Extremely high alkaline protease from a deep-subsurface bacterium, Alkaliphilus transvaalensis

A new high-alkaline protease (ALTP) was purified to homogeneity from a culture of the strictly anaerobic and extremely alkaliphilic Alkaliphilus transvaalensis. The molecular mass was 30 kDa on sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The enzyme showed the maximal caseinolytic activity higher than pH 12.6 in KCl–NaOH buffer at 40°C. Hydrolysis of the oxidized insulin B-chain followed by mass spectrometric analysis of the cleaved products revealed that as many as 24 of the total 29 peptide bonds are hydrolyzed in a block-cutting manner, suggesting that ALTP has a widespread proteolytic functions. Calcium ion had no effect on the activity and stability of ALTP, unlike known subtilisins. The deduced amino acid sequence of the enzyme comprised 279 amino acids plus 97 prepropeptide amino acids. The amino acid sequence of mature ALTP was confirmed by capillary liquid chromatography coupled to tandem mass spectrometry, which was the 93% coverage of the deduced amino acid sequence. The mature enzyme showed moderate homology to subtilisin LD1 from the alkaliphilic Bacillus sp. strain KSM-LD1 with 64% identity, and both enzymes formed a new subcluster at an intermediate position among true subtilisins and high-alkaline proteases in a phylogenetic tree of subtilase family A. ALTP is the first high-alkaline protease reported from a strict anaerobe in this family.     

Critical negative regulation of type 1 T cell immunity and immunopathology by signaling adaptor DAP12 during intracellular infection

Transmembrane signaling adaptor DAP12 has increasingly been recognized for its important role in innate responses. However, its role in the regulation of antimicrobial T cell responses has remained unknown. In our current study, we have examined host defense, T cell responses, and tissue immunopathology in models of intracellular infection established in wild-type and DAP12-deficient mice. During mycobacterial infection, lack of DAP12 leads to pronounced proinflammatory and Th1 cytokine responses, overactivation of Ag-specific CD4 and CD8 T cells of type 1 phenotype, and heightened immunopathology both in the lung and lymphoid organs. DAP12-deficient airway APC display enhanced NF-kappaB activation and cytokine responses upon TLR stimulation or mycobacterial infection in vitro. Of importance, adoptive transfer of Ag-loaded DAP12-deficient APC alone could lead to overactivation of transferred transgenic or endogenous wild-type T cells in vivo. We have further found that the immune regulatory role by DAP12 is not restricted only to intracellular bacterial infection, since lack of this molecule also leads to uncontrolled type 1 T cell activation and severe immunopathology and tissue injury during intracellular viral infection. Our study thus identifies DAP12 as an important novel immune regulatory molecule that acts, via APC, to control the level of antimicrobial type 1 T cell activation and immunopathology.     

Critical role of the Fc receptor gamma-chain on APCs in the development of allergen-induced airway hyperresponsiveness and inflammation

The FcR common gamma-chain (FcRgamma) is an essential component of the receptors FcepsilonRI, FcgammaRI, and FcgammaRIII, which are expressed on many inflammatory cell types. The role of these receptors in the initiation or maintenance of allergic inflammation has not been well defined. FcRgamma-deficient (FcRgamma(-/-)) and control (wild-type (WT)) mice were sensitized and subsequently challenged with OVA. Following sensitization and challenge to OVA, FcRgamma-deficient (FcRgamma(-/-)) mice developed comparable levels of IgE and IgG1 as WT mice. However, numbers of eosinophils, levels of IL-5, IL-13, and eotaxin in bronchoalveolar lavage fluid, and mononuclear cell (MNC) proliferative responses to OVA were significantly reduced, as was airway hyperresponsiveness (AHR) to inhaled methacholine. Reconstitution of FcRgamma(-/-) mice with whole spleen MNC from WT mice before sensitization restored development of AHR and the numbers of eosinophils in bronchoalveolar lavage fluid; reconstitution after sensitization but before OVA challenge only partially restored these responses. These responses were also restored when FcRgamma(-/-) mice received T cell-depleted MNC, T and B cell-depleted MNC, or bone marrow-derived dendritic cells before sensitization from FcR(+/+) or FcgammaRIII-deficient but not FcRgamma(-/-) mice. The expression levels of FcgammaRIV on bone marrow-derived dendritic cells from FcR(+/+) mice were found to be low. These results demonstrate that expression of FcRgamma, most likely FcgammaRI, on APCs is important during the sensitization phase for the development of allergic airway inflammation and AHR.     

