A functional model for pMMO (particulate methane monooxygenase): Hydroxylation of alkanes with H2O2 catalyzed by beta-diketiminatocopper(II) complexes

The reaction of copper(II) complexes supported by a series of beta-diketiminate ligands ((R1,R2)L, [(Dipp)N-C(R(2))-C(R(1))-C(R(2))-N(Dipp)](-), Dipp=2,6-diisopropylphenyl; see ) and H(2)O(2) has been examined spectroscopically at a low temperature. The beta-diketiminatocopper(II) complexes with R(2)=H (no substituent on the beta-carbon) provided a copper-oxygen intermediate that exhibited the same spectroscopic features as those of the bis(mu-oxo)dicopper(III) complex generated by the reaction of corresponding beta-diketiminatocopper(I) complex and O(2). On the other hand, the beta-diketiminatocopper(II) complexes with methyl substituent on the beta-carbon (R(2)=Me) did not produce such an intermediate in the same reaction. The beta-diketiminatocopper(II) complexes carrying an electron-withdrawing substituent on the alpha-carbon (R(1)=NO(2) or CN) but no beta-substituent (R(2)=H) exhibited a high catalytic activity in the oxygenation reaction of alkanes with H(2)O(2). Mechanism of the catalytic oxygenation reaction as well as the substituent effects of the ligands on the copper(II)-H(2)O(2) reactivity is discussed.     

Role of Shiga toxin 2 (Stx2)-binding protein,human serum amyloid P component (HuSAP), in Shiga toxin-producing Escherichia coli infections: assumption from in vitro and in vivo study using HuSAP and anti-Stx2 humanized monoclonal antibody TMA-15

Shiga toxin 2 (Stx2) is a major pathogenic factor in Shiga toxin-producing Escherichia coli (STEC) infections. Some factor that neutralizes Stx2 in vitro had been shown to be specifically present in human serum and we recently identified it as human serum amyloid P component (HuSAP). Here, we report the role of HuSAP in STEC infections. HuSAP could not rescue Stx2-challenged mice from death, and it instead reduced the efficacy of the Stx2-neutralizing humanized monoclonal antibody TMA-15 when a lower dose of TMA-15 was injected to the mice. By contrast, the efficacy of TMA-15 at a higher dose was uninfluenced by the presence of HuSAP. These findings suggest that HuSAP acts as a carrier protein of Stx2 rather than as a Stx2-neutralizing factor in the human circulation and that passive immune therapy with Stx2-neutralizing antibodies such as TMA-15 is useful to prevent severe complications associated with STEC infections even in the presence of HuSAP.     

Vasoactive intestinal peptide stimulates mucus secretion, but nitric oxide has no effect on mucus secretion in the ferret trachea.

Vasoactive intestinal peptide (VIP) and nitric oxide (NO) are neurotransmitters involved in the regulation of bronchial and pulmonary vascular tone. Published studies of the effects of VIP on airway mucus secretion have yielded conflicting results. The purpose of this study was to determine the effect of VIP on mucus secretion in the ferret trachea and if this effect was influenced by NO. We used a sandwich enzyme-linked lectin assay to measure mucin secretion and a turbidimetric assay to measure lysozyme (serous cell) secretion from ferret tracheal segments. VIP (10(-7) M) increased mucin secretion over 2 h. VIP (10(-9) to 10(-5) M) stimulated mucin secretion in a dose-dependent fashion. VIP-induced mucin secretion was partially blocked by a VIP receptor antagonist (a chimeric VIP-pituitary adenylate cyclase-activating peptide analog, VIP receptor antagonist) at a 10-fold excess concentration. At all concentrations tested, neither NG-nitro-L-arginine methyl ester, an inhibitor of NO synthase, nor S-nitroso-N-acetyl-penicillamine, an NO donor, had any significant effect on constitutive or VIP-induced mucus secretion. We conclude that VIP-stimulated mucin and lysozyme secretion was both time dependent and dose dependent and that NO neither stimulates nor inhibits mucus secretion in the ferret trachea.     

