Biomedical subjects

J C Lacal

Publications and source records attributed to J C Lacal.

At least 55 records · Page 3Linked to original sources

PubMed1994

Progesterone but not ras requires MPF for in vivo activation of MAPK and S6 KII: MAPK is an essential conexion point of both signaling pathways.

Induction of mitosis in Xenopus laevis oocytes by hormones and the oncogenic ras-p21 protein has been shown to correlate with a cascade of phosphorylations of the Ser/Thr family of kinases. However, the exact hierarchy of enzymes and their mutual interdependency has not been fully elucidated yet. We have used the Xenopus laevis system to investigate the mechanism of activation of the Ser/Thr kinases cascade and their relationship. Comparison between progesterone-induced germinal vesicle breakdown (GVBD), a hallmark of mitosis in oocytes, to that triggered by ras-p21, revealed the existence of at least two independent mechanisms to activate the MAP kinase enzyme in vivo. While progesterone function is dependent of cdc2 protein kinase activity, ras-p21 is independent of this enzyme. However, both progesterone and ras-p21 converge at the MAP kinase level, and depletion of MAP kinase activity inhibits the GVBD and S6 kinase II activation induced by both progesterone and ras-p21. These results provides further evidence that MAP kinase is a critical step for regulation of the cell cycle in oocytes and a critical point where ras and progesterone signaling converge.

2-Aminopurine↗

PubMed1994

Activation of type D phospholipase by serum stimulation and ras-induced transformation in NIH3T3 cells.

Mitogenic stimulation of NIH3T3 fibroblasts with growth factors or ras oncogenes is associated with an increase in the levels of phosphorylcholine and diacylglycerol. Both metabolites could be generated as a result of direct activation of a phosphatidylcholine-specific phospholipase C (PC-PLC) or by a more complex pathway, involving activation of phospholipase D followed by choline kinase and phosphatidic acid-hydrolase. We show evidence indicating that the generation of phosphorylcholine and diacylglycerol follow independent mechanisms in both serum-treated and in ras-transformed NIH3T3 cells. No significant activation of a PC-PLC enzyme was observed. Instead, activation of a phosphatidylcholine-specific phospholipase D (PC-PLD) was detected. Moreover, while a fivefold constitutive activation of the endogenous PLD activity and a twofold increase on the levels of phosphatidic acid were observed in ras-transformed cells, very small alterations on these parameters were detected at late times after serum stimulation of quiescent cells. Thus, cell proliferation induced by ras oncogenes in fibroblasts cells may be functionally linked to activation of a PC-PLD enzyme. The differences found in the activation of this enzyme between ras-transformed and normal cells may constitute an important difference in mitogenic signalling between normal and transformed cells.

3T3 Cells↗

PubMed1993

Acylphosphatase synergizes with progesterone during maturation of Xenopus laevis oocytes.

Xenopus laevis oocytes are physiologically arrested in the G2/M phase border of the first meiotic division. A number of different stimuli can trigger off the re-entry into the cell cycle as a consequence of activation of either membrane-dependent or -independent intracellular signals. This system has been widely used to study signal transduction mechanisms induced by hormones. Among those more intensively researched, special attention has been devoted to elucidate the mechanism of activation induced by progesterone. However, despite intense efforts to understand the intracellular signalling mechanism of progesterone, a clear notion of the most relevant events involved in this process has not yet been elucidated. We provide evidence that acylphosphatase, an enzyme responsible for the regulation of membrane pumps in eukaryotic cells, synergizes with progesterone for induction of oocyte maturation. We deduced that this synergism may be related to the regulation of intracellular Ca2+ levels for several reasons: (1) maturation of oocytes by extracellular Ca2+ is blocked by acylphosphatase; (2) both progesterone and acylphosphatase drastically reduced Ca2+ uptake; (3) progesterone-induced maturation does not depend on a rise in intracellular Ca2+, since microinjection of EGTA, a calcium chelator, does not affect maturation induced by progesterone.

Acid Anhydride Hydrolases↗

PubMed1993

Microinjection of acylphosphatase blocks Xenopus laevis oocytes maturation induced by ras-p21.

