The regulation of integrin-linked kinase in human platelets: evidence for involvement in the regulation of integrin alpha(2)beta(1)

Background: Activation of the platelet integrin alpha(2)beta(1) is closely regulated due to the high thrombogenicity of its ligand. As a beta(1) interacting kinase, ILK represents a candidate intracellular regulator of alpha(2)beta(1) in human platelets. Objectives We investigated the regulation of ILK in human platelets and the role of ILK in regulating alpha(2)beta(1) activation in HEL cells, a megakaryocytic cell line. Methods: An in-vitro kinase assay was used to determine the effect of platelet agonists on ILK kinase activity together with the contribution of PI3K and PKC on ILK activation. Interaction of ILK with beta(1)-integrin subunits was investigated by coimmunoprecipitation and the role of ILK in regulating alpha(2)beta(1) function assessed by overexpression studies in HEL cells. Results: We report that collagen and thrombin modulate ILK kinase activity in human platelets in an aggregation-independent manner. Furthermore, ILK activity is dually regulated by PI3K and PKC in thrombin-stimulated platelets and regulated by PI3K in collagen-stimulated cells. ILK associates with the beta(1)-integrin subunits immunoprecipitated from platelet cell lysates, an association which increased upon collagen stimulation. Overexpression of ILK in HEL cells enhanced alpha(2)beta(1)-mediated adhesion whereas overexpression of kinase-dead ILK reduced adhesion, indicating a role for this kinase in the positive regulation of alpha(2)beta(1). Conclusions: Our findings that ILK regulates alpha(2)beta(1) in HEL cells, is activated in platelets and associates with beta(1)-integrins, raise the possibility that it may play a key role in adhesion events upon agonist stimulation of platelets.

Platelet adhesion to collagen and collagen-related peptide under flow. Roles of the alpha(2)beta(1) integrin, GPVI, and Src tyrosine kinases

Objective - Platelet stimulation by collagen and collagen-related peptides (CRPs) is associated with activation of protein tyrosine kinases. In the present study, we investigated the role of Src family tyrosine kinases in the initial adhesion events of human platelets to collagen and cross-linked CRP. Methods and Results - Under arterial flow conditions, a glycoprotein VI - specific substrate, cross-linked CRP, caused rapid (<2 second) platelet retention and protein tyrosine phosphorylation that were markedly decreased by the Src family kinase inhibitor pyrozolopyrimidine (PP2) or by aggregation inhibitor GRGDSP. CRP-induced platelet retention was transient, and 90% of single platelets or aggregates detached within seconds. PP2, although having no effect on RGD peptide-binding to CRP, completely blocked aggregation and tyrosine phosphorylation of Syk and phospholipase Cγ2 (PLCγ2). In contrast, PP2 weakly (<30%) suppressed firm adhesion to collagen mediated primarily by the alpha(2)beta(1) integrin. Although PP2 prevented activation of Syk and PLCgamma2 in collagen-adherent platelets, tyrosine phosphorylation of several unidentified protein bands persisted, as did autophosphorylation of pp125(FAK). Conclusions - These findings indicate that activation of Src-tyrosine kinases Syk and PLCgamma2 is not required for the initial stable attachment of human platelets to collagen and for FAK autophosphorylation. However, Src-tyrosine kinases are critical for glycoprotein VI - mediated signaling leading to platelet aggregation.

Regioselective synthesis of mannobiose and mannotriose by reverse hydrolysis using a novel 1,6-alpha-D-mannosidase from Aspergillus phoenicis

A novel 1,6-alpha-D-mannosidase was produced by Aspergillus phoenicis grown on a commercial manno-oligosaccharide preparation in liquid culture. The enzyme hydrolysed only alpha-D-Manp-(1 --> 6)-D-Manp and did not act on alpha-D-Manp-(1 --> 2)-D-Manp, or alpha-D-Manp-(1 --> 3)-D-Manp. The 1,6-alpha-D-mannosidase was used for synthesis of manno-oligosaccharides by reverse hydrolysis reaction. The highest yields, expressed as percentages (w/w) of total sugar, were similar to21% mannobiose and similar to5% mannotriose, and they were obtained with 45% (w/w) initial mannose concentration at pH 4.5 after 12 days incubation at 55 degreesC. The disaccharide and trisaccharide products were separated and their structures determined by methylation analysis. Only 1-6 linkages were found in both of them. (C) 2003 Elsevier B.V. All rights reserved.

A new entry to 4,6-O-benzylidene glucal from phenyl 1-seleno-alpha-D-mannopyranoside

The two-step synthesis of 4,6-O-benzylidene glucal, in 59% overall yield, from phenyl 1-seleno-alpha-D-mannopyranoside is described. (c) 2005 Elsevier Ltd. All rights reserved.

