Biomedical subjects

S Roche

Publications and source records attributed to S Roche.

At least 19 recordsLinked to original sources

PubMed2002

Sex chromosome mosaicism in couples undergoing intracytoplasmic sperm injection.

BACKGROUND: Several studies have shown an increased frequency of constitutional chromosome aberrations in male and female partners of couples examined prior to ICSI. We conducted a cohort study to determine whether there was an increase in numerical sex chromosome mosaicism among couples undergoing ICSI compared with fertile couples. METHODS: Cytogenetic investigations were performed in 228 females and 208 males seen for ICSI between January 1997 and March 2001. They were matched to control females and males. RESULTS: Sex chromosome loss or gain was observed in at least one cell from 24.1% of ICSI women in comparison with 22% of controls (not significant). A significant difference between these two groups was found when X chromosome loss in at least two cells was considered, 9.6% for ICSI females versus 4.8% for controls (P = 0.01). No significant difference was observed between male groups concerning loss or gain of the X or Y chromosome. CONCLUSION: Our results support previously published studies indicating that the loss of an X chromosome in a single cell in females undergoing ICSI is probably an artefact. However, they suggest that a woman could have true sex chromosome mosaicism when two 45,X0 cells are found.

Adult↗

PubMed2002

[Breech presentation at term: a survey on obstetrical practice in France and a search for a homogeneous attitude associated with lower neonatal risk].

OBJECTIVES: 1- In order to define a "consensual attitude" in case of breech presentation, we queried the AUDIPOG network on obstetrical practice in search for the more frequently accepted criteria for normal delivery. 2- For consensual practices that could be identified, early neonatal complications were compared between women cared for in maternity wards applying these practices and those cared for in maternity wards not applying these practices. MATERIALS AND METHODS: 1- A confidential survey was sent to participating obstetrics units to determine their practical attitude in case of breech presentation. A consensual attitude was established on the basis of their responses. 2- Crude and adjusted comparisons were made concerning neonatal results between the consensual and non consensual groups of obstetrics units. RESULTS: Response rate was 85% for 175 obstetrics departments consulted. Six criteria were identified leading to classification of 42% of the units as consensual obstetrics units. The risk of major neonatal complications was lower in the consensual group than in the non-consensual group: adjusted OR=0.27 (95% CI: 0.09-0.85). CONCLUSION: A questionnaire on obstetrical practice can help define consensual attitudes associated with lower risk of neonatal complications.

Attitude of Health Personnel↗

PubMed2001

Magnetoresistance of carbon nanotubes: from molecular to mesoscopic fingerprints.

A magnetoresistance study of carbon nanotubes demonstrates that, for small diameters, the location of the chemical potential and the orientation of the magnetic field are parameters that enable tuning from positive to negative magnetoresistance, a phenomenon not related with weak localization. For larger diameters (> or =10 nm), the conventional mesoscopic behavior of magnetotransport is recovered.

Journal Article↗

PubMed2001

The hepatitis B virus HBx protein induces adherens junction disruption in a src-dependent manner.

Chronic hepatitis B virus infection is strongly associated with the development of hepatocellular carcinoma (HCC). Epithelial tumors are frequently characterized by loss of cadherin expression or function. Cadherin-dependent adhesion prevents the acquisition of a migratory and invasive phenotype, and loss of its function is itself enough for the progression from adenoma to carcinoma. The HBx protein of hepatitis B virus is thought to contribute to the development of the carcinoma, however, its role in the oncogenic and metastatic processes is far from being fully understood. We report herein the ability of HBx to disrupt intercellular adhesion in three different cell lines stably transfected with an inducible HBx expression vector. The linkage between the actin cytoskeleton and cadherin complex, which is essential for its function, is disrupted in the presence of HBx, as indicated by detergent solubility and immunoprecipitation experiments. In addition, beta-catenin was tyrosine phosphorylated in HBx-expressing cells. Inhibition of the src family of tyrosine kinases resulted in the prevention of the disruption of adherens junctions. These results suggest that HBx is able to disrupt intercellular adhesion in a src-dependent manner, and provide a novel mechanism by which HBx may contribute to the development of HCC.

Adherens Junctions↗

PubMed2001

A specific role of phosphatidylinositol 3-kinase gamma. A regulation of autonomic Ca(2)+ oscillations in cardiac cells.

