RESUMEN
Recently, it has been reported that IL-4 production by murine Th2 cell lines is insensitive to inhibition by E-type prostaglandins. In the present study, IL-4 production in vitro by freshly isolated concanavalin A (Con A)-stimulated murine spleen cells was readily suppressed by PGE2 with an I50 of 2 nM. Comparable suppression by PGE2 was seen after priming by anti-CD3 epsilon antibody instead of Con A or with other changes in the culture conditions. PGE2 was an effective inhibitor after elimination of Ly2.2+ T cells, consistent with a direct effect on Th2 cells. In the absence of added prostaglandins, IL-4 production was enhanced 1.5- to 7.0-fold by 0.2-2.0 microM indomethacin, indicating that endogenous arachidonate metabolites such as PGE2 and PGI2 regulate IL-4 production in our usual culture system. The inhibition of Th2 cell secretion by PGE2 in vitro may have physiologic and pharmacologic implications for the regulation of Th2 cell function and IgE production in vivo.
Asunto(s)
Dinoprostona/fisiología , Regulación de la Expresión Génica/efectos de los fármacos , Interleucina-4/biosíntesis , Bazo/citología , Células Th2/efectos de los fármacos , Animales , Secuencia de Bases , Bucladesina/farmacología , Células Cultivadas , Concanavalina A/farmacología , Cruzamientos Genéticos , Dinoprost/farmacología , Dinoprostona/biosíntesis , Dinoprostona/genética , Dinoprostona/farmacología , Epoprostenol/biosíntesis , Epoprostenol/genética , Epoprostenol/fisiología , Femenino , Indometacina/farmacología , Interleucina-4/genética , Activación de Linfocitos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Endogámicos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Prostaglandina D2/farmacología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Bazo/inmunología , Células Th2/metabolismoRESUMEN
Two Rhizobium strains were cultured on a defined medium; one was a normal strain of the cowpea group (ANU240) while the other (IC3342) was an unusual but related strain of the same group which induced abnormal shoot development, including proliferation of lateral buds, in nodulated plants. Culture supernatants were examined for the presence of cytokinins by mass spectrometry using deuterium-labelled internal standards and by radioimmunoassay. In culture supernatants of both strains a range of cytokinins was detected and quantified, but N6-(2-isopentenyl)adenine (iP) and zeatin (Z) were the dominant cytokinins. The levels of Z and iP in supernatants of strain IC3342 were 26 and 8 times, respectively, those in supernatants of the strain ANU240. These results appear to provide the first unambiguous identifications of cytokinins in Rhizobium culture media. The cytokinin level in xylem sap of pigeonpea plants inoculated with strain IC3342 was markedly greater than that in plants inoculated with a normal nodulating strain. The abnormal proliferation of lateral buds in the former plants is probably linked to the elevation of cytokinin level in xylem sap caused by strain IC3342.
Asunto(s)
Citocininas/biosíntesis , Rhizobium/metabolismo , Adenina/análogos & derivados , Adenina/biosíntesis , Adenina/química , Citocininas/química , Cromatografía de Gases y Espectrometría de Masas , Isopenteniladenosina , Estructura Molecular , Zeatina/análogos & derivados , Zeatina/química , Zeatina/metabolismoRESUMEN
A uniquely abnormal shoot development (shoot tip-bending, leaf curling, release from apical dominance, and stunted growth) in pigeonpea (Cajanus cajan Millsp) induced by a nodulating Rhizobium strain, IC3342, is thought to be due to a hormonal imbalance. Amaranthus betacyanin bioassay indicated that xylem exudate and leaf extracts from pigeonpea plants with Rhizobium-induced leaf curl symptoms contained high concentrations of cytokinin relative to those in normal plants. Radioimmunoassay (RIA) of samples purified with high performance liquid chromatography revealed that zeatin riboside (ZR) and dihydrozeatin riboside (DZR) concentrations in xylem sap from plants with leaf curl symptoms were 7 to 9 times higher than those in the sap from symptomless, nodulated plants. The sap from symptomless plants nodulated by a Curl(-) mutant had ZR and DZR concentrations comparable to those in the normal plant sap. RIA indicated that the respective concentrations of zeatin and N(6)-isopenteny-ladenine in culture filtrates of the curl-inducing strain IC3342 were 26 and 8 times higher than those in filtrates of a related normal nodulating strain (ANU240). Gas chromatographic-mass spectrometric analyses revealed similar differences. Gene-specific hybridization and sequence comparisons failed to detect any homology of IC3342 DNA to Agrobacterium tumefaciens or Pseudomonas savastanoi genetic loci encoding enzymes involved in cytokinin biosynthesis.
