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S Grinstein

Publications and source records attributed to S Grinstein.

At least 91 records · Page 5Linked to original sources
PubMed1997

Cytosolic alkalinization increases stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) activity and p38 mitogen-activated protein kinase activity by a calcium-independent mechanism.

Stress-activated protein kinases (SAPK) are stimulated by a variety of agents and conditions that also activate the Na+/H+ exchanger (NHE). Activation of the exchanger results in a rapid increase in intracellular pH (pHi), raising the possibility that cytosolic alkalinization may contribute to SAPK activation. This hypothesis was tested by manipulating the pHi of U937 cells using permeant weak bases. Three different bases increased pHi and caused a 4-12-fold increase in SAPK activity with a time course that paralleled intracellular alkalinization. p38, a related stress kinase, was also stimulated by the weak bases. Stimulation of the stress kinases was not accompanied by changes in cytosolic free calcium nor was the activation of SAPK achieved when calcium was elevated by thapsigargin or calcium ionophores. Weak bases not only alter the pH of the cytosol but also alkalinize endomembrane compartments such as endosomes and lysosomes. However, the latter do not appear to mediate the stimulation of SAPK, since neither bafilomycin A1 nor desipramine, agents that neutralize acidic endomembrane compartments, activated the kinase. Because hyperosmolarity acutely activates the NHE, we considered whether the resulting cytosolic alkalinization mediates the activation of SAPK upon cell shrinkage. The addition of amiloride or the omission of Na+, which were verified to inhibit NHE, did not prevent the osmotically induced activation of SAPK. We conclude that cytosolic alkalinization increases the activity of SAPK and p38 by a calcium-independent mechanism that does not involve acidic intracellular organelles. In addition, even though cell shrinkage is accompanied by alkalinization due to the activation of NHE, the increased pHi is not the main cause of the observed stimulation of SAPK upon hyperosmotic challenge.

Ammonium Chloride↗
PubMed1997

Phosphorylation and subcellular redistribution of pleckstrin in human neutrophils.

Pleckstrin, originally described as a major substrate of protein kinase C (PKC) in platelets, was found to be highly expressed in human neutrophils (intracellular concentration, approximately 15 microM). As PKC isoforms play an important role in mediating neutrophil antimicrobial responses, we studied the regulation of pleckstrin phosphorylation in response to inflammatory stimuli. Following treatment of neutrophils with FMLP, 12-O-tetradecanoylphorbol-13-acetate, or opsonized zymosan, pleckstrin was rapidly phosphorylated, which resulted in a shift in its electrophoretic mobility. Several lines of evidence suggest that pleckstrin is phosphorylated in part by a nonconventional PKC following stimulation by FMLP: 1) chelation of intracellular Ca2+ had only a partial inhibitory effect; 2) diacylglycerol kinase inhibitors shortened the duration of phosphorylation, while the phosphatidic acid phosphohydrolase antagonist propranolol extended it; and 3) wortmannin and erbstatin blocked the phosphorylation of pleckstrin. These results suggest that nonconventional PKC isoforms, possibly delta or zeta, mediate the phosphorylation of pleckstrin. Both PKCdelta and -zeta are expressed in human neutrophils. Increased association of pleckstrin with both microsomes and with the cytoskeleton was observed in stimulated cells. These findings suggest that phosphorylation by nonconventional PKC isoforms induces a conformational change in pleckstrin that promotes its interaction with membranes and/or with the cytoskeleton. Such a translocation may serve to target proteins or lipids recognized by pleckstrin homology domains to sites where they can contribute to the microbicidal response.

Androstadienes↗
PubMed1997

Calreticulin is essential for integrin-mediated calcium signalling and cell adhesion.

