Hyperthermophilic DNA methyltransferase M.PabI from the archaeon Pyrococcus abyssi

Genome sequence comparisons among multiple species of Pyrococcus, a hyperthermophilic archaeon, revealed a linkage between a putative restriction-modification gene complex and several large genome polymorphisms/rearrangements. From a region apparently inserted into the Pyrococcus abyssi genome, a hyperthermoresistant restriction enzyme [PabI; 5'-(GTA/C)] with a novel structure was discovered. In the present work, the neighboring methyltransferase homologue, M.PabI, was characterized. Its N-terminal half showed high similarities to the M subunit of type I systems and a modification enzyme of an atypical type II system, M.AhdI, while its C-terminal half showed high similarity to the S subunit of type I systems. M.PabI expressed within Escherichia coli protected PabI sites from RsaI, a PabI isoschizomer. M.PabI, purified following overexpression, was shown to generate 5'-GTm6AC, which provides protection against PabI digestion. M.PabI was found to be highly thermophilic; it showed methylation at 95 degrees C and retained at least half the activity after 9 min at 95 degrees C. This hyperthermophilicity allowed us to obtain activation energy and other thermodynamic parameters for the first time for any DNA methyltransferases. We also determined the kinetic parameters of kcat, Km, DNA, and Km, AdoMet. The activity of M.PabI was optimal at a slightly acidic pH and at an NaCl concentration of 200 to 500 mM and was inhibited by Zn2+ but not by Mg2+, Ca2+, or Mn2+. These and previous results suggest that this unique methyltransferase and PabI constitute a type II restriction-modification gene complex that inserted into the P. abyssi genome relatively recently. As the most thermophilic of all the characterized DNA methyltransferases, M.PabI may help in the analysis of DNA methylation and its application to DNA engineering.     

Liberation of zinc-containing L31 (RpmE) from ribosomes by its paralogous gene product, YtiA, in Bacillus subtilis

We have found that alternative localization of two types of L31 ribosomal protein, RpmE and YtiA, is controlled by the intracellular concentration of zinc in Bacillus subtilis. The detailed mechanisms for the alternation of L31 proteins under zinc-deficient conditions were previously unknown. To obtain further information about this regulatory mechanism, we have studied the stability of RpmE in vivo and the binding affinity of these proteins to ribosomes in vitro, and we have found that liberation of RpmE from ribosomes is triggered by the expression of ytiA, which is induced by the derepression of Zur under zinc-deficient conditions.     

Atomic structure of the sweet-tasting protein thaumatin I at pH 8.0 reveals the large disulfide-rich region in domain II to be sensitive to a pH change

Thaumatin, an intensely sweet-tasting plant protein, elicits a sweet taste at 50 nM. Although the sweetness remains when thaumatin is heated at 80 °C for 4 h under acid conditions, it rapidly declines when heating at a pH above 6.5. To clarify the structural difference at high pH, the atomic structure of a recombinant thaumatin I at pH 8.0 was determined at a resolution of 1.0 Å. Comparison to the crystal structure of thaumatin at pH 7.3 and 7.0 revealed the root-mean square deviation value of a Cα atom to be substantially greater in the large disulfide-rich region of domain II, especially residues 154–164, suggesting that a loop region in domain II to be affected by solvent conditions. Furthermore, B-factors of Lys137, Lys163, and Lys187 were significantly affected by pH change, suggesting that a striking increase in the mobility of these lysine residues, which could facilitate a reaction with a free sulfhydryl residue produced via the β-elimination of disulfide bonds by heating at a pH above 7.0. The increase in mobility of lysine residues as well as a loop region in domain II might play an important role in the heat-induced aggregation of thaumatin above pH 7.0.     

Association of major histocompatibility complex class I haplotypes with disease progression after simian immunodeficiency virus challenge in burmese rhesus macaques

Nonhuman primate AIDS models are essential for the analysis of AIDS pathogenesis and the evaluation of vaccine efficacy. Multiple studies on human immunodeficiency virus and simian immunodeficiency virus (SIV) infection have indicated the association of major histocompatibility complex class I (MHC-I) genotypes with rapid or slow AIDS progression. The accumulation of macaque groups that share not only a single MHC-I allele but also an MHC-I haplotype consisting of multiple polymorphic MHC-I loci would greatly contribute to the progress of AIDS research. Here, we investigated SIVmac239 infections in four groups of Burmese rhesus macaques sharing individual MHC-I haplotypes, referred to as A, E, B, and J. Out of 20 macaques belonging to A(+) (n = 6), E(+) (n = 6), B(+) (n = 4), and J(+) (n = 4) groups, 18 showed persistent viremia. Fifteen of them developed AIDS in 0.5 to 4 years, with the remaining three at 1 or 2 years under observation. A(+) animals, including two controllers, showed slower disease progression, whereas J(+) animals exhibited rapid progression. E(+) and B(+) animals showed intermediate plasma viral loads and survival periods. Gag-specific CD8(+) T-cell responses were efficiently induced in A(+) animals, while Nef-specific CD8(+) T-cell responses were in A(+), E(+), and B(+) animals. Multiple comparisons among these groups revealed significant differences in survival periods, peripheral CD4(+) T-cell decline, and SIV-specific CD4(+) T-cell polyfunctionality in the chronic phase. This study indicates the association of MHC-I haplotypes with AIDS progression and presents an AIDS model facilitating the analysis of virus-host immune interaction.     