Asunto(s)
Inflamación/fisiopatología , Neutrófilos/fisiología , Animales , Ácido Araquidónico , Ácidos Araquidónicos/metabolismo , Movimiento Celular , Humanos , Inflamación/metabolismo , Leucotrienos/metabolismo , Leucotrienos/fisiología , Pulmón/patología , Pulmón/fisiopatología , Neutrófilos/metabolismo , Oxígeno/metabolismoAsunto(s)
Hipersensibilidad Inmediata/inmunología , Inmunoglobulina E/inmunología , Estrés Fisiológico/inmunología , Adaptación Fisiológica , Frío/efectos adversos , Exposición a Riesgos Ambientales , Humanos , Inmunoglobulina E/análisis , Inmunoglobulina E/biosíntesis , Hipersensibilidad Respiratoria/inmunología , Inanición/inmunologíaRESUMEN
The profile of endogenous cytokinins in a genetic tumor line of tobacco, namely, Nicotiana glauca (Grah.) x Nicotiana langsdorffii (Weinm.), following 1 to 10 weeks of growth on solid medium was determined by radioimmunoassay. (3)H-labeled cytokinins of high specific activity were added during tissue extraction to correct for the purification losses. Following subculture (of 4-week-old tissues when their cytokinin content is high) onto fresh medium the total cytokinin content continued to be high during the first week (1470 picomoles per gram fresh weight) when the tissue fresh weight remained essentially unchanged (lag phase). The cytokinin levels then declined by about half in 2- and 3-week-old tissues (626 and 675 picomoles per gram fresh weight, respectively), a period when rapid increase in tissue fresh weight was recorded. Increments of 840% and 2780% over initial fresh weight were obtained in 2- and 3-week-old cultures, respectively. The cytokinin content then increased to initial high levels in 4-week-old tissues (1384 picomoles per gram fresh weight) after which it gradually declined with tissue age. The lowest cytokinin levels (432 picomoles per gram fresh weight) were observed in 10-week-old tissues. Maximal tissue fresh weight (4030% increase over initial fresh weight) was recorded in 5-week-old cultures after which it decreased slowly to 77.5% of the highest tissue fresh weight in 10-week-old cultures. Zeatin appeared to be the dominant endogenous cytokinin in tissues of all ages. Other cytokinins quantified were dihydrozeatin, zeatin riboside, and dihydrozeatin riboside; the values may include contributions from aglucones derived from the hydrolysis of corresponding O-glucosides, since the entire basic fraction was treated with beta-glucosidase before analysis. In addition the levels of isopentenyladenine, isopentenyladenosine, and the nucleotides of zeatin riboside, dihydrozeatin riboside, and isopentenyladenosine were also determined.
RESUMEN
A preliminary characterization of the protein phosphotyrosine phosphatase (PTPase) activity in human peripheral blood lymphocytes (PBL) has been made using two tyrosine-containing peptides and the epidermal growth factor receptor from A-431 cells as substrates. High PTPase activity with a pH optimum near 7.4 was observed in both the membrane and the cytosolic fractions. At least three distinct fractions with PTPase activity were separated on DEAE cellulose columns, indicating that the enzyme is heterogeneous. Vanadate, molybdate, and salts of zinc, copper, and mercury were all effective enzyme inhibitors, although the inhibition was generally incomplete and showed some variation with the enzyme preparation. The difficulty in completely inhibiting PTPase activity in lymphocytes may help explain the variation between laboratories in studies of tyrosine phosphorylation in these cells. Studies with highly purified T lymphocytes obtained by filtration of PBL through nylon wool columns indicated that the activity is present in T cells. Absorption with agarose containing anti-HLe-1, a mouse monoclonal IgG1 antibody specific for the leukocyte-specific surface protein T-200 (CD45), removed up to 40% of the PTPase activity. Enzyme activity was recovered on the immunoadsorbent after extensive washing, confirming that the enzyme was being bound to the beads. Immunoabsorbents containing other mouse IgG1 antibodies failed to bind PTPase activity, indicating that the binding to beads with anti-HLe-1 antibody is specific. Further characterization of the CD45 and PTPase activities in lymphocytes may provide a better understanding of the role of protein tyrosine phosphorylation in the regulation of proliferation and differentiation in these cells.