Integrins are important mediators of cell adhesion to extracellular ligands and can transduce biochemical signals both into and out of cells. The cytoplasmic domains of integrins interact with several structural and signalling proteins and consequently participate in the regulation of cell shape, motility, growth and differentiation. It has been shown that calreticulin associates with the cytoplasmic domains of integrin alpha-subunits and that this interaction can influence integrin-mediated cell adhesion to extracellular matrix. We have now developed calreticulin-deficient embryonic stem (ES) cells and isolated embryonic fibroblasts from calreticulin mutant mice. We find that in both cell types integrin-mediated adhesion is severely impaired, although integrin expression is unaltered. Expression of recombinant calreticulin in double knockout ES cells by complementary DNA transfection rescued integrin-mediated adhesion. In wild-type cells, engagement of surface integrins induced a transient elevation in cytosolic calcium concentration owing to influx of extracellular calcium. This calcium transient was absent in calreticulin-deficient cells. In contrast, the amount of calcium in endomembrane stores, which is sensitive to both inositol 1,4,5-trisphosphate and thapsigargin, was indistinguishable in the two cell types. Our results indicate that calreticulin is an essential modulator both of integrin adhesive functions and integrin-initiated signalling, but that it may not play a significant role in the storage of luminal calcium.

Animals↗
PubMed1997

Chronic extracellular acidosis induces plasmalemmal vacuolar type H+ ATPase activity in osteoclasts.

Proton extrusion into an extracellular resorption compartment is an essential component of bone degradation by osteoclasts. Chronic metabolic acidosis is known to induce negative calcium balance and bone loss by stimulating osteoclastic bone resorption, but the underlying mechanism is not known. The present studies were undertaken to evaluate whether chronic acidosis affects proton extrusion mechanisms in osteoclasts cultured on glass coverslips. Acidosis, mimicked experimentally by maintaining the cells at extracellular pH 6.5, rapidly lowered intracellular pH to 6.8. However, after 2 hours, a proportion of cells demonstrated the capacity to restore intracellular pH to near normal levels. To define the mechanism responsible for this recovery, the activity of individual H+ transport pathways was analyzed. We found that chronic acid treatment for up to 6 h did not significantly affect the cellular buffering power or Na+/H+ antiport activity. In contrast, chronic acidosis activated vacuolar H+ pumps in the osteoclasts. Although only approximately 5% of the control cells displayed proton pump activity, about 40% of cells kept at extracellular pH 6. 5 for 4-6 h were able to recover from the acute acid load by means of bafilomycin A1-sensitive proton extrusion. Conversely, the H+-selective conductance recently described in the plasma membrane of osteoclasts was clearly inhibited in the cells exposed to chronic acidosis. Following acid treatment, the activation threshold of the H+ conductance was shifted to more positive potentials, and the current density was significantly reduced. Considered together, these results suggest that induction of plasmalemmal vacuolar type ATPase activity by chronic acidosis, generated either systemically due to metabolic disease or locally at sites of inflammation, is likely to stimulate osteoclastic bone resorption and thus to promote bone loss.

Acidosis↗
PubMed1997

Functional expression and apical localization of the cystic fibrosis transmembrane conductance regulator in MDCK I cells.

The gene product affected in cystic fibrosis, the cystic fibrosis transmembrane conductance regulator (CFTR), is a chlorideselective ion channel that is regulated by cAMP-dependent protein kinase-mediated phosphorylation, ATP binding and ATP hydrolysis. Mutations in the CFTR gene may result in cystic fibrosis characterized by severe pathology (e.g. recurrent pulmonary infection, male infertility and pancreatic insufficiency) involving organs expressing the CFTR. Interestingly, in the kidney, where expression of the CFTR has been reported, impaired ion transport in patients suffering from cystic fibrosis could not be observed. To understand the role of the CFTR in chloride transport in the kidney, we attempted to identify an epithelial cell line that can serve as a model. We demonstrate that the CFTR is expressed constitutively in Madine-Darby canine kidney (MDCK) type I cells, which are thought to have originated from the distal tubule of the dog nephron. We show expression at the mRNA level, using reverse transcriptase-PCR, and at the protein level, using Western blot analysis with three different monoclonal antibodies. Iodide efflux measurements indicate that CFTR expression confers a plasma membrane anion conductance that is responsive to stimulation by cAMP. The cAMP-stimulated iodide release is sensitive to glybenclamide, diphenylamine carboxylic acid and 5-nitro-2-(3-phenylpropylamino)benzoic acid, but not to 4,4'-di-isothiocyanostilbene-2,2'-disulphonic acid, an inhibitor profile characteristic of the CFTR chloride channel. Finally, the polarized localization of the CFTR to the apical plasma membrane was established by iodide efflux measurements and cell-surface biotinylation on MDCK I monolayers. Interestingly, MDCK type II cells, which are thought to have originated from the proximal tubule of the kidney, lack CFTR protein expression and cAMP-stimulated chloride conductance. In conclusion, we propose that MDCK type I and II cells can serve as convenient model systems to study the physiological role and differential expression of CFTR in the distal and proximal tubule respectively.

