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Biomedical subjects

A Ragab

Publications and source records attributed to A Ragab.

At least 19 recordsLinked to original sources
PubMed2003

Platelet aggregation induced by the C-terminal peptide of thrombospondin-1 requires the docking protein LAT but is largely independent of alphaIIb/beta3.

Thrombospondin-1 (TSP1) is abundantly secreted during platelet activation and plays a role in irreversible platelet aggregation. A peptide derived from the C-terminal domain of TSP1, RFYVVMWK (RFY) can activate human platelets at least in part via its binding to integrin-associated protein. Although integrin-associated protein is known to physically interact with alphaIIb/beta3, we found that this major platelet integrin had only a partial implication in RFY-mediated platelet aggregation. Accordingly, RFY induced a significant Glanzmann type I thrombasthenic platelet aggregation. The alphaIIb/beta3-dependent part of platelet aggregation induced by RFY was mainly due to secreted ADP and thromboxane A2. In the absence of alphaIIb/beta3 and fibrinogen, RFY stimulated a rapid tyrosine phosphorylation of a set of proteins, including Syk, linker for activation of T cells (LAT) and phospholipase Cgamma2. This signaling pathway was critical for RFY-mediated platelet activation as revealed by the use of pharmacological inhibitors as well as LAT-deficient mouse platelets. Phosphoinositide 3-kinase activation was also required for RFY-mediated platelet aggregation. Our results unravel a new alphaIIb/beta3 and fibrinogen-independent mechanism for platelet aggregation in response to the active peptide from the C-terminal domain of TSP1.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Differential regulation of phosphoinositide metabolism by alphaVbeta3 and alphaVbeta5 integrins upon smooth muscle cell migration.

Smooth muscle cell migration is a key step of atherosclerosis and angiogenesis. We demonstrate that alpha(V)beta(3) and alpha(V)beta(5) integrins synergistically regulate smooth muscle cell migration onto vitronectin. Using an original haptotactic cell migration assay, we measured a strong stimulation of phosphoinositide metabolism in migrating vascular smooth muscle cells. Phosphatidic acid production and phosphoinositide 3-kinase IA activation were triggered only upon alpha(V)beta(3) engagement. Blockade of alpha(V)beta(3) engagement or phospholipase C activity resulted in a strong inhibition of smooth muscle cell spreading on vitronectin. By contrast, blockade of alpha(V)beta(5) reinforced elongation and polarization of cell shape. Moreover, Pyk2-associated tyrosine kinase and phosphoinositide 4-kinase activities measured in Pyk2 immunoprecipitates were stimulated upon cell migration. Blockade of either alpha(V)beta(3) or alpha(V)beta(5) function, as well as inhibition of phospholipase C activity, decreased both Pyk2-associated activities. We demonstrated that the Pyk2-associated phosphoinositide 4-kinase corresponded to the beta isoform. Our data point to the metabolism of phosphoinositides as a regulatory pathway for the differential roles played by alpha(V)beta(3) and alpha(V)beta(5) upon cell migration and identify the Pyk2-associated phosphoinositide 4-kinase beta as a common target for both integrins.

1-Phosphatidylinositol 4-Kinase↗
PubMed2001

Enterophilins, a new family of leucine zipper proteins bearing a b30.2 domain and associated with enterocyte differentiation.

Enterocyte terminal differentiation occurs at the crypt-villus junction through the transcriptional activation of cell-specific genes, many of which code for proteins of the brush border membrane such as intestinal alkaline phosphatase, sucrase-isomaltase, or the microvillar structural protein villin. Several studies have shown that this sharp increase in specific mRNA levels is intimately associated with arrest of cell proliferation. We isolated several clones from a guinea pig intestine cDNA library. They encode new proteins characterized by an original structure associating a carboxyl-terminal B30.2/RFP-like domain and a long leucine zipper at the amino terminus. The first member of this novel gene family codes for a 65-kDa protein termed enterophilin-1, which is specifically expressed in enterocytes before their final differentiation. Enterophilin-1 is the most abundant in the small intestine but is still present in significant amounts in colonic enterocytes. In Caco-2 cells, a similar 65-kDa protein was recognized by a specific anti-enterophilin-1 antibody, and its expression was positively correlated with cell differentiation status. In addition, transfection of HT-29 cells with enterophilin-1 full-length cDNA slightly inhibited cell growth and promoted an increase in alkaline phosphatase activity. Taken together, these data identify enterophilins as a new family of proteins associated with enterocyte differentiation.

