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131.
Koji Ohashi Takashi Enomoto Yusuke Joki Rei Shibata Yasuhiro Ogura Yoshiyuki Kataoka Yuuki Shimizu Takahiro Kambara Yusuke Uemura Daisuke Yuasa Kazuhiro Matsuo Satoko Hayakawa Mizuho Hiramatsu-Ito Toyoaki Murohara Noriyuki Ouchi 《The Journal of biological chemistry》2014,289(20):14132-14144
Strategies to stimulate revascularization are valuable for cardiovascular diseases. Here we identify neuron-derived neurotrophic factor (NDNF)/epidermacan as a secreted molecule that is up-regulated in endothelial cells in ischemic limbs of mice. NDNF was secreted from cultured human endothelial cells, and its secretion was stimulated by hypoxia. NDNF promoted endothelial cell network formation and survival in vitro through activation of Akt/endothelial NOS (eNOS) signaling involving integrin αvβ3. Conversely, siRNA-mediated knockdown of NDNF in endothelial cells led to reduction of cellular responses and basal Akt signaling. Intramuscular overexpression of NDNF led to enhanced blood flow recovery and capillary density in ischemic limbs of mice, which was accompanied by enhanced phosphorylation of Akt and eNOS. The stimulatory actions of NDNF on perfusion recovery in ischemic muscles of mice were abolished by eNOS deficiency or NOS inhibition. Furthermore, siRNA-mediated reduction of NDNF in muscles of mice resulted in reduction of perfusion recovery and phosphorylation of Akt and eNOS in response to ischemia. Our data indicate that NDNF acts as an endogenous modulator that promotes endothelial cell function and ischemia-induced revascularization through eNOS-dependent mechanisms. Thus, NDNF can represent a therapeutic target for the manipulation of ischemic vascular disorders. 相似文献
132.
Yoshimitsu Hamano Naoko Kito Akihiro Kita Yuuki Imokawa Kazuya Yamanaka Chitose Maruyama Hajime Katano 《Applied and environmental microbiology》2014,80(16):4993-5000
ε-Poly-l-lysine (ε-PL), consisting of 25 to 35 l-lysine residues with linkages between the α-carboxyl groups and ε-amino groups, is produced by Streptomyces albulus NBRC14147. ε-PL synthetase (Pls) is a membrane protein with six transmembrane domains (TM1 to TM6) as well as both an adenylation domain and a thiolation domain, characteristic of the nonribosomal peptide synthetases. Pls directly generates ε-PL chain length diversity (25- to 35-mer), but the processes that control the chain length of ε-PL during the polymerization reaction are still not fully understood. Here, we report on the identification of Pls amino acid residues involved in the regulation of the ε-PL chain length. From approximately 12,000 variants generated by random mutagenesis, we found 8 Pls variants that produced shorter chains of ε-PL. These variants have one or more mutations in two linker regions connecting the TM1 and TM2 domains and the TM3 and TM4 domains. In the Pls catalytic mechanism, the growing chain of ε-PL is not tethered to the enzyme, implying that the enzyme must hold the growing chain until the polymerization reaction is complete. Our findings reveal that the linker regions are important contributors to grasp the growing chain of ε-PL. 相似文献
133.
Takumi Ota Masao Doi Fumiyoshi Yamazaki Daisuke Yarimizu Kazuki Okada Iori Murai Hida Hayashi Sumihiro Kunisue Yuuki Nakagawa Hitoshi Okamura 《Molecular and cellular biology》2014,34(20):3880-3894
The 3β-hydroxysteroid dehydrogenase (3β-HSD) is an enzyme crucial for steroid synthesis. Two different 3β-HSD isoforms exist in humans. Classically, HSD3B2 was considered the principal isoform present in the adrenal. However, we recently showed that the alternative isoform, HSD3B1, is expressed specifically within the adrenal zona glomerulosa (ZG), where aldosterone is produced, raising the question of why this isozyme needs to be expressed in this cell type. Here we show that in both human and mouse, expression of the ZG isoform 3β-HSD is rapidly induced upon angiotensin II (AngII) stimulation. AngII is the key peptide hormone regulating the capacity of aldosterone synthesis. Using the human adrenocortical H295R cells as a model system, we show that the ZG isoform HSD3B1 differs from HSD3B2 in the ability to respond to AngII. Mechanistically, the induction of HSD3B1 involves de novo protein synthesis of the nuclear orphan receptors NGFIB and NURR1. The HSD3B1 promoter contains a functional NGFIB/NURR1-responsive element to which these proteins bind in response to AngII. Knockdown of these proteins and overexpression of a dominant negative NGFIB both reduce the AngII responsiveness of HSD3B1. Thus, the AngII-NGFIB/NURR1 pathway controls HSD3B1. Our work reveals HSD3B1 as a new regulatory target of AngII. 相似文献
134.