Asunto(s)
Antígenos de Diferenciación/metabolismo , Antígenos de Histocompatibilidad/metabolismo , Linfocitos/enzimología , Fosfoproteínas Fosfatasas/metabolismo , Antígenos de Diferenciación/análisis , Antígenos de Histocompatibilidad/análisis , Humanos , Concentración de Iones de Hidrógeno , Antígenos Comunes de Leucocito , Fosfoproteínas Fosfatasas/análisis , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Proteínas Tirosina Fosfatasas , Fracciones Subcelulares/enzimologíaAsunto(s)
Leucotrienos/fisiología , Prostaglandinas/fisiología , Animales , Ácido Araquidónico , Ácidos Araquidónicos/metabolismo , Comunicación Celular , Eicosanoides/biosíntesis , Humanos , Sistema Inmunológico/citología , Inflamación/inmunología , Inflamación/metabolismo , Leucocitos/metabolismo , Linfocitos T/metabolismoRESUMEN
The metabolism of the polyphosphoinositides, diphosphoinositide (DPI) and triphosphoinositide (TPI), was studied during mediator release from rat mast cells. Serosal mast cells were purified by density gradient centrifugation and prelabeled with 32PO4. Incorporation of 32PO4 into DPI and TPI was determined by thin-layer chromatography on oxalic acid impregnated silica gel plates. 32PO4 incorporation into DPI and TPI was increased by Concanavalin A (ConA) or compound 48/80. The concentration of ConA causing a half-maximal increase in DPI labeling was less than that required for a comparable change in histamine release. The increases in DPI labeling and histamine release in response to ConA were enhanced by phosphatidylserine. The addition of alpha-methylmannoside to mast cells after challenge with ConA rapidly halted DPI and TPI labeling. The results of these studies indicate that changes in the metabolism of polyphosphoinositides may be an intrinsic part of the biochemical mechanisms that control mediator release from mast cells.
Asunto(s)
Liberación de Histamina , Mastocitos/metabolismo , Fosfatidilinositoles/metabolismo , Animales , Concanavalina A/farmacología , Liberación de Histamina/efectos de los fármacos , Técnicas In Vitro , Cinética , Masculino , Fosfatos/metabolismo , Fosfatos de Fosfatidilinositol , Fosfatidilserinas/farmacología , Ratas , Ratas EndogámicasRESUMEN
When [(3)H]dihydrozeatin riboside and [(3)H]zeatin riboside were supplied to soybean (Glycine max L.) explants (comprising one leaf, associated pods, and subtending stem) via the xylem at mid to late podfill, 0.1% of the supplied (3)H was extracted from the seeds. The distribution of (3)H in the explants was similar to that bound previously following uptake of [(3)H]zeatin riboside at earlier stages of pod development. Metabolites formed in the explants from (3)H-labeled zeatin, zeatin riboside, and dihydrozeatin riboside were identified and related to the endogenous cytokinins shown to be present. When zeatin riboside and zeatin were supplied for 1 hour, zeatin nucleotide was the principal metabolite formed and this appeared to be the precursor of the other metabolites detected subsequently. Explants supplied with zeatin riboside or dihydrozeatin riboside for 1 hour, and then transferred to water for 20 to 24 hours, yielded leaf blades in which the main metabolites were O-glucosyldihydrozeatin, adenosine, and adenine. The metabolism of zeatin riboside in blades of explants at pre-podfill, early podfill, and mid to late podfill did not differ appreciably. The results are discussed in relation to leaf senescence and seed development.