Modulatory effect of fatty acids on fungicidal activity, respiratory burst and TNF-alpha and IL-6 production in J774 murine macrophages

The reported effects of different families of fatty acids (FA; SFA, MUFA, n-3 and n-6 PUFA) on human health and the importance of macrophage respiratory burst and cytokine release to immune defence led us to examine the influence of palmitic acid (PA), oleic acid (OA), linoleic acid, arachidonic acid, EPA and DHA on macrophage function. We determined fungicidal activity, reactive oxygen species (ROS) and cytokine production after the treatment of J774 cells with non-toxic concentrations of the FA. PA had a late and discrete stimulating effect on ROS production, which may be associated with the reduced fungicidal activity of the cells after treatment with this FA. OA presented a sustained stimulatory effect on ROS production and increased fungicidal activity of the cells, suggesting that enrichment of diets with OA may be beneficial for pathogen elimination. The effects of PUFA on ROS production were time-and dose-dependently regulated, with no evident differences between n-3 and n-6 PUFA. It was worth noting that most changes induced after stimulation of the cells with lipopolysaccharide were suppressed by the FA. The present results suggest that supplementation of the diet with specific FA, not classes of FA, might enable an improvement in host defence mechanisms or a reduction in adverse immunological reactions.

Projectile breakup dynamics for (6)Li+(59)Co: Kinematical analysis of alpha-d coincidences

A study of the kinematics of the alpha-d coincidences in the (6)Li + (59)Co system at a bombarding energy of E(lab) = 29.6MeV is presented. With exclusive measurements performed over different angular intervals it is possible to identify the respective contributions of the sequential and direct projectile breakup components. The angular distributions of both breakup components are fairly well described by the Continuum-Discretized Coupled-Channels framework (CDCC). Furthermore, a careful analysis of these processes using a semiclassical approach provides information on both their lifetime and their distance of occurrence with respect to the target. Breakup to the low-lying (near-threshold) continuum is delayed, and happens at large internuclear distances. This suggests that the influence of the projectile breakup on the complete fusion process can be related essentially to the direct breakup to the (6)Li high-lying continuum spectrum.

IL-6 and TNF-alpha production during active canine visceral leishmaniasis

We investigated the production of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) during canine visceral leishmaniasis (VL) to gain a better understanding of the role of such multi-functional cytokines in parasite resistance. IL-6 and TNF-alpha levels were measured by capture ELISA in sera from 8 healthy dogs from a non-endemic area (control group) and in sera from 16 dogs from Aracatuba, SP, Brazil, an area endemic for leishmaniosis. The dogs from the endemic area were selected by positive ELISA serology against total Leishmania chagasi antigen, positive spleen imprints for Leishmania, and the presence of at least three clinical signs associated with active visceral leishmaniasis (fever, dermatitis, lymphoadenopathy, onychogryphosis, weight loss, cachexia, locomotory difficulty, conjunctivitis, epistaxis, hepatosplenomegaly, edema, and apathy).Enhanced systemic IL-6 production was found in sera from dogs with the active disease compared to healthy dogs (t-test, P < 0.05). In contrast, TNF-alpha did not differ between the two groups studied. There was no correlation between IL-6 production and anti-leishmanial antibody titers in the sera. Our findings suggest that IL-6 is a good marker of active disease during leishmaniasis, and that other cytokines may be involved in the hypergammaglobulinemia characteristic of canine visceral leishmaniasis. (c) 2006 Published by Elsevier B.V.

MKK3/6-p38 MAPK signaling is required for IL-1 beta and TNF-alpha-induced RANKL expression in bone marrow stromal cells

Coupled bone turnover is directed by the expression of receptor-activated NF-kappa B ligand (RANKL) and its decoy receptor, osteoprotegerin (OPG). Proinflammatory cytokines, such as interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) induce RANKL expression in bone marrow stromal cells. Here, we report that IL-1 beta and TNF-alpha-induced RANKL requires p38 mitogen-activating protein kinase (MAPK) pathway activation for maximal expression. Real-time PCR was used to assess the p38 contribution toward IL-1 beta and TNF-alpha-induced RANKL mRNA expression. Steady-state RANKL RNA levels were increased approximately 17-fold by IL-1 beta treatment and subsequently reduced similar to 70%-90% when p38 MAPK was inhibited with SB203580. RANKL mRNA stability data indicated that p38 MAPK did not alter the rate of mRNA decay in IL-1 beta-induced cells. Using a RANKL-luciferase cell line receptor containing a 120-kB segment of the 5' flanking region of the RANKL gene, reporter expression was stimulated 4-5-fold by IL-1 beta or TNF-alpha treatment. IL-1 beta-induced RANKL reporter expression was completely blocked with specific p38 inhibitors as well as dominant negative mutant constructs of MAPK kinase-3 and -6. In addition, blocking p38 signaling in bone marrow stromal cells partially inhibited IL-1 beta and TNF-alpha-induced osteoclastogenesis in vitro. Results from these studies indicate that p38 MAPK is a major signaling pathway involved in IL-1 beta and TNF-alpha-induced RANKL expression in bone marrow stromal cells.

