. This is of particular relevance to phagocytic cells within the acidic inflammatory microenvironment where the pHi tends to be acid loaded. We have previously reported that a vacuolar-type H(+)-ATPase (V-ATPase) situated in the plasma membrane of macrophages and poised to extrude protons from the cytoplasmic to the extracellular space is an important pHi regulatory mechanism within the inflammatory milieu. Since this microenvironment is frequently characterized by the influx of cells known to release inflammatory cytokines, we performed studies to examine the effect of one such mediator molecule, interleukin-1 (IL-1), on pHi regulation in peritoneal macrophages. IL-1 caused a time- and dose-dependent increase in macrophage pHi recovery from an acute acid load. This effect was specific to IL-1 and was due to enhanced plasmalemmal V-ATPase activity. The increased V-ATPase activity by IL-1 occurred following a lag period of several hours and required de novo protein and mRNA synthesis. However, Northern blot analysis revealed that IL-1 did not exert its effect via alterations in the levels of mRNA transcripts for the A or B subunits of the V-ATPase complex. Finally, stimulation of both cAMP-dependent protein kinase and protein kinase C was required for the stimulatory effect of IL-1 on V-ATPase activity. Thus, cytokines present within the inflammatory milieu are able to modulate pHi regulatory mechanisms. These data may represent a novel mechanism whereby cytokines may improve cellular function at inflammatory sites.

Animals↗
PubMed1996

Endogenous reactive oxygen intermediates activate tyrosine kinases in human neutrophils.

In response to invading microorganisms, neutrophils produce large amounts of superoxide and other reactive oxygen intermediates (ROI) by assembly and activation of a multicomponent enzyme complex, the NADPH oxidase. While fulfilling a microbicidal role, ROI have also been postulated to serve as signaling molecules, because activation of the NADPH oxidase was found to be associated with increased tyrosine phosphorylation (Fialkow, L., Chan, C. K., Grinstein, S., and Downey, G.P. (1993) J. Biol. Chem. 268, 17131-17137). The mechanism whereby ROI induces phosphotyrosine accumulation was investigated using electroporated neutrophils stimulated with guanosine 5'-O-3-thiotriphosphate in order to bypass membrane receptors. In vitro immune complex assays and immunoblotting were used to identify five tyrosine kinases present in human neutrophils. Of these, p56/59hck, p72syk, and p77btk were activated during production of ROI. Interestingly, the in vitro autophosphorylation activities of p53/56lyn and p59fgr were found to decline with ROI production. The mode of regulation of p56/59hck was explored in detail. Oxidizing agents were unable to activate p56/59hck in vitro and, once activated in situ, reducing agents failed to inactivate it, suggesting that the effects of ROI are indirect. Tyrosine phosphorylation of p56/59hck paralleled its activation, and dephosphorylation in vitro reversed the stimulation. We therefore conclude that tyrosine phosphorylation is central to the regulation of p56/59hck and likely also of p72syk, which is similarly phosphorylated upon activation of the oxidase. Because ROI have been shown to reduce the activity of tyrosine phosphatases, we suggest that this inhibition allows constitutively active kinases to auto/transphosphorylate on stimulatory tyrosine residues, leading to an increase in their catalytic activity. Enhanced phosphotyrosine accumulation would then result from the combined effects of increased phosphorylation with decreased dephosphorylation.

Enzyme Activation↗
PubMed1996

Exudative neutrophils show impaired pH regulation compared with circulating neutrophils.

OBJECTIVE: To investigate the effects of neutrophil (PMN) transmigration into inflammatory sites on cytosolic pH (pHi) regulation. DESIGN: Exudative PMNs were obtained by bronchoalveolar lavage from rats sustaining endotoxin-induced lung injury. Circulating PMNs were purified with density-gradient centrifugation. Cytosolic pH was measured with single-cell fluorescence imaging using the pH-sensitive dye biscarboxyethyl-carboxyfluorescein. RESULTS: Exudative PMNs showed impaired pHi recovery from an induced acid load compared with circulating PMNs. Under conditions of extracellular acidosis, exudative PMNs showed impaired pHi homeostasis and produced decreased superoxide compared with circulating cells. Inhibition of the sodium-proton exchanger attenuated the differences in pHi recovery, suggesting a mechanism underlying the pHi regulatory dysfunction. All cells had comparable adenosine triphosphate levels and superoxide production at physiologic extracellular pH. CONCLUSION: Impaired pHi regulation of exudative cells may mediate cellular dysfunction and impaired resolution of infection at inflammatory sites.