Ras proteins induce germinal vesicle breakdown (GVBD) when microinjected into Xenopus laevis oocytes. The mechanism of action is still unresolved, although several hypotheses have been proposed. Acylphosphatase is a cytosolic enzyme that specifically catalyses the hydrolysis of the carboxylphosphate bond of acylphosphate for the removal of acylphosphate residues of various membrane pumps. A direct effect of acylphosphatase on the regulation of ionic balance of a cell by interaction with ionic membrane pumps has been proposed. We have analyzed the effect of microinjecting acylphosphatase, by itself or along with ras-p21 proteins or progesterone, into oocytes. The enzyme alone is unable to induce GVBD, but increases oocyte maturation induced by progesterone. By contrast, acylphosphatase blocked GVBD induced by microinjection of oncogenic ras-p21. These data suggest that acylphosphatase acts synergistically or antagonistically with factors involved in proliferating signals by altering the intracellular ionic conditions of the cell, conforming the hypothesis that the intracellular ionic condition of the cell is important in the induction of proliferating signals, and that its perturbation may have a serious effect on signal transduction.

Acid Anhydride Hydrolases↗

PubMed1993

Localization of rap1 and rap2 proteins in the gelatinase-containing granules of human neutrophils.

The subcellular localization of rap proteins in resting human neutrophils was investigated by immunoblot analysis with specific anti-rap2 and anti-rap1 antibodies of the membrane proteins obtained from distinct subcellular fractions. Rap2 protein was mainly located in gelatinase-containing granules, whereas rap1 protein was detected both in gelatinase-containing granules and in fractions enriched in plasma membrane. Neither rap1 nor rap2 proteins were found in the cytosol or in azurophilic granules. Rap2B, not rap2A, appeared to be the major rap2 protein in human neutrophils. The identification and subcellular localization of rap1 and rap2 proteins at the membranes of gelatinase-rich granules suggest that these proteins could play a role in the regulation of the rapid and selective mobilization of gelatinase-containing granules in human neutrophils.

Amino Acid Sequence↗

PubMed1993

Phospholipase-induced maturation of Xenopus laevis oocytes: mitogenic activity of generated metabolites.

Signal transduction induced by generation of second messengers from membrane phospholipids is considered a major regulatory mechanism in control of cell proliferation. We report here that in the Xenopus laevis oocytes model, microinjection of the three most relevant types of phospholipases acting on membrane phospholipids (A2, C, and D) are capable of inducing oocyte maturation with similar efficiencies. This effect is mediated by the generation of known second messengers such as lyso-phospholipids, arachidonic acid, diacylglycerol, and phosphatidic acid. Specific inhibitors of protein kinase C made it possible to identify alternative independent signalling pathways for induction of oocyte maturation. Our results indicate that while phospholipase C seems to be dependent on protein kinase C (PKC), phospholipase A2, and phospholipase D are completely independent of protein kinase C function. Thus, the oocyte system is a powerful tool for the analysis of the potential mitogenic activity of lipid metabolites. It is also an excellent tool for unravelling the different routes involved in the regulation of cell growth.

Alkaloids↗

PubMed1993

Phosphorylcholine: a novel second messenger essential for mitogenic activity of growth factors.

Growth factor stimulation of quiescent cells induces a series of intracellular early and late events that ultimately lead to DNA synthesis and cell division. We describe here that production of phosphorylcholine is an essential component of the late events involved in the induction of DNA synthesis by platelet-derived growth factor (PDGF-BB), a prototype mitogen for fibroblasts. Moreover, phosphorylcholine itself is mitogenic when added exogenously to NIH3T3 cells, further indicating its role as a crucial intracellular messenger for DNA synthesis. Choline kinase, the first step in the route of phosphatidylcholine synthesis appears to be the critical regulatory enzyme in phosphorylcholine production, indicating that regulation of choline kinase represents a key step during mitogenic stimulation. We also describe that several growth factors (PDGF-AA, basic FGF, EGF and phorbol esters) rely on their ability to generate phosphorylcholine for their proliferating activity. In contrast, DNA synthesis induced by serum did not require phosphorylcholine. Moreover, the requirement for phosphorylcholine production in PDGF-stimulated cells can be over-ruled by addition of insulin. Thus, cell proliferation in NIH3T3 cells can be triggered off by alternative pathways and one of them involves generation of phosphorylcholine.

3T3 Cells↗

PubMed1993

Tumorigenic activity of rho genes from Aplysia californica.

rho genes have been found in both lower and higher eucaryotes. They code for proteins of 21 kDa, highly conserved in evolution, which belong to the superfamily of ras GTPases. Among the members of this superfamily there are proteins with a regulatory function, such as ras, and proteins involved in vesicular trafficking, such as the family of rab proteins. We have investigated the putative role of rho proteins from Aplysia californica as transforming GTPases utilizing the wild-type and a Val-14 mutant, equivalent to the oncogenic Val-12 mutation of ras genes found in animal and human tumors. Over-expression of either rho gene was sufficient to confer anchorage- and serum-independent growth. Moreover, when introduced into nude mice, selected clones generated from either gene were able to induce tumors, although those carrying the mutated version were more efficient. Pathological analysis indicated that generated tumors corresponded to well-differentiated fibrosarcomas with distinct and intersecting bundles and spindle cells. By contrast, ras-induced tumors were poorly differentiated fibrosarcomas. Thus, our results indicate that under appropriate conditions rho genes function as oncogenes and may have a role in the regulation of proliferation in fibroblast cells.

