Eugenol and its liposome-based nano carrier reduce anxiety by inhibiting glyoxylase-1 expression in mice.

The most common form of psycho-social dysfunction is anxiety with depression being related closely without any age bar. They are present with combined state of sadness, confusion, stress, fear etc. Glyoxalase system contains enzyme named glyoxalase 1 (GLO1).It is a metabolic pathway which detoxifies alpha-oxo-aldehydes, particularly methylglyoxal (MG). Methylglyoxal is mainly made by the breakdown of the glycolytic intermediates, glyceraldehyde-3-phosphates and dihydroxyacetone phosphate. Glyoxylase-1 expression is also related with anxiety behavior. A casual role or GLO-1 in anxiety behavior by using viral vectors for over expression in the anterior cingulate cortex was found and it was found that local GLO-1 over expression increased anxiety behavior. The present study deals with the molecular mechanism of protective activity of eugenol against anxiolytic disorder. A pre-clinical animal study was performed on 42 BALB/c mice. Animals were given stress through conventional restrain model. The mRNA expression of GLO-1 was analyzed by real time RT-PCR. Moreover, the GLO-1 protein expression was also examined by immunohistochemistry in whole brain and mean density was calculated. The mRNA and protein expressions were found to be increased in animals given anxiety as compared to the normal control. Whereas, the expressions were decreased in the animals treated with eugenol and its liposome-based nanocarriers in a dose dependent manner. However, the results were better in animals treated with nanocarriers as compared to the compound alone. It is concluded that the eugenol and its liposome-based nanocarriers exert anxiolytic activity by down-regulating GLO-1 protein expression in mice.

Eugenol and its liposome-based nano carrier reduce anxiety by inhibiting glyoxylase-1 expression in mice / Eugenol e seu nanocarreador à base de lipossoma reduzem a ansiedade ao inibir a expressão de glioxalase 1 em camundongos

The most common form of psycho-social dysfunction is anxiety with depression being related closely without any age bar. They are present with combined state of sadness, confusion, stress, fear etc. Glyoxalase system contains enzyme named glyoxalase 1 (GLO1).It is a metabolic pathway which detoxifies alpha-oxo-aldehydes, particularly methylglyoxal (MG). Methylglyoxal is mainly made by the breakdown of the glycolytic intermediates, glyceraldehyde-3-phosphates and dihydroxyacetone phosphate. Glyoxylase-1 expression is also related with anxiety behavior. A casual role or GLO-1 in anxiety behavior by using viral vectors for over expression in the anterior cingulate cortex was found and it was found that local GLO-1 over expression increased anxiety behavior. The present study deals with the molecular mechanism of protective activity of eugenol against anxiolytic disorder. A pre-clinical animal study was performed on 42 BALB/c mice. Animals were given stress through conventional restrain model. The mRNA expression of GLO-1 was analyzed by real time RT-PCR. Moreover, the GLO-1 protein expression was also examined by immunohistochemistry in whole brain and mean density was calculated. The mRNA and protein expressions were found to be increased in animals given anxiety as compared to the normal control. Whereas, the expressions were decreased in the animals treated with eugenol and its liposome-based nanocarriers in a dose dependent manner. However, the results were better in animals treated with nanocarriers as compared to the compound alone. It is concluded that the eugenol and its liposome-based nanocarriers exert anxiolytic activity by down-regulating GLO-1 protein expression in mice.

A forma mais comum de disfunção psicossocial é a ansiedade intimamente relacionada com a depressão, sem qualquer barreira de idade. Elas estão presentes em um estado combinado de tristeza, confusão, estresse, medo etc. O sistema de glioxalase contém uma enzima chamada glioxalase 1 (GLO1). É uma via metabólica que desintoxica alfa-oxo-aldeídos, particularmente metilglioxal (MG). O metilglioxal é produzido principalmente pela quebra dos intermediários glicolíticos, gliceraldeído-3-fosfatos e fosfato de diidroxiacetona. A expressão da glioxalase 1 também está relacionada ao comportamento de ansiedade. Um papel casual ou GLO1 no comportamento de ansiedade usando vetores virais para superexpressão no córtex cingulado anterior foi encontrado e descobriu-se que a superexpressão local de GLO1 aumentava o comportamento de ansiedade. O presente estudo trata do mecanismo molecular da atividade protetora do eugenol contra o transtorno ansiolítico. Um estudo pré-clínico em animais foi realizado em 42 camundongos BALB / c. Os animais foram submetidos ao estresse por meio do modelo de contenção convencional. A expressão de mRNA de GLO1 foi analisada por RT-PCR em tempo real. Além disso, a expressão da proteína GLO1 também foi examinada por imuno-histoquímica em todo o cérebro e a densidade média foi calculada. Verificou-se que as expressões de mRNA e proteínas estavam aumentadas em animais que receberam ansiedade em comparação com o controle normal. Considerando que as expressões foram diminuídas nos animais tratados com eugenol e seus nanocarreadores baseados em lipossomas de forma dependente da dose. No entanto, os resultados foram melhores em animais tratados com nanocarreadores em comparação com o composto sozinho. Conclui-se que o eugenol e seus nanocarreadores baseados em lipossomas exercem atividade ansiolítica por regulação negativa da expressão da proteína GLO1 em camundongos.