Jian Xu Kaito Yoshimura Hiroaki Mon Zhiqing Li Li Zhu Kazuhiro Iiyama Takahiro Kusakabe Jae Man Lee 《Molecular biotechnology》2014,56(3):193-198
The systemic RNA interference defective-1 (SID-1) can transport double-stranded RNA (dsRNA) into cytosol across the cytoplasmic membrane. We report here that ectopic expression of Caenorhabditis elegans SID-1 allows BmN4 cells to import extracellular plasmid dsDNA into cells via the direct soaking method. Interestingly, BmN4-SID1 cells incorporate dsRNA and plasmid DNA simultaneously. Furthermore, the ectopic SID-1 allows us to establish a stably transformed cell line by the simple soaking method. Our results provide an alternative method for silkworm gene function analysis with low cost and low cell toxicity. 相似文献
135.
Staphylococcus aureus forms giant colonies on soft-agar surfaces, which is called colony-spreading. In the present study, we searched for host factors that influence S. aureus colony-spreading activity. The addition of calf serum, porcine serum, or silkworm hemolymph to soft-agar medium stimulated S. aureus colony-spreading activity. Gel filtration column chromatography of calf serum produced a high molecular weight fraction and a low molecular weight fraction, both of which exhibited colony-spreading stimulatory activity. In the low molecular weight fraction, we identified the stimulatory factor as bovine serum albumin. The stimulatory fraction in the high molecular weight fraction was identified as high-density lipoprotein (HDL) particles. Delipidation of HDL abolished the stimulatory activity of HDL. Phosphatidylcholine, which is the major lipid component in HDL particles, stimulated the colony-spreading activity. Other phosphatidylcholine-containing lipoprotein particles, low-density lipoprotein and very low-density lipoprotein, also showed colony-spreading stimulatory activity. These findings suggest that S. aureus colony-spreading activity is stimulated by albumin and lipoprotein particles in mammalian serum. 相似文献
136.
Miyashita A Iyoda S Ishii K Hamamoto H Sekimizu K Kaito C 《FEMS microbiology letters》2012,333(1):59-68
Studies of enterohemorrhagic Escherichia coli (EHEC) infection mechanisms using mammals require large numbers of animals and are both costly and associated with ethical problems. Here, we evaluated the pathogenic mechanisms of EHEC in the silkworm model. Injection of a clinically isolated EHEC O157:H7 Sakai into either the silkworm hemolymph or intraperitoneal fluid of mice killed the host animals. EHEC O157:H7 Sakai deletion mutants of the rfbE gene, which encodes perosamine synthetase, a monosaccharide component synthetase of the O-antigen, or deletion mutants of the waaL gene, which encodes O-antigen ligase against the lipid A-core region of lipopolysaccharide (LPS), had attenuated killing ability in both silkworms and mice. Introduction of the rfbE gene or the waaL gene into the respective mutants restored the killing ability in silkworms. Growth of both mutants was inhibited by a major antimicrobial peptide in the silkworm hemolymph, moricin. The viability of both mutants was decreased in swine serum. The bactericidal effect of swine serum against both mutants was inactivated by heat treatment. These findings suggest that the LPS O-antigen of EHEC O157:H7 plays an important defensive role against antimicrobial factors in the host body fluid and is thus essential to the lethal effects of EHEC in animals. 相似文献
137.