Animals↗
PubMed1997

Modulation of the retinoic acid and retinoid X receptor signaling pathways in P19 embryonal carcinoma cells by calreticulin.

Calreticulin is a widely expressed calcium binding protein that can bind to an amino acid sequence motif, KXGFFKR, which is present in the cytoplasmic domain of all integrin alpha-subunits. Closely related sequences, KXFFKR and KXFFRR, are encoded in the DNA-binding domain of all members of the steroid/thyroid/retinoid receptor superfamily and it has recently been demonstrated that calreticulin inhibits their activity both in vitro and in vivo. Here we present novel evidence that calreticulin can interfere directly with the retinoic acid (RARs) and retinoid X (RXRs) receptor pathways. Calreticulin exhibits the ability to inhibit DNA-binding activity of both heterodimeric RAR/RXR and homodimeric RXR complexes in vitro. Inhibition of RXR binding to DNA is achieved with a concentration of calreticulin that is approximately fourfold lower than that required for inhibition of RAR/RXR binding to a cognate binding site. Coprecipitation experiments suggest a direct protein:protein interaction between calreticulin and retinoid receptors. Stable overexpression of calreticulin in P19 embryonal carcinoma cells significantly decreases the rapid activation of the endogenous RA-responsive RARbeta gene, abrogates the ability of endogenous RAR/RXR complexes to bind to DNA, and inhibits the emergence of the RA-induced differentiated phenotype. These data demonstrate that calreticulin can interfere with the two distinct retinoid signaling pathways through a mechanism likely involving direct protein:protein interactions and that disruption of the retinoid signal alters biological processes in vivo.

Amino Acid Sequence↗
PubMed1997

Regulation of Src homology 2-containing tyrosine phosphatase 1 during activation of human neutrophils. Role of protein kinase C.

The tyrosine phosphorylation of several proteins induced in neutrophils by soluble and particulate stimuli is thought to be crucial for initiating antimicrobial responses. Although activation of tyrosine kinases is thought to mediate this event, the role of tyrosine phosphatases in the initiation and modulation of neutrophil responses remains largely undefined. We investigated the role of Src homology 2-containing tyrosine phosphatase 1 (SHP-1; also known as protein tyrosine phosphatase 1C (PTP1C), hematopoetic cell phosphatase, PTP-N6, and SHPTP-1), a phosphatase expressed primarily in hemopoietic cells, in the activation of human neutrophils. SHP-1 mRNA and protein were detected in these cells, and the enzyme was found to be predominantly localized to the cytosol in unstimulated cells. Following stimulation with neutrophil agonists such as phorbol ester, chemotactic peptide, or opsonized zymosan, a fraction of the phosphatase redistributed to the cytoskeleton. Agonist treatment also induced significant decreases (30-60%) in SHP-1 activity, which correlated temporally with increases in the cellular phosphotyrosine content. Phosphorylation of SHP-1 on serine residues was associated with the inhibition of its enzymatic activity, suggesting a causal relationship. Accordingly, both the agonist-evoked phosphorylation of SHP-1 and the inhibition of its catalytic activity were blocked by treatment with bisindolylmaleimide I, a potent and specific inhibitor of protein kinase C (PKC) activity. Immunoprecipitated SHP-1 was found to be phosphorylated efficiently by purified PKC in vitro. Such phosphorylation also caused a decrease in the phosphatase activity of SHP-1. Together, these data suggest that inhibition of SHP-1 by PKC-mediated serine phosphorylation plays a role in facilitating the accumulation of tyrosine-phosphorylated proteins following neutrophil stimulation. These findings provide a new link between the PKC and tyrosine phosphorylation branches of the signaling cascade that triggers antimicrobial responses in human neutrophils.