Acidosis↗
PubMed1996

Na+/H+ exchange activity during phagocytosis in human neutrophils: role of Fcgamma receptors and tyrosine kinases.

In neutrophils, binding and phagocytosis facilitate subsequent intracellular killing of microorganisms. Activity of Na+/H+ exchangers (NHEs) participates in these events, especially in regulation of intracellular pH (pHi) by compensating for the H+ load generated by the respiratory burst. Despite the importance of these functions, comparatively little is known regarding the nature and regulation of NHE(s) in neutrophils. The purpose of this study was to identify which NHE(s) are expressed in neutrophils and to elucidate the mechanisms regulating their activity during phagocytosis. Exposure of cells to the phagocytic stimulus opsonized zymosan (OpZ) induced a transient cytosolic acidification followed by a prolonged alkalinization. The latter was inhibited in Na+-free medium and by amiloride analogues and therefore was due to activation of Na+/H+ exchange. Reverse transcriptase PCR and cDNA sequencing demonstrated that mRNA for the NHE-1 but not for NHE-2, 3, or 4 isoforms of the exchanger was expressed. Immunoblotting of purified plasma membranes with isoform-specific antibodies confirmed the presence of NHE-1 protein in neutrophils. Since phagocytosis involves Fcgamma (FcgammaR) and complement receptors such as CR3 (a beta2 integrin) which are linked to pathways involving alterations in intracellular [Ca2+]i and tyrosine phosphorylation, we studied these pathways in relation to activation of NHE-1. Cross-linking of surface bound antibodies (mAb) directed against FcgammaRs (FcgammaRII > FcgammaRIII) but not beta2 integrins induced an amiloride-sensitive cytosolic alkalinization. However, anti-beta2 integrin mAb diminished OpZ-induced alkalinization suggesting that NHE-1 activation involved cooperation between integrins and FcgammaRs. The tyrosine kinase inhibitors genistein and herbimycin blocked cytosolic alkalinization after OpZ or FcgammaR cross-linking suggesting that tyrosine phosphorylation was involved in NHE-I activation. An increase in [Ca2+]i was not required for NHE-1 activation because neither removal of extracellular Ca2+ nor buffering of changes in [Ca2+]i inhibited alkalinization after OpZ or Fc-gammaR cross-linking. In summary, Fc-gammaRs and beta2 integrins cooperate in activation of NHE-1 in neutrophils during phagocytosis by a signaling pathway involving tyrosine phosphorylation.

Amiloride↗
PubMed1996

Intracellular pH regulation during spreading of human neutrophils.