Eugenol and its liposome-based nano carrier reduce anxiety by inhibiting glyoxylase-1 expression in mice

Abstract The most common form of psycho-social dysfunction is anxiety with depression being related closely without any age bar. They are present with combined state of sadness, confusion, stress, fear etc. Glyoxalase system contains enzyme named glyoxalase 1 (GLO1).It is a metabolic pathway which detoxifies alpha-oxo-aldehydes, particularly methylglyoxal (MG). Methylglyoxal is mainly made by the breakdown of the glycolytic intermediates, glyceraldehyde-3-phosphates and dihydroxyacetone phosphate. Glyoxylase-1 expression is also related with anxiety behavior. A casual role or GLO-1 in anxiety behavior by using viral vectors for over expression in the anterior cingulate cortex was found and it was found that local GLO-1 over expression increased anxiety behavior. The present study deals with the molecular mechanism of protective activity of eugenol against anxiolytic disorder. A pre-clinical animal study was performed on 42 BALB/c mice. Animals were given stress through conventional restrain model. The mRNA expression of GLO-1 was analyzed by real time RT-PCR. Moreover, the GLO-1 protein expression was also examined by immunohistochemistry in whole brain and mean density was calculated. The mRNA and protein expressions were found to be increased in animals given anxiety as compared to the normal control. Whereas, the expressions were decreased in the animals treated with eugenol and its liposome-based nanocarriers in a dose dependent manner. However, the results were better in animals treated with nanocarriers as compared to the compound alone. It is concluded that the eugenol and its liposome-based nanocarriers exert anxiolytic activity by down-regulating GLO-1 protein expression in mice.

Resumo A forma mais comum de disfunção psicossocial é a ansiedade intimamente relacionada com a depressão, sem qualquer barreira de idade. Elas estão presentes em um estado combinado de tristeza, confusão, estresse, medo etc. O sistema de glioxalase contém uma enzima chamada glioxalase 1 (GLO1). É uma via metabólica que desintoxica alfa-oxo-aldeídos, particularmente metilglioxal (MG). O metilglioxal é produzido principalmente pela quebra dos intermediários glicolíticos, gliceraldeído-3-fosfatos e fosfato de diidroxiacetona. A expressão da glioxalase 1 também está relacionada ao comportamento de ansiedade. Um papel casual ou GLO1 no comportamento de ansiedade usando vetores virais para superexpressão no córtex cingulado anterior foi encontrado e descobriu-se que a superexpressão local de GLO1 aumentava o comportamento de ansiedade. O presente estudo trata do mecanismo molecular da atividade protetora do eugenol contra o transtorno ansiolítico. Um estudo pré-clínico em animais foi realizado em 42 camundongos BALB / c. Os animais foram submetidos ao estresse por meio do modelo de contenção convencional. A expressão de mRNA de GLO1 foi analisada por RT-PCR em tempo real. Além disso, a expressão da proteína GLO1 também foi examinada por imuno-histoquímica em todo o cérebro e a densidade média foi calculada. Verificou-se que as expressões de mRNA e proteínas estavam aumentadas em animais que receberam ansiedade em comparação com o controle normal. Considerando que as expressões foram diminuídas nos animais tratados com eugenol e seus nanocarreadores baseados em lipossomas de forma dependente da dose. No entanto, os resultados foram melhores em animais tratados com nanocarreadores em comparação com o composto sozinho. Conclui-se que o eugenol e seus nanocarreadores baseados em lipossomas exercem atividade ansiolítica por regulação negativa da expressão da proteína GLO1 em camundongos.

Eugenol and its liposome-based nano carrier reduce anxiety by inhibiting glyoxylase-1 expression in mice / Eugenol e seu nanocarreador à base de lipossoma reduzem a ansiedade ao inibir a expressão de glioxalase 1 em camundongos

Abstract The most common form of psycho-social dysfunction is anxiety with depression being related closely without any age bar. They are present with combined state of sadness, confusion, stress, fear etc. Glyoxalase system contains enzyme named glyoxalase 1 (GLO1).It is a metabolic pathway which detoxifies alpha-oxo-aldehydes, particularly methylglyoxal (MG). Methylglyoxal is mainly made by the breakdown of the glycolytic intermediates, glyceraldehyde-3-phosphates and dihydroxyacetone phosphate. Glyoxylase-1 expression is also related with anxiety behavior. A casual role or GLO-1 in anxiety behavior by using viral vectors for over expression in the anterior cingulate cortex was found and it was found that local GLO-1 over expression increased anxiety behavior. The present study deals with the molecular mechanism of protective activity of eugenol against anxiolytic disorder. A pre-clinical animal study was performed on 42 BALB/c mice. Animals were given stress through conventional restrain model. The mRNA expression of GLO-1 was analyzed by real time RT-PCR. Moreover, the GLO-1 protein expression was also examined by immunohistochemistry in whole brain and mean density was calculated. The mRNA and protein expressions were found to be increased in animals given anxiety as compared to the normal control. Whereas, the expressions were decreased in the animals treated with eugenol and its liposome-based nanocarriers in a dose dependent manner. However, the results were better in animals treated with nanocarriers as compared to the compound alone. It is concluded that the eugenol and its liposome-based nanocarriers exert anxiolytic activity by down-regulating GLO-1 protein expression in mice.