Amino Acid Sequence↗
PubMed2000

FcgammaRIIA requires a Gi-dependent pathway for an efficient stimulation of phosphoinositide 3-kinase, calcium mobilization, and platelet aggregation.

FcgammaRIIA, the only Fcgamma receptor present in platelets, is involved in heparin-associated thrombocytopenia (HIT). Recently, adenosine diphosphate (ADP) has been shown to play a major role in platelet activation and aggregation induced by FcgammaRIIA cross-linking or by sera from HIT patients. Herein, we investigated the mechanism of action of ADP as a cofactor in FcgammaRIIA-dependent platelet activation, which is classically known to involve tyrosine kinases. We first got pharmacologic evidence that the ADP receptor coupled to Gi was required for HIT sera or FcgammaRIIA clustering-induced platelet secretion and aggregation. Interestingly, the signaling from this ADP receptor could be replaced by triggering another Gi-coupled receptor, the alpha(2A)-adrenergic receptor. ADP scavengers did not significantly affect the tyrosine phosphorylation cascade initiated by FcgammaRIIA cross-linking. Conversely, the Gi-dependent signaling pathway, initiated either by ADP or epinephrine, was required for FcgammaRIIA-mediated phospholipase C activation and calcium mobilization. Indeed, concomitant signaling from Gi and FcgammaRIIA itself was necessary for an efficient synthesis of phosphatidylinositol 3,4,5-trisphosphate, a second messenger playing a critical role in the process of phospholipase Cgamma2 activation. Altogether, our data demonstrate that converging signaling pathways from Gi and tyrosine kinases are required for platelet secretion and aggregation induced by FcgammaRIIA.

Adenosine Diphosphate↗
PubMed1999

Phosphoinositide 3-kinase and integrin signalling are involved in activation of Bruton tyrosine kinase in thrombin-stimulated platelets.

Bruton tyrosine kinase (Btk) plays a crucial role in the differentiation of B lymphocytes and belongs to the group of Tec kinases, which are characterised by the presence of a pleckstrin homology domain. Here we show that Btk is activated and undergoes tyrosine phosphorylation upon challenge of platelet thrombin receptor, these responses requiring engagement of alphaIIb/beta3 integrin and phosphoinositide 3-kinase activity. These data unravel a novel signalling pathway involving Btk downstream of an adhesive receptor via a complex regulation implicating the products of phosphoinositide 3-kinase, which might act to anchor Btk at the membrane.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1998

Ectopic epididymal expression of guinea pig intestinal phospholipase B. Possible role in sperm maturation and activation by limited proteolytic digestion.

Guinea pig intestinal phospholipase B is a calcium-independent phospholipase hydrolyzing sequentially the acyl ester bonds at sn-2 and sn-1 positions of glycerophospholipids, promoting the formation of sn-glycero-3-phosphocholine from phosphatidylcholine. This 140-kDa glycoprotein from the brush border membrane of differentiated enterocytes contributes to lipid digestion as an ectoenzyme. The cDNA coding for guinea pig phospholipase B was revealed to be the homologue of AdRab-B, an mRNA appearing in rabbit upon intestine development. The sequence predicts a polypeptide of 1463 amino acids displaying four homologous repeats, two of them containing the lipase consensus sequence GXSXG. A 5-kilobase transcript was particularly abundant in mature ileal and jejunal enterocytes but was also detected in epididymis, where phospholipase B displayed a higher molecular mass (170 kDa versus 140 kDa in intestine), with no obvious evidence for enzyme activity. Trypsin treatment of phospholipase B immunoprecipitated from epididymal membranes reduced its size to 140 kDa, coinciding with the appearance of a significant phospholipase A2 activity. The same results were obtained in COS cells transfected with phospholipase B cDNA. Since sn-glycero-3-phosphocholine present at high concentrations in seminal plasma mainly stems from epididymis, this suggests a possible role of phospholipase B in male reproduction. This novel localization also unravels a mechanism of phospholipase B activation by limited proteolysis involving either trypsin in the intestinal lumen or a trypsin-like endopeptidase in the male reproductive tract.

Amino Acid Sequence↗
PubMed1997

Integrin-dependent tyrosine phoshorylation and cytoskeletal translocation of Tec in thrombin-activated platelets.