Partial amino-acid sequence of ECP31, a carrot embryogenic-cell protein,and enhancement of its accumulation by abscisic acid in somatic embryos

ECP31, an embryogenic-cell protein from carrot (Daucus carota L.), was purified by sequential column-chromatographic steps and digested by V8 protease on a nitrocellulose membrane. The resultant peptides were separated by reverse-phased column chromatography and sequenced. The sequences obtained were 70–80% homologous to those of a late-embryogenesis-abundant protein (D34) from cotton (Baker et al, 1988, Plant Mol. Biol. 11, 227–291). The level of ECP31 in somatic embryos of carrot was increased by treatment of the embryos with 3.7 · 10^–6 M abscisic acid (ABA) for 48 h, and there was no change in this enhanced level for up to 192 h in the presence of ABA. No similar enhancing effect of ABA was observed on the level of ECP31 in embryogenic callus or segments of carrot hypocotyls. In an immunohistochemical analysis, ECP31 was found in epidermal tissue and in the vascular system of ABA-treated somatic embryos.Abbreviations ABA abscisic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - LEA protein late-embryogenesis-abundant protein To whom correspondence should be addressedThis work was supported in part by a grant-in-aid for Special Research in Priority Areas (Project No. 02242102) from the Ministry of Education, Science and Culture, Japan, and by Special Coordination Funds of the Science and Technology Agency of the Japanese Government.     

Mammalian Bcnt/Cfdp1, a potential epigenetic factor characterized by an acidic stretch in the disordered N-terminal and Ser250 phosphorylation in the conserved C-terminal regions

The BCNT (Bucentaur) superfamily is classified by an uncharacteristic conserved sequence of ∼80 amino acids (aa) at the C-terminus, BCNT-C (the conserved C-terminal region of Bcnt/Cfdp1). Whereas the yeast Swc5 and Drosophila Yeti homologues play crucial roles in chromatin remodelling organization, mammalian Bcnt/Cfdp1 (craniofacial developmental protein 1) remains poorly understood. The protein, which lacks cysteine, is largely disordered and comprises an acidic N-terminal region, a lysine/glutamic acid/proline-rich 40 aa sequence and BCNT-C. It shows complex mobility on SDS/PAGE at ∼50 kDa, whereas its calculated molecular mass is ∼33 kDa. To characterize this mobility discrepancy and the effects of post-translational modifications (PTMs), we expressed various deleted His–Bcnt in E. coli and HEK cells and found that an acidic stretch in the N-terminal region is a main cause of the gel shift. Exogenous BCNT/CFDP1 constitutively expressed in HEK clones appears as a doublet at 49 and 47 kDa, slower than the protein expressed in Escherichia coli but faster than the endogenous protein on SDS/PAGE. Among seven in vivo phosphorylation sites, Ser²⁵⁰, which resides in a region between disordered and ordered regions in BCNT-C, is heavily phosphorylated and detected predominantly in the 49 kDa band. Together with experiments involving treatment with phosphatases and Ser²⁵⁰ substitutions, the results indicate that the complex behaviour of Bcnt/Cfdp1 on SDS/PAGE is caused mainly by an acidic stretch in the N-terminal region and Ser²⁵⁰ phosphorylation in BCNT-C. Furthermore, Bcnt/Cfdp1 is acetylated in vitro by CREB-binding protein (CBP) and four lysine residues including Lys²⁶⁸ in BCNT-C are also acetylated in vivo, revealing a protein regulated at multiple levels.     