Resumo A forma mais comum de disfunção psicossocial é a ansiedade intimamente relacionada com a depressão, sem qualquer barreira de idade. Elas estão presentes em um estado combinado de tristeza, confusão, estresse, medo etc. O sistema de glioxalase contém uma enzima chamada glioxalase 1 (GLO1). É uma via metabólica que desintoxica alfa-oxo-aldeídos, particularmente metilglioxal (MG). O metilglioxal é produzido principalmente pela quebra dos intermediários glicolíticos, gliceraldeído-3-fosfatos e fosfato de diidroxiacetona. A expressão da glioxalase 1 também está relacionada ao comportamento de ansiedade. Um papel casual ou GLO1 no comportamento de ansiedade usando vetores virais para superexpressão no córtex cingulado anterior foi encontrado e descobriu-se que a superexpressão local de GLO1 aumentava o comportamento de ansiedade. O presente estudo trata do mecanismo molecular da atividade protetora do eugenol contra o transtorno ansiolítico. Um estudo pré-clínico em animais foi realizado em 42 camundongos BALB / c. Os animais foram submetidos ao estresse por meio do modelo de contenção convencional. A expressão de mRNA de GLO1 foi analisada por RT-PCR em tempo real. Além disso, a expressão da proteína GLO1 também foi examinada por imuno-histoquímica em todo o cérebro e a densidade média foi calculada. Verificou-se que as expressões de mRNA e proteínas estavam aumentadas em animais que receberam ansiedade em comparação com o controle normal. Considerando que as expressões foram diminuídas nos animais tratados com eugenol e seus nanocarreadores baseados em lipossomas de forma dependente da dose. No entanto, os resultados foram melhores em animais tratados com nanocarreadores em comparação com o composto sozinho. Conclui-se que o eugenol e seus nanocarreadores baseados em lipossomas exercem atividade ansiolítica por regulação negativa da expressão da proteína GLO1 em camundongos.

Phosphatidic acid elicits calcium mobilization and actin polymerization through a tyrosine kinase-dependent process in human neutrophils: a mechanism for induction of chemotaxis.

Phospholipids mediate important effects as extracellular messengers in diverse biological systems. We investigated the effects of phosphatidic acid, a biologically active phospholipid potentially involved in the inflammatory process, on calcium mobilization and actin polymerization in human neutrophils and correlated these effects with induction of chemotactic migration. Intermediate-chain length phosphatidic acid (DiC10-PA) induced a biphasic increase in intracellular Ca2+ characterized by a rapid rise commencing immediately upon addition of stimulus followed by a secondary increase which, unlike the initial response, was eliminated by chelation of extracellular Ca2+. Neither of these responses were induced by C10-lysophosphatidic acid or diacylglycerol. The tyrosine kinase inhibitor herbimycin-A (5-10 microg/ml) completely blunted the initial but not the delayed response effected by DiC10-PA. Long-chain phosphatidic acid (DiC18:1) induced only an initial rapid increase in intracellular Ca2+ and this response was similarly markedly attenuated by herbimycin-A. Among several physiologically relevant phospholipids, only phosphatidic acid was able to induce Ca2+ mobilization; phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol -- used individually or in mixed liposomes -- were without effect. Phosphatidic acid conferred calcium-mobilizing activity upon inactive liposome preparations and phosphatidic acid-enriched cellular plasma membranes possessed similar calcium-mobilizing activity. Both DiC10-PA and DiC18:1-PA induced actin polymerization in neutrophils at rates which mirrored the influence of each agent on Ca2+ mobilization. Herbimycin-A blunted the initial increase in actin polymerization effected by phosphatidic acid but had no effect on the delayed, EGTA-sensitive phase. DiC10-PA and DiC18:1-PA also induced neutrophil migration along a concentration gradient. Phospholipids that failed to induce a calcium transient, including phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, and phosphatidylinositol, likewise failed to induce either actin polymerization or chemotactic migration. Unlike chemotaxis induced by zymosan-activated human serum, phosphatidate-induced chemotaxis was strongly inhibited by pretreatment of cells with herbimycin-A. Consistent with these observations, phosphatidic acid induced the tyrosine phosphorylation of several proteins as early as 10 s after stimulation. Phosphorylation of two distinct proteins with approximate molecular sizes of 72 and 82 kDa was inhibited by levels of herbimycin A used to effectively inhibit calcium mobilization, actin polymerization and chemotaxis. Thus, in neutrophilic leukocytes, extracellular phosphatidic acid induces a unique tyrosine kinase-based signalling pathway that results in calcium mobilization and actin polymerization. These processes may promote directed cellular migration as a consequence of the interaction of phosphatidic acid with neutrophil plasma membranes.