3T3 Cells↗

PubMed1990

rap1B, a cAMP-dependent protein kinase substrate, associates with the platelet cytoskeleton.

rap1B is a member of the ras superfamily of low molecular weight GTP binding proteins which constitutes a focal point of GTP and cAMP signal transduction systems. Like other members of this superfamily, rap1B is membrane-associated in resting platelets, presumably through polyisoprenylation. The studies presented here were undertaken to determine the subcellular changes in rap1B localization during cell activation. Activated and unactivated platelets were fractionated by Triton X-100 lysis followed by differential centrifugation to obtain a 10,000 x g cytoskeleton fraction, a 100,000 x g membrane skeleton fraction, and a 100,000 x g supernatant fraction containing solubilized proteins. In unactivated platelets, rap1B was present in the 100,000 x g supernatant fraction. In contrast, in platelets activated with 1 unit/ml alpha-thrombin or with the calcium ionophore, A23187, rap1B was quantitatively recovered in the 10,000 x g cytoskeleton fraction. rap1B was absent from the 100,000 x g fraction containing the membrane skeleton and could not be detected in the 100,000 x g supernatant containing cytosolic proteins and solubilized membrane components. These results indicate that rap1B associates with the cytoskeleton during cell activation.

Amino Acid Sequence↗

PubMed1990

Differential effects of phorbol ester on the in vitro invasiveness of malignant and non-malignant human fibroblast cells.

The effect of the phorbol ester tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) on cell invasion was studied using an in vitro assay for cell invasion through a reconstituted basement membrane matrix (Matrigel). TPA inhibited the invasiveness of malignant human fibrosarcoma HT1080 cells. In contrast, WI-38 lung fibroblasts, which show a very low invasive capacity, were stimulated (3-fold) to invade Matrigel after exposure to TPA for 48 hours. The inhibitory or stimulatory effects of TPA on cell invasion were correlated with a decrease or an increase in cell motility and collagenase IV activity, respectively. Synthetic diacylglycerols partially mimicked the inhibitory action of TPA on HT1080 cells but failed to stimulate WI-38 cell invasion. Immunoblots demonstrated that in both cell lines the alpha and beta isoforms of protein kinase C were equally down-regulated after a 5 hour exposure to TPA despite the basal low level of protein kinase C polypeptide in the malignant cells. Thus, whereas in WI-38 cells induction of an invasive behavior could be observed in the absence of protein kinase C, in the malignant cells disappearance of the kinase was associated with a non-invasive phenotype.

Blotting, Western↗

PubMed1990

Potentiation of oncogenic N-ras-induced neurite outgrowth and ornithine decarboxylase activity by phorbol dibutyrate and protein kinase inhibitor H-8.

A recombinant N-ras oncogene, under the transcriptional control of a corticosteroid-inducible mouse mammary tumor virus (MMTV) promoter, has been stably transfected into a PC12 rat pheochromocytoma subline. This cell line, designated UR61, undergoes N-ras-induced neurite outgrowth and cessation of division when treated with dexamethasone (Guerrero et al.: Biochemical and Biophysical Research Communications 150:1185-1192, 1988). We have employed the UR61 cell line as a model for ras oncogene-induced neuronal differentiation. In UR61 cells, dexamethasone-induced expression of the recombinant N-ras gene resulted in time-dependent expression of ornithine decarboxylase enzyme (ODC) activity. Prompted by recent reports of possible functional (Lacal et al.: Molecular and Cellular Biology 7:4146-4149, 1987; Wolfman and Macara: Nature 325: 359-361, 1987) and direct (Jeng et al.: Biochemical and Biophysical Research Communications 145:782-788, 1987) interactions between oncogene ras-coded p21 and protein kinase C (PK-C; Ca++/phospholipid-dependent protein kinase), we employed the protein kinase inhibitor H-8 (N-[2-(methylamino)ethyl]-5-isoquinoline sulfonamide dihydrochloride) and phorbol 12,13-dibutyrate (PDBu) to investigate this putative interaction in the UR61 cells, where ODC activity and neurite outgrowth were used as indicators of oncogenic N-ras action. Treatment of UR61 cells with PDBu depleted cells of PK-C and failed to promote neurite outgrowth but enhanced N-ras-induced neurite outgrowth and ODC activity. H-8, which suppressed ODC induction by forskolin and phorbol myristate acetate, enhanced both N-ras-induced ODC activity and neurite outgrowth. Inhibition of ODC activity by difluoromethylornithine (DFMO) did not suppress oncogenic ras-induced neurite outgrowth, suggesting that these two ras-triggered events are mechanistically independent. These findings suggest that certain actions of N-ras can occur in cells depleted of PK-C, and thus, the role of PK-C in ras-induced differentiation differs from its role in ras-induced mitogenesis and transformation.

