Biomedical subjects

G Gacon

Publications and source records attributed to G Gacon.

At least 37 records · Page 2Linked to original sources

PubMed1992

Inhibition of superoxide production in B lymphocytes by rac antisense oligonucleotides.

Rac1 and Rac2 gene products are small GTP-binding proteins showing 92% homology to each other. According to recent studies performed in cell-free systems, Rac1 and Rac2 proteins may be involved in the activation of NADPH-oxidase, the superoxide-generating enzymatic complex active in phagocytes. Epstein-Barr virus (EBV) transformed B lymphocytes, which express rac1 and rac2 genes, also efficiently release superoxide anions when triggered by various cell surface stimuli. To investigate the regulatory role of Rac proteins in living cells, we analyzed superoxide production in response to cross-linking of surface immunoglobulins or phorbol ester treatment in human EBV-transformed B lymphocytes pretreated with Rac sense and antisense oligonucleotides. We report here that (i) the rac protein content estimated by immunoblotting can be decreased by 60% in Rac antisense pretreated cells and (ii) a strong (50-60%), dose-dependent inhibition of superoxide production is observed in antisense pretreated cells whereas cells pretreated with sense oligonucleotide are unaffected. The data presented show, for the first time in whole cells, that superoxide production is modulated by the Rac protein content, thus demonstrating the physiological role of Rac proteins in the regulation of NADPH-oxidase.

B-Lymphocytes↗

PubMed1992

ADP-ribosylation of the ras-related, GTP-binding protein RhoA inhibits lymphocyte-mediated cytotoxicity.

The Rho proteins are identified as a subgroup of the Ras superfamily of low molecular weight GTP-binding proteins. We have studied the expression of these proteins in human cytotoxic natural killer cells and found that RhoA is the most abundantly expressed member of the Rho family. The Rho proteins are specific substrates for ADP-ribosylation catalyzed by the C3 exoenzyme from Clostridium botulinum. We report here that introduction of recombinant C3 in electropermeabilized natural killer cells or in cytotoxic T lymphocytes resulted in a dose-dependent inhibition of their cytolytic function. Furthermore, a single substrate is efficiently ADP-ribosylated by C3 in extracts from cytotoxic cells. Biochemical analyses indicate that this substrate is RhoA, and subcellular fractionation experiments demonstrate that it is essentially present in the cytosol of the cells. Western blot analysis, however, revealed that a small proportion of the Rho protein can be found associated with the cell membrane as well as with the cytotoxic granules. These results indicate that the low molecular weight GTP-binding protein RhoA is present in cytotoxic lymphocytes and plays a critical role in cell-mediated cytotoxicity.

ADP Ribose Transferases↗

PubMed1991

Phosphorylation of the lymphoid cell kinase p56lck is stimulated by micromolar concentrations of Zn2+.

In particulate fractions from LSTRA lymphoma cells, tyrosine phosphorylation of the lymphoid specific tyrosine kinase p56lck is elicited by Zn2+ in the absence of other divalent cations. Zn2+ alone also induces autophosphorylation of immunoprecipitated p56lck. The effect of Zn2+ is dose dependent; it is detected at concentrations of Zn2+ as low as 5 microM and reaches a maximum at 100 microM Zn2+. Among other divalent cations tested, Mn2+, and Co2+ to a lesser extent, were also effective. Zn2+ also stimulated p56lck phosphorylation in the presence of Mg2+ ions at physiological concentration, whereas orthovanadate had no effect. These results suggest that Zn2+ activates the autophosphorylation of p56lck; this fact could be related with the stimulating effect of Zn2+ in the activation of T lymphocytes.

Animals↗

PubMed1991

A hemopoietic specific gene encoding a small GTP binding protein is overexpressed during T cell activation.