Purinergic stimulation of cardiomyocytes turns on a Src family tyrosine kinase-dependent pathway that stimulates PLCgamma and generates IP(3), a breakdown product of phosphatidylinositol 4,5-bisphosphate (PIP2). This signaling pathway closely regulates cardiac cell autonomic activity (i.e., spontaneous cell Ca(2+) spiking). PIP2 is phosphorylated on 3' by phosphoinositide 3-kinases (PI3Ks) that belong to a broad family of kinase isoforms. The product of PI3K, phosphatidylinositol 3,4,5-trisphosphate, regulates activity of PLCgamma. PI3Ks have emerged as crucial regulators of many cell functions including cell division, cell migration, cell secretion, and, via PLCgamma, Ca(2+) homeostasis. However, although PI3Kalpha and -beta have been shown to mediate specific cell functions in nonhematopoietic cells, such a role has not been found yet for PI3Kgamma. We report that neonatal rat cardiac cells in culture express PI3Kalpha, -beta, and -gamma. The purinergic agonist predominantly activates PI3Kgamma. Both wortmannin and LY294002 prevent tyrosine phosphorylation, and membrane translocation of PLCgamma as well as IP(3) generation in ATP-stimulated cells. Furthermore, an anti-PI3Kgamma, but not an anti-PI3Kbeta, injected in the cells prevents the effect of ATP on cell Ca(2+) spiking. A dominant negative mutant of PI3Kgamma transfected in the cells also exerts the same action. The effect of ATP was observed on spontaneous Ca(2+) spiking of wild-type but not of PI3Kgamma(2/2) embryonic stem cell-derived cardiomyocytes. ATP activates the Btk tyrosine kinase, Tec, and induces its association with PLCgamma. A dominant negative mutant of Tec blocks the purinergic effect on cell Ca(2+) spiking. Tec is translocated to the T-tubes upon ATP stimulation of cardiac cells. Both an anti-PI3Kgamma antibody and a dominant negative mutant of PI3Kgamma injected or transfected into cells prevent the latter event. We conclude that PI3Kgamma activation is a crucial step in the purinergic regulation of cardiac cell spontaneous Ca(2+) spiking. Our data further suggest that Tec works in concert with a Src family kinase and PI3Kgamma to fully activate PLCgamma in ATP-stimulated cardiac cells. This cluster of kinases provides the cardiomyocyte with a tight regulation of IP(3) generation and thus cardiac autonomic activity.

Adenosine Triphosphate↗

PubMed2001

The COOH-terminal Src kinase Csk is a tumor antigen in human carcinoma.

The cytoplasmic tyrosine kinase cSrc is involved in the regulation of many important cellular functions including cell growth and transformation, and its activity is down-regulated by phosphorylation of the Tyr530 residue by the COOH-terminal Src tyrosine kinase, Csk. Because cSrc was previously found overexpressed, activated, and in some cases mutated in carcinoma, we investigated whether it could act as a tumor antigen. We show that whereas no autoantibodies were found against cSrc or its relative Fyn, up to 20% of patients with carcinoma had high-affinity autoantibodies against Csk. Immunity mainly resulted from a secondary response, as indicated by the presence of IgG1 in the sera. Antibodies were linked to the cancer because they were not detected in healthy subjects nor in patients with unrelated diseases, and their levels decreased in the sera of patients after surgical resection. Furthermore, they behaved as early markers of epithelial transformation because they were present in sera of patients with early-stage tumors and precancerous lesions such as colorectal polyps and in sera of patients that were scored negative for other cancer serological markers (CEA, CA15-3, CA19-9, p53 antibodies). Finally the presence of these antibodies was attributed, at least in part, to a substantial elevation of Csk protein levels in the corresponding tumors. However a strong increase in Src activity was also observed in these tissues, which suggested that Csk cannot regulate Src-like activity in carcinoma. Taken together, these data demonstrate that Csk acts as an autoantigen, and the detection of anti-Csk antibodies may have potential diagnostic usefulness in the early detection and postoperative follow-up of patients with carcinoma.

Adenocarcinoma↗

PubMed2001

Native and DPPA cross-linked collagen sponges seeded with fetal bovine epiphyseal chondrocytes used for cartilage tissue engineering.

