PubMed Health⌕ Search

Biomedical subjects

J Posada

Publications and source records attributed to J Posada.

At least 19 recordsLinked to original sources
PubMed2005

Functional analysis of the early chlorosis factor gene.

Chlorosis is one of the symptoms of bacterial spot disease caused by Xanthomonas campestris pv. vesicatoria, which induces chlorosis before any other symptoms appear on tomato. We report characterization of a 2.1-kb gene called early chlorosis factor (ecf). The gene ecf encodes a hydrophobic protein with similarity to four other proteins in plant pathogens, including HolPsyAE, and uncharacterized gene products from X. campestris pv. campestris and X. axonopodis pv. citri, and, at the tertiary structure level, to colicin Ia from Escherichia coli. We demonstrate that the associated phenotype is hrp dependent, and that the ecf gene product appears to be translocated to host cells. The gene ecf has no impact on electrolyte leakage or on bacterial growth in planta in response to infection. Concentrated culture filtrates do not produce chlorosis. Study of its role in Xanthomonas spp.-tomato interactions will forward our understanding of symptom production by plant pathogens and allows further investigation into the mechanisms of bacterial virulence and production of symptoms.

Amino Acid Sequence↗
PubMed2000

Death and injury from motor vehicle crashes in Colombia.

We report data on the distribution and determinants of road deaths and injuries for all victims in Colombia, with the aim of defining targets and priorities for highway death prevention in that country and other rapidly urbanizing nations. Using information from Colombia's Fund for the Prevention of Road Injury and the national death registry, we studied data on deaths and injuries from 1991 to 1995 for the nation as a whole and for the country's two largest cities, Santa Fe de Bogotá and Medellín. Deaths and injuries are rising in the nation as a whole. Of the deaths, 75% occur in urban areas, and 80% are in males. Pedestrians aged 15-34 are a peak subgroup. Thirty-four percent of deaths are attributable to speeding and/or alcohol consumption. Death tolls are highest at night and on weekends. Specific priority targets for intervention are indicated by the fact that 75% of road deaths in Colombia occur in urban areas and that 80% of all victims are males.

Accidents, Traffic↗
PubMed1999

Transmembrane helix 7 of the endothelin B receptor regulates downstream signaling.

Endothelin is a 21-amino acid peptide with a striking diversity of important biological responses, including, vasoconstriction, bronchoconstriction, and mitogenesis. Endothelin-1 binding to the endothelin B receptor (ETB), a member of the superfamily of G-protein-coupled receptors, was associated with catalytic activation of the extracellular-regulated kinase 2 (ERK2) and stimulation of AP-1 transcriptional reporter activity. A panel of single point mutations in transmembrane helix 6 (TM6), intracellular loop 3, and transmembrane helix 7 (TM7) were developed to study the structural requirements for ETB activation. Point mutations within highly conserved regions of TM6 and intracellular loop 3 were without effect on agonist-stimulated ERK activation. However, mutations within TM7 of the ETB significantly impacted ligand-stimulated downstream signaling. For example, nine point mutations within TM7 of the ETB were identified that prevented endothelin-stimulated ERK activation. Interestingly, the TM7 mutants fell into two classes; several exhibited greatly decreased AP-1 activity, relative to wild type ETB, whereas others displayed augmented endothelin-stimulated AP-1 transcriptional activity relative to wild type ETB. Our results suggest that TM7 of the ETB is involved in its activation mechanism and regulates agonist-stimulated ERK activation.

Amino Acid Sequence↗
PubMed1999

Endothelin-stimulated ERK activation in airway smooth-muscle cells requires calcium influx and Raf activation.

Endothelin (ET)-1 is a 21-amino-acid peptide that is a potent vasoconstrictor and mitogen. By binding to its G-protein coupled receptor, ET-1 stimulates the proliferation of airway smooth-muscle (ASM) cells, which may be involved in the pathogenesis of asthma. The ETB receptor stimulates activation of the extracellular regulated kinase 2 (ERK2), which is thought to be required for proliferation of ASM cells. Our findings reveal that ET rapidly activates Raf, and that dominant-negative Raf interferes with ET-induced ERK activation in ASM cells. Expression of the amino-terminal Ras-binding domain of Raf inhibited ET-induced ERK activation, suggesting that ET-stimulated Raf activation is a Ras-dependent process. Furthermore, ET-stimulated ERK and Raf activation in ASM cells require calcium influx; chelating extracellular calcium or preventing calcium influx through calcium channels inhibited ET-stimulated, but not phorbol ester-stimulated, ERK and Raf activation.

Animals↗
PubMed1997

Inhibition of ERK activation attenuates endothelin-stimulated airway smooth muscle cell proliferation.