Complete genome sequence of Sphingobium sp. strain SYK-6, a degrader of lignin-derived biaryls and monoaryls

Sphingobium sp. strain SYK-6 is able to grow on an extensive variety of lignin-derived biaryls and monoaryls, and the catabolic genes for these compounds are useful for the production of industrially valuable metabolites from lignin. Here we report the complete nucleotide sequence of the SYK-6 genome which consists of the 4,199,332-bp-long chromosome and the 148,801-bp-long plasmid.     

Biocompatible and biodegradable dual-drug release system based on silk hydrogel containing silk nanoparticles

We developed a facile and quick ethanol-based method for preparing silk nanoparticles and then fabricated a biodegradable and biocompatible dual-drug release system based on silk nanoparticles and the molecular networks of silk hydrogels. Model drugs incorporated in the silk nanoparticles and silk hydrogels showed fast and constant release, respectively, indicating successful dual-drug release from silk hydrogel containing silk nanoparticles. The release behaviors achieved by this dual-drug release system suggest to be regulated by physical properties (e.g., β-sheet contents and size of the silk nanoparticles and network size of the silk hydrogels), which is an important advantage for biomedical applications. The present silk-based system for dual-drug release also demonstrated no significant cytotoxicity against human mesenchymal stem cells (hMSCs), and thus, this silk-based dual-drug release system has potential as a versatile and useful new platform of polymeric materials for various types of dual delivery of bioactive molecules.     

Crucial Involvement of Tumor-Associated Neutrophils in the Regulation of Chronic Colitis-Associated Carcinogenesis in Mice

Ulcerative colitis (UC) is a major form of chronic inflammation that can frequently progress to colon cancer. Several studies have demonstrated massive infiltration of neutrophils and macrophages into the lamina propria and submucosa in the progression of UC-associated colon carcinogenesis. Macrophages contribute to the development of colitis-associated colon cancer (CAC). However, the role of neutrophils is not well understood. To better understand the involvement of tumor-associated neutrophils (TANs) in the regulation of CAC, we used a mouse CAC model produced by administering azoxymethane (AOM), followed by repeated dextran sulfate sodium (DSS) ingestion. This causes severe colonic inflammation and subsequent development of multiple tumors in mice colon. We observed that colorectal mucosal inflammation became increasingly severe with AOM and DSS treatment. Macrophages infiltrated the lamina propria and submucosa, together with a marked increase in neutrophil infiltration. The chemokine CXCL2 increased in the lamina propria and submucosal regions of the colons of the treated mice, together with the infiltration of neutrophils expressing CXCR2, a specific receptor for CXCL2. This process was followed by neoplastic transformation. After AOM and DSS treatment, the mice showed enhanced production of metalloproteinase (MMP)-9 and neutrophil elastase (NE), accompanied by excessive vessel generation and cell proliferation. Moreover, CXCL2 promoted neutrophil recruitment and induced neutrophils to express MMP-9 and NE in vitro. Furthermore, administration of neutrophil-neutralizing antibodies after the last DSS cycle markedly reduced the number and size of tumors and decreased the expression of CXCR2, CXCL2, MMP-9, and NE. These observations indicate a crucial role for TANs in the initiation and progression of CAC and suggest that the CXCL2–CXCR2 axis might be useful in reducing the risk of UC-associated colon cancer.     

MURC, a muscle-restricted coiled-coil protein that modulates the Rho/ROCK pathway, induces cardiac dysfunction and conduction disturbance

We identified a novel muscle-restricted putative coiled-coil protein, MURC, which is evolutionarily conserved from frog to human. MURC was localized to the cytoplasm with accumulation in the Z-line of the sarcomere in the murine adult heart. MURC mRNA expression in the heart increased during the developmental process from the embryonic stage to adulthood. In response to pressure overload, MURC mRNA expression increased in the hypertrophied heart. Using the yeast two-hybrid system, we identified the serum deprivation response (SDPR) protein, a phosphatidylserine-binding protein, as a MURC-binding protein. MURC induced activation of the RhoA/ROCK pathway, which modulated serum response factor-mediated atrial natriuretic peptide (ANP) expression and myofibrillar organization. SDPR augmented MURC-induced transactivation of the ANP promoter in cardiomyocytes, and RNA interference of SDPR attenuated the action of MURC on the ANP promoter. Transgenic mice expressing cardiac-specific MURC (Tg-MURC) exhibited cardiac contractile dysfunction and atrioventricular (AV) conduction disturbances with atrial chamber enlargement, reduced thickness of the ventricular wall, and interstitial fibrosis. Spontaneous episodes of atrial fibrillation and AV block were observed in Tg-MURC mice. These findings indicate that MURC modulates RhoA signaling and that MURC plays an important role in the development of cardiac dysfunction and conduction disturbance with increased vulnerability to atrial arrhythmias.