Using a specific polyclonal anti-Tec antibody, we have shown that Tec is expressed in human platelets. In addition, Tec was found to undergo tyrosine phosphorylation during platelet activation. The phosphorylation increased after 1 min and remained stable after 3 min of thrombin treatment. The tetrapeptide RGDS inhibited more than 90% of thrombin-induced tyrosine phosphorylation of Tec and blocked its translocation to the cytoskeleton. These results suggest that Tec participates in platelet signaling downstream of integrin activation.

Biological Transport↗
PubMed1996

G-protein beta gamma subunits mediate specific phosphorylation of the protein-tyrosine phosphatase SH-PTP1 induced by lysophosphatidic acid.

SH-PTP1 is a protein-tyrosine phosphatase preferentially expressed in hematopoietic cells and bearing two SH2 (src homology-2) domains. In the human megakaryocytic cell line Dami, lysophosphatidic acid (LPA) promoted a rapid increase in SH-PTP1 phosphorylation on both serine and tyrosine residues. Only tyrosine phosphorylation was significantly inhibited by pertussis toxin and by the protein kinase C inhibitor GF109203X. Moreover, SH-PTP1 was phosphorylated upon challenge with other agonists acting via G-protein-coupled receptors such as alpha-thrombin, epinephrine, and ADP, whereas the closely related protein-tyrosine phosphatase SH-PTP2 failed to share such a regulation in Dami cells. We developed an in vitro assay that reproduced LPA-dependent phosphorylation of SH-PTP1 in a cell-free system. The fusion protein glutathione S-transferase-beta-adrenergic receptor kinase 1-(495-689) or the transducin subunit Galphat-GDP, which act as specific antagonists of Gbetagamma, inhibited SH-PTP1 phosphorylation. Moreover, purified transducin Gbetagamma subunits mimicked the effect of LPA. Finally, stable expression of beta-adrenergic receptor kinase 1-(495-689) in Dami cells resulted in the inhibition of SH-PTP1 as a specific target of protein kinases linked to G-protein-coupled receptors via Gbetagamma subunits.

Cells, Cultured↗
PubMed1996

Regional distribution of organochlorine insecticide residues in human milk from Egypt.

The organochlorine insecticide residues in 60 human milk samples from 20 Egyptian governorates were analyzed by gas chromatography/electron capture and gas chromatography/mass spectrometry. The results showed that the main detected organochlorine insecticides were p,p'-DDE and lindane. Other HCH isomers, p,p'-DDT and endosulfan I residues were detected in some samples, but aldrin and endrin were not detected in most of the samples. The mean values for p,p'-DDE, lindane, endosulfan I and p,p'-DDT levels in the milk samples of the 20 governorates studied were 21.37 ppb, 8.42 ppb, 4.84 ppb and 2.93 ppb respectively, which are lower than the levels reported, a number of years ago, in most of the developed countries. Relatively higher levels of organochlorine insecticide residues were recorded in the samples from intensive agricultural activity regions.

Chromatography, Gas↗
PubMed1995

Increase in receptor-like protein tyrosine phosphatase activity and expression level on density-dependent growth arrest of endothelial cells.

Protein tyrosine phosphatase (PTPase) activity was examined in two cell lines: human umbilical vein endothelial (HUVE) cells, which display contact inhibition of cell growth, and A427 human adenocarcinoma cells, which have lost this ability. HUVE cells harvested at high density displayed a 10-fold increase in membrane-associated PTPase activity. A427 cells exhibited no such phenomenon. Moreover, modification of HUVE cell growth rate by a stimulating agent such as basic fibroblast growth factor or by blocking compounds such as thymidine or suramin resulted in no change in PTPase activity, suggesting that the observed increase in membrane-associated activity at confluency was specific for cell-cell-contact-directed growth arrest. The expression of various PTPase mRNAs was examined in HUVE and A427 cells. Of the receptor-like PTPases tested, two were exclusively expressed in HUVE cells (PTP gamma and HPTP beta). Only HPTP beta, which is structurally similar in its extracellular region to cell-adhesion receptors of the immunoglobulin superfamily, displayed a pattern of expression related to the increase in PTPase activity. Competitive PCR was used to quantify its expression during cell culture. A 12-fold increase in HPTP beta mRNA expression was detected and it parallelled the time course of PTPase activity. This observation strongly implicates receptor-like PTPases in density-dependent growth arrest.