Enzyme Activation↗
PubMed1997

Chemotactic peptide N-formyl-met-leu-phe activation of p38 mitogen-activated protein kinase (MAPK) and MAPK-activated protein kinase-2 in human neutrophils.

Activation of polymorphonuclear leukocytes (PMN) by chemotactic peptides initiates a series of functional responses that serve to eliminate pathogens. The intermediate steps that link engagement of the chemoattractant receptor to the microbicidal responses involve protein kinases that have yet to be identified. In this study we detected in human PMN the presence of p38 mitogen-activated protein kinase (MAPK), which became rapidly tyrosine phosphorylated and activated in response to the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP). Pretreatment of PMN with wortmannin, a phosphatidylinositol 3-kinase inhibitor, or bis-indolylmaleimide, a protein kinase C antagonist, resulted in partial inhibition of p38 phosphorylation upon fMLP stimulation. Similarly, phosphorylation of p38 was only partially inhibited when the fMLP-induced cytosolic calcium transient was prevented. Stimulation of PMN by the chemoattractant also resulted in the rapid phosphorylation and activation of MAPK-activated protein kinase-2 (MAPKAPK-2), which was completely inhibited by the specific p38 inhibitor, SB203580. The physical interaction of p38 with MAPKAPK-2 was studied by coimmunoprecipitation. These two kinases were found to be associated in unstimulated PMN but dissociated upon activation of the cells by fMLP. Together these findings demonstrate the activation of p38 by chemotactic peptides in human PMN by a process involving phosphatidylinositol 3-kinase, protein kinase C, and calcium. p38, in turn, is an upstream activator of MAPKAPK-2.

Androstadienes↗
PubMed1997

Muscarinic agonists induce phosphorylation-independent activation of the NHE-1 isoform of the Na+/H+ antiporter in salivary acinar cells.

Cholinergic agonists stimulate isotonic fluid secretion in the parotid gland. This process is driven by the apical exit of Cl-, which enters the cells partly via Cl-/HCO-3 exchange across the basolateral membrane. Acidification of the cytosol by the extrusion of HCO-3 is prevented by the concomitant activation of the Na+/H+ exchanger (NHE), which is directly activated by cholinergic stimulation. Multiple isoforms of the NHE have been described in mammalian cells, but the particular isoform(s) present in salivary glands and their mechanism of activation have not been defined. Reverse transcriptase-polymerase chain reaction with isoform-specific primers was used to establish that NHE-1 and NHE-2, but not NHE-3 or NHE-4, are expressed in parotid glands. The presence of NHE-1 was confirmed by immunoblotting and immunofluorescence, which additionally demonstrated that this isoform is abundant in the basolateral membrane of acinar cells. The predominant role of NHE-1 in carbachol-induced Na+/H+ exchange was established pharmacologically using HOE694, an inhibitor with differential potency toward the individual isoforms. Because muscarinic agonists induce stimulation of protein kinases in acinar cells, we assessed the role of phosphorylation in the activation of the antiport. Immunoprecipitation experiments revealed that, although NHE-1 was phosphorylated in the resting state, no further phosphorylation occurred upon treatment with carbachol. Similar phosphopeptide patterns were observed in control and carbachol-treated samples. Together, these findings indicate that NHE-1, the predominant isoform of the antiporter in the basolateral membrane of acinar cells, is activated during muscarinic stimulation by a phosphorylation-independent event. Other processes, such as association of Ca2+-calmodulin complexes to the cytosolic domain of the antiporter, may be responsible for the activation of Na+/H+ exchange.