The regulation of the intracelluar pH (pHi) during spreading of human neutrophils was studied by a combination of fluorescence imaging and video microscopy. Spreading on adhesive substrates caused a rapid and sustained cytosolic alkalinization. This pHi increase was prevented by the omission of external Na+, suggesting that it results from the activation of Na+/H+ exchange. Spreading-induced alkalinization was also precluded by the compound HOE 694 at concentrations that selectively block the NHE-1 isoform of the Na+H+ antiporter. Inhibition of Na+/H+ exchange by either procedure unmasked a sizable cytosolic acidification upon spreading, indicative of intracellular acid production. The excess acid generation was caused, at least in part, by the activation of the respiratory burst, since the acidification closely correlated with superoxide production, measured in single spreading neutrophils with dihydrorhodamine-123, and little acid production was observed in the presence of diphenylene iodonium, a blocker of the NADPH oxidase. Moreover, neutrophils from chronic granulomatous disease patients, which do not produce superoxide, failed to acidify. Comparable pHi changes were observed when beta 2 integrins were selectively activated during spreading on surfaces coated with anti-CD18 antibodies. When integrin engagement was precluded by pretreatment with soluble anti-CD18 antibody, the pHi changes associated with spreading on fibrinogen were markedly reduced. Inhibition of microfilament assembly with cytochalasin D precluded spreading and concomitantly abolished superoxide production and the associated pHi changes, indicating that cytoskeletal reorganization and/or an increase in the number of adherence receptors engaged are required for the responses. Neutrophils spread normally when the oxidase was blocked or when pHi was clamped near physiological values with nigericin. Spreading, however, was strongly inhibited when pHi was clamped at acidic values. Our results indicate that neutrophils release superoxide upon spreading, generating a burst of intracellular acid production. The concomitant activation of the Na+/H+ antiport not only prevents the deleterious effects of the acid released by the NADPH oxidase, but induces a net cytosolic alkalinization. Since several functions of neutrophils are inhibited at an acidic pHi, the coordinated activation of pHi regulatory mechanisms along with the oxidase is essential for sustained microbicidal activity.

Actin Cytoskeleton↗
PubMed1996

Dynamic measurement of the pH of the Golgi complex in living cells using retrograde transport of the verotoxin receptor.

The B subunit of verotoxin (VT1B) from enterohemorrhagic Escherichia coli is responsible for the attachment of the holotoxin to the cell surface, by binding to the glycolipid, globotriaosyl ceramide. After receptor-mediated endocytosis, the toxin is targeted to the Golgi complex by a process of retrograde transport. We took advantage of this unique property of VT1B to measure the pH of the Golgi complex in intact live cells. Purified recombinant VT1B was labeled with either rhodamine or fluorescein for subcellular localization by confocal microscopy. After 1 h at 37 degrees C, VT1B accumulated in a juxtanuclear structure that colocalized with several Golgi markers, including alpha-mannosidase II, beta-COP, and NBD-ceramide. Moreover, colchicine and brefeldin A induced dispersal of the juxtanuclear staining, consistent with accumulation of VT1B in the Golgi complex. Imaging of the emission of fluorescein-labeled VT1B was used to measure intra-Golgi pH (pHG), which was calibrated in situ with ionophores. In intact Vero cells, pHG averaged 6.45 +/- 0.03 (standard error). The acidity of the Golgi lumen dissipated rapidly upon addition of bafilomycin A1, a blocker of vacuolar-type ATPases, pHG remained constant despite acidification of the cytosol by reversal of the plasmalemmal Na+/H+ antiport. Similarly, pHG was unaffected by acute changes in cytosolic calcium. Furthermore, pHG recovered quickly toward the basal level after departures imposed with weak bases. These findings suggest that pHG is actively regulated, despite the presence of a sizable H+ "leak" pathway. The ability of VT1B to target the Golgi complex should facilitate not only studies of acid-base regulation, but also analysis of other ionic species.

Amino Acid Sequence↗
PubMed1996

Intracellular pH regulation in leukocytes: mechanisms and functional significance.

The regulation of the intracellular pH (pHi) of phagocytic cells is critical to their function and viability. The acidic nature of the abscesses to which these cells migrate and the burden of acid that they generate in the activated state tend to perturb the pHi outside of the physiological range. Failure to maintain pHi homeostasis results in decreased cellular enzyme activity, cellular migration, and microbicidal function. Several pHi regulatory mechanisms, including the sodium-proton exchanger, proton conductive pathways, and a vacuolar type H(+)-ATPase exist in leukocytes and play an important role in preventing deviations in the pHi away from the physiological range. The last of these mechanisms is central to the regulation of cytoplasmic pH, and is modulated by mediator molecules in the inflammatory mileu.

Acids↗
PubMed1996

Detection and characterization of antigens in urine of patients with acute, congenital, and chronic Chagas' disease.