Adenocarcinoma↗
PubMed1995

Tyrosine phosphorylation of an SH2-containing protein tyrosine phosphatase is coupled to platelet thrombin receptor via a pertussis toxin-sensitive heterotrimeric G-protein.

SH-PTP1 is a protein tyrosine phosphatase (PTP) predominantly expressed in haematopoietic cells and containing two src homology-2 (SH2) domains. Here we report that SH-PTP1 is phosphorylated on both serine and tyrosine residues in response to thrombin or phorbol myristate acetate (PMA), which increased by 60 and 40%, respectively, SH-PTP1 activity. Thrombin-induced phosphorylation of SH-PTP1 is an early signalling event (maximal within 10 s) involving neither integrin signalling, nor calcium, nor release of ADP or thromboxane A2. Moreover, in contrast with PMA, the effect of thrombin on the tyrosine phosphorylation of SH-PTP1 was hardly affected by GF109203X, a specific protein kinase C (PKC) inhibitor. Finally, phosphorylation of SH-PTP1 could be provoked in permeabilized platelets by thrombin or GTP gamma S. This was abolished by pertussis toxin, the specificity of this effect being verified with the megakaryocytic cell line Dami cell. Our data thus identify SH-PTP1 as an in vivo substrate of a putative protein tyrosine kinase linked to the thrombin receptor by a Gi protein. This might offer some clue to unravel the mechanism of thrombin not only in platelets but also in nucleated cells, where its mitogenic effect is known to involve pertussis toxin-sensitive G-proteins, tyrosine phosphorylation and the ras pathway.

Binding Sites↗
PubMed1995

Secretory phospholipase A2 generates the novel lipid mediator lysophosphatidic acid in membrane microvesicles shed from activated cells.

Nonpancreatic secretory phospholipase A2 (sPLA2) displays proinflammatory properties; however, its physiological substrate is not identified. Although inactive toward intact cells, sPLA2 hydrolyzed phospholipids in membrane microvesicles shed from Ca(2+)-loaded erythrocytes as well as from platelets and from whole blood cells challenged with inflammatory stimuli. sPLA2 was stimulated upon degradation of sphingomyelin (SPH) and produced lysophosphatidic acid (LPA), which induced platelet aggregation. Finally, lysophospholipid-containing vesicles and sPLA2 were detected in inflammatory fluids in relative proportions identical to those used in vitro. We conclude that upon loss of phospholipid asymmetry, cell-derived microvesicles provide a preferential substrate for sPLA2. SPH hydrolysis, which is provoked by various cytokines, regulates sPLA2 activity, and the novel lipid mediator LPA can be generated by this pathway.

Animals↗
PubMed1994

Translocation of an SH2-containing protein tyrosine phosphatase (SH-PTP1) to the cytoskeleton of thrombin-activated platelets.

A significant protein tyrosine phosphatase (PTP) activity was found to be associated with the cytoskeleton of thrombin-stimulated platelets. Translocation of the enzyme became maximal within 1-2 min of thrombin stimulation and was suppressed by cytochalasin D or upon inhibition of aggregation. Immunoblotting as well as immunoprecipitation revealed that a PTP with two SH2 domains (SH-PTP1) displayed the same behaviour, translocation to the cytoskeleton showing the same time course as that observed for pp60c-src. We conclude that SH-PTP1 might represent a critical enzyme in the complex interplay between the various proteins regulating protein tyrosine phosphorylation in the cytoskeletal matrix.

Biological Transport↗
PubMed1994

Neonatal rhabdomyosarcoma: the IRS experience.

Neonatal rhabdomyosarcoma is rare, and the characteristics and optimal therapy for patients with this condition have not been well described. Of 3,217 eligible patients entered in the Intergroup Rhabdomyosarcoma Study (IRS), 14 were less than 30 days old at the time of diagnosis. Among these patients, male gender, Caucasian race, caudal tumors, and embryonal/botryoid and undifferentiated histology predominate. Although half the neonates survived, neither histology, tumor size, nor type of surgery were predictive of outcome. The presence of necrosis and small round cell configuration coincides with a poor prognosis, regardless of histological diagnosis. Neonatal rhabdomyosarcoma of caudal origin has a favorable prognosis.

Age Factors↗
PubMed1994

Renal toxicity of ifosfamide in pilot regimens of the intergroup rhabdomyosarcoma study for patients with gross residual tumor.