Phosphatidylinositol 3'-kinase-mediated calcium mobilization regulates chemotaxis in phosphatidic acid-stimulated human neutrophils.

Phosphatidylinositol 3'-kinase (PI 3'-kinase) plays an important role in the migration of hepatocytes, endothelial cells and neoplastic cells to agonists which activate cellular tyrosine kinases. We examined the PI 3'-kinase-dependent chemotactic responses of neutrophilic leukocytes induced by phosphatidic acid (PA) in order to clarify mechanisms by which the enzyme potentially influences cellular migration. Western analysis of immunoprecipitates indicated that PA induced the tyrosine phosphorylation of three distinct proteins involved in functional activation which co-immunoprecipitated in PA-stimulated cells. These proteins were identified as lyn, syk and the 85 kDa regulatory subunit of PI 3'-kinase. Chemotactic responses to PA but not to several other neutrophil agonists were inhibited by the PI 3'-kinase inhibitors wortmannin and LY294002. Chemotactic inhibition resulted from upstream inhibition of calcium mobilization. Chelation of extracellular calcium by ethylene glycol-bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) did not affect the PA-induced chemotaxis, whereas chelation of intracellular calcium by 1, 2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA) attenuated this response. Thus, changes in intracellular Ca(2+) levels that can be effected by Ca(2+) mobilized from intracellular stores in the absence of Ca(2+) influx regulate PA-induced chemotaxis. Furthermore, PI 3'-kinase inhibition blunted the agonist-dependent generation of inositol 1,4,5-trisphosphate (IP(3)), suggesting that PI 3'-kinase exerted its effects on calcium mobilization from intracellular sources by mediating activation of phospholipase C (PLC) in PA-stimulated cells. Moreover, the PI 3'-kinase inhibitor LY294002 also inhibited phosphorylation of syk in PA-stimulated cells. We, therefore, propose that products of PI 3'-kinase confined to the inner leaflet of the plasma membrane play a role in activation of syk, calcium mobilization and induction of chemotactic migration.

Docosahexaenoic acid induces apoptosis in Jurkat cells by a protein phosphatase-mediated process.

Docosahexaenoic acid (DHA) is an omega-3 fatty acid under intense investigation for its ability to modulate cancer cell growth and survival. This research was performed to study the cellular and molecular effects of DHA. Our experiments indicated that the treatment of Jurkat cells with DHA inhibited their survival, whereas similar concentrations (60 and 90 microM) of arachidonic acid and oleic acid had little effect. To explore the mechanism of inhibition, we used several measures of apoptosis to determine whether this process was involved in DHA-induced cell death in Jurkat cells. Caspase-3, an important cytosolic downstream regulator of apoptosis, is activated by death signals through proteolytic cleavage. Incubation of Jurkat cells with 60 and 90 microM DHA caused proteolysis of caspase-3 within 48 and 24 h, respectively. DHA treatment also caused the degradation of poly-ADP-ribose polymerase and DNA fragmentation as assayed by flow cytometric TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) assay. These results indicate that DHA induces apoptosis in Jurkat leukemic cells. DHA-induced apoptosis was effectively inhibited by tautomycin and cypermethrin at concentrations that affect protein phosphatase 1 (PP1) and protein phosphatase 2B (PP2B) activities, respectively, implying a role for these phosphatases in the apoptotic pathway. Okadaic acid, an inhibitor of protein phosphatase 2A, had no effect on DHA-induced apoptosis. These results suggest that one mechanism through which DHA may control cancer cell growth is through apoptosis involving PP1/PP2B protein phosphatase activities.

Prevention of docosahexaenoic acid-induced cytotoxicity by phosphatidic acid in Jurkat leukemic cells: the role of protein phosphatase-1.

The present investigation explores the role of phosphatidic acid (PA), a specific protein phosphatase-1 (PP1) inhibitor, in cytotoxicity induced by docosahexaenoic acid (DHA). The cytotoxicity of DHA was assayed by quantifying cell survival using the trypan blue exclusion method. A dose-response effect demonstrated that 5 or 10 microM DHA has no effect on Jurkat cell survival; however, 15 microM DHA rapidly decreased cell survival to 40% within 2 h of treatment. Cytotoxicity of 15 microM DHA was prevented by PA. Structurally similar phospholipids (lysophosphatidic acid, sphingosine 1-phosphate, sphingosine, and sphingosine phosphocholine) or metabolites of PA (lyso-PA and diacylglycerol) did not prevent DHA-induced cytotoxicity. PA did not produce micelles alone or in combination with DHA as examined spectrophotometrically, indicating that PA did not entrap DHA and therefore did not affect the amount of DHA available to the cells. Supporting this observation, the uptake or incorporation of [1-14C]DHA in Jurkat cells was not affected by the presence of PA. However, PA treatment reduced the amount of DHA-induced inorganic phosphate released from Jurkat leukemic cells and also inhibited DHA-induced dephosphorylation of cellular proteins. These observations indicate that PA has exerted its anti-cytotoxic effects by causing inhibition of protein phosphatase activities. Cytotoxicity of DHA on Jurkat cells was also blocked by the use of a highly specific caspase-3 inhibitor (N-acetyl-ala-ala-val-ala-leu-leu-pro-ala-val-leu-leu-ala-leu-leu-ala-pro-asp-glu-val-asp-CHO), indicating that the cytotoxic effects of DHA were due to the induction of apoptosis though activation of caspase-3. Consistent with these data, proteolytic activation of procaspase-3 was also evident when examined by immunoblotting. PA prevented procaspase-3 degradation in DHA-treated cells, indicating that PA causes inhibition of DHA-induced apoptosis in Jurkat leukemic cells. Since DHA-induced apoptosis can be inhibited by PA, we conclude that the process is mediated through activation of PP1.