6-[omega-arylalkenyl]-5,6-dihydro-alpha-pyrones from Cryptocarya moschata (Lauraceae)

Eleven 6-[omega-arylalkenyl]-5,6-dihydro-alpha-pyrones, cryptomoscatones D2, E1, E2, E3 and F1 and cryptopyranmoscatones A1, A2, A3, B1, B2 and B4, in addition to goniothalamin and cryptofolione, were isolated from branch and stem bark of Cryotocarya moschata, Lauraceae. Their structures were established by spectroscopic methods. (C) 2000 Elsevier B.V. Ltd. All rights reserved.

MKK3/6-p38 MAPK negatively regulates murine MMP-13 gene expression induced by IL-1 beta and TNF-alpha in immortalized periodontal ligament fibroblasts

Matrix metalloprotease-13 (MMP-13) or collagenase-3 is involved in a number of pathologic processes such as tumor metastasis and angiogenesis, osteoarthritis, rheumatoid arthritis and periodontal diseases. These conditions are associated with extensive degradation of both connective tissue and bone. This report examines gene regulation mechanisms and signal transduction pathways involved in Mmp-13 expression induced by proinflammatory cytokines in periodontal ligament (PDL) fibroblasts. Mmp-13 mRNA expression was increased 10.7 and 9.5 fold after stimulation with IL-1 beta (5 ng/mL) and TNF-alpha (10 ng/mL), respectively. However, inhibition of p38 MAPKinase with SB203580 resulted in significant (p < 0.001) induction (23.2 and 18.1 fold, respectively) of Mmp-13 mRNA as assessed by real time PCR. Negative regulation of IL-1 induced Mmp-13 expression was confirmed by inhibiting p38 MAPK gene expression with siRNA. Transient transfection of dominant negative forms of MKK3 and MKK6 also resulted in increased levels of Mmp-13 mRNA after IL-1 beta stimulation. Mmp-13 mRNA expression induced by TNF-alpha was decreased by JNK and ERK inhibition. Western blot and zymogram analysis indicated that Mmp-13 protein expression induced by the proinflammatory cytokines were also upregulated by inhibition of p38 MAPK. Reporter gene experiments using stable cell lines harboring 660-bp sequence of the murine Mmp-13 proximal promoter indicated that transcriptional mechanisms were at least partially involved in this negative regulation of Mmp-13 expression by p38 MAPK and upstream MKK3/6. These results suggest a negative transcriptional regulatory mechanism mediated by p38 MAPK and upstream MKK3/6 on Mmp-13 expression induced by proinflammatory cytokines in PDL fibroblasts. (c) 2005 Elsevier B.V./International Society of Matrix Biology. All rights reserved.

Recombinant disintegrin targets alpha(v) beta(3) integrin and leads to mediator production

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Terpinen-4-ol and alpha-terpineol (tea tree oil components) inhibit the production of IL-1 beta, IL-6 and IL-10 on human macrophages

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

TNF-alpha and IL-6 immunohistochemistry in rat renal tissue experimentaly infected with Leptospira interrogans serovar Canicola

Leptospirosis is a public health problem worldwide and its etiology remains unclear. Its pathogenesis involves a complex interaction between host and infecting microorganism. The inflammatory reaction that controls the infection process also underscores many pathophysiological events occurring in leptospirosis. We investigated the presence of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) in renal tissues by immunohistochemical and histopathological examination in animals experimentally inoculated with Leptospira serovar Canicola. All the tests were carried out 2, 7, 14, 21 or 28 days after inoculation. Although TNF-alpha and IL-6 had been detected in tissues throughout the observation period, these cytokines appeared more intensely during the initial phase of infection. Therefore, both TNF-alpha and IL-6 were associated with the immunopathogenesis of leptospirosis. This profile suggests a high immunocellular response throughout the early infection stages followed by subsequent humoral response.

alpha,beta-Unsaturated Diazoketones as Platforms in the Asymmetric Synthesis of Hydroxylated Alkaloids. Total Synthesis of 1-Deoxy-8,8a-diepicastanospermine and 1,6-Dideoxyepicastanospermine and Formal Synthesis of Pumiliotoxin 251D

A versatile and concise approach for the stereoselective synthesis of mono-, di-, and trihydroxylated indolizidines is presented in four to six steps from Cbz-prolinal and a diazophosphonate. The key steps involved a Wolff rearrangement, followed by a stereoselective dihydroxylation/epoxidation reaction, from an alpha,beta-unsaturated diazoketone. The strategy also permits extension to the synthesis of many natural hydroxylated indolizidine alkaloids as demonstrated in the formal synthesis of pumiliotoxin 251D.