Animals↗

PubMed1990

Thrombolamban, the 22-kDa platelet substrate of cyclic AMP-dependent protein kinase, is immunologically homologous with the Ras family of GTP-binding proteins.

Platelet inhibition by agents that increase intracellular levels of cAMP is associated with cAMP-dependent phosphorylation of specific intracellular proteins, including a membrane-associated 22-kDa microsomal protein called thrombolamban. In view of recent studies suggesting that platelets also contain 22-kDa GTP-binding proteins that are homologous with ras-encoded p21 proteins, the present work was undertaken to examine the possibility that thrombolamban and the Ras-like proteins were the same. Platelet microsomes were labeled with [gamma-32P]ATP and the labeled proteins were examined by autoradiography of sodium dodecyl sulfate/polyacrylamide gels. On Western blots of both one-dimensional and two-dimensional gels, thrombolamban immunoreacted with M90, a monoclonal antibody that recognizes the GTP-binding domain of Ras p21 proteins, but not with Y13-259, a monoclonal antibody that recognizes another domain and is specific for Ras proteins. Overlay experiments with unlabeled platelet microsomes demonstrated numerous low molecular weight proteins that bound [alpha-32P]GTP, although none could be identified as thrombolamban. Finally, thrombolamban was immunoprecipitated by M90. These studies show that thrombolamban is a low molecular weight protein that is immunologically related to the Ras family of GTP-binding proteins.

Adenosine Triphosphate↗

PubMed1990

Diacylglycerol production in Xenopus laevis oocytes after microinjection of p21ras proteins is a consequence of activation of phosphatidylcholine metabolism.

Microinjection of p21Ha-ras proteins into Xenopus laevis oocytes induces a rapid increase of 1,2-diacylglycerol (DAG) levels. The observed alterations in DAG levels were consistent with the ability of the protein to induce maturation, measured by germinal vesicle breakdown (GVBD). Both the increase in DAG levels and GVBD activity were dependent on the ability of the proteins to undergo membrane translocation. Alterations of DAG levels or GVBD activity did not correlate with changes in the levels of inositol phosphates. However, at minimal doses sufficient to achieve maximal biological response, a biphasic increase in the amounts of phosphocholine and CDP-choline was observed. The first burst of phosphocholine and CDP-choline preceded the increase in DAG levels. The second peak paralleled the appearance of DAG. Choline kinase activity was also increased in oocyte extracts after p21ras microinjection. These results suggest that both the synthesis and degradation of phosphatidylcholine are activated after microinjection of ras proteins into Xenopus oocytes, resulting in a net production of DAG.

Animals↗

PubMed1990

Regulation of protein kinase C activity in neuronal differentiation induced by the N-ras oncogene in PC-12 cells.

Expression of the N-ras oncogene under the control of the glucocorticoid-responsive promoter in the pheochromocytoma cell line UR61, a subline of PC-12 cells, has been used to investigate the differentiation process to neuronal cells triggered by ras oncogenes (I. Guerrero, A. Pellicer, and D. E. Burstein, Biochem. Biophys. Res. Commun. 150:1185-1192, 1988). Using ras-inducible cell lines, we observed that expression of the oncogenic N-ras p21 protein interferes with the ability of phorbol esters to induce downregulation of protein kinase C. This effect was associated with the appearance of immunologically detectable protein kinase C as well as the activity of the enzyme as analyzed either by binding of [3H]phorbol-12,13-dibutyrate in intact cells or by in vitro kinase activity. These results indicate a relationship between ras p21 and protein kinase C in neuronal differentiation in this model system. Comparison to the murine fibroblast system suggests that this relationship may be functional.

Adrenal Gland Neoplasms↗

PubMed1989

Agonist-induced phosphorylation of an immunologically ras-related protein in human erythroleukemia cells.

