Coordination chemical studies on metalloenzymes. II. Kinetic behavior of various types of chelating agents towards bovine carbonic anhydrase.

In order to investigate the kinetics and mechanism of the removal of zinc ions from bovine carbonic anhydrase [EC 4.2.1.1] (BCA), several chelating agents with various stability constants were used to remove zinc from BCA. The second-order rate constants (kaap) of zinc removal from BCA were found to be in the following order; 2,6-pyridinedicarboxylic acid greater than 2-pyridinecarboxylic acid greater than 2,4-pyridinedicarboxylic acid greater than 2,3-pyridinedicarboxylic acid greater than or approximately 1,10-phenanthroline greater than or approximately 5-methyl-1,10-phenanthroline greater than 2,2'-bipyridine. With similar chelating agents the greater the stability constant, the faster was the rate of removal of zinc ions from BCA. With EDTA, trans-1,2-cyclohexanediaminetetraacetic acid, and nitrilotriacetic acid, the rate of zinc ion removal from the native enzyme was governed by the rate of spontaneous dissociation of zinc enzyme. The rate constants for the removal of zinc ions from BCA were governed by the affinity of the chelating agents for the metal ion and the conformation of the chelating agents. Based on these findings, reaction pathways for various chelating agents are proposed.     

Nitrification and nitrogen accumulation in the early stages of primary succession on Mt. Fuji

The relationship between nitrification potential and nitrogen accumulation was studied in an early successional sere on Mt. Fuji. Soil organic nitrogen accumulated with the invasion ofPolygonum cuspidatum and successively withMiscanthus oligostachyus and other species. Laboratory incubation experiments showed a higher nitrification potential at theM. oligostachyus state. The numbers of nitrifying bacteria increased with the progress of succession. No significant difference in nitrate reductase activity was found between pioneer and succeeding species. The soil solution at theM. oligostachyus stage contained a lower level of nitrate than rainwater, while that of the bare ground and theP. cuspidatum stage contained a higher nitrate level than rainwater. It was concluded that the high nitrate levels in the soil solution of the bare ground and theP. cuspidatum stage were due to lower nitrate-absorbing activity, leading to loss of nitrogen with precipitation, while the lower nitrate levels at theM. oligostachyus stage when higher nitrification activity occurred were due to higher nitrate-absorbing activity, preventing net loss of nitrogen from the ecosystem.     

Role of Nrf2 in host defense against influenza virus in cigarette smoke-exposed mice

Influenza virus is a common respiratory tract viral infection. Although influenza can be fatal in patients with chronic pulmonary diseases such as chronic obstructive pulmonary disease, its pathogenesis is not fully understood. The Nrf2-mediated antioxidant system is essential to protect the lungs from oxidative injury and inflammation. In the present study, we investigated the role of Nrf2 in protection against influenza virus-induced pulmonary inflammation after cigarette smoke exposure with both in vitro and in vivo approaches. For in vitro analyses, peritoneal macrophages isolated from wild-type and Nrf2-deficient mice were treated with poly(I:C) and/or cigarette smoke extract. For in vivo analysis, these mice were infected with influenza A virus with or without exposure to cigarette smoke. In Nrf2-deficient macrophages, NF-κB activation and the induction of its target inflammatory genes were enhanced after costimulation with cigarette smoke extract and poly(I:C) compared with wild-type macrophages. The induction of antioxidant genes was observed for the lungs of wild-type mice but not those of Nrf2-deficient mice after cigarette smoke exposure. Cigarette smoke-exposed Nrf2-deficient mice showed higher rates of mortality than did wild-type mice after influenza virus infection, with enhanced peribronchial inflammation, lung permeability damage, and mucus hypersecretion. Lung oxidant levels and NF-κB-mediated inflammatory gene expression in the lungs were also enhanced in Nrf2-deficient mice. Our data indicate that the antioxidant pathway controlled by Nrf2 is pivotal for protection against the development of influenza virus-induced pulmonary inflammation and injury under oxidative conditions.     

Characterization of phytochelatin synthase produced by the primitive red alga Cyanidioschyzon merolae

Phytochelatins (PCs), non-protein peptides with the general structure [(γ-Glu-Cys)n-Gly (n≥ 2)], are involved in the detoxification of toxic heavy metals mainly in higher plants. The synthesis of the peptides is mediated by phytochelatin synthase (PCS), which is activated by a range of heavy metals. CmPCS, a PCS-like gene found in the genomic DNA of the primitive red alga Cyanidioschyzon merolae, was isolated and a recombinant protein (rCmPCS) fused with a hexahistidine tag at the N-terminus of CmPCS was produced. The finding that this protein mediated PC synthesis from glutathione in a metal-dependent way clearly establishes that rCmPCS is functional. The maximum activity was attained at a reaction temperature of 50 °C, considerably higher than the temperature required for the maximal activity of PCS isolated from the higher plant Silene cucubalus, probably due to the alga being a thermophile. CmPCS showed optimal pH in a slightly higher region than higher plant PCSs, probably due to the less effective charge relay network in the catalytic triad. In addition, the pattern of enzyme activation by metal ions was specific to rCmPCS, with Ag+, Cu2+, and Hg2+ showing only limited activation. In contrast to other eukaryotic PCSs, CmPCS has an extra domain in the N-terminal region from residues 1 to 109, and contains fewer cysteine residues in the C-terminal domain. These differences may be responsible for the metal specificity of the activation of CmPCS. Although the enzyme preparation lost PCS activity progressively when stored at 4 °C, the inclusion of Cd2+ in the preparation effectively prevented the reduction of activity. Furthermore, Cd2+ effectively restored the activity of the inactivated enzyme. These results indicate that Cd2+ ions bind the enzyme to maintain the structural integrity of the peptides.     