Collagen-based biomaterials in the form of sponges (bovine type I collagen, both native and cross-linked by treatment with diphenylphosphorylazide, noted control and DPPA sponges respectively) were tested as three-dimensional scaffolds to support chondrocyte proliferation with maintenance of the phenotype in order to form neocartilage. Control and DPPA sponges were initially seeded with 10(6) or 10(7) foetal bovine epiphyseal chondrocytes and maintained for 4 weeks in culture under static conditions in RPMI/NCTC medium with 10% FCS and without addition of fresh ascorbic acid. Both supports were always present during the study and a partial decrease in size and weight was detected only with control sponges, both seeded and unseeded. Cell proliferation was only noted in the 10(6) cells-seeded sponges (4-fold increase after 4 weeks of culture). Specific cartilage collagens (types II and XI) were deposited in the matrix throughout the culture and traces of type I collagen were noticed only in the culture medium after 2-3 weeks and 4 weeks in the case of 10(6) and 10(7) cells-seeded sponges, respectively. Glycosaminoglycans accumulated in the matrix, up to 1.8 and 9.8% of total dry weight after one month with both seeding conditions, which was much lower than in the natural tissue. In the 10(7) cells-seeded sponges, mineral deposition, observed with unseeded sponges, was significantly decreased (2- to 3-fold). These in vitro results indicate that both collagen matrices can support the development of tissue engineered cartilage.

Amino Acids↗

PubMed2000

A critical role for phosphoinositide 3-kinase upstream of Gab1 and SHP2 in the activation of ras and mitogen-activated protein kinases by epidermal growth factor.

Although the mechanisms involved in the activation of mitogen-activated protein kinases (MAPK) by receptor tyrosine kinases do not display an obvious role for phosphoinositide 3-kinases (PI3Ks), we have observed in the nontransformed cell line Vero stimulated with epidermal growth factor (EGF) that wortmannin and LY294002 nearly abolished MAPK activation. The effect was observed under strong stimulation and was independent of EGF concentration. In addition, three mutants of class Ia PI3Ks were found to inhibit MAPK activation to an extent similar to their effect on Akt/protein kinase B activation. To determine the importance of PI3K lipid kinase activity in MAPK activation, we have used the phosphatase PTEN and the pleckstrin homology domain of Tec kinase. Overexpression of these proteins, but not control mutants, was found to inhibit MAPK activation, suggesting that the lipid products of class Ia PI3K are necessary for MAPK signaling. We next investigated the location of PI3K in the MAPK cascade. Pharmacological inhibitors and dominant negative forms of PI3K were found to block the activation of Ras induced by EGF. Upstream from Ras, although association of Grb2 with its conventional effectors was independent of PI3K, we have observed that the recruitment of the tyrosine phosphatase SHP2 required PI3K. Because SHP2 was also essential for Ras activation, this suggested the existence of a PI3K/SHP2 pathway leading to the activation of Ras. In addition, we have observed that the docking protein Gab1, which is involved in PI3K activation during EGF stimulation, is also implicated in this pathway downstream of PI3K. Indeed, the association of Gab1 with SHP2 was blocked by PI3K inhibitors, and expression of Gab1 mutant deficient for binding to SHP2 was found to inhibit Ras stimulation without interfering with PI3K activation. These results show that, in addition to Shc and Grb2, a PI3K-dependent pathway involving Gab1 and SHP2 is essential for Ras activation under EGF stimulation.

Animals↗

PubMed2000

Identification of a novel immunoreceptor tyrosine-based activation motif-containing molecule, STAM2, by mass spectrometry and its involvement in growth factor and cytokine receptor signaling pathways.