Endothelin is a small peptide that is a potent bronchoconstrictor, mitogen for airway smooth muscle (ASM), and is believed to be involved in the pathogenesis of asthma. To understand how endothelin stimulates the proliferation of ASM cells in culture, we evaluated the relationship between mitogen activated protein (MAP) kinase activation and cell proliferation. Endothelin is a potent stimulator of the extracellular regulated kinase 2 (ERK2) subgroup of MAP kinases, and ERK2 activation was tightly correlated with the proliferation of rat ASM cells. PD98059, a small molecule inhibitor of MEK (MAP or ERK kinase) was used to establish the role of ERK2 activation in the endothelin-stimulated signal transduction pathway leading to cell proliferation. While PD98059 significantly inhibited the ability of endothelin to activate ERK, the drug did not appear to effect the catalytic activity of an activated MEK mutant, or ERK in vitro. The data suggest that the mechanism of PD98059 inhibition of the ERK2 pathway in ASM cells may involve inhibition of MEK activation. The endothelin signal transduction pathway that culminates in ERK2 activation was dependent on protein kinase C (PKC), since depletion of PKC significantly inhibited the ability of endothelin to activate ERK2. Taken together, the data imply that activation of ERK is a critical endpoint in the endothelin signal transduction pathway since inhibition of this kinase inhibits endothelin-induced ASM cell proliferation.

Animals↗
PubMed1996

Activation of multiple mitogen-activated protein kinase signal transduction pathways by the endothelin B receptor requires the cytoplasmic tail.

Endothelin is a 21-amino acid peptide with remarkably diverse biological properties, including potent vasoconstriction, induction of mitogenesis, and a role in the development of blood vessels. In the present study, stimulation of the endothelin B receptor was found to activate three distinct mitogen-activated protein kinase signal transduction pathways, the extracellular regulated kinase (ERK) 2, c-Jun N-terminal kinase 1 (JNK), and p38 kinase. These mitogen-activated protein kinase isozymes are thought to mediate very different biological outcomes, suggesting that the observed pattern of kinases activation may be important for the diverse biological properties of endothelin. The cytoplasmic tail of the endothelin B receptor was found to be required for activation of all three mitogen-activated protein kinases and stimulation of intracellular calcium levels. An endothelin B receptor truncated at the C-terminal tail was not able to stimulate the mitogen-activated protein kinases or increase cytosolic free calcium. Furthermore, ectopic expression of the cytoplasmic tail attenuated signaling through the wild type receptor. The observed ERK activation appeared to be mediated by heterotrimeric G proteins, since ectopic expression of a transducin alpha-subunit inhibited endothelin-stimulated ERK activation. The data suggest that the cytosolic tail of the endothelin B receptor is involved in calcium mobilization and mitogen-activated protein kinase activation via a G protein-dependent mechanism.

Animals↗
PubMed1996

Asbestos causes stimulation of the extracellular signal-regulated kinase 1 mitogen-activated protein kinase cascade after phosphorylation of the epidermal growth factor receptor.

Asbestos fibers are human carcinogens with undefined mechanisms of action. In studies here, we examined signal transduction events induced by asbestos in target cells of mesothelioma and potential cell surface origins for these cascades. Asbestos fibers, but not their nonfibrous analogues, induced protracted phosphorylation of the mitogen-activated protein (MAP) kinases and extracellular signal-regulated kinases (ERK) 1 and 2, and increased kinase activity of ERK2. ERK1 and ERK2 phosphorylation and activity were initiated by addition of exogenous epidermal growth factor (EGF) and transforming growth factor-alpha, but not by isoforms of platelet-derived growth factor or insulin-like growth factor-1 in mesothelial cells. MAP kinase activation by asbestos was attenuated by suramin, which inhibits growth factor receptor interactions, or tyrphostin AG 1478, a specific inhibitor of EGF receptor tyrosine kinase activity (IC50 = 3 nM). Moreover, asbestos caused autophosphorylation of the EGF receptor, an event triggering the ERK cascade. These studies are the first to establish that a MAP kinase signal transduction pathway is initiated after phosphorylation of a peptide growth factor receptor following exposure to asbestos fibers.

Animals↗
PubMed1993

Phosphorylation of mitogen-activated protein kinases is involved in the epidermal growth factor and phorbol ester, but not in the thyrotropin/cAMP, thyroid mitogenic pathway.

