Aim: To clarify whether there is any association between the extent of Chlamydia pneumoniae (C. pneumoniae) infection and plaque instability or post–directional coronary atherectomy (DCA) restenosis, we determined the frequency of C. pneumoniae infection and its localization in symptomatic coronary atherosclerotic plaques using specimens obtained from DCA. Methods and results: Immunohistochemistry (IHC) and real-time polymerase chain reaction (RT-PCR) revealed the existence of C. pneumoniae in all 50 specimens of coronary atherosclerotic plaques obtained by DCA. C. pneumoniae–positive cell ratio determined with IHC or copy numbers of C. pneumoniae DNA detected by RT-PCR did not differ significantly between patients with stable angina pectoris and those with acute coronary syndrome (IHC: 16.4 ± 7.6% vs 18.0 ± 7.1%, P = .42; RT-PCR: no. of cases with high copy numbers 12/25 vs 10/25, P = .78), or between patients with subsequent post-DCA restenosis and those without (IHC: 17.1 ± 8.0% vs 18.0 ± 7.4%, P = .74; RT-PCR: 5/12 vs 10/21, P = 1.00). Conclusions: C. pneumoniae was highly prevalent in coronary atherosclerotic plaques of patients who underwent DCA. However, the extent of C. pneumoniae infection in coronary atherosclerotic plaques was not associated with plaque instability or post-DCA restenosis.

Percutaneous devices—indwelling catheters—related infections are serious clinical incidents. It is accordingly necessary to develop anti-infective coating materials suitable for the devices for long-term effectiveness. In our research group, highly dispersible and crystalline hydroxyapatite (HAp) nanoparticles doped with metallic or halogen ions possessing antibacterial activities have been developed. In this study, antibacterial, dispersible, and crystalline zinc (Zn)-doped hydroxyapatite [Zn(15)-HAp] nanoparticles substituted with 13.5% Zn content [Zn/(Zn + Ca) × 100] were prepared by a wet chemical method using an anti-sintering agent through calcination. Antibacterial activities of Zn(15)-HAp nanoparticles were evaluated using Escherichia coli ( E. coli) and Staphylococcus aureus. The survival rates of the bacteria on Zn(15)-HAp nanoparticles were significantly lower than that on normal HAp (nHAp) coated surfaces, while no influences were observed on proliferation of L929 cells. Even after soaking Zn(15)-HAp nanoparticles in PBS for 2 weeks, the antibacterial activities against E. coli were maintained at a similar level to a 20 min soaking. The bacterial death was related to not only ion-exchange phenomenon between Zn and magnesium ions but also accumulation of reactive oxygen species (ROS) in the cells. Allergic-like reactions—anaphylactoid reactions—might not readily occur with Zn(15)-HAp nanoparticles because the amounts of histamine released from HMC-1 cells co-cultured with nanoparticles were not significantly different to that of nHAp, but were statistically much lower than that of chlorhexidine.

Abstract Halomonas species are halophilic and alkaliphilic bacteria, which exhibit potential for industrial production of a variety of chemicals, such as polyhydroxyalkanoates and ectoine, by fermentation because of their favorable characteristics, including high-density culturing capacity and low risk of contamination. However, genetic tools to modify the metabolism of Halomonas for suitable fermentation performance are limited. In this study, we developed