In an effort to clone novel tyrosine-phosphorylated substrates of the epidermal growth factor receptor, we have initiated an approach coupling affinity purification using anti-phosphotyrosine antibodies to mass spectrometry-based identification. Here, we report the identification of a signaling molecule containing a Src homology 3 domain as well as an immunoreceptor tyrosine-based activation motif (ITAM). This molecule is 55% identical to a previously isolated molecule designated signal transducing adaptor molecule (STAM) that was identified as an interleukin (IL)-2-induced phosphoprotein and is therefore designated STAM2. Tyrosine phosphorylation of STAM2 is induced by growth factors such as epidermal growth factor and platelet-derived growth factor as well as by cytokines like IL-3. Several of the deletion mutants tested except the one containing only the amino-terminal region underwent tyrosine phosphorylation upon growth factor stimulation, implying that STAM2 is phosphorylated on several tyrosine residues. STAM2 is downstream of the Jak family of kinases since coexpression of STAM2 with Jak1 or Jak2 but not an unrelated Tec family kinase, Etk, resulted in its tyrosine phosphorylation. In contrast to epidermal growth factor receptor-induced phosphorylation, this required the ITAM domain since mutants lacking this region did not undergo tyrosine phosphorylation. Finally, overexpression of wild type STAM2 led to an increase in IL-2-mediated induction of c-Myc promoter activation indicating that it potentiates cytokine receptor signaling.

Adaptor Proteins, Signal Transducing↗

PubMed2000

A specific function for phosphatidylinositol 3-kinase alpha (p85alpha-p110alpha) in cell survival and for phosphatidylinositol 3-kinase beta (p85alpha-p110beta) in de novo DNA synthesis of human colon carcinoma cells.

We have previously shown an important function of phosphatidylinositol 3-kinase (PI3K)alpha(p85alpha-p110alpha) and PI3Kbeta (p85-alpha-p110beta) for DNA synthesis induced by various mitogens in non transformed fibroblasts and we now report a specific role of these enzymes in human colon cancer cell growth. Using antibodies specific to p110alpha and to p110beta catalytic subunits, increase in PI3Kalpha and PI3Kbeta activities was detected in 15/19 human tumour biopsies relative to adjacent normal mucosa of human colon and bladder. Increase in such activities was also observed in adenocarcinoma cell lines CaCo2, CO115, HCT 116, LS 174T and WiDr relative to non-transformed fibroblasts. Maximal PI3Kalpha activity was observed for LS 174T and PI3Kbeta activity for WiDr cells. This was partly correlated with an increase in p110alpha and p110beta protein levels both in some primary tumours and established cell lines, suggesting that PI3K overexpression is involved in enzymatic deregulation. Functional consequence of such activation was assessed by a microinjection approach. An injection of neutralizing antibody specific to p110beta in WiDr, HCT116 and CO 115 cells inhibited de novo DNA synthesis, whereas antibodies specific to p110gamma had no effect. Neutralizing antibodies specific to p110alpha induced apoptosis, a response that was reverted by treating cells with the caspase inhibitor z-VAD-fmk. However anti-p110beta and anti-p110gamma antibodies did not affect cell survival. We concluded that PI3Kalpha and PI3Kbeta play important roles in human colon cancer cell growth with a specific function for PI3Kbeta in de novo DNA synthesis and an involvement of PI3Kalpha in cell survival.

3T3 Cells↗

PubMed2000

Deregulation of the cytoplasmic tyrosine kinase cSrc in the absence of a truncating mutation at codon 531 in human bladder carcinoma.

The involvement of the cytoplasmic tyrosine kinase cSrc was investigated in human bladder carcinogenesis. Kinase activity was determined in tissue lysates from bladder transitional cell carcinoma (TCC) relative to normal epithelia. Strong kinase activation was observed at all stages of carcinogenesis with a peak at the stage pT1, where tumor cells disrupt the basement membrane and invade the submucosa. In agreement with a role for cSrc in cell invasion, immunocytochemistry analysis showed a strong staining of invading cells. An increase in cSrc protein level were also found in most tumor samples, however, it did not correlate with an increase in activity (r = 0.44) suggesting that cSrc is deregulated in these tumors. Indeed, high Src activity was affinity-purified from a column (IRSVSSDGHE(p)YIYVDP-Affigel 10) that specifically retains active Src. Enzymatic regulation involves the C-terminus, recently found mutated at codon 531 in a subset of advanced human colon cancers. However, no such mutations were detected in TCC, suggesting the existence of other mechanisms for kinase activation.

Base Sequence↗

PubMed2000

Controlling substitution chemistry in ruthenium(II) systems. Synthesis of heteroleptic complexes incorporating the [Ru([9]aneS3)]2+ metal center.