PURPOSE: The purpose of this review is to characterize the nephrotoxicity noted in newly diagnosed patients under 21 years of age after treatment with ifosfamide-containing chemotherapy regimens and local irradiation for localized gross residual rhabdomyosarcoma or undifferentiated sarcoma. PATIENTS AND METHODS: From 1987 to 1991, 194 previously untreated patients received vincristine and ifosfamide plus dactinomycin or etoposide for 1-2 years. Ifosfamide was given at 1.8 g/m2/day for 5 days with sodium mercaptoethane sulfonate, or 9 g/m2 of ifosfamide per course. The three-drug regimen was repeated every 3-4 weeks. RESULTS: Twenty-eight patients (14%) developed renal toxicity: 19 had renal tubular dysfunction (RTD) characterized by low serum phosphate (< or = 3 mg/dl) or bicarbonate (< 20 or = mEq/L) levels, five had decreased glomerular function (DGF), and four had both RTD and DGF. When nine or more courses of ifosfamide (> 72 g/m2) were given, children < 3 years of age had a higher incidence of RTD than did children > or = 3 years of age (34% versus 6%; p < 0.001). A similar age difference was observed even when eight or fewer courses (< or = 72 g/m2) were given (p = 0.03). A matched case-control comparison showed that renal abnormalities at diagnosis, chiefly hydronephrosis, also increased the risk of renal tubular injury by ifosfamide by a factor of 13 (p < 0.001). Patients with DGF tended to be older than those with RTD, and all but one received > 72 g/m2 of ifosfamide. CONCLUSIONS: Patients who are < 3 years of age who receive more than eight courses (> 72 g/m2) of ifosfamide and who have a preexisting renal abnormality have an increased risk of RTD and DGF. The renal function of patients being considered for ifosfamide treatment must be carefully monitored. Ifosfamide should be avoided in patients with renal abnormalities at diagnosis unless the potential benefit clearly exceeds the risk of further renal impairment.

Acute Kidney Injury↗
PubMed1994

Thrombin-induced redistribution of protein-tyrosine-phosphatases to the cytoskeletal complexes in human platelets.

Human platelets provide an attractive model for studying the regulation of tyrosine phosphorylations and cell-cell adhesion. Major non-receptor tyrosine-kinases are suggested to be responsible for an increase in protein tyrosine phosphorylation following platelet stimulation. Agonist-induced platelet activation triggers also the reorganization of the cytoskeleton with association of multiple signalling proteins. To understand if protein-tyrosine-phosphatases (PTPs) were involved in platelet aggregation, we have investigated the subcellular distribution of these enzymes in resting and thrombin-stimulated platelets. A high level of PTP activity in human resting cells is distributed for 65% and 35%, respectively, in cytosolic and particular fractions. About 10% of this activity are redistributed to the cytoskeletal network during platelet activation. This translocation is dependent on actin polymerization as proved by the disappearance of this phenomenon in cells pretreated by cytochalasin D. Moreover, immunoblotting using anti-PTP polyclonal antibodies indicates that two PTPs, SH-PTP1 and p58 related to HPTP beta, translocate from membranes to Triton X-100 insoluble fractions after platelet activation. This translocation is correlated with the redistribution of several signalling proteins suggesting the possible regulation between these molecules and PTPs.

Biological Transport, Active↗
PubMed1994

Implication of a protein-tyrosine-phosphatase in human lung cancer.

Protein tyrosyl phosphorylation plays an essential role in regulating cellular events such as proliferation, differentiation and oncogenesis. The recent characterization of the family of protein tyrosine phosphatases (PTPases) suggests that dephosphorylation might be a crucial event in these phenomena. One of the functions of PTPases is to reverse the effect of protein tyrosine kinases (PTKases), many of which are oncogenes, suggesting that they may act as tumor suppressors as described for HPTP gamma. In order to investigate the implication in lung cancer of HPTP beta, a receptor PTPase, we have developed a semi-quantitative method derived from primer-directed reverse transcription (RT) and subsequent polymerase chain reaction (PCR) with 32P-labelled nucleotide. We have demonstrated that the expression of HPTP beta mRNA was dramatically decreased in lung adenocarcinomas and lung malpighian carcinomas as compared to normal lung tissue. In addition, HPTP beta was not expressed in the pulmonar adenocarcinoma cell line A427, which proliferates in a deregulated way. These results suggest that the loss of expression of HPTP beta might play a role in neoplasic transformation and thus this molecule could act as a tumor suppressor factor.

Adenocarcinoma↗