We have isolated, from a human B cell line cDNA library, a cDNA (Gx) encoding a small G protein identical to rac 2, a member of the ras superfamily. Gx/rac 2 gene is expressed as a unique mRNA of 1,7 Kb in peripheral blood lymphocytes, in purified B and T cells, in thymus as well as in several B and T cell lines. It is not expressed in many other tissues analysed including liver, brain, lung, heart and kidney. Upon in vitro stimulation with phytohemagglutinin A, peripheral blood lymphocytes show a clear increase of the Gx/rac 2 mRNA after 6 hours; a 30-50 fold accumulation is reached at 24 hours and persists thereafter. Purified T lymphocytes exhibit a similar increase in Gx/rac 2 mRNA expression upon mitogenic stimulation. Therefore, the expression of the Gx/rac 2 gene appears to be restricted to cells of the hemopoietic lineage and to be strongly stimulated during T cell activation. Gx/rac 2 protein must fulfill a specific role in activated T cells that could provide a new model for studying the function of small G proteins.

Amino Acid Sequence↗

PubMed1990

[Stress fractures of the tibia: uncommon mechanical complication of osteoarthritis of the knee. Report of 3 cases].

Three cases are reported of spontaneous fracture of the tibia complicating osteoarthritis of the knee with genu varum. These fractures, which are very rare, with only eight cases published in the literature, seem to be associated with severe osteoarthritis in small, obese women. They usually progress to union with simple relief from weight-bearing but non-union may develop which makes the treatment of the causative osteoarthritis more difficult. A review of the published cases helped the indications for treatment to be defined.

Aged↗

PubMed1989

Interleukin 2 stimulates tyrosine phosphorylation in T cell membrane fractions.

Interleukin 2 is a growth factor secreted by T lymphocytes upon antigenic stimulation and inducing the proliferation of T cells bearing at their surface the heterodimeric high-affinity form of its receptor. No enzymatic function has so far been demonstrated in the receptor subunits. In an attempt to elucidate the biochemical pathway of signal transduction, we investigated the capacity of interleukin 2 to modulate tyrosine phosphorylation in T cell membranes. Membrane-rich fractions from T cells were tested for their ability to phosphorylate tyrosine in the presence or absence of added recombinant interleukin 2. Using as substrate a synthetic polymer of glutamic acid and tyrosine, we demonstrated a 3-4-fold stimulation of tyrosine phosphorylation in the presence of interleukin 2; this stimulating effect appeared to be well correlated with interleukin 2 function since (a) it was not observed in insensitive cells, (b) it required the presence of the high-affinity form of the receptor and (c) it was dose-dependent. Confirmatory results were obtained by phosphorylating membrane-rich fractions with [gamma-32P]ATP and by analysing the resulting phosphoproteins: only in fractions from cells with the high-affinity form of the receptor were several membrane proteins specifically phosphorylated on tyrosine residues in response to interleukin 2. At least two proteins of 115 and 58 kDa were consistently hyperphosphorylated on tyrosine in an interleukin-2-dependent manner. This stimulation was strongly dependent on the presence of the protein tyrosine phosphatase inhibitor, sodium orthovanadate. Thus, we propose that interleukin 2 enhances tyrosine phosphorylation by stimulating a tyrosine kinase activity. The nature of the enzyme involved remains to be determined.

Cell Fractionation↗

PubMed1989

The inhibitory effect of PGE2 on T cell activation is not associated with inhibition of PKC translocation.

We have recently reported on the effect of PGE2 on T cell activation and suggested that their immunosuppressive effect may involve the PKC activation pathway. In the present study, we further investigated the potential interference of PGE2 with PHA induced signaling in T lymphocytes. We demonstrate that the PHA mediated increase in IP3, the putative mobilizer of intracellular Ca2+, is slightly affected following cell incubation with PGE2. Treatment of cell culture with the tumor promoter TPA abrogates the suppressive activity of PGE2 whereas exogenous diacylglycerol (1,2-diolein) has only a marginal effect. This suggests that PGE2 affect PKC activity at sites distal to IP3 and DG generation. We also demonstrate that the PGE2 suppressive effect on T lymphocyte activation is not related to an inhibition of PKC translocation.