The complex [Ru(py)3([9]aneS3)][PF6]2, 1 (py = pyridine), has proved to be a suitable starting material for the synthesis of heteroleptic Ru(II) complexes. By exploiting unfavorable steric interactions between 2-H and 6-H hydrogens of coordinated pyridyl ligands, we have synthesized half-sandwich complexes incorporating the thiocrown [9]aneS3 and a variety of facially coordinated N-donor ligands. Such complexes are easily prepared: Stirring 1 at room temperature in the presence of a suitable nitrile ligand leads to the exclusive substitution of one py ligand to produce complexes such as [([9]aneS3)Ru(py)2(NCMe)][PF6]2, 2. However, if the same reaction is carried out at higher temperatures, two py ligands are substituted, leading to complexes such as [([9]aneS3)Ru(py)(NCMe)2][PF6]2, 3. An alternative approach to such heteroleptic species has also been developed which exploits the restricted ability of thioethers to neutralize positive charges through sigma-donation. This phenomenon allows the synthesis of heteroleptic complexes in a two-step procedure via monocationic species. By variation of the donor/acceptor properties of ligands incorporated into the [Ru([9]aneS3)]2+ metal center, it is possible to tune the Ru(III)/Ru(II) redox couple over a range of > 700 mV. The solid-state structures of 1-3 were confirmed by X-ray crystallography studies. Crystal data: C22H30F12N4O2P2RuS3 (1.CH3NO2), monoclinic, Cc, a = 23.267(5) A, b = 11.5457(18) A, c = 26.192(5) A, alpha = 90 degrees, beta = 114.836(10) degrees, gamma = 90 degrees, Z = 8; C18H25F12N3P2RuS3 (2), triclinic, P1, a = 11.3958(19) A, b = 11.4280(19) A, c = 11.930(2) A, alpha = 100.518(3) degrees, beta = 100.542(3) degrees, gamma = 112,493(3) degrees, Z = 2; C15H23F12N3P2RuS3 (3), orthorhombic, Pna2(1)), a = 14.748(5) A, b = 18.037(18) A, c = 10.341(5) A, alpha = 90 degrees, beta = 90 degrees, gamma = 90 degrees, Z = 4.

Journal Article↗

PubMed2000

Slap negatively regulates Src mitogenic function but does not revert Src-induced cell morphology changes.

Src-like adapter protein (Slap) is a recently identified protein that negatively regulates mitogenesis in murine fibroblasts (S. Roche, G. Alonso, A. Kazlausakas, V. M. Dixit, S. A. Courtneidge, and A. Pandey, Curr. Biol. 8:975-978, 1998) and comprises an SH3 and SH2 domain with striking identity to the corresponding Src domains. In light of this, we sought to investigate whether Slap could be an antagonist of all Src functions. Like Src, Slap was found to be myristylated in vivo and largely colocalized with Src when coexpressed in Cos7 cells. Microinjection of a Slap-expressing construct into quiescent NIH 3T3 cells inhibited platelet-derived growth factor (PDGF)-induced DNA synthesis, and the inhibition was rescued by the transcription factor c-Myc but not by c-Jun/c-Fos expression. Fyn (or Src) overexpression overrides the G(1)/S block induced by both SrcK- and a Slap mutant with a deletion of its C terminus (SlapDeltaC), but not the block induced by Slap or SlapDeltaSH3, implying that the C terminus is a noncompetitive inhibitor of Src mitogenic function. Furthermore, a chimeric adapter comprising SrcDeltaK fused to the Slap C terminus (Src/SlapC) also inhibited Src function during the PDGF response in a noncompetitive manner, as Src coexpression could not rescue PDGF signaling. Slap, however, did not reverse deregulated Src-induced cell transformation, as it was unable to inhibit depolymerization of actin stress fibers while still being able to inhibit SrcY527F-induced DNA synthesis. This was attributed to a distinct Slap SH3 binding specificity, since the chimeric Slap/SrcSH3 molecule, in which the Slap SH3 was replaced by the Src SH3 sequence, substantially restored stress fiber formation. Indeed, three amino acids important for ligand binding in Src SH3 were replaced in the Slap SH3 sequence; Slap SH3 did not bind to the Src SH3 partners p85alpha, Shc, and Sam68 in vitro, and the chimeric tyrosine kinase Slap/SrcK, composed of SlapDeltaC fused to the SH2 linker kinase sequence of Src, was not regulated in vivo. Furthermore, the Src SH3 domain is required for signaling during mitogenesis and since Slap/SrcK behaved as a dominant negative in the PDGF mitogenic response when microinjected into quiescent fibroblasts. We conclude that Slap is a negative regulator of Src during mitogenesis involving both the SH2 and the C terminus domains in a noncompetitive manner, but it does not regulate all Src function due to specific SH3 binding substrates.