Monoclonal antibody M90 recognizes a specific epitope of the ras-encoded p21 protein. This region comprises amino acids 107-130 containing the residues 116-119, which are related to GTP binding. This antibody strongly reacts on Western Blots with a 22kDa protein from human erythroleukemia (HEL) cells. Treatment of HEL cells with iloprost, an agonist that increases cellular cyclic AMP levels, produces the appearance of a protein with an apparent molecular mass of 24kDa. This protein is also recognized by antiserum M90 on Western Blots; its appearance parallels a decrease of the 22kDa protein, and it can be labeled with 32P. This effect is also observed with dibutyryl cyclic AMP, which indicates phosphorylation of the 22kDa protein by cyclic AMP-dependent protein kinase. This phosphorylation produces an electrophoretic mobility change of the 22kDa protein to a 24kDa region on gels. The change of mobility of the 22kDa protein induced by iloprost in HEL cells is also observed when the protein is labeled with [35S]methionine and immunoprecipitated with antiserum M90. This information indicates a coupling mechanism involving phosphorylation of an oncogene product in HEL cells.

Antibodies, Monoclonal↗

PubMed1989

Identification of rho as a substrate for botulinum toxin C3-catalyzed ADP-ribosylation.

Recombinant Aplysia rho and a GTP-binding protein purified from human neutrophil membranes (G22K) were ADP-ribosylated by botulinum toxin C3 with stoichiometries of 0.8 and 0.6, respectively. Rho and G22K appeared to be different proteins since (i) rho migrated faster on polyacrylamide gels, (ii) unlike G22K, rho did not require the presence of cytosol to be ADP-ribosylated, (iii) G22K was not recognized by an anti-rho antiserum, and (iv) antibody 142-24E05 recognized G22K effectively but only poorly cross reacted with rho. ADP-ribosylation had no effect on the ability of rho to bind or hydrolyse GTP. Therefore, it appears that there are multiple botulinum toxin C3 substrates and that the toxin exerts its effects on cell function by a mechanism other than modulating the GTPase activity of rho.

Adenosine Diphosphate Ribose↗

PubMed1989

A ras-related protein is phosphorylated and translocated by agonists that increase cAMP levels in human platelets.

The antigenicity of platelet proteins was assayed against various monoclonal antibodies (mAbs) that recognize specific epitopes of the ras-encoded p21 protein. mAb M90, which detects the region of p21 protein within amino acids 107-130 and inhibits its GTP-binding activity, strongly reacted with a 22-kDa protein present in the particulate fraction of human platelets. Other mAbs against ras-encoded proteins, including Y13-259, which efficiently detects ras proteins from a variety of organisms, did not recognize the platelet 22-kDa protein. Transfer of the platelet 22-kDa protein to nitrocellulose paper showed that the protein binds [alpha-32P]GTP. Moreover, preincubation of the transferred protein with mAb M90 drastically reduced its GTP-binding activity. Treatment of platelets with iloprost, a prostacyclin analog, caused (i) a time-dependent increase of a 24-kDa protein that is recognized by mAb M90 in particulate and cytosolic fractions and (ii) the gradual decrease of the 22-kDa protein from the particulate fraction. When platelets were labeled with 32P and then treated with iloprost, the 24-kDa protein was found to be phosphorylated. The 32P-labeled 24-kDa protein was specifically immunoprecipitated by mAb M90. These results suggest that appearance of the 24-kDa protein results from phosphorylation of the 22-kDa protein, which shifts its mobility to a higher molecular mass area.

Antibodies, Monoclonal↗

PubMed1989

Development of an enzyme-linked immunosorbent assay for equine infectious anemia virus detection using recombinant Pr55gag.

To provide more sensitive and convenient methods for the detection of equine infectious anemia virus (EIAV), we developed an enzyme-linked immunosorbent assay (ELISA) employing the EIAV gag precursor (Pr55gag) produced by using recombinant DNA techniques. The antigenic reactivity of the recombinant EIAV Pr55gag was found to be equivalent to that of the virion p24gag and elicited high-titered antiserum in rabbits. When a large number of horse sera were analyzed for the presence of antibodies to EIAV by this ELISA, a radioimmunoassay for EIAV p15gag, or the standard agar gel immunodiffusion test, there was 98.7% concordance among the assays. By using the ELISA it was possible to specifically detect antibodies earlier after experimental infection of horses with EIAV than with the other two tests. A competition ELISA developed in order to detect EIAV gag antigens was found to be approximately 15 times more sensitive than the radioimmunoassay for EIAV p15gag. Antigens of other animal lentiviruses as well as those of the prototype oncovirus failed to compete in this assay.

Animals↗