Cells, Cultured↗

PubMed1988

Comparison of interleukin 2 and 12-O-tetradecanoylphorbol 13-acetate as signals for protein kinase C activation in purified human T lymphocytes.

Interleukin 2 (IL2) and 12-O-tetradecanoylphorbol 13-acetate (TPA) have been compared for their ability to induce translocation of protein kinase C (PKC) in T lymphocytes prestimulated with anti-CD3 monoclonal antibody (mAb), either in the presence or absence of monocytes. TPA alone did not promote purified T cell growth, but it was able to induce a transient, within 30 min, translocation of PKC activity. The profiles of PKC association with the membrane of the T cells under TPA stimulation were quite similar when either the anti-CD3 mAb or the fixed monocytes, or both, were added to the T cells. The decrease of cytosolic PKC under TPA stimulation was less pronounced for the purified T cells stimulated with anti-CD3 mAb, fixed monocytes alone or both than for unstimulated purified T cells. Even in the absence of monocytes, the addition of exogenous IL2 to the anti-CD3 mAb-treated T cells resulted in PKC translocation, with a transient increase in the PKC activity found in both the particulate and cytosolic fractions. When exogenous IL2 was added to the proliferating T cells, the association of PKC with the membrane was prolonged and the activity did not reach a plateau during the first 2 h after the IL2 stimulation. In parallel, the level of PKC associated with the membrane was higher in proliferating cells than in resting cells even 4 days after stimulation. These results suggest that activation of PKC by IL2 might be different from the direct activation of PKC by TPA and that a specific activation pathway, at least kinetically distinct from the classical phosphatidyl inositol diphosphate degradation by phospholipase C, might be involved during IL2 stimulation of T lymphocytes through high-affinity IL2 receptors.

Antigens, Differentiation, T-Lymphocyte↗

PubMed1985

Vesicular stomatitis virus produced from infected LSTRA lymphoma cells bear tyrosine protein kinase activity (p56).

Murine LSTRA lymphoma cells contain a very active tyrosine protein kinase of 56 kDa (p56) which is not related to any of the other known tyrosine kinases. In the past the purification and characterization of the p56 have been hampered because of the low amount of this protein in LSTRA membranes. In this study, we have utilized a different approach for purification which consisted of trapping the protein in the membrane of vesicular stomatitis virus. Incubation of the virions with [gamma-32P]ATP resulted in the phosphorylation of p56 on tyrosine residues. Moreover, the phosphopeptide digest profile of vesicular stomatitis virus-p56 was identical to that observed with authentic LSTRA-p56. The p56 from such virions could be resolved from other proteins by two-dimensional gels, and furthermore, such virions have been used to prepare several antisera directed against the p56.

Amino Acids↗

PubMed1985

Phosphoinositides are not phosphorylated by the very active tyrosine protein kinase from the murine lymphoma LSTRA.

We studied the ability to phosphorylate phosphoinositides by 3 different subcellular preparations, and immunopurified tyrosine protein kinase (TPK) from two murine lymphoma cell lines induced by the Moloney murine leukemia virus: LSTRA with a very active TPK and MBL2 without significant TPK activity. We could not find any difference in the phosphorylation of phosphoinositides by these preparations. The TPK purified with two antibodies which phosphorylate actively tyrosine on exogenous substrate were unable to phosphorylate phosphoinositides.

Animals↗

PubMed1985

Modulation of adenylate cyclase by guanine nucleotides and Kirsten sarcoma virus mediated transformation.