Efficacy of 100 mg of double-bolus alteplase in achieving complete perfusion in the treatment of acute myocardial infarction.

OBJECTIVES: The purpose of this study was to assess the efficacy of 150 mg of aspirin plus 100 mg of alteplase, administered as two intravenous bolus injections of 50 mg each given 30 min apart, and followed by intravenous heparin, on infarct-related coronary artery patency (Thrombolysis in Myocardial Infarction [TIMI] flow grade 3). BACKGROUND: Previous workers have shown in animals that reducing the duration of an infusion of recombinant tissue-type plasminogen activator increases the initial rate of thrombolysis, resulting in high early infarct-related coronary artery patency rates. The logical progression of this idea is bolus administration. METHODS: Consecutive patients presenting up to 6 h from the onset of symptoms were recruited for the study. Angiography was performed at 60 and 90 min after the first bolus and between 19 to 48 h after study entry. Patients were followed up for 1 month. RESULTS: At 60 min, angiography revealed infarct-related coronary artery patency of TIMI flow grade 3 in 55 (86%) of 64 patients (95% confidence interval [CI] 75% to 93%) and TIMI flow grade 2 or 3 in 58 (91%) of 64 patients (95% CI 81% to 97%). At 90 min, infarct-related artery patency of TIMI flow grade 3 was achieved in 74 (88%) of 84 patients (95% CI 79% to 94%) and TIMI flow grade 2 or 3 in 78 (93%) of 84 patients (95% CI 85% to 97%). Two patients (2.4%) had early angiographic reocclusion whereas 10 (11.9%) had late reinfarction. Bleeding episodes were mostly minor, and there was no cerebrovascular bleeding. Five patients (6.0%) died within 1 month of the acute myocardial infarction. CONCLUSIONS: In 84 patients with acute myocardial infarction, administration of 100 mg of double-bolus (2 x 50 mg) alteplase, aspirin and heparin is associated with remarkably high early infarct-related coronary artery patency rates (TIMI flow grade 3) of 86% and 88%, respectively, at 60 and 90 min.

Phosphatidic acid binding to human neutrophils: effects on tyrosine kinase-regulated intracellular Ca2+ mobilization.

Neutrophils provide an attractive model with which to characterize cellular effects of phosphatidic acid (PA) independently of effects triggered by lysophosphatidic acid (LPA), since these cells lack LPA receptors. We developed a novel method to quantitate binding of PA to neutrophils and neutrophil plasma membranes. Intact cells or subcellular fractions were immobilized on nitrocellulose membranes and incubated in a bath containing [32P]PA under various conditions, followed by rapid rinsing with a mild detergent (0.05% Tween 20) to minimize non-specific binding. With this method, dioctanoyl PA specifically ligated plasma-membrane binding sitesin a time- and temperature-dependent manner. Specific binding of (DiC8-PA was markedly potentiated by pre-treatment of cells or membranes with ecto-phosphatidic acid phosphohydrolase (PAPase) inhibitor dimethylsphingosine (DMS). Optimum binding of DiC8-PA to PAPase-inhibited cells occurred within 10 min at room temperature, increased linearly with the cell concentration used, and was not significantly affected by alteration of pH over the range of 5.5-8.5. Of several phosphatidic acid species examined, optimal specific binding to immobilized neutrophils was observed with DiC8-PA and dicapryl (DiC10) PA; dicaproyl (DiC6) PA bound weakly, whereas dimyristoyl (DiC14) PA and dipalmitoyl (DiC16) PA did not bind. Dioleoyl (DiC18:1) PA bound to immobilized cells, but this binding was essentially non-specific, in that it was not reduced by excess non-radioactive ligand. Various LPA preparations, including [32P] lyso-octanoyl (C8) PA and [32P] lyso-oleoyl (C18:1) PA, showed very low specific binding to neutrophils in this system. Specific binding of DiC8-PA and DiC10-PA preparations correlated well with the ability of each to effect the mobilization of intracellular Ca2+ in neutrophils. Ca2+ mobilization was characterized by two distinct phases; a rapid rise that was inhibited in the presence of the tyrosine kinase inhibitor herbimycin-A, followed by a sustained increase that was eliminated in the presence of EGTA. The results are consistent with the hypothesis that neutrophils have specific binding sites for phosphatidic acid, the occupation of which leads to rapid mobilization of intracellular free Ca2+ via activation of tyrosine kinases. The methods described in this report may facilitate the identification and characterization of functional phosphatidic acid receptors on neutrophil plasma membranes.