3T3 Cells↗

PubMed1999

An epidermal growth factor receptor/Gab1 signaling pathway is required for activation of phosphoinositide 3-kinase by lysophosphatidic acid.

Phosphoinositide 3-kinase (PI3K) has been shown to play an essential role in G protein-induced signaling even in non-myeloid cells where few agonists of G protein-coupled receptors are known to activate PI3K. We have identified adherent cell lines where lysophosphatidic acid (LPA) strongly and rapidly activates the accumulation of PI3K lipid products. The process is not modified by expression of a kinase-dead mutant of the Gbetagamma-responsive PI3K p110gamma. In contrast, it is inhibited by genistein or expression of a dominant negative mutant of p85 and potentiated by overexpressing wild-type p110alpha or -beta but not -gamma. By using a specific chemical inhibitor of the epidermal growth factor receptor (EGFR) and expression of a dominant negative mutant, we have observed that recruitment of p85/p110 PI3Ks occurs through transactivation of the EGFR by LPA and downstream mobilization of the docking protein Gab1 that associates with p85 upon LPA stimulation. Finally, we show that LPA cannot activate PI3K in cell lines lacking the EGFR/Gab1 pathway, including cells that transactivate the PDGF receptor. Altogether, these results demonstrate that activation of PI3K by LPA is conditioned by the ability of LPA to transactivate an EGFR/Gab1 signaling pathway.

Adaptor Proteins, Signal Transducing↗

PubMed1999

Regulation of growth, protein synthesis, and maturation of fetal bovine epiphyseal chondrocytes grown in high-density culture in the presence of ascorbic acid, retinoic acid, and dihydrocytochalasin B.

Phenotypic expression of chondrocytes can be modulated in vitro by changing the culture technique and by agents such vitamins and growth factors. We studied the effects of ascorbic acid, retinoic acid (0.5 and 10 microM), and dihydrocytochalasin B (3, 10, 20 microM DHCB), separately or in combination (ascorbic acid + retinoic acid or ascorbic acid + DHCB), on the induction of maturation of fetal bovine epiphyseal chondrocytes grown for up to 4 weeks at high density in medium containing 10% fetal calf serum and the various agents. In the absence of any agent or with retinoic acid or DHCB alone, the metabolic activity of the cells remained very low after day 6, with no induction of type I or X collagen synthesis nor increase in alkaline phosphatase activity. Chondrocytes treated with fresh ascorbic acid showed active protein synthesis associated with expression of types I and X after 6 and 13 days, respectively. This maturation was not accompanied by obvious hypertrophy of the cells or high alkaline phosphatase activity. Addition of retinoic acid to the ascorbic acid-treated cultures decreased the level of type II collagen synthesis and delayed the induction of types I and X collagen, which were present only after 30 days. A striking increase in alkaline phosphatase activity (15-20-fold) was observed in the presence of both ascorbic acid and the highest dose of retinoic acid (10 microM). DHCB was also a potent inhibitor of the maturation induced by treatment with ascorbic acid, as the chondrocytes maintained their rounded shape and synthesized type II collagen without induction of type I or X collagen. The pattern of protein secretion was compared under all culture conditions by two-dimensional gel electrophoresis. The different regulations of chondrocyte differentiation by ascorbic acid, retinoic acid, and DHCB were confirmed by the important qualitative and quantitative changes in the pattern of secreted proteins observed by two-dimensional gel electrophoresis along the study.

Animals↗

PubMed1998

Src-like adaptor protein (Slap) is a negative regulator of mitogenesis.