Certain tumour cells contain activated ras genes that code for 21 000 dalton proteins (p21). These proteins associate with the inner face of the plasma membrane and bind guanine nucleotides specifically. In order to determine whether p21s have functions similar to other GTP binding proteins, we investigated the regulation, by guanine nucleotides, of adenylate cyclase (AC) activity in membrane preparations isolated from fibroblasts (C127) transformed by a temperature sensitive mutant of Kirsten sarcoma virus (Ts 371). The degree of AC stimulation by GMP P(NH)P increased when these cells were shifted from the permissive temperature (33 degrees C) to the non-permissive temperature (39 degrees C). This effect was more pronounced at low Mg++ and low GMP P(NH)P concentrations. AC stimulation remained unchanged in rat fibroblasts infected with a temperature sensitive mutant of Rous Sarcoma virus. AC activity was depressed in C127 cells infected with wild type KiMSV. Our data illustrate the feasibility of correlating alterations in the AC system with ras gene expression and using such experimental approaches to elucidate the physiological functions of the p21 proteins.

Adenylyl Cyclases↗

PubMed1985

Acute myeloblastic leukemia with active tyrosine protein kinase.

High level of tyrosine protein kinase activity was found in a membrane fraction isolated from an acute myeloblastic leukemia, out of 24 leukemias of different origin investigated. The major substrate for tyrosine phosphorylation in vitro was a 58 kDA protein (p58). The phosphorylation proceeded actively at 0 degrees C and was strongly stimulated by Mn2+ ions. Comparison by partial proteolysis of the p58 with similar phosphoproteins from a T-lymphoma line (KE37) and from lectin stimulated lymphocytes showed high similarity. The possible role of the tyrosine kinase activity in this leukemia is discussed.

Gastrins↗

PubMed1985

Detection of tyrosine-specific protein kinases with gastrin as exogenous substrate.

Gastrin was recently shown to be phosphorylated on its single tyrosine by the epidermal growth factor (EGF)-stimulated tyrosine protein kinase (TPK). The TPK previously detected in the murine lymphoma (LSTRA) induced by the Moloney murine leukemia virus phosphorylates gastrin, the apparent Km is 65 microM and the maximum rate 1900 pmol/min per mg; the kinase is more efficient with MnCl2 than with MgCl2, is stimulated by NaVO3 and inhibited by ZnCl2. Gastrin phosphorylation is observed only when a TPK is expressed by the cell: extracts of fibroblasts infected with a temperature-sensitive mutant of the Rous sarcoma virus had no gastrin kinase activity when grown at the non-permissive temperature whereas cells grown at the permissive temperature were transformed and disclosed a clear gastrin kinase activity. Gastrin kinases were detected in various transformed cells: human lymphomas, K562 cells, cells from a patient with acute proliferative leukemia, and normal cells: human T and B lymphocytes.

Animals↗

PubMed1984

Stimulation of tyrosine phosphorylation in lectin treated human lymphocytes.

Large increases in tyrosine phosphorylation have been detected in subcellular matrixes isolated from lectin treated human lymphocytes. In lectin stimulated cells proteins of molecular weight 105, 75, 58 and 35 kDa contained phosphotyrosine (P-tyr) whereas non-stimulated cells had no 105 and low levels of P-tyr in proteins of 75, 58 and 35 kDa. In stimulated cells increased tyrosine kinase activity was also shown using gastrin as substrate. In both stimulated and non-stimulated cells the 58 kDa phosphoprotein was the most heavily labelled, after partial proteolysis of the 58 kDa different phosphopeptides were generated. A peptide with a sequence analogous to the autophosphorylated tyrosine site of pp60src inhibited tyrosine phosphorylation in stimulated cells. The lymphocyte system provides a useful tool to study normal tyrosine protein kinases and their role in cellular proliferation.

Detergents↗

PubMed1984

Identification of a 58,000 daltons phosphoprotein with tyrosine protein kinase activity in a murine lymphoma cell line.