Animals↗
PubMed1997

Inhibition of neutrophil oxidative burst and granule secretion by wortmannin: potential role of MAP kinase and renaturable kinases.

Exposure of neutrophils to a variety of agonists including soluble chemoattractant peptides and cytokines results in degranulation and activation of the oxidative burst (effector functions) that are required for bacterial killing. At present, the signaling pathways regulating these important functions are incompletely characterized. Mitogen-activated protein (MAP) kinases (MAPK) as well as members of a family of "renaturable kinases" are rapidly activated in neutrophils in response to diverse physiological agonists, suggesting that they may regulate cell activation. Antagonists of phosphatidyl inositol-3-(OH) kinase (PI3-kinase) such as wortmannin (Wtmn) inhibit these effector responses as well as certain of the above-mentioned kinases, leading to the suggestion that these enzymes lie downstream of PI3-kinase in the pathway regulating the oxidative burst and granule secretion. However, an apparent discrepancy exists in that, while virtually obliterating activity of PI3-kinase and the oxidase at low concentrations (ID50 < 20 nM), Wtmn has only variable inhibitory effects on MAPK even at substantially higher concentrations (75-100 nM). This raises the possibility that the inhibitory effects of Wtmn are mediated via other enzyme systems. The purpose of the current study was therefore to compare the effects of Wtmn on PI3-kinase activity and on the chemoattractant-activated kinases, and to determine the potential relationship of these pathways to microbicidal responses. In human neutrophils, both the oxidative burst and granule secretion induced by fMLP were inhibited by Wtmn but at markedly different concentrations: the oxidative burst was inhibited with an ID50 of < 5 nM while granule secretion was only partially inhibited at concentrations exceeding 75 nM. Activation of both MEK-1 and MAPK in response to fMLP was only partially inhibited by high doses of Wtmn (ID50 of > 100 nM and approximately 75 nM, respectively). In contrast, Wtmn potently inhibited fMLP-induced activation of the 63 and 69 kDa renaturable kinases (ID50 approximately 5-10 nM). We speculate that the renaturable kinases may be involved in the regulation of the oxidative burst, whereas the MAPK pathway may play a role in other neutrophil functions such as granule secretion.

Androstadienes↗
PubMed1997

Role of intracellular pH in proliferation, transformation, and apoptosis.

Both cellular proliferation and apoptosis (programmed cell death) have been claimed to be modulated, perhaps even triggered by, changes in intracellular pH. In this review, we summarize the evidence that gave rise to these hypotheses. To facilitate a critical appraisal of the existing data, we briefly review the main pathways involved in cytosolic pH homeostasis and their regulation by mitogens and by apoptosis-inducing agents. The information available at present suggests that cytosolic pH plays a permissive role in cellular growth and proliferation, but is neither a trigger nor an essential step in the mitogenic signal transduction cascade. Concerning apoptosis, it is clear that lowering the pH in vitro can activate DNase II. However, the evidence linking cytosolic acidification with DNA degradation in vivo is presently not convincing. We conclude that the cytosolic pH, an essential physiological parameter that is tightly controlled by multiple, complementary, or redundant systems, is unlikely to play a role in signalling either cell growth or death.

Apoptosis↗
PubMed1997

ATP dependence of Na+/H+ exchange. Nucleotide specificity and assessment of the role of phospholipids.