Identification of a TPX2-like microtubule-associated protein in Drosophila

Chromosome segregation during mitosis and meiosis relies on the spindle and the functions of numerous microtubule-associated proteins (MAPs). One of the best-studied spindle MAPs is the highly conserved TPX2, which has been reported to have characteristic intracellular dynamics and molecular activities, such as nuclear localisation in interphase, poleward movement in the metaphase spindle, microtubule nucleation, microtubule stabilisation, microtubule bundling, Aurora A kinase activation, kinesin-5 binding, and kinesin-12 recruitment. This protein has been shown to be essential for spindle formation in every cell type analysed so far. However, as yet, TPX2 homologues have not been found in the Drosophila genome. In this study, I found that the Drosophila protein Ssp1/Mei-38 has significant homology to TPX2. Sequence conservation was limited to the putative spindle microtubule-associated region of TPX2, and intriguingly, D-TPX2 (Ssp1/Mei-38) lacks Aurora A- and kinesin-5-binding domains, which are highly conserved in other animal and plant species, including many insects such as ants and bees. D-TPX2 uniformly localised to kinetochore microtubule-enriched regions of the metaphase spindle in the S2 cell line, and it had microtubule binding and bundling activities in vitro. In comparison with other systems, the contribution of D-TPX2 to cell division seems to be minor; live cell imaging of microtubules and chromosomes after RNAi knockdown identified significant delay in chromosome congression in only 18% of the cells. Thus, while this conserved spindle protein is present in Drosophila, other mechanisms may largely compensate for its spindle assembly and chromosome segregation functions.     

The C-type lectin receptor Clec1A plays an important role in the development of experimental autoimmune encephalomyelitis by enhancing antigen presenting ability of dendritic cells and inducing inflammatory cytokine IL-17

Clec1A, a member of C-type lectin receptor family, has a carbohydrate recognition domain in its extracellular region, but no known signaling motif in the cytoplasmic domain. Clec1a is highly expressed in endothelial cells and weakly in dendritic cells. Although this molecule was reported to play an important role in the host defense against Aspergillus fumigatus by recognizing 1,8-dihydroxynaphthalene-melanin on the fungal surface, the roles of this molecule in un-infected animals remain to be elucidated. In this study, we found that Clec1a^−/− mice develop milder symptoms upon induction of experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. The maximum disease score was significantly lower, and demyelination and inflammation of the spinal cord were much milder in Clec1a^−/− mice compared to wild-type mice. No abnormality was detected in the immune cell composition in the draining lymph nodes and spleen on day 10 and 16 after EAE induction. Recall memory T cell proliferation after restimulation with myelin oligodendrocyte glycoprotein peptide (MOG_35–55) in vitro was decreased in Clec1a^−/− mice, and antigen presenting ability of Clec1a^−/− dendritic cells was impaired. Interestingly, RNA-Seq and RT-qPCR analyses clearly showed that the expression of inflammatory cytokines including Il17a, Il6 and Il1b was greatly decreased in Clec1a^−/− mice after induction of EAE, suggesting that this reduced cytokine production is responsible for the amelioration of EAE in Clec1a^−/− mice. These observations suggest a novel function of Clec1A in the immune system.     

Dysregulation of suppressor of cytokine signaling 3 in keratinocytes causes skin inflammation mediated by interleukin-20 receptor-related cytokines

Homeostatic regulation of epidermal keratinocytes is controlled by the local cytokine milieu. However, a role for suppressor of cytokine signaling (SOCS), a negative feedback regulator of cytokine networks, in skin homeostasis remains unclear. Keratinocyte specific deletion of Socs3 (Socs3 cKO) caused severe skin inflammation with hyper-production of IgE, epidermal hyperplasia, and S100A8/9 expression, although Socs1 deletion caused no inflammation. The inflamed skin showed constitutive STAT3 activation and up-regulation of IL-6 and IL-20 receptor (IL-20R) related cytokines, IL-19, IL-20 and IL-24. Disease development was rescued by deletion of the Il6 gene, but not by the deletion of Il23, Il4r, or Rag1 genes. The expression of IL-6 in Socs3 cKO keratinocytes increased expression of IL-20R-related cytokines that further facilitated STAT3 hyperactivation, epidermal hyperplasia and neutrophilia. These results demonstrate that skin homeostasis is strictly regulated by the IL-6-STAT3-SOCS3 axis. Moreover, the SOCS3-mediated negative feedback loop in keratinocytes has a critical mechanistic role in the prevention of skin inflammation caused by hyperactivation of STAT3.