The Src-like adaptor protein (Slap) is a recently identified adaptor protein containing Src homology 3 (SH3) and SH2 domains. Slap is found in a wide range of cell types and was shown to interact with the Eck receptor tyrosine kinase in a yeast two-hybrid interaction screen [1]. Here, we found that Slap is expressed in NIH3T3 cells and could associate with the activated platelet-derived growth factor (PDGF) receptor. Using mutated versions of the PDGF receptor and phosphopeptide competition experiments, we determined that Slap has the highest affinity for the Src-binding site of the PDGF receptor. Our inability to produce cell lines that stably expressed Slap suggested that Slap inhibited cell growth. We further investigated this issue by transiently expressing Slap by microinjection. Overexpression of Slap by this method inhibited DNA synthesis induced by PDGF and serum, whereas overexpression of the adaptor proteins Grb2 and Shc did not. Finally, microinjection of a Slap antibody into NIH3T3 cells that had been stimulated with suboptimal doses of growth factors potentiated the effects of the growth factors. These data suggest that, unlike other adaptor proteins, Slap is a negative regulator of signalling initiated by growth factors.

3T3 Cells↗

PubMed1998

Src family tyrosine kinase regulates intracellular pH in cardiomyocytes.

The Anion Cl-/HCO3- Exchangers AE1, AE2, and AE3 are membrane pH regulatory ion transporters ubiquitously expressed in vertebrate tissues. Besides relieving intracellular alkaline and CO2 loads, the AEs have an important function during development and cell death and play a central role in such cellular properties as cell shape, metabolism, and contractility. The activity of AE(s) are regulated by neurohormones. However, little is known as to the intracellular signal transduction pathways that underlie this modulation. We show here that, in cardiomyocytes that express both AE1 and AE3, the purinergic agonist, ATP, triggers activation of anion exchange. The AE activation is observed in cells in which AE3 expression was blocked but not in cells microinjected with neutralizing anti-AE1 antibodies. ATP induces tyrosine phosphorylation of AE1, activation of the tyrosine kinase Fyn, and association of both Fyn and FAK with AE1. Inhibition of Src family kinases in vivo by genistein, herbimycin A, or ST638 prevents purinergic activation of AE1. Microinjection of either anti-Cst.1 antibody or recombinant CSK, both of which prevent activation of Src family kinase, significantly decreases ATP-induced activation of AE. Microinjection of an anti-FAK antibody as well as expression in cardiomyocytes of Phe397 FAK dominant negative mutant, also prevents purinergic activation of AE. Therefore, tyrosine kinases play a key role in acute regulation of intracellular pH and thus in cell function including excitation-contraction coupling of the myocardium.

Adenosine Triphosphate↗

PubMed1998

Effects of oxyhemoglobin in vitro in cerebral arteries from normal animals and animals subject to subarachnoid hemorrhage or indomethacin treatment.

Experiments were performed to test the hypothesis that subarachnoid hemorrhage (SAH) causes functionally relevant perturbations of cyclooxygenase activity in cerebral arteries. Four groups of rabbits were formed: (I) controls; (II) sham injected animals (2 ml physiological solution in the cisterna magna); (III) SAH group (2 ml blood in cisterna magna); (IV) indomethacin group (4 mg/kg i.v. 30 min before sacrifice). Animals of groups II and III were used 3 days after injection. The basilar arteries (BAs) were removed and perfused at a constant flow rate (after electrocoagulation of all branches) in vitro in a 2-ml bath at 37 degrees C. After 45 min equilibration, the arteries were subjected to a fixed protocol: first, in Krebs solution, contraction to increasing extraluminal concentrations of histamine (HA), followed by a single maximal extraluminal concentration of acetylcholine (ACh); then, after 30 min rest, the same tests were repeated in oxyhemoglobin (oxyHb) solution (extraluminal, 10-4 M). Perfusion pressure changes reflected changes in artery resistance. Although oxyHb alone increased pressure, indicating contraction of the arteries, its most important effect was to increase contraction to HA (in groups II, III, and IV but not controls) and to strongly inhibit ACh-induced relaxation in the SAH (-66.3%) and indomethacin (-46.9%) groups (III and IV) but not the control (-27.6%) group. The latter result suggests that a relaxing factor released by ACh in oxyHb solution in the control group was not present in groups III and IV. In conjunction with the results on HA, which is known to normally release prostacyclin (PGI2) from the endothelium, it is concluded that PGI2 was not or little released from arteries of the SAH group when they bathed in oxyHb solution. Alternatively, in the SAH group constrictor prostaglandins were released in response to HA and ACh in place of PGI2.

Acetylcholine↗