Roles of chymase in stenosis occurring after polytetrafluoroethylene graft implantations

Chymase is an important enzyme for the generation of angiotensin (Ang) II and in the activation of transforming growth factor (TGF)-beta1. Therefore, chymase may be involved in the hemodialysis access dysfunction, which is caused by intimal hyperplasia that occurs after polytetrafluoroethylene (PTFE) graft implantations. Bilateral U-shaped PTFE grafts were placed between the femoral vein and artery in dogs. Chymase inhibitor (NK3201, 1 mg/kg per day, p.o.) treatments were initiated 3 days before the operation. After the implantation, the stenosis by neointima proliferation was most frequently observed in the venous side of the PTFE grafts. In the hyperplastic neointima, myofibroblasts were the main cellular components. On the other hand, fibroblasts only occupied cellular components in a much smaller proportion in the neointima. However, these cells seem to be rich in the properties of proliferation and migration. After PTFE graft implantations, extensive accumulations of chymase-positive mast cells were found mainly in the tissue surrounding the grafts. The Ang II- and TGF-beta-positive cells were found in an adjacent section that was in close proximity to the chymase-positive cells. In contrast, the AT(1) receptors, as well as TGF-beta type II receptors, were expressed either in the neointima or in the outside adventitia of the PTFE grafts. Chymase inhibitor treatment resulted in a reduction of chymase, Ang II and TGF-beta1 expression, leading to a significant inhibition of neointimal formation. These findings indicating that an increase of chymase via promoting Ang II and TGF-beta1 generation plays a pivotal role in the neointimal formation after the implantation of PTFE grafts and also suggesting that chymase inhibition may be a new strategy that can be used to prevent PTFE graft dysfunctions in clinical settings.     

Protein kinase C delta regulates the phosphorylation of heat shock protein 27 in human hepatocellular carcinoma

We have recently reported that attenuated phosphorylation of heat shock protein (HSP) 27 correlates with tumor progression in patients with hepatocellular carcinoma (HCC). In the present study, we investigated what kind of kinase regulates phosphorylation of HSP27 in human HCC-derived HuH7 cells. 12-O-tetradecanoylphorbol-13-acetate (TPA) and 1-oleoyl-2-acetylglycerol, direct activators of protein kinase C (PKC), markedly strengthened the phosphorylation of HSP27. Bisindorylmaleimide I, an inhibitor of PKC, suppressed the TPA-induced levels of HSP27 phosphorylation in addition to its basal levels. Knock down of PKCdelta suppressed HSP27 phosphorylation, as well as p38 mitogen-activated protein kinase (MAPK) phosphorylation. SB203580, an inhibitor of p38 MAPK, suppressed the TPA-induced HSP27 phosphorylation. Our results strongly suggest that activation of PKCdelta regulates the phosphorylation of HSP27 via p38 MAPK in human HCC.     

Design and synthesis of new potent dipeptidyl peptidase IV inhibitors with enhanced ex vivo duration

A series of 5beta-methylprolyl-2-cyanopyrrolidine analogs were synthesized and evaluated as DPP-IV inhibitors, and the duration of their ex vivo activity was assessed. Comparison of their potency and duration of action was done among three different species. The mode of binding was investigated, and the effect on the plasma glucose level was evaluated. Structure-activity relationships are also presented.     

Loss of calcineurin homologous protein-1 in chicken B lymphoma DT40 cells destabilizes Na+/H+ exchanger isoform-1 protein

NHE1/SLC9A1 is a ubiquitous isoform of vertebrate Na⁺/H⁺ exchangers (NHEs) functioning in maintaining intracellular concentrations of Na⁺ and H⁺ ions. Calcineurin homologous protein-1 (CHP1) binds to the hydrophilic region of NHE1 and regulates NHE1 activity but reportedly does not play a role in translocating NHE1 from the endoplasmic reticulum to the plasma membrane. However, an antiport function of NHE1 requiring CHP1 remains to be clarified. Here we established CHP1-deficient chicken B lymphoma DT40 cells by gene targeting to address CHP1 function. CHP1-deficient cells showed extensive decreases in Na⁺/H⁺ activities in intact cells. Although NHE1 mRNA levels were not affected, NHE1 protein levels were significantly reduced not only in the plasma membrane but in whole cells. The expression of a CHP1 transgene in CHP1-deficient cells rescued NHE1 protein expression. Expression of mutant forms of CHP1 defective in Ca²⁺ binding or myristoylation also partially decreased NHE1 protein levels. Knockdown of CHP1 also caused a moderate decrease in NHE1 protein in HeLa cells. These data indicate that CHP1 primarily plays an essential role in stabilization of NHE1 for reaching of NHE1 to the plasma membrane and its exchange activity. membrane protein; transporter; antiporter; quality control; degradation     

A new method of defense response analysis using a transient expression system in rice protoplasts

We established a new plant defense response assay using a transient expression system in rice protoplasts. The assay system sensitively detected defense induction by flagellin, which had previously been assigned to a specific elicitor. Our assay system provides a rapid and efficient way to dissect rice defense mechanisms.