Activation of GPVI by collagen is regulated by alpha(2)beta(1) and secondary mediators

We have compared the roles of adenosine diphosphate (ADP), thromboxanes and the integrin alpha(2)beta(1) in the activation of washed platelets by collagen in the presence of the alpha(IIb)beta3 antagonist lotrafiban. The stimulation of protein tyrosine phosphorylation by a collagen suspension is markedly delayed in the presence of the above inhibitors but shows substantial recovery with time. In comparison, activation of phospholipase C (PLC), Ca2+ elevation and dense granule secretion are more severely suppressed by the above inhibitors. blockade has a slightly greater inhibitory effect on all of the above responses than a combination of ADP receptor antagonists and cyclooxygenase inhibitor. Platelets exposed to a collagen monolayer show robust elevation of Ca2+ that is delayed in the presence of the above inhibitors and which is accompanied by of-granule secretion. These results demonstrate that secondary mediators and alpha(2)beta(1) modulate collagen-induced intracellular signaling but have negligible effect on GPVI signaling induced by the specific agonist convulxin. This work supports the postulate that the major role of of alpha(2)beta(1) is to increase the avidity of collagen for the platelet surface and by doing so enhance activation of GPVI. Therefore we propose an important role of secondary mediators in collagen-induced signaling is the indirect regulation of GPVI signaling via activation of alpha(2)beta(1).

Distinct roles of GPVI and integrin alpha(2)beta(1) in platelet shape change and aggregation induced by different collagens

1 Various platelet membrane glycoproteins have been proposed as receptors for collagen, in some cases as receptors For specific collagen types. In this study we have compared the ability of a range of collagen types to activate platelets.

Connective tissue growth factor antagonizes transforming growth factor-beta 1/Smad signalling in renal mesangial cells

The critical involvement of TGF-beta 1 (transforming growth factor-beta 1) in DN (diabetic nephropathy) is well established. However, the role of CTGF (connective tissue growth factor) in regulating the complex interplay of TGF-beta 1 signalling networks is poorly understood. The purpose of the present study was to investigate co-operative signalling between CTGF and TGF-beta 1 and its physiological significance. CTGF was determined to bind directly to the T beta RIII (TGF-beta type III receptor) and antagonize TGF-beta 1-induced Smad phosphorylation and transcriptional responses via its N-terminal half. Furthermore, TGF-beta 1 binding to its receptor was inhibited by CTGF. A consequent shift towards non-canonical TGF-beta 1 signalling and expression of a unique profile of differentially regulated genes was observed in CTGF/TGF-beta 1-treated mesangial cells. Decreased levels of Smad2/3 phosphorylation were evident in STZ (streptozotocin)-induced diabetic mice, concomitant with increased levels of CTGF Knockdown of T beta RIII restored TGF-beta 1-mediated Smad signalling and cell contractility, suggesting that T beta RIII is key for CTGF-mediated regulation of TGF-beta 1. Comparison of gene expression profiles from CTGF/TGF-beta 1-treated mesangial cells and human renal biopsy material with histological diagnosis of DN revealed significant correlation among gene clusters. In summary, mesangial cell responses to TGF-beta 1 are regulated by cross-talk with CTGF, emphasizing the potential utility of targeting CTGF in DN.

Novel anti-inflammatory compound SEN1176 alleviates behavioural deficits induced following bilateral intrahippocampal injection of aggregated amyloid-beta(1-42).

Behavioral effects of a novel anti-inflammatory SEN1176 were investigated. This pyrrolo[3,2-e][1,2,4]triazolo[1,5-a]pyrimidine suppresses amyloid-ß (Aß)1-42-induced macrophage production of nitric oxide, TNF-a, IL-1ß, and IL-6 in a dose-dependent fashion, an activity profile consistent with SEN1176 being a neuroinflammation inhibitor. Using male Sprague-Dawley rats, SEN1176 was examined relative to detrimental behavioral effects induced following bilateral intrahippocampal (IH) injections of aggregated Aß1-42. The rats were trained to respond under an alternating-lever cyclic-ratio (ALCR) schedule of food reinforcement, enabling measurement of parameters of operant performance that reflect aspects of learning and memory. Under the ALCR schedule, orally administered SEN1176 at 5, 20, or 30 mg/kg was effective in reducing the behavioral deficit caused by bilateral IH aggregated Aß1-42 injections in a dose-related manner over a 90-day treatment period. SEN1176 at 20 and 30 mg/kg significantly reduced lever switching errors and, at doses of 5, 10, and 30 mg/kg, significantly reduced incorrect lever perseverations, indicating a reduction of the behavioral deficit induced as a result of inflammation following IH Aß1-42 injections. When treatment with SEN1176 was instigated 30 days after IH Aß1-42 injections, it resulted in progressive protection, and withdrawal of SEN1176 treatment 60 days after IH Aß1-42 injections revealed partial retention of the protective effect. SEN1176 also significantly reduced numbers of activated astrocytes adjacent to the aggregated Aß1-42 injection sites. These results indicate the potential of SEN1176 for alleviating chronic neuroinflammatory processes related to brain Aß deposition that affect learning and memory in Alzheimer's disease.

Gingival fibroblast response to cyclosporin A and transforming growth factor beta(1)

This study investigates a potential role for TGF beta(1), in the pathogenesis of cyclosporin A-induced gingival overgrowth (CsA-OG). TGF beta(1) was localized immunohistochemically in the connective tissue of both normal gingiva and CsA-OG. Intense staining for TGF beta(1) was detected at the tips of the dermal papillae of the overgrown gingiva. In addition, fibroblasts derived from healthy gingiva and fibroblasts derived from CsA-OG were cultured both as monolayers or embedded in a 3D-collagen gel. Fibroblast activity was monitored in terms of protein and collagen production in the presence of (i) 1 ng/ml TGF beta(1), (ii) 500 ng/ml CsA, or (iii) 500 ng/ml CsA and 1 ng/ml TGF beta(1). In monolayer culture TGF beta(1) significantly increased protein and collagen production in all cell strains (p

Structural and kinetic characterization of the LPS biosynthetic enzyme D-alpha,beta-D-heptose-1,7-bisphosphate phosphatase (GmhB) from Escherichia coli

Lipopolysaccharide is a major component of the outer membrane of gram-negative bacteria and provides a permeability barrier to many commonly used antibiotics. ADP-heptose residues are an integral part of the LPS inner core, and mutants deficient in heptose biosynthesis demonstrate increased membrane permeability. The heptose biosynthesis pathway involves phosphorylation and dephosphorylation steps not found in other pathways for the synthesis of nucleotide sugar precursors. Consequently, the heptose biosynthetic pathway has been marked as a novel target for antibiotic adjuvants, which are compounds that facilitate and potentiate antibiotic activity. D-alpha,beta-D-heptose-1,7-bisphosphate phosphatase (GmhB) catalyzes the third essential step of LPS heptose biosynthesis. This study describes the first crystal structure of GmhB and enzymatic analysis of the protein. Structure-guided mutations followed by steady state kinetic analysis, together with established precedent for HAD phosphatases, suggest that GmhB functions through a phosphoaspartate intermediate. This study provides insight into the structure-function relationship of GmhB, a new target for combatting gram-negative bacterial infection.

The wbbD gene of E. coli strain VW187 (O7:K1) encodes a UDP-Gal:GlcNAc{alpha}-pyrophosphate-R {beta}1,3-galactosyltransferase involved in the biosynthesis of O7-specific lipopolysaccharide

In this work, we demonstrate that the wbbD gene of the O7 lipopolysaccharide (LPS) biosynthesis cluster in Escherichia coli strain VW187 (O7:K1) encodes a galactosyltransferase involved in the synthesis of the O7-polysaccharide repeating unit. The galactosyltransferase catalyzed the transfer of Gal from UDP-Gal to the GlcNAc residue of a GlcNAc-pyrophosphate-lipid acceptor. A mutant strain with a defective wbbD gene was unable to form O7 LPS and lacked this specific galactosyltransferase activity. The normal phenotype was restored by complementing the mutant with the cloned wbbD gene. To characterize the WbbD galactosyltransferase, we used a novel acceptor substrate containing GlcNAcalpha-pyrophosphate covalently bound to a hydrophobic phenoxyundecyl moiety (GlcNAc alpha-O-PO(3)-PO(3)-(CH(2))(11)-O-phenyl). The WbbD galactosyltransferase had optimal activity at pH 7 in the presence of 2.5 mM MnCl(2). Detergents in the assay did not increase glycosyl transfer. Digestion of enzyme product by highly purified bovine testicular beta-galactosidase demonstrated a beta-linkage. Cleavage of product by pyrophosphatase and phosphatase, followed by HPLC and NMR analyses, revealed a disaccharide with the structure Gal beta1-3GlcNAc. Our results conclusively demonstrate that WbbD is a UDP-Gal: GlcNAcalpha-pyrophosphate-R beta1,3-galactosyltransferase and suggest that the novel synthetic glycolipid acceptor may be generally applicable to characterize other bacterial glycosyltransferases.

Diabetes downregulates large-conductance Ca2+-activated potassium beta 1 channel subunit in retinal arteriolar smooth muscle

Retinal vasoconstriction and reduced retinal blood flow precede the onset of diabetic retinopathy. The pathophysiological mechanisms that underlie increased retinal arteriolar tone during diabetes remain unclear. Normally, local Ca(2+) release events (Ca(2+)-sparks), trigger the activation of large-conductance Ca(2+)-activated K(+)(BK)-channels which hyperpolarize and relax vascular smooth muscle cells, thereby causing vasodilatation. In the present study, we examined BK channel function in retinal vascular smooth muscle cells from streptozotocin-induced diabetic rats. The BK channel inhibitor, Penitrem A, constricted nondiabetic retinal arterioles (pressurized to 70mmHg) by 28%. The BK current evoked by caffeine was dramatically reduced in retinal arterioles from diabetic animals even though caffeine-evoked [Ca(2+)](i) release was unaffected. Spontaneous BK currents were smaller in diabetic cells, but the amplitude of Ca(2+)-sparks was larger. The amplitudes of BK currents elicited by depolarizing voltage steps were similar in control and diabetic arterioles and mRNA expression of the pore-forming BKalpha subunit was unchanged. The Ca(2+)-sensitivity of single BK channels from diabetic retinal vascular smooth muscle cells was markedly reduced. The BKbeta1 subunit confers Ca(2+)-sensitivity to BK channel complexes and both transcript and protein levels for BKbeta1 were appreciably lower in diabetic retinal arterioles. The mean open times and the sensitivity of BK channels to tamoxifen were decreased in diabetic cells, consistent with a downregulation of BKbeta1 subunits. The potency of blockade by Pen A was lower for BK channels from diabetic animals. Thus, changes in the molecular composition of BK channels could account for retinal hypoperfusion in early diabetes, an idea having wider implications for the pathogenesis of diabetic hypertension.

Immunological homologues of the Arabidopsis thaliana beta 1 tubulin are polyglutamylated in Nicotiana tabacum

Peptide-specific antibody AABI, raised to the C-terminal 13 amino acids of Arabidopsis thaliana beta 1 tubulin, identifies a single electrophoretically separable beta-tubulin on 2-D-gel Western blots of total protein extracts from A. thaliana seedlings. We show that AABI crossreacts with two of the eight polyglutamylated beta-tubulin isoforms present in purified Nicotiana tabacum tubulin fractionated by high-resolution isoelectric focussing. Immunolocalisation studies using AAB1 revealed that the two N. tabacum polyglutamylated beta 1-tubulin isoforms are utilised in all four plant microtubule arrays (the interphase cortical array, the preprophase band, the spindle and the phragmoplast) indicating that there is no apparent subcellular sorting of these isotypes.

MIC-1 (a multifunctional modulator of dendritic cell phenotype and function) is produced by decidual stromal cells and trophoblasts

Macrophage inhibitory cytokine-1 (MIC-1) is a multifunctional cytokine produced in high amounts by placental tissue. Inhibiting trophoblast invasion and suppressing inflammation through inhibition of macrophage activation, MIC-1 is thought to provide pleiotropic functions in the establishment and maintenance of pregnancy. So far, little is known about the decidual cell subsets producing MIC-1 and the effect of this cytokine on dendritic cells (DCs), which are known to play a distinct role in the development of pro-fetal tolerance in pregnancy.