In dog thyroid epithelial cells (thyrocytes) in primary culture, thyrotropin (TSH) acting through cAMP induces proliferation and differentiation expression, whereas epidermal growth factor (EGF) and tumor-promoting phorbol esters induce proliferation and dedifferentiation. In these cells we have demonstrated mitogen-activated protein (MAP) kinase phosphorylation by 32P labeling and two-dimensional gel electrophoresis and by immunodetection with anti-MAP kinase and anti-phosphotyrosine antibodies after one- or two-dimensional gel electrophoresis. MAP kinase localization was demonstrated by immunochemical staining. We show the following results. (i) As in other systems, EGF and phorbol esters induced p42 and p44 MAP kinases phosphorylation on tyrosine, serine, and threonine. This effect was rapid, peaking after 5 and 15 min, respectively, followed by a slow decline thereafter. It preceded a translocation of MAP kinase immunoreactivity from cytoplasm to nucleus. (ii) Carbamylcholine, a potent stimulator of the Ca(2+)-phosphatidylinositol cascade which is unable to induce DNA synthesis, stimulated MAP kinases phosphorylation and nuclear staining with kinetics similar to those observed after EGF action, indicating that MAP kinase phosphorylation was not sufficient for mitogenesis. (iii) The cAMP-dependent mitogenic cascade elicited by TSH and forskolin did not involve the phosphorylation and nuclear translocation of p42 and p44 MAP kinases at any time during the entire prereplicative phase. Activation of MAP kinases by phosphorylation is therefore not a necessary step in the G0-G1 transition in this mitogenic cascade.

Animals↗
PubMed1993

p42 mitogen-activated protein kinase in brain: prominent localization in neuronal cell bodies and dendrites.

Neurotransmitters and growth factors can trigger activation of a newly described family of mitogen-activated protein kinases. To help define the role of this kinase family in signal transduction in the nervous system, we have conducted immunohistochemical studies to localize p42 mitogen-activated protein kinase in rat brain sections. Light-microscopic studies revealed staining in neuronal cell bodies and dendrites that is particularly prominent in superficial layers of the neocortex, the hippocampal CA3 region and dentate gyrus, as well as cerebellar Purkinje cells. Discrete staining of oligodendrocytes was also apparent in fiber tracts, indicating expression of p42 mitogen-activated protein kinase in both neuronal and glial cell types. Electron-microscopic studies demonstrated that staining in dendrites is closely associated with microtubules. In the cell bodies, prominent staining was associated with the Golgi apparatus. In contrast, immunolabeling of synaptic terminals was not detected. Previous studies have demonstrated that p42 mitogen-activated protein kinase responds to neuronal stimulation. Immunohistochemical studies presented in this paper demonstrate prominent staining for this kinase in neuronal cell bodies and dendrites. Therefore, this kinase is likely to play a key role in postsynaptic signal transduction. As both p42 mitogen-activated protein kinase and microtubule-associated protein 2, an in vitro substrate of p42 mitogen-activated kinase, are associated with dendritic microtubules, this kinase may mediate effects of growth factors or neurotransmitters on the dendritic cytoskeleton.

Amino Acid Sequence↗
PubMed1993

Mos stimulates MAP kinase in Xenopus oocytes and activates a MAP kinase kinase in vitro.

Several protein kinases, including Mos, maturation-promoting factor (MPF), mitogen-activated protein (MAP) kinase, and MAP kinase kinase (MAPKK), are activated when Xenopus oocytes enter meiosis. De novo synthesis of the Mos protein is required for progesterone-induced meiotic maturation. Recently, bacterially synthesized maltose-binding protein (MBP)-Mos fusion protein was shown to be sufficient to initiate meiosis I and MPF activation in fully grown oocytes in the absence of protein synthesis. Here we show that MAP kinase is rapidly phosphorylated and activated following injection of wild-type, but not kinase-inactive mutant, MBP-Mos into fully grown oocytes. MAP kinase activation by MBP-Mos occurs within 20 min, much more rapidly than in progesterone-treated oocytes. The MBP-Mos fusion protein also activates MPF, but MPF activation does not occur until approximately 2 h after injection. Extracts from oocytes injected with wild-type but not kinase-inactive MBP-Mos contain an activity that can phosphorylate MAP kinase, suggesting that Mos directly or indirectly activates a MAPKK. Furthermore, activated MBP-Mos fusion protein is able to phosphorylate and activate a purified, phosphatase-treated, rabbit muscle MAPKK in vitro. Thus, in oocytes, Mos is an upstream activator of MAP kinase which may function through direct phosphorylation of MAPKK.

Animals↗
PubMed1992

Purification and characterization of mitogen-activated protein kinase activator(s) from epidermal growth factor-stimulated A431 cells.

Two peaks of mitogen-activated protein (MAP) kinase activator activity are resolved upon ion exchange chromatography of cytosolic extracts from epidermal growth factor-stimulated A431 cells. Two forms of the activator (1 and 2) have been purified from these peaks, using chromatography on Q-Sepharose, heparin-agarose, hydroxylapatite, ATP-agarose, Sephacryl S-300, Mono S, and Mono Q. The two preparations each contained one major protein band with an apparent molecular mass of 46 or 45 kDa, respectively, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Evidence identifying the MAP kinase activators as the 46- and 45-kDa proteins is presented. Using inactive mutants of MAP kinase as potential substrates, it was found that each preparation of MAP kinase activator catalyzes phosphorylation of the regulatory residues, threonine 188 and tyrosine 190, of Xenopus MAP kinase. These results support the concept that the MAP kinase activators are protein kinases. These MAP kinase kinases demonstrate an apparent high degree of specificity toward the native conformation of MAP kinase, although slow autophosphorylation on serine, threonine, and tyrosine residues and phosphorylation of myelin basic protein on serine and threonine residues is detected as well.