Interleukin-11 induces phosphatidic acid formation and activates MAP kinase in mouse 3T3-L1 cells.

Interleukin-11 (IL-11) stimulated [3H]phosphatidic acid (PA) formation in [3H]arachidonic acid (AA) prelabelled quiescent mouse 3T3-L1 cells. When IL-11 stimulated 3T3-L1 cells were incubated with NaF, a phosphatidic acid phosphohydrolase (PAP) inhibitor, increased PA formation was observed. In the presence of ethanol, phosphatidylethanol accumulated at the expense of PA. These results indicated that the formation of PA upon IL-11 stimulation was a result of phospholipase D (PLD) activation. Endogenous accumulation of PA by NaF treatment or exogenously added PA enhanced tyrosine phosphorylation of two proteins of 44 KDa (p44) and 47 KDa (p47) whereas tyrosine phosphorylation of other proteins was not affected. Among various PA species, dipalmitoyl PA was found to be most effective in enhancing tyrosine phosphorylation of these proteins. p44 and p47 cross reacted with anti-MAP kinase monoclonal antibody (MoAb) in both immunoprecipitation and western blot analysis. Lysates from IL-11-induced or PA-induced cells stimulated phosphorylation of a synthetic peptide substrate for MAP kinase, indicating the activation of MAP kinase in the induced cells. These studies suggest that one of the cellular signalling mechanisms of IL-11 in 3T3-L1 cells involves the activation of phospholipase D to produce the second messenger PA. The increased level of PA enhances tyrosine phosphorylation of p44 and p47 which belong to the members of MAP kinase family and thus transduces some of the mitogenic signals of IL-11 in this cell line.

Phorbol ester-induced priming of superoxide generation by phosphatidic acid-stimulated neutrophils and granule-free neutrophil cytoplasts.

This study was undertaken to examine the mechanisms involved in polymorphonuclear leukocyte superoxide release stimulated by exogenous phosphatidic acid (PA). Unlike the immediate burst of superoxide release affected by membrane-permeable dioctanoylglycerol (DiC8-DAG), dioctanoyl phosphatidic acid (DiC8-PA) induced superoxide release after a lag period of 5-20 min. This period was considerably reduced or eliminated when cells were primed by substimulatory levels of phorbol myristate acetate (PMA). Granule-depleted neutrophil cytoplasts also responded to DiC8-PA with a burst of superoxide generation. Activation of the cytoplast superoxide generating system in response to DiC8-PA was also significantly faster after cells had been preexposed to substimulatory levels of PMA, indicating that at least a portion of the priming mechanism was independent of PMA-induced degranulation. To further examine the potential mechanism of PMA priming of responses to PA, we evaluated the activity of neutrophil ecto-phosphatidic acid phosphohydrolase (ecto-PA phosphohydrolase), which generates diacylglycerol from exogenous PA. PMA priming had no discernable effect on the activity of this enzyme. In addition, propranolol, an inhibitor of PA phosphohydrolase, did not selectively inhibit PMA priming of neutrophil responses to DiC8-PA, indicating that priming did not result from acceleration of DiC8-PA hydrolysis. We therefore investigated the possibility that activation of protein kinase C was the basis of the primed response. Several semiselective protein kinase C inhibitors (calphostin C, H-7, and acylmethylglycerol) inhibited DiC8-DAG- and DiC8-PA-induced superoxide release as well as PMA-primed responses to approximately the same extent. These results are consistent with the hypothesis that neutrophil responses to phosphatidate are mediated by diglyceride generated by the action of ecto-PA phosphohydrolase. PMA priming does not result from increased catalytic activity of ecto-PA phosphohydrolase but rather seems to result from potentiation of an intermediate involved in the cells' response to multiple stimuli.

Genetic selection for insulin-like growth factor-1 in growing mice is associated with altered growth.

Substantial responses in the 6-week and mature body-weights of mice occurred after 7 generations of selection for or against plasma levels of Insulin-like Growth Factor-1 (IGF-1). Plasma levels of IGF-1 were also significantly different after 7 generations of selection (high line = 85 +/- 2 ng/ml, low line = 58 +/- 2 ng/ml). The average 6-week weight in the line selected for high plasma IGF-1 was 22.5 +/- .2 g compared with 18.5 +/- .2 g in the low plasma IGF-1 line, after 7 generations of selection. The difference between lines was maintained at 20 weeks of age. These data provide further evidence for the roles of IGF-1 in the regulation of somatic growth and as a mediator of a genetic component of growth.

Insulin-like growth factor-1 (IGF-1) in mice reduces weight loss during starvation.