We studied the ATP dependence of NHE-1, the ubiquitous isoform of the Na+/H+ antiporter, using the whole-cell configuration of the patch-clamp technique to apply nucleotides intracellularly while measuring cytosolic pH (pHi) by microfluorimetry. Na+/H+ exchange activity was measured as the Na(+)-driven pHi recovery from an acid load, which was imposed via the patch pipette. In Chinese hamster ovary (CHO) fibroblasts stably transfected with NHE-1, omission of ATP from the pipette solution inhibited Na+/H+ exchange. Conversely, ATP perfusion restored exchange activity in cells that had been metabolically depleted by 2-deoxy-D-glucose and oligomycin. In cells dialyzed in the presence of ATP, no "run-down" was observed even after extended periods, suggesting that the nucleotide is the only diffusible factor required for optimal NHE-1 activity. Half-maximal activation of the antiporter was obtained at approximately 5 mM Mg-ATP. Submillimolar concentrations failed to sustain Na+/H+ exchange even when an ATP regenerating system was included in the pipette solution. High ATP concentrations are also known to be required for the optimal function of other cation exchangers. In the case of the Na/Ca2+ exchanger, this requirement has been attributed to an aminophospholipid translocase, or "flippase.". The involvement of this enzyme in Na+/H+ exchange was examined using fluorescent phosphatidylserine, which is actively translocated by the flippase. ATP depletion decreased the transmembrane uptake of NBD-labeled phosphatidylserine (NBD-PS), indicating that the flippase was inhibited. Diamide, an agent reported to block the flippase, was as potent as ATP depletion in reducing NBD-PS uptake. However, diamide had no effect on Na+/H+ exchange, implying that the effect of ATP is not mediated by changes in lipid distribution across the plasma membrane. K-ATP and ATP gamma S were as efficient as Mg-ATP in sustaining NHE-1 activity, while AMP-PNP and AMP-PCP only partially substituted for ATP. In contrast, GTP gamma S was ineffective. We conclude that ATP is the only soluble factor necessary for optimal activity of the NHE-1 isoform of the antiporter. Mg2+ does not appear to be essential for the stimulatory effect of ATP. We propose that two mechanisms mediate the activation of the antiporter by ATP: one requires hydrolysis and is likely an energy-dependent event. The second process does not involve hydrolysis of the gamma-phosphate, excluding mediation by protein or lipid kinases. We suggest that this effect is due to binding of ATP to an as yet unidentified, nondiffusible effector that activates the antiporter.

Adenosine Triphosphate↗
PubMed1997

NO3--induced pH changes in mammalian cells. Evidence for an NO3--H+ cotransporter.

The effect of NO3- on intracellular pH (pHi) was assessed microfluorimetrically in mammalian cells in culture. In cells of human, hamster, and murine origin addition of extracellular NO3- induced an intracellular acidification. This acidification was eliminated when the cytosolic pH was clamped using ionophores or by perfusing the cytosol with highly buffered solutions using patch-pipettes, ruling out spectroscopic artifacts. The NO3-- induced pH change was not due to modulation of Na+/H+ exchange, since it was also observed in Na+/H+ antiport-deficient mutants. Though NO3- is known to inhibit vacuolar-type (V) H+-ATPases, this effect was not responsible for the acidification since it persisted in the presence of the potent V-ATPase inhibitor bafilomycin A1. NO3-/HCO3- exchange as the underlying mechanism was ruled out because acidification occurred despite nominal removal of HCO3-, despite inhibition of the anion exchanger with disulfonic stilbenes and in HEK 293 cells, which seemingly lack anion exchangers (Lee, B. S., R.B. Gunn, and R.R. Kopito. 1991. J. Biol. Chem. 266:11448- 11454). Accumulation of intracellular NO3-, measured by the Greiss method after reduction to NO2-, indicated that the anion is translocated into the cells along with the movement of acid equivalents. The simplest model to explain these observations is the cotransport of NO3- with H+ (or the equivalent counter-transport of NO3- for OH-). The transporter appears to be bi-directional, operating in the forward as well as reverse directions. A rough estimate of the fluxes of NO3- and acid equivalents suggests a one-to-one stoichiometry. Accordingly, the rate of transport was unaffected by sizable changes in transmembrane potential. The cytosolic acidification was a saturable function of the extracellular concentration of NO3- and was accentuated by acidification of the extracellular space. The putative NO3--H+ cotransport was inhibited markedly by ethacrynic acid and by alpha-cyano-4-hydroxycinnamate, but only marginally by 4, 4'-diisothiocyanostilbene-2,2' disulfonate or by p-chloromercuribenzene sulfonate. The transporter responsible for NO3--induced pH changes in mammalian cells may be related, though not identical, to the NO3--H+ cotransporter described in Arabidopsis and Aspergillus. The mammalian cotransporter may be important in eliminating the products of NO metabolism, particularly in cells that generate vast amounts of this messenger. By cotransporting NO3- with H+ the cells would additionally eliminate acid equivalents from activated cells that are metabolizing actively, without added energetic investment and with minimal disruption of the transmembrane potential, inasmuch as the cotransporter is likely electroneutral.