Animals↗
PubMed1992

Requirements for phosphorylation of MAP kinase during meiosis in Xenopus oocytes.

Mitogen-activated protein (MAP) kinases are activated in response to a variety of extracellular stimuli by phosphorylation on tyrosine and threonine residues. Xp42 is a Xenopus laevis MAP kinase that is activated during oocyte maturation. Modified forms of Xp42 that lacked enzymatic activity or either of the phosphorylation sites were expressed in Xenopus oocytes. When meiotic maturation was induced with progesterone, each mutant Xp42 was phosphorylated, indicating that at least one kinase was activated that can phosphorylate Xp42 on tyrosine and threonine. Phosphorylation of one residue is not strictly dependent on phosphorylation of the other.

Amino Acid Sequence↗
PubMed1991

Tyrosine phosphorylation and activation of homologous protein kinases during oocyte maturation and mitogenic activation of fibroblasts.

Meiotic maturation of Xenopus and sea star oocytes involves the activation of a number of protein-serine/threonine kinase activities, including a myelin basic protein (MBP) kinase. A 44-kDa MBP kinase (p44mpk) purified from mature sea star oocytes is shown here to be phosphorylated at tyrosine. Antiserum to purified sea star p44mpk was used to identify antigenically related proteins in Xenopus oocytes. Two tyrosine-phosphorylated 42-kDa proteins (p42) were detected with this antiserum in Xenopus eggs. Xenopus p42 chromatographs with MBP kinase activity on a Mono Q ion-exchange column. Tyrosine phosphorylation of Xenopus p42 approximately parallels MBP kinase activity during meiotic maturation. These results suggest that related MBP kinases are activated during meiotic maturation of Xenopus and sea star oocytes. Previous studies have suggested that Xenopus p42 is related to the mitogen-activated protein (MAP) kinases of culture mammalian cells. We have cloned a MAP kinase relative from a Xenopus ovary cDNA library and demonstrate that this clone encodes the Xenopus p42 that is tyrosine phosphorylated during oocyte maturation. Comparison of the sequences of Xenopus p42 and a rat MAP kinase (ERK1) and peptide sequences from sea star p44mpk indicates that these proteins are close relatives. The family members appear to be tyrosine phosphorylated, and activated, in different contexts, with the murine MAP kinase active during the transition from quiescence to the G1 stage of the mitotic cell cycle and the sea star and Xenopus kinases being active during M phase of the meiotic cell cycle.

Amino Acid Sequence↗
PubMed1989

Protein kinase C in adriamycin action and resistance in mouse sarcoma 180 cells.

Adriamycin has a wide variety of biological actions on susceptible cells, several of which may be integrally involved in cytotoxicity. In this paper, we present evidence that one of the alterations in cell function that occurs in the presence of Adriamycin is an elevation in the production of diacylglycerol. The effect is rapid, reaches a peak within 10 min of exposure of Sarcoma 180 cells to Adriamycin, and can thus be classified among the earliest alterations that occur in cells damaged by Adriamycin. Concomitant with the rise in diacylglycerol is an increase in cytosolic protein kinase C activity. Although Adriamycin does not appear to modulate the activity of this enzyme by direct binding, drug-exposed Sarcoma 180 cells have a 56% increase in intrinsic cytosolic protein kinase C (PKC) activity, with no change in the activity of the membrane form. Experiments with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate suggest that the PKC effect is linked to Adriamycin action, since activation of the enzyme by short 12-O-tetradecanoylphorbol-13-acetate exposure enhances Adriamycin's cytotoxicity as well as its ability to provoke DNA damage (measured by alkaline elution). Likewise, down-regulation of PKC by extended 12-O-tetradecanoylphorbol-13-acetate exposure partially protects the cells from Adriamycin-induced cytotoxicity as well as from DNA damage. Thus, the ability of cells to be injured by Adriamycin appears to be correlated with the activity of PKC. Multidrug-resistant subline Sarcoma 180A10 cells have the same total quantity of membrane-recruitable PKC as the sensitive parent Sarcoma 180 cells, as determined by [3H]phorbol-12,13-dibutyrate binding. However, the resistant cells have a significantly higher intrinsic PKC activity and an altered ability to translocate the enzyme to the cell surface. Taken together, the results raise the possibility that cell signaling mechanisms, particularly those involving protein kinase C, may play an important role in mediating the biological action of the anticancer drug Adriamycin.

Animals↗