Expression pattern of protease activated receptors in lymphoid cells.

Protease-activated receptors (PARs) are a subfamily of four G-protein-coupled receptors mediating multiple functions. PARs expression was studied in subpopulations of human lymphocytes. Our results indicate that natural killer cells expressed mRNA for PAR1, PAR2 and PAR3, CD4+ T cells expressed PAR1 and PAR2, while γδ and CD8+ T cells only expressed PAR1. PAR4 was absent at mRNA level and B cells did not express any PAR. Analyses of the cell surface PARs expression by flow cytometry were consistent with the mRNA data and also between different donors. PAR1 is the most abundant member of the PAR family present in lymphocytes.

Antinociceptive and anti-inflammatory effects of Croton malambo bark aqueous extract.

Croton malambo (K.) bark aqueous extract, popularly known in Venezuela as "palomatias" or "torco" was tested for acute toxicity and for its anti-inflammatory and antinociceptive effects using tail flick and writhing syndrome tests models, respectively. Croton malambo aqueous extract (6.15 mg/kg i.p.) administered intraperitoneally had a significant antinociceptive and anti-inflammatory effects compared to acetylsalicylic acid (200mg/kg p.o.) and sodium diclofenac (5.64 mg/kg p.o.). Studies to determine correlation between chemical composition and pharmacological activity are underway.

Thrombin selectively induces transcription of genes in human monocytes involved in inflammation and wound healing.

Thrombin is essential for blood coagulation but functions also as a mediator of cellular signalling. Gene expression microarray experiments in human monocytes revealed thrombin-induced upregulation of a limited subset of genes, which are almost exclusively involved in inflammation and wound healing. Among these, the expression of F3 gene encoding for tissue factor (TF) was enhanced indicating that this physiological initiator of coagulation cascade may create a feed-forward loop to enhance blood coagulation. Activation of protease-activated receptor type 1 (PAR1) was shown to play a main role in promoting TF expression. Moreover, thrombin induced phosphorylation of ERK1/2, an event that is required for expression of thrombin-regulated genes. Thrombin also increased the expression of TF at the protein level in monocytes as evidenced by Western blot and immunostaining. Furthermore, FXa generation induced by thrombin-stimulated monocytes was abolished by a TF blocking antibody and therefore it is entirely attributable to the expression of tissue factor. This cellular activity of thrombin provides a new molecular link between coagulation, inflammation and wound healing.

Apoptotic-like activity of staurosporine in axenic cultures of Trypanosoma evansi.

Trypanosoma evansi is a blood protozoan parasite of the genus Trypanosoma which is responsible for surra (Trypanosomosis) in domestic and wild animals. This study addressed apoptotic-like features in Trypanosoma evansi in vitro. The mechanism of parasite death was investigated using staurosporine as an inducing agent. We evaluated its effects through several cytoplasmic features of apoptosis, including cell shrinkage, phosphatidylserine exposure, maintenance of plasma membrane integrity, and mitochondrial trans-membrane potential. For access to these features we have used the flow cytometry and fluorescence microscopy with cultures in the stationary phase and adjusted to a density of 10(6) cells/mL. The apoptotic effect of staurosporine in T. evansi was evaluated at 20 nM final concentration. There was an increase of phosphatidylserine exposure, whereas mitochondrial potential was decreased. Moreover, no evidence of cell permeability increasing with staurosporine was observed in this study, suggesting the absence of a necrotic process. Additional studies are needed to elucidate the possible pathways associated with this form of cell death in this hemoparasite.

Apoptotic-like activity of staurosporine in axenic cultures of Trypanosoma evansi / Actividad proapoptótica de la estaurosporina en cultivos axénicos de Trypanosoma evansi

Trypanosoma evansi is a blood protozoan parasite of the genus Trypanosoma which is responsible for surra (Trypanosomosis) in domestic and wild animals. This study addressed apoptotic-like features in Trypanosoma evansi in vitro. The mechanism of parasite death was investigated using staurosporine as an inducing agent. We evaluated its effects through several cytoplasmic features of apoptosis, including cell shrinkage, phosphatidylserine exposure, maintenance of plasma membrane integrity, and mitochondrial trans-membrane potential. For access to these features we have used the flow cytometry and fluorescence microscopy with cultures in the stationary phase and adjusted to a density of 10(6) cells/mL. The apoptotic effect of staurosporine in T. evansi was evaluated at 20 nM final concentration. There was an increase of phosphatidylserine exposure, whereas mitochondrial potential was decreased. Moreover, no evidence of cell permeability increasing with staurosporine was observed in this study, suggesting the absence of a necrotic process. Additional studies are needed to elucidate the possible pathways associated with this form of cell death in this hemoparasite.

Trypanosoma evansi es un hemoparásito, el cual es el agente causal de la surra (tripanosomiasis) en mamíferos, perteneciente al orden Kinetoplastidae. Este estudio se oriento a caracterizar la muerte celular similar a apoptosis en cultivos in vitro de Trypanosoma evansi a través del uso del inductor esturosporina. Este efecto se evaluó a través de diversos aspectos fenotípicos de la apoptosis: el encogimiento celular, la exposición de fosfatidilserina, el mantenimiento de la integridad de la membrana plasmática y el potencial de membrana mitocondrial. Para evaluar estas características se utilizaron técnicas de citometría de flujo y microscopía de fluorescencia con cultivos en fase estacionaria ajustados a una densidad de 10(6) células/mL. El efecto apoptótico de la estaurosporina en Trypanosoma evansi fue evaluado a una concentración de 20 nM. Se evidenció un aumento de la exposición a fosfatidilserina, mientras que el potencial mitocondrial disminuyó. Por otra parte, no hay evidencias de aumento de la permeabilidad celular con estaurosporina, sugiriendo la ausencia de un proceso necrótico. Estudios adicionales son necesarios para dilucidar las posibles vías asociadas con esta forma de muerte celular en este hemoparásito.

Coupling of M3 acetylcholine receptor to Gq16 activates a natriuretic peptide receptor guanylyl cyclase.

Muscarinic activation of tracheal smooth muscle (TSM) involves a M(3)AChR/heterotrimeric-G protein/NPR-GC coupling mechanism. G protein activators Mastoparan (MAS) and Mastoparan-7 stimulated 4- and 10-fold the NPR-GC respectively, being insensitive to PTX and antibodies against Galpha(i/o) subfamily. Muscarinic and MAS stimulation of NPR-GC was blocked by antibodies against C-terminal of Galpha(q16), whose expression was confirmed by RT-PCR. However, synthetic peptides from C-terminal of Galpha(q15/16) stimulated the NPR-GC. Coupling of alpha(q16) to M(3)AChR is supported by MAS decreased [(3)H]QNB binding, being abolished after M(3)AChR-4-DAMP-alkylation. Anti-i(3)M(3)AChR antibodies blocked the muscarinic activation of NPR-GC, and synthetic peptide from i(3)M(3)AChR (M(3)P) was more potent than MAS increasing GTPgamma [(35)S] and decreasing the [(3)H]QNB activities. Coupling between NPR-GC and Galpha(q16) was evaluated by using trypsin-solubilized-fraction from TSM membranes, which displayed a MAS-sensitive-NPR-GC activity, being immunoprecipitated with anti-Galpha(q16), also showing an immunoreactive heterotrimeric-G-beta-subunit. These data support the existence of a novel transducing cascade, involving Galpha(q16)beta gamma coupling M(3)AChR to NPR-GC.