Monoclonal antibodies raised against purified Trypanosoma cruzi urinary antigens were used in an enzyme-linked immunosorbent assay (ELISA) capture test for parasite antigens present in urine specimens of Argentinean and Brazilian patients with Chagas' disease. At diagnosis, antigenuria was demonstrated by ELISA in all acutely and congenitally infected infants studied. Moreover, T. cruzi urinary antigens were detected in samples from three of five patients with acute infections and four of five patients with congenital infections following chemotherapy. At least one ELISA-positive urine specimen from each individual was recorded in a longitudinal survey of 12 chronic chagasic patients. The same parasitic antigens (90 to 80 kDa, pI 5.7 to 6.0; 70 to 65 kDa, pI 4.9 to 4.5; 50 to 45 kDa, pI 5.3 to 5.1; and 40 to 35 kDa, pI 4.8 to 4.5) were identified by immunoprecipitation and two-dimensional polyacrylamide gel electrophoresis analysis of urine samples from patients with different forms of chagasic infection. The 90- to 80-kDa urinary protein resembles a trypomastigote-shed antigen. Determination of antigenuria proved valuable for early diagnosis of Chagas' disease and also for diagnosis of chronic cases with conflicting serology.

Acute Disease↗
PubMed1996

Coimmunoprecipitation of a 24-kDa protein with NHE1, the ubiquitous isoform of the Na+/H+ exchanger.

Ancillary proteins have been proposed to account for phosphorylation-independent regulation of the Na+/H+ exchanger (NHE), but such putative proteins have not been identified. Here we describe the specific association of NHE1 with a protein of approximately 24 kDa (p24). Immunoprecipitation of NHE1 from lysates of [35S] cysteine- and/or methionine-labeled cells with the use of an anti-NHE1 antibody demonstrated specific coimmunoprecipitation of p24 with NHE1. The stoichiometry of p24 relative to NHE1, assessed by their radiolabel content, was consistent between experiments and among cell types. Immunoblotting demonstrated that p24 is not a proteolytic product of NHE1. Internal deletion mutants and chimeras of NHE1/NHE3 suggest that p24 binds to residues 515-566 or 695-815 of NHE1 or to the transmembrane region of both NHE1 and NHE3. Protein p24 is not constitutively phosphorylated nor could phosphorylation be induced by serum or phorbol ester treatment. Binding of p24 to NHE1 is Ca2+ independent. Protein p24 failed to bind [gamma-32P]GTP in a blot-overlay assay, suggesting that it is not a low-molecular-weight GTP-binding protein. Identification of the p24:NHE1 interaction may contribute to our understanding of antiporter regulation.

Animals↗
PubMed1995

Subcellular distribution of docking/fusion proteins in neutrophils, secretory cells with multiple exocytic compartments.

Neutrophils contain at least four distinct types of secretory organelles, which undergo exocytosis during infection and inflammation. The signaling pathways leading to secretion of individual granules and their kinetics of exocytosis vary greatly, causing temporal and regional differences in docking and fusion with the plasma membrane. As a step toward understanding the processes underlying differential granular secretion in neutrophils, we assessed the presence and distribution of a number of proteins reported to be involved in vesicular docking and/or fusion in other systems. Specific Abs were used for immunoblotting of cells fractionated by density gradients and free-flow electrophoresis, and for localization by confocal immunofluorescence and electron microscopy. Syntaxin 1, VAMP (vesicle-associated membrane protein)-1, synaptosome-associated protein-25 (SNAP-25), synaptophysin, and cellubrevin were not detectable in human neutrophils. In contrast, syntaxin 4, VAMP-2, and the 39-kDa isoform of secretory carrier membrane protein (SCAMP) were present. SCAMP was found mainly in secondary and tertiary granules and in a fraction containing secretory vesicles, but was virtually absent from the primary (lysosomal) granules. This profile is consistent with the proposed "post-Golgi" distribution of SCAMP. VAMP-2 was largely absent from primary and secondary granules, but concentrated in tertiary granules and secretory vesicles. This pattern of distribution parallels the increasing sensitivity of these exocytic compartments to intracellular free calcium. Accordingly, ionomycin induced translocation of VAMP-2 toward the plasma membrane. Syntaxin 4 was found almost exclusively in the plasma membrane, and it accumulated in lamellipodia of migrating cells. This regional accumulation may contribute to localized secretion into the phagosomal lumen.