Adenosine Triphosphatases↗
PubMed1997

The role of tyrosine phosphorylation in lipopolysaccharide- and zymosan-induced procoagulant activity and tissue factor expression in macrophages.

The expression of surface procoagulants by exudative macrophages represents an important mechanism underlying local fibrin deposition at sites of extravascular inflammation. The present studies investigated the contribution of tyrosine phosphorylation to the generation of macrophage procoagulant activity (PCA) and tissue factor expression in response to proinflammatory stimuli. Both lipopolysaccharide (LPS) and zymosan rapidly stimulated tyrosine phosphorylation in elicited murine peritoneal macrophages. This effect was prevented by the tyrosine kinase inhibitors genistein and herbimycin and augmented by the addition of the phosphotyrosine phosphatase inhibitor vanadate. The vanadate-mediated rise in phosphotyrosine accumulation was abrogated by the use of diphenylene iodonium, an inhibitor of the respiratory burst oxidase, suggesting a role for peroxides of vanadate as contributors to the tyrosine phosphorylation. This notion was supported by the finding that vanadyl hydroperoxide markedly increased the accumulation of phosphotyrosine residues. To define the role of tyrosine phosphorylation in the induction of macrophage PCA by LPS, the effects of tyrosine kinase inhibition by genistein and herbimycin were investigated. Both agents inhibited the expression of macrophage PCA. Further, Northern blot analysis with the cDNA probe for murine tissue factor indicated that the inhibition occurred at the mRNA level or earlier. Since vanadate augmented phosphotyrosine accumulation, it was hypothesized that it might enhance generation of macrophage products. However, vanadate reduced induction of PCA in response to LPS. By contrast, vanadate augmented basal prostaglandin E2 (PGE2) release and stimulated PGE2 release by macrophages. Indomethacin prevented the increase in PGE2 but only partially restored normal levels of PCA. The effect of vanadate on tissue factor expression appeared to be posttranscriptional. These studies thus demonstrate, by functional Western blotting and Northern blotting techniques, that tyrosine phosphorylation plays a role in the regulation of macrophage PCA and tissue factor expression in response to proinflammatory stimuli.

Animals↗
PubMed1997

Epstein Barr virus in Argentine pediatric Hodgkin's disease.

Since Hodgkin's disease (HD) is an heterogeneous condition with diverse histological and epidemiological subgroups, it seemed worthwhile to investigate the Argentine pediatric pattern. Moreover, the presence of Epstein Barr virus (EBV) infection occurs at different ages depending on the development status of the country. Thus, it was interesting to assess the relation between EBV and HD in the Argentine pediatric population. The age distribution profile of our pediatric HD patients showed a peak in early childhood which declined towards adolescence, closely resembling EBV infection pattern. Male:female ratio of the studied population was 3.2:1 and the histological subtype distribution disclosed that mixed cellularity HD (MCHD) was the most common, an epidemiological profile shared with other developing countries. Fifty percent of assessed HD cases were associated with EBV, showing a significantly higher prevalence in the 3-6 years-old group, indicating a non-random distribution. EBV was also present in most of MCHD cases and in some nodular lymphocyte predominance HD (nLPHD) but entirely absent in nodular sclerosis HD (NSHD). Both EBV subtypes, namely EBV-1 and EBV-2, were detected in studied HD cases. EBV-HD association in the Argentine pediatric population reveals typical epidemiological features indicating EBV as the aetiologic agent or, alternatively as a cofactor in a considerable percentage of such HD cases.