Human DNA replication-related element binding factor (hDREF) self-association via hATC domain is necessary for its nuclear accumulation and DNA binding

We previously demonstrated that hDREF, a human homologue of Drosophila DNA replication-related element binding factor (dDREF), is a DNA-binding protein predominantly distributed with granular structures in the nucleus. Here, glutathione S-transferase pulldown and chemical cross-linking assays showed that the carboxyl-terminal hATC domain of hDREF, highly conserved among hAT transposase family members, possesses self-association activity. Immunoprecipitation analyses demonstrated that hDREF self-associates in vivo, dependent on hATC domain. Moreover, analyses using a series of hDREF mutants carrying amino acid substitutions in the hATC domain revealed that conserved hydrophobic amino acids are essential for self-association. Immunofluorescence studies further showed that all hDREF mutants lacking self-association activity failed to accumulate in the nucleus. Self-association-defective hDREF mutants also lost association with endogenous importin beta1. Moreover, electrophoretic gel-mobility shift assays revealed that the mutations completely abolished the DNA binding activity of hDREF. These results suggest that self-association of hDREF via the hATC domain is necessary for its nuclear accumulation and DNA binding. We also found that ZBED4/KIAA0637, another member of the human hAT family, also self-associates, again dependent on the hATC domain, with deletion resulting in loss of efficient nuclear accumulation. Thus, hATC domains of human hAT family members appear to have conserved functions in self-association that are required for nuclear accumulation.     

Microbial community diversity in the phycosphere of natural populations of the toxic alga, Alexandrium fundyense

The dinoflagellate Alexandrium fundyense is the major causative organism of paralytic shellfish poisoning in the Gulf of Maine. While laboratory studies have shown that A. fundyense population dynamics can be affected dramatically by co-occurring bacteria, little is known about these interactions in nature. Because A. fundyense is typically a minor Gulf of Maine phytoplankton community member, analyses of the bulk community cannot be used to address bacterium-A. fundyense associations. Therefore, an immunomagnetic bead method was used to selectively capture A. fundyense cells, and the bacteria attached to them, from complex natural samples. Bulk particle-associated and free-living bacterial communities were collected simultaneously. DNA was extracted from all sample types and subjected to 16S rRNA gene fragment amplification, denaturing gradient gel electrophoresis (DGGE) and sequence analysis. Ordination analysis of DGGE profiles confirmed that A. fundyense-associated bacteria community profiles were distinct from bulk bacterial community profiles, indicating selection of specific phylotypes in the A. fundyense phycosphere. Phylogenetic analyses confirmed that Alexandrium-associates were distinct from bulk particle-associated bacteria and that they included a greater prevalence and broader diversity of Gammaproteobacteria than previously thought to be associated with toxic algae. Phylogenetic groups known to be associated with dinoflagellates were also found, including members of the families Alteromonadaceae, Pseudoalteromonadaceae, Rhodobacteraceae and Flavobacteraceae.     

Molecular Dissection of the Rho-associated Protein Kinase (p160ROCK)-regulated Neurite Remodeling in Neuroblastoma N1E-115 Cells

A critical role for the small GTPase Rho and one of its targets, p160ROCK (a Rho-associated coiled coil-forming protein kinase), in neurite remodeling was examined in neuroblastoma N1E-115 cells. Using wild-type and a dominant-negative form of p160ROCK and a p160ROCK-specific inhibitor, Y-27632, we show here that p160ROCK activation is necessary and sufficient for the agonist-induced neurite retraction and cell rounding. The neurite retraction was accompanied by elevated phosphorylation of myosin light chain and the disassembly of the intermediate filaments and microtubules. Y-27632 blocked both neurite retraction and the elevation of myosin light chain phosphorylation in a similar concentration-dependent manner. On the other hand, suppression of p160ROCK activity by expression of a dominant-negative form of p160ROCK induced neurites in the presence of serum by inducing the reassembly of the intermediate filaments and microtubules. The neurite outgrowth by the p160ROCK inhibition was blocked by coexpression of dominant-negative forms of Cdc42 and Rac, indicating that p160ROCK constitutively and negatively regulates neurite formation at least in part by inhibiting activation of Cdc42 and Rac. The assembly of microtubules and intermediate filaments to form extended processes by inhibitors of the Rho–ROCK pathway was also observed in Swiss 3T3 cells. These results indicate that Rho/ROCK-dependent tonic inhibition of cell process extension is exerted via activation of the actomysin-based contractility, in conjunction with a suppression of assembly of intermediate filaments and microtubules in many cell types including, but not exclusive to, neuronal cells.