RESUMEN
A monocyte-rich preparation from the adherent cell fraction of human peripheral blood leukocytes was incubated for 1-8 h with [35S]methionine or [3H]leucine in the presence and absence of bacterial lipopolysaccharide (LPS). The macromolecules released into the supernatant were analysed by two-dimensional gel electrophoresis and radioautography. A complex labelling pattern involving at least 20 easily demonstrable and apparently distinct products with a broad range of molecular masses and isoelectric points was observed. LPS or LPS plus actinomycin in combination markedly stimulated the labelling and release of at least twelve different macromolecules ranging in apparent Mr from 12,000 to 46,000. Studies with monocytes that had been additionally purified by centrifugal elutriation and with the monocyte-like human cell line U-937 indicated that monocytes rather than contaminating cells were the source of these products. The majority of the secreted products were unique and did not cross-react with antibodies to interleukin 1 or tumour necrosis factor. The high resolving capacity of two-dimensional gel electrophoresis may be useful to define further the diverse biological activities and potential monokines released from monocytes at various stages of their differentiation and activation.
Asunto(s)
Proteínas Sanguíneas/metabolismo , Lipopolisacáridos/farmacología , Monocitos/metabolismo , Línea Celular , Dactinomicina/farmacología , Electroforesis en Gel de Poliacrilamida , Humanos , Técnicas In Vitro , Interleucina-1/metabolismo , Cinética , Monocitos/efectos de los fármacos , Neuraminidasa/farmacología , Inhibidores de Proteasas/farmacología , Factor de Necrosis Tumoral alfa/sangreRESUMEN
Even though therapeutic approaches using specific receptor antagonists are attractive as a means of controlling allergic and nonallergic inflammation in the lung, rapidly increasing evidence that allergic mediators are involved in the overall control of immune responsiveness raises concerns as to possible unexpected effects of such therapy. Agents directed toward specific enzymes involved either in the release or metabolism of arachidonic acid raise additional possibilities in regard to possible interference with normal intracellular signaling processes where lipids, including some of arachidonatic metabolites themselves, appear to participate at a number of different levels. Studies with anti-inflammatory agents therefore need to be interpreted cautiously with due cognizance of the possible complexities of agent action, of possible interactions between mediators, and of longer term changes in immune function and resistance that may be being initiated.
Asunto(s)
Inflamación/fisiopatología , Lípidos/fisiología , Animales , Formación de Anticuerpos , Comunicación Celular , Humanos , Hipersensibilidad/inmunología , Hipersensibilidad/fisiopatología , Inflamación/enzimología , Inflamación/terapia , Membranas Intracelulares , Metabolismo de los LípidosRESUMEN
We have previously shown that serotonin (5-HT) suppresses interferon-gamma (IFN-gamma)-induced Ia expression. In the present report, we show that 5-HT as well as other monoamines, histamine and dopamine, modulate IFN-gamma-induced phagocytosis in murine bone marrow macrophages. The effect of 5-HT on IFN-gamma-induced phagocytosis varied according to the concentration of IFN-gamma to which the macrophages were exposed. At low concentrations of IFN-gamma, 5-HT augmented phagocytosis, whereas at high concentrations of IFN-gamma, 5-HT suppressed phagocytosis. At both low and high IFN-gamma concentrations the response to 5-HT was dose-related and occurred at physiologic concentrations; the half-maximal effect was 6 X 10(-7) M and 3 X 10(-7) M for low and high IFN-gamma concentrations, respectively. Both histamine and dopamine also augmented IFN-gamma (1 U/ml) induced phagocytosis, at half-maximal augmenting concentrations of 7 X 10(-8) M and 4 X 10(-7) M, respectively. The 5-HT effects were blocked by the 5-HT antagonists spiperone, ketanserin, LY53857, mCPP, and PAPP, but not by the histamine antagonists pyrilamine, chlorpheniramine, or cimetidine. Histamine augmentation of IFN-gamma-induced phagocytosis was blocked by the H1 antagonists pyrilamine and chlorpheniramine, but not by the H2 antagonist cimetidine. The dopamine effect was blocked by spiperone and pyrilamine, both of which have been shown to block dopaminergic effects in other systems. This data provides functional evidence that at least part of the modulation of IFN-gamma-induced phagocytosis by 5-HT occurs through a 5-HT receptor-mediated mechanism, and 5-HT, dopamine, and histamine modulate IFN-gamma-induced phagocytosis independently through their respective receptors.