Cells, Cultured↗
PubMed1995

Presence of Epstein-Barr virus and strain type assignment in Argentine childhood Hodgkin's disease.

Epstein-Barr virus (EBV) has been implicated in the etiology of a large number of malignancies. Most recently several studies have linked EBV to Hodgkin's disease. In this report, formalin-fixed, paraffin-embedded tissues were collected retrospectively from 41 children with Hodgkin's disease treated at our hospital. Lymph node biopsies were examined for the presence of two virus-encoded latent proteins: latent membrane protein (LMP) and Epstein-Barr nuclear antigen-2 (EBNA-2), in Reed-Sternberg (RS) and Hodgkin (H) cells, by peroxidase immunolabeling. Nonisotopic Epstein-Barr encoded RNAs (EBERs) in situ hybridization was also performed and positive labeling in malignant cells was detected. Twenty specimens were EBER+/LMP+, 2 were EBER+/LMP-, and 19 were EBER-/LMP-. However, none of the 41 cases expressed EBNA-2. Twenty-two of 41 (54%) cases were EBV positive including 2 of 6 with lymphocyte predominance, 19 of 25 with mixed cellularity, 0 of 9 with nodular sclerosis, and 1 of 1 with lymphocyte depletion. In the age range of 2 to 6 years, 14 of 17 (82%) samples were EBV-positive, whereas only 8 of 24 (33%) samples from the age range of 7 to 15 years contained EBV. (P = .004), a two-tailed Fisher's test). In 17 samples, polymerase chain reaction amplification was performed using strain specific primers for exon sequences of the EBNA-3C gene of EBV. From 12 positive samples, 8 contained EBV-A and 4 EBV-B. These results support the hypothesis that EBV contributes to the pathogenesis of pediatric Hodgkin's disease, particularly in mixed cellularity Hodgkin's disease and in the younger group.

Adolescent↗
PubMed1995

Regulation of cytoplasmic pH in osteoclasts. Contribution of proton pumps and a proton-selective conductance.

Osteoclasts resorb bone by secreting protons into an extracellular resorption zone through vacuolar-type proton pumps located in the ruffled border. The present study was undertaken to evaluate whether proton pumps also contribute to intracellular pH (pHi) regulation. Fluorescence imaging and photometry, and electrophysiological methods were used to characterize the mechanisms of pH regulation in isolated rabbit osteoclasts. The fluorescence of single osteoclasts cultured on glass coverslips and loaded with a pH-sensitive indicator was measured in nominally HCO(3-)-free solutions. When suspended in Na(+)-rich medium, the cells recovered from an acute acid load primarily by means of an amiloride-sensitive Na+/H+ antiporter. However, rapid recovery was also observed in Na(+)-free medium when K+ was used as the substitute. Bafilomycin-sensitive, vacuolar-type pumps were found to contribute marginally to pH regulation and no evidence was found for K+/H+ exchange. In contrast, pHi recovery in high K+ medium was largely attributed to a Zn(2+)-sensitive proton conductive pathway. The properties of this conductance were analyzed by patch-clamping osteoclasts in the whole-cell configuration. Depolarizing pulses induced a slowly developing outward current and a concomitant cytosolic alkalinization. Determination of the reversal potential during ion substitution experiments indicated that the current was due to H+ (equivalent) translocation across the membrane. The H+ current was greatly stimulated by reducing pHi, consistent with a homeostatic role of the conductive pathway during intracellular acidosis. These results suggest that vacuolar-type proton pumps contribute minimally to the recovery of cytoplasmic pH from intracellular acid loads. Instead, the data indicate the presence of a pH- and membrane potential-sensitive H+ conductance in the plasma membrane of osteoclasts. This conductance may contribute to translocation of charges and acid equivalents during bone resorption and/or generation of reactive oxygen intermediates by osteoclasts.

Animals↗