Plasma concentrations of IGF-1 decrease markedly during starvation secondary to a reduction in somatotropic receptors in the liver. We investigated whether IGF-1 administration during starvation in mice inhibits the catabolic state normally observed. Plasma concentrations of IGF-1 in starved mice receiving IGF-1 therapy were similar to values from non-starved mice, whereas bGH treatment failed to increase plasma IGF-1 levels. The degree of weight loss during 36 hours of starvation was reduced (p less than 0.01) by frequent treatment with subcutaneous IGF-1 but not by bGH therapy. The effect was restricted to the period 28 to 36 hours after commencement of the fast. These results suggest that a fall in circulating IGF-1 may play a role in the metabolic adaptation during malnutrition.

Chemistry and mechanism of urease inhibition.

Studies on enzyme inhibition remain an important area of pharmaceutical research since these studies have led to the discoveries of drugs useful in a variety of physiological conditions. The enzyme inhibitors can interact with enzymes and block their activity towards natural substrates. Urease inhibitors have recently attracted much attention as potential new anti-ulcer drugs. Ironically, urease was the first enzyme crystallized but its mechanism of action is still largely misunderstood. This chapter therefore reviews comprehensive developments in the field of urease inhibitors. Inhibitors of urease can be broadly classified into two categories: (1) active site directed (substrate-like), (2) mechanism-based directed. We present here the examples of selected inhibitors along with their mechanisms of action to characterize their mode of urease inhibition. The observations that urease due to its high substrate (urea) specificity can only bind to a few inhibitors with a similar binding mode as urea is also discussed. Several non-covalent interactions including hydrogen bonds and hydrophobic contacts stabilize the enzyme-inhibitor complex. Regardless of the class of compound, it is reported that only a few functional groups with electronegative atoms such as oxygen, nitrogen and sulfur act either as bidentate (mostly), tridentate (rarely), or as ligand-chelator to form octahedral complexes with two slightly distorted octahedral Ni ions of the enzyme. Bulky groups attached to the pharmacophore were found to decrease the activity of inhibitors, since the lack of a bulky attachment makes it easier for urease inhibitors to enter the substrate-binding pocket as well as avoid unfavorable steric interactions with amino acid residues in its vicinity. This review is intended to provide highlights of the inhibition of urease by hydroxamic acids (HXAs), phosphorodiamidates (PPDs), imidazoles, phosphazene and related compounds. These compounds are compared to previously reported urease inhibitors for the catalytic models proposed for urease activity. The differences in inhibition of urease activities from plants and of bacterial origin by various inhibitors and physiological implications of urease inhibition are discussed.

Interleukin-8: An autocrine inflammatory mediator.

Interleukin-8 (IL-8), a pro-inflammatory chemokine, induces trafficking of neutrophils across the vascular wall. The release of IL-8 is triggered by inflammatory signals from a large variety of cells. The diversity in the cellular source indicates pleiotropy of its functions. IL-8 plays a key role in host defense mechanism through its effects on neutrophil activation, but a continued presence of IL-8 in circulation in response to inflammatory conditions may lead to a variable degree of tissue damage. Like most of the peptide hormones or mediators, IL-8 transmits its signals through distinct cell surface receptors. The membrane spanning heptahelical IL-8 receptor is coupled with the effector enzyme(s) through the intermediacy of heterotrimeric GTP-binding regulatory proteins. A growing number of studies demonstrated regulation of IL-8 activity by pertussis toxin treatment, implying a role of pertussis toxin sensitive G proteins (Gi), in IL-8 induced effects. IL-8 induced activation of G-protein results in activation of phospholipase C b2 (PLCb2). This enzyme catalyzes the hydrolysis of membrane phosphoinositides to yield diacylglycerol (DAG) and inositol 1,4,5 trisphosphate (IP3), which in turn activates protein kinase C (PKC) and mobilizes the intracellular Ca2+, respectively. Neutrophils activation of phospholipase D (PLD) and superoxide generation in response to IL-8 have also been demonstrated. Furthermore, IL-8-mediated activation of mitogen activating protein kinase (MAPK) and tyrosine phosphorylation of cellular proteins have been observed. It appears that the signalling pathways induced by IL-8 are subject to fine modulations by the demand and presence of IL-8. The presence of IL-8 in various pathophysiological condition implies that blockade of its actions could be exploited for therapeutic purposes.

Developmental patterns of plasma insulin-like growth factor-I (IGF-I) and body growth in mice from lines divergently selected on the basis of plasma IGF-I.

A study was conducted to investigate developmental patterns of plasma concentrations of insulin-like growth factor-I (IGF-I), body growth and body composition in mice from lines selected for seven generations on the basis of low (L) or high (H) plasma IGF-I, and in a random-bred control (C) line. Litter size was standardized to eight individuals with equal sex ratios (as far as possible) within 48 h of birth. Pups were weaned at an average of 21 days and separated on the basis of sex. Blood samples were collected from one male and one female of each litter on days, 21, 42, 63 and 105 for analysis of plasma concentrations of IGF-I. The animals were then killed and analysed for water, fat and crude protein content. The plasma concentration of IGF-I was influenced by line (P less than 0.05) but not by sex. Significant (P less than 0.001) differences in liveweight between mice from L and H lines were first evident at 21 days of age. From 28 until 105 days of age the H line was significantly (P less than 0.001) heavier than both L and C lines, but differences between C and L lines were inconsistent and mostly non-significant. The growth velocity of the H line was significantly greater than that of C or L lines between 14 and 42 days of age, but differences in growth velocities of C compared with L lines were generally non-significant.(ABSTRACT TRUNCATED AT 250 WORDS)

PLA2-independent induction of CSF-1 gene expression in growth-arrested fibroblasts.