Seiya Watanabe Daichi Morimoto Fumiyasu Fukumori Hiroto Shinomiya Hisashi Nishiwaki Miyuki Kawano-Kawada Yuuki Sasai Yuzuru Tozawa Yasuo Watanabe 《The Journal of biological chemistry》2012,287(39):32674-32688
l-Hydroxyproline (4-hydroxyproline) mainly exists in collagen, and most bacteria cannot metabolize this hydroxyamino acid. Pseudomonas putida and Pseudomonas aeruginosa convert l-hydroxyproline to α-ketoglutarate via four hypothetical enzymatic steps different from known mammalian pathways, but the molecular background is rather unclear. Here, we identified and characterized for the first time two novel enzymes, d-hydroxyproline dehydrogenase and Δ1-pyrroline-4-hydroxy-2-carboxylate (Pyr4H2C) deaminase, involved in this hypothetical pathway. These genes were clustered together with genes encoding other catalytic enzymes on the bacterial genomes. d-Hydroxyproline dehydrogenases from P. putida and P. aeruginosa were completely different from known bacterial proline dehydrogenases and showed similar high specificity for substrate (d-hydroxyproline) and some artificial electron acceptor(s). On the other hand, the former is a homomeric enzyme only containing FAD as a prosthetic group, whereas the latter is a novel heterododecameric structure consisting of three different subunits (α4β4γ4), and two FADs, FMN, and [2Fe-2S] iron-sulfur cluster were contained in αβγ of the heterotrimeric unit. These results suggested that the l-hydroxyproline pathway clearly evolved convergently in P. putida and P. aeruginosa. Pyr4H2C deaminase is a unique member of the dihydrodipicolinate synthase/N-acetylneuraminate lyase protein family, and its activity was competitively inhibited by pyruvate, a common substrate for other dihydrodipicolinate synthase/N-acetylneuraminate lyase proteins. Furthermore, disruption of Pyr4H2C deaminase genes led to loss of growth on l-hydroxyproline (as well as d-hydroxyproline) but not l- and d-proline, indicating that this pathway is related only to l-hydroxyproline degradation, which is not linked to proline metabolism. 相似文献
138.
We previously identified the cvfA gene (SA1129) as a novel virulence regulator in Staphylococcus aureus using the silkworm infection model. The cvfA gene, which is conserved among various pathogenic bacteria, contributes to the expression of the agr locus, a global virulence regulator that controls the expression of genes encoding various exoproteins, such as hemolysin. CvfA protein has a transmembrane domain, an RNA binding domain (KH domain), and a metal-dependent phosphohydrolase domain (HD domain). We report here the purification of recombinant CvfA protein from a membrane fraction of Escherichia coli by measuring its phosphodiesterase activity. Purified CvfA protein hydrolyzed the phosphodiester linkage of 2',3'-cyclic AMP, 2',3'-cyclic GMP, and 2',3'-cyclic phosphate at the 3'-terminal of RNA in the presence of Mn(2+). CvfA mutant proteins with amino acid substitutions in the HD domain had significantly decreased phosphodiesterase activity. Furthermore, mutated cvfA genes encoding proteins with low phosphodiesterase activity did not complement the decreased hemolysin production or the attenuated killing ability against silkworms in the cvfA deletion mutant. These results suggest that the phosphodiesterase activity of CvfA protein is required for virulence in S. aureus. 相似文献
139.
Chikara Kaito Yosuke Omae Yasuhiko Matsumoto Makiko Nagata Hiroki Yamaguchi Taiji Aoto Teruyo Ito Keiichi Hiramatsu Kazuhisa Sekimizu 《PloS one》2008,3(12)
Staphylococcus aureus colonies can spread on soft agar plates. We compared colony spreading of clinically isolated methicillin-sensitive S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA). All MSSA strains showed colony spreading, but most MRSA strains (73%) carrying SCCmec type-II showed little colony spreading. Deletion of the entire SCCmec type-II region from these MRSA strains restored colony spreading. Introduction of a novel gene, fudoh, carried by SCCmec type-II into Newman strain suppressed colony spreading. MRSA strains with high spreading ability (27%) had no fudoh or a point-mutated fudoh that did not suppress colony spreading. The fudoh-transformed Newman strain had decreased exotoxin production and attenuated virulence in mice. Most community-acquired MRSA strains carried SCCmec type-IV, which does not include fudoh, and showed high colony spreading ability. These findings suggest that fudoh in the SCCmec type-II region suppresses colony spreading and exotoxin production, and is involved in S. aureus pathogenesis. 相似文献
140.
We collected two gobies, Apocryptodon punctatus and Acentrogobius sp. A, and four alpheid shrimps from an estuary in southern Japan, to identify the symbiotic shrimps with gobies and to understand their partner specificity and dependency. Analysis based on generalized linear models identified two specific partners for each goby. A. punctatus frequently coexisted with partners and showed similar habitats, suggesting that this goby might be an obligate mutualist. Acentrogobius sp. A had lower rates of coexistence and appeared in habitats different from one of its partners, suggesting that this goby might be a facultative mutualist. 相似文献