A very high level of tyrosine protein kinase (TPK) activity has been recently detected in a murine lymphoma, induced by Moloney murine leukemia virus. A major endogenous substrate for tyrosine phosphorylation in vitro is a protein of Mr 55-60,000 (p58) associated with the detergent insoluble matrix of LSTRA cells; in the present work p58 was solubilized, isolated by anion exchange chromatography and then precipitated by antiphosphotyrosine antibodies. Through these steps of isolation, TPK activity was measured by the use of a simplified gastrin phosphorylation assay. It is demonstrated that the TPK activity copurifies with p58, which leads to the conclusion that p58 bears itself the enzymatic activity. Although functionally similar to other enzymes of this group, this newly characterized TPK does not seem to be closely related to one of the previously documented TPK. This suggests either that this protein is the product of a so far unrecognized cellular TPK gene or that it derives from a rearrangement of one of the previously described TPK genes.

Animals↗

PubMed1983

Tyrosine phosphorylation in human T lymphoma cells.

A high level of tyrosine protein kinase (TPK) has been recently detected in the murine lymphoma LSTRA. The main substrate for tyrosine phosphorylation in this cell line is a Mr 55 000 protein associated with the insoluble matrix of the cell. These findings prompted the search for TPK activities in human lymphoid cells. Three human T lymphoma cell lines (i.e. Molt. 4, JM, and Ke 37) and control lymphocytes were examined. After in vitro phosphorylation of detergent insoluble extracts from human T lymphoma cells, 2 major phosphotyrosine containing proteins with Mr 55 000 and 35 000 can be detected in all three T lymphoma lines, whereas an additional species with Mr 78 000 is present only in the Ke 37 cell line. Similar size proteins are weakly phosphorylated in normal lymphocytes. Tyrosine phosphorylation in these proteins proceeds actively at 0 degrees C, and is dramatically stimulated by Mn++ ions. Partial proteolysis mappings of the Mr 55 000 phosphoproteins from murine and human lymphomas revealed a strong homology among these molecules. The function of this protein in transformed lymphocytes is discussed.

Animals↗

PubMed1982

High level of tyrosine protein kinase in a murine lymphoma cell line induced by Moloney leukemia virus.

Several sarcoma-inducing viruses encode protein kinases that phosphorylate tyrosine residues. Such enzymatic activities can be detected within the detergent-insoluble matrix of transformed fibroblasts. We have analysed the protein kinase activities in two murine lymphoma cell lines ( MBL2 and LSTRA) induced by Moloney murine leukemia virus (Mo-MuLV). After incubation of the detergent-insoluble matrix of these cells with [gamma-32P]ATP, several alkali-resistant phosphoproteins, including a very heavily labelled 55 000 mol. wt. protein ( p55 ), have been detected in LSTRA, reflecting the activity of a protein kinase specific to this cell line. This protein kinase activity shares some of the distinctive properties of the protein kinases of transforming viruses, i.e., specificity for tyrosine residues, association with membranous and/or cytoskeletal structures, and inhibition by a synthetic peptide derived from the phosphorylation site of pp60src. In view of the absence of a transforming gene in MoMuLV , it is likely that the high level of protein kinase detected in the LSTRA cell line arises from the expression of a cellular gene.

Amino Acids↗

PubMed1982

Phosphorylations of the subcellular matrix in cells transformed by Rous' sarcoma virus.

The transforming protein of Rous' sarcoma virus (RSV) is a phosphoprotein of Mr 60 000 (pp60src) which displays protein kinase activity specific for tyrosine residues; pp60src is associated with the plasma membrane and is recovered in the detergent-insoluble material which represents the subcellular matrix of the cell. After phosphorylation of this material of RSV-transformed cells with [gamma-32P]ATP, five phosphoproteins have been detected which are not seen in normal cells. These proteins (Mr = 135 000, 125 000, 75 000, 70 000, 60 000) contain phosphotyrosine. Their phosphorylation is strongly inhibited by anti-pp60src antibodies. In cells transformed by a temperature-sensitive mutant of RSV, these phosphoproteins, present at the permissive temperature, are no longer detected at the non-permissive temperature. It is concluded that these phosphorylations are mediated by pp60src protein kinase activity. This supports a possible role of the phosphorylation of cytoskeletal proteins in the transformation process.

Avian Sarcoma Viruses↗