At the site of a wound or an infection, localized production of colony-stimulating factor-1 (CSF-1) by resident macrophages is chemotactic for circulating monocytes. Several intracellular signaling pathways, including those initiated in response to activation of phospholipase A2 (PLA2) have been proposed to play a role in the regulation of CSF-1 gene expression. The goal of these studies was to determine whether PLA2 is required for the initial increase in CSF-1 gene expression in serum- or IL-1 alpha-stimulated growth-arrested fibroblasts. IL-1 alpha- or serum-stimulation of growth-arrested fibroblasts had no effect on PLA2 enzyme activity and inhibitors of cytosolic or Ca(2+)-independent PLA2 activity had no effect on IL-1 alpha- or serum-mediated increases in CSF-1 mRNA levels. High concentrations of the PLA2 inhibitors, 4-bromophenacyl lactone and quinacrine, resulted in a generalized decrease in cellular mRNA levels. Our results, obtained in fibroblasts, suggest treatment with 4-bromophenacyl lactone or quinacrine, instead of inhibiting PLA2 activity specifically, results in a generalized depression of cellular mRNA levels. These data demonstrate that the initial increase in CSF-1 gene expression in growth-arrested fibroblasts treated with serum or IL-1 alpha occurs through a PLA2-independent mechanism.

Messenger functions of phosphatidic acid.

Under physiological conditions, phosphatidic acid (PA) is an anionic phospholipid with moderate biological reactivity. Some of its biological effects can be attributed to lyso-PA and diacylglycerol generated by the action of cellular hydrolases. However, it is clear that the parent compound exhibits biological activities of its own. Early studies implicated PA in the transport of Ca++ across plasma membranes as well as in the mobilization of intracellular stored calcium. Both responses may be induced as a consequence of other cellular processes activated by PA, as opposed to being directly mediated by the lipid. PA may be involved in the activation of certain functions confined to specialized groupings of cells, such as the neutrophil superoxide-generating enzyme or actin polymerization. Recent studies implicate PA as an activator of intracellular protein kinases, and a PA-dependent superfamily of kinases involved in cellular signalling has been hypothesized. Deployed on the outer surface of the plasma membrane, PA potentially provides a method of communication between cells in direct contact. This review will explore the known functions of PA as an intracellular mediator and extracellular messenger of biological activities and address ways in which these functions are potentially regulated by cellular enzymes which hydrolyse the phospholipid.

Activation of endothelial cell phospholipase D by migrating neutrophils.

BACKGROUND: Vascular endothelium plays a critical role in regulating the integrity of intercellular adhesive and junctional contacts in response to migrating neutrophils during the inflammatory process. Biochemical responses induced in endothelial cells by adherent, migrating neutrophils are poorly understood. This study was undertaken to explore the possibility that endothelial cell phospholipase D (PLD) is activated when neutrophils migrate through endothelial cell monolayers in response to chemotactic stimuli. METHODS: Bovine pulmonary artery endothelial cells (BPAEC) were cultured on polycarbonate filters at the base of inserts in Transwell chambers (Costar, Cambridge, MA) and subsequently labeled with 3H-myristic acid. Human neutrophil migration through BPAEC monolayers was measured in response to a chemoattractant gradient produced by leukotriene B4 (LTB4). After neutrophils were induced to migrate through endothelial monolayers in the presence of 0.1% ethanol, the filters were excised, and cellular lipids were extracted. Levels of labeled phosphatidylethanol (PEt), an index of activation of endothelial cell PLD, were measured in chromatographs of resolved phospholipids. RESULTS: When neutrophils migrated through endothelial monolayers in response to LTB4, endothelial cells accumulated significant levels of PEt, indicating activation of endothelial cell PLD. Kinetic analysis demonstrated that PEt generation closely paralleled chemotactic migration over a 2-hour time period. Direct contact of neutrophils with the endothelium failed to induce PEt formation in the absence of neutrophil chemotaxis, and LTB4 was an ineffective stimulus of endothelial cell PLD activity in the absence of migrating neutrophils. Neutrophil migration-dependent endothelial PLD activation was observed when chemotaxis was induced by an unrelated chemoattractant, serum activated by exposure to Escherichia coli. However, neutrophil migration alone could not account for activation of endothelial cell PLD, since the peptide chemoattractant f-met-leu-phe (FMLP) induced comparable migration of neutrophils through endothelial monolayers but did not induce PEt generation. CONCLUSIONS: Our study indicates that chemotactic migration of neutrophils through endothelial monolayers results in endothelial cell PLD activation. This process may amplify both target and effector cell reactivity during the inflammatory response.