Asunto(s)
Dopamina/farmacología , Histamina/farmacología , Interferón gamma/antagonistas & inhibidores , Activación de Macrófagos/efectos de los fármacos , Macrófagos/efectos de los fármacos , Fagocitosis/efectos de los fármacos , Serotonina/farmacología , Animales , Células de la Médula Ósea , Antagonistas de Dopamina , Antagonistas de los Receptores Histamínicos/farmacología , Interferón gamma/farmacología , Macrófagos/fisiología , Masculino , Ratones , Ratones Endogámicos , Proteínas Recombinantes/farmacología , Antagonistas de la Serotonina/farmacologíaRESUMEN
The metabolism of [(3)H]6-benzylamino purine was studied in presenescent and early senescent soybean (Glycine max [L.] Merr.) leaves. In both types of leaves, the metabolism was essentially the same. The principal metabolite was identified as beta-(6-benzylaminopurin-9-yl)alanine by mass spectral studies, which included discharge ionization-secondary ion mass spectrometry and pulsed positive ion-negative ion-chemical ionization mass spectrometry. Conversion to this alanine conjugate was found to be inhibited 2,4-dichlorophenoxyacetic acid and 5,7-dichloroindoleacetic acid.
Asunto(s)
Activación de Linfocitos , Linfocitos/inmunología , Mitógenos , División Celular , Células Cultivadas , Técnicas de Cultivo/métodos , Metabolismo Energético , Enzimas/metabolismo , Humanos , Linfocitos/clasificación , Linfocitos/metabolismo , Monocitos/inmunología , Biosíntesis de ProteínasRESUMEN
When rat mast cells are prelabeled with 32PO4 and exposed to non-immunologic or immunologic stimuli under conditions that lead to mediator release from granules, they show rapid increases in labeling of a number of high molecular weight proteins. To determine if granule membrane proteins are subject to protein phosphorylation and perhaps participate in this response, granules with intact or broken membranes were isolated from sonicated, purified rat serosal mast cells on a Percoll gradient. When the granules with broken membranes were incubated with [gamma-32P]ATP and Mg2+ in the absence of exogenous protein kinases, one major radioactive band was recovered in the 44K area after electrophoresis in sodium dodecyl sulfate/polyacrylamide gels. The phosphorylation reaction with ATP required Mg2+, was enhanced by 0.05 to 0.5 microM cyclic AMP, and was inhibited by Ca2+ (0.5 mM and higher). The initial reaction was rapid, and the maximal response was seen at 30 degrees. The 44K band was absent in granules with intact membranes incubated with [gamma-32P]ATP but present when intact granules were lysed with distilled water before adding the [gamma-32P]ATP. These observations indicate that granules have an endogenous phosphorylating system and that the phosphorylation response is on the inner surface of the granule membranes. The possibility was not excluded that a portion of the phosphorylating activity was derived from the cytosol and became firmly associated with broken granules when the intact cells were sonicated. Analysis for possible phosphorylated amino acids in the 44K band after acid hydrolysis showed both phosphoserine and phosphothreonine, indicating that the radioactivity was in a phosphorylated protein or glycoprotein. The 44K phosphorylated protein was made up of several components ranging in pI from approximately 7.6 to 6.6. While the identity of the phosphorylated 44K polypeptide is uncertain, one important possibility is that it is part of an autophosphorylated cAMP dependent protein kinase. The cyclic AMP dependent phosphorylating activity present in granules provides a mechanism by which the granules might respond rapidly to cyclic AMP during mediator release.