Adolescent↗
PubMed1997

Low-risk human papillomavirus types 6 and 11 associated with carcinomas of the genital and upper aero-digestive tract.

BACKGROUND: Human papillomavirus (HPVs) types 6 and 11, are frequently found in non-malignant anogenital condylomas and laryngeal papillomas. However, in an ongoing epidemiological study in Argentina in which 163 anogenital and aero-respiratory cancer biopsies were analyzed, several cases infected with low-risk HPVs could be detected. METHODS: Tumor samples were analyzed by Southern blot hybridization, using HPVs 6, 11, 16, 18, 30, 31, 33 and 35 as probes, and polymerase chain reaction (PCR) with type-specific primers and probes. RESULTS: Four invasive carcinomas containing HPV 6 or 11 could be detected (2.4%) by Southern blot technique. HPV 6 DNA was found in a penile carcinoma, a groin carcinoma, and in a tonsillar carcinoma. One cervical carcinoma harbored HPV 11 DNA. Using HPVs 6, 11, 16, 18 and 33 anticontamination primers and probes in PCR, no additional high-risk HPV types could be detected in these four cancers. CONCLUSION: This report shows the presence of low-risk HPVs (HPV 6 and 11) associated to malignant tumors in a frequency higher than usually observed. The data raise questions about which are the circumstances which may favor low-risk HPV related oncogenesis.

Blotting, Southern↗
PubMed1997

Involvement of the actin network in insulin signalling.

The purpose of the studies included in this chapter was to examine the role of the actin network in the propagation of insulin action leading to stimulation of glucose transport and activation of the mitogen-activated protein kinase cascade. The active insulin receptor phosphorylates tyrosine residues of intracellular proteins such as the insulin receptor substrate-1 (IRS-1) which acts as docking sites for molecules containing Src homology 2 (SH2) domains. One such molecule is phosphatidylinositol 3-kinase (PI 3-kinase) which becomes activated by binding to IRS-1. PI 3-kinase activity is required for the insulin-stimulation of glucose transport and glycogen synthesis. Grb2, a small adaptor molecule, can bind IRS-1 and, through the guanine nucleotide exchange factor Sos, leads to the activation of the small GTP binding protein Ras. Through a cascade of protein kinases, activation of Ras results in activation of the Erk 1 and 2 mitogen-activated protein kinases (MAPKs) which appear to control important nuclear and metabolic events. To investigate the role of the actin network in the propagation of insulin action leading to stimulation of glucose transport and the activation of the Erk MAPKs, we used the fungal metabolite cytochalasin D which disassembles the actin network. Actin disassembly abolished almost completely the ability of insulin to increase the rate of glucose transport into L6 muscle cells (myotubes) through prevention of the insulin-induced recruitment of glucose transporters to the plasma membrane which is the event that mediates the increase in the rate of transport. Actin disassembly did not affect either the insulin-mediated phosphorylation of IRS-1, the association of PI 3-kinase with this molecule, or the activation of IRS-1-associated PI 3-kinase. These results were also verified in another insulin responsive cell line, the 3T3-L1 adipocytes. In these cells, actin disassembly inhibited the insulin-induced recruitment of PI 3-kinase to intracellular membranes containing glucose transporters. Moreover, actin disassembly abolished the insulin-mediated phosphorylation of the Erk MAPKs. We conclude that the cellular actin network of insulin responsive cells is not required for the activation of PI 3-kinase but prevents its cellular redistribution. In contrast, intact actin filaments are essential for the propagation of insulin signals leading to the the activation of the MAPKs.

3T3 Cells↗