Anti‐inflammatory effects of activated protein C on human dendritic cells

Activated protein C (APC) has an anticoagulant action and plays an important role in blood coagulation homeostasis. In addition to its anticoagulant action, APC is known to have cytoprotective effects, such as anti‐apoptotic action and endothelial barrier protection, on vascular endothelial cells and monocytes. However, the effects of APC on DCs have not been clarified. To investigate the effects of APC on human DCs, monocytes were isolated from peripheral blood and DC differentiation induced with LPS. APC significantly inhibited the production of inflammatory cytokines TNF‐α and IL‐6 during differentiation of immature DCs to mature DCs, but did not inhibit the production of IL‐12 and anti‐inflammatory cytokine IL‐10. Interestingly, treatment with 5 μg/mL, but not 25 μg/mL, of APC significantly enhanced production of IL‐10. In addition, protein C, which is the zymogen of APC, did not affect production of these cytokines. On the other hand, flow cytometric analysis of DC's surface molecules indicated that APC does not significantly affect expression of CD83, a marker of mDC differentiation, and the co‐stimulatory molecules CD40, CD80 and CD86. These results suggest that APC has anti‐inflammatory effects on human DCs and may be effective against some inflammatory diseases in which the pathogenesis involves TNF‐α and/or IL‐6 production.     

Impact of the genome wide supported NRGN gene on anterior cingulate morphology in schizophrenia

Background

The rs12807809 single-nucleotide polymorphism in NRGN is a genetic risk variant with genome-wide significance for schizophrenia. The frequency of the T allele of rs12807809 is higher in individuals with schizophrenia than in those without the disorder. Reduced immunoreactivity of NRGN, which is expressed exclusively in the brain, has been observed in Brodmann areas (BA) 9 and 32 of the prefrontal cortex in postmortem brains from patients with schizophrenia compared with those in controls.

Methods

Genotype effects of rs12807809 were investigated on gray matter (GM) and white matter (WM) volumes using magnetic resonance imaging (MRI) with a voxel-based morphometry (VBM) technique in a sample of 99 Japanese patients with schizophrenia and 263 healthy controls.

Results

Although significant genotype-diagnosis interaction either on GM or WM volume was not observed, there was a trend of genotype-diagnosis interaction on GM volume in the left anterior cingulate cortex (ACC). Thus, the effects of NRGN genotype on GM volume of patients with schizophrenia and healthy controls were separately investigated. In patients with schizophrenia, carriers of the risk T allele had a smaller GM volume in the left ACC (BA32) than did carriers of the non-risk C allele. Significant genotype effect on other regions of the GM or WM was not observed for either the patients or controls.

Conclusions

Our findings suggest that the genome-wide associated genetic risk variant in the NRGN gene may be related to a small GM volume in the ACC in the left hemisphere in patients with schizophrenia.     

<Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Cry11Ba works synergistically with Cry4Aa but not with Cry11Aa for toxicity against mosquito <Emphasis Type="Italic">Culex pipiens</Emphasis> (Diptera: Culicidae) larvae

A 2,175-bp modified gene (cry11Ba-S1) encoding Cry11Ba from Bacillus thuringiensis subsp. jegathesan was designed and the recombinant protein was expressed as a fusion protein with glutathione S-transferase in Escherichia coli. The recombinant Cry11Ba was highly toxic against Culex pipiens mosquito larvae, being nine and 17 times more toxic than mosquitocidal Cry4Aa and Cry11Aa from Bacillus thuringiensis subsp. israelensis, respectively. Interestingly, a further increase in the toxicity of the recombinant Cry11Ba was achieved by mixing with Cry4Aa, but not with Cry11Aa. These findings suggested that Cry11Ba worked synergistically with Cry4Aa, but not with Cry11Aa, in exhibiting toxicity against C. pipiens larvae. On the other hand, the amount of Cry toxin bound to brush border membrane vesicles (BBMVs) did not significantly change between individual toxins and the toxin mixtures, suggesting that the increase in toxins binding to BBMVs was not a reason for the observed synergistic effect. It is generally accepted that synergism of toxins is a potentially powerful tool for enhancing insecticidal activity and managing Cry toxin resistance in mosquitoes. The mixture of Cry4Aa and Cry11Ba in order to increase toxicity would be very valuable in terms of mosquito control.     

CC chemokine receptor 7 contributes to Gi-dependent T cell motility in the lymph node

Naive T cells migrate extensively within lymph node (LN) T zones to scan for Ag-bearing dendritic cells. However, the extracellular signals controlling T cell motility in LNs are not well defined. In this study, by real-time imaging of LNs, we show that the inhibition of Gi signaling in T cells severely impairs their migration. The chemokine CCL21, a ligand of CCR7, strongly induces chemokinesis in vitro, and T cell motility in LNs from CCR7 ligand-deficient plt/plt mice was reduced. CCR7-deficient T cells in wild-type LNs showed a similar reduction in motility, and antagonism of CXCR4 function did not further decrease their motility. The effect of CCR7 or CCR7-ligand deficiency could account for approximately 40% of the Gi-dependent motility. These results reveal a role for CCR7 in promoting T cell migration within lymphoid organ T zones, and they suggest the additional involvement of novel Gi-coupled receptors in promoting T cell motility at these sites.     

Paxillin family members function as Csk-binding proteins that regulate Lyn activity in human and murine platelets

SFKs (Src family kinases) contribute importantly to platelet function in haemostasis. SFK activity is controlled by Csk (C-terminal Src kinase), which phosphorylates a C-terminal tyrosine residue on SFKs, resulting in inhibition of SFK activity. Csk is recruited to sites of SFK activity by tyrosine-phosphorylated Csk-binding proteins. Paxillin, a multidomain adaptor protein, has been shown to act as a Csk-binding protein and to inhibit Src activity during growth factor signalling. Human platelets express Hic-5, a member of the paxillin family; however, its ability to act as a Csk-binding protein has not been characterized. We sought to identify and characterize the ability of paxillin family members to act as Csk-binding proteins during platelet activation. We found that murine and human platelets differ in the complement of paxillin family members expressed. Human platelets express Hic-5, whereas murine platelets express paxillin and leupaxin in addition to Hic-5. In aggregating human platelets, Hic-5 was tyrosine phosphorylated and recruited Csk via its SH2 domains. In aggregating murine platelets, however, Csk bound preferentially to paxillin, even though both paxillin and Hic-5 were abundantly present and became tyrosine phosphorylated. The SFK Lyn, but not Src or Fyn, was associated with paxillin family members in resting and aggregated human and murine platelets. Lyn, however, was phosphorylated on its C-terminal inhibitory tyrosine residue only following platelet aggregation, which was coincident with recruitment of Csk to paxillin and/or Hic-5 in a manner dependent on prior alpha(IIb)beta3 engagement. These observations support the notion that Hic-5 and paxillin function as negative feedback regulators of SFKs in aggregated platelets and that, when both are present, paxillin is preferentially used.     

Induction and subsequent repair of DNA damage by fast neutrons in cultured mammalian cells

The induction and repair of DNA damage were studied by a DNA unwinding method in mouse L5178Y cells exposed to fast neutrons. DNA lesions induced by fast neutrons were classified into three types from their repair profiles: fast-reparable breaks (T1/2 = 3-5 min), slow-reparable breaks (T1/2 = 70 min), and nonreparable breaks. The repair rates of both fast-reparable and slow-reparable breaks were almost the same as those of corresponding damage induced by low-LET radiation. Neutrons induced a smaller amount of fast-reparable damage, an almost equal amount of slow-reparable damage, and a larger amount of nonreparable damage than those induced by equal doses of gamma rays or X rays. RBEs for fast- and slow-reparable damage were 0.3 and 0.9, respectively. The RBE for nonreparable damage was dose dependent and was 1.4 at the level of 100 breaks/10(12) Da DNA. Among the three types of lesions, only the nonreparable damage levels correlated with the linear-quadratic shape of the survival curves and with the enhanced killing effectiveness of neutrons (RBE = 1.7 at D0).     

Regulation of Hyaluronan (HA) Metabolism Mediated by HYBID (Hyaluronan-binding Protein Involved in HA Depolymerization,KIAA1199) and HA Synthases in Growth Factor-stimulated Fibroblasts

Regulation of hyaluronan (HA) synthesis and degradation is essential to maintenance of extracellular matrix homeostasis. We recently reported that HYBID (HYaluronan-Binding protein Involved in hyaluronan Depolymerization), also called KIAA1199, plays a key role in HA depolymerization in skin and arthritic synovial fibroblasts. However, regulation of HA metabolism mediated by HYBID and HA synthases (HASs) under stimulation with growth factors remains obscure. Here we report that TGF-β1, basic FGF, EGF, and PDGF-BB commonly enhance total amount of HA in skin fibroblasts through up-regulation of HAS expression, but molecular size of newly produced HA is dependent on HYBID expression levels. Stimulation of HAS1/2 expression and suppression of HYBID expression by TGF-β1 were abrogated by blockade of the MAPK and/or Smad signaling and the PI3K-Akt signaling, respectively. In normal human skin, expression of the TGF-β1 receptors correlated positively with HAS2 expression and inversely with HYBID expression. On the other hand, TGF-β1 up-regulated HAS1/2 expression but exerted only a slight suppressive effect on HYBID expression in synovial fibroblasts from the patients with osteoarthritis or rheumatoid arthritis, resulting in the production of lower molecular weight HA compared with normal skin and synovial fibroblasts. These data demonstrate that although TGF-β1, basic FGF, EGF, and PDGF-BB enhance HA production in skin fibroblasts, TGF-β1 most efficiently contributes to production of high molecular weight HA by HAS up-regulation and HYBID down-regulation and suggests that inefficient down-regulation of HYBID by TGF-β1 in arthritic synovial fibroblasts may be linked to accumulation of depolymerized HA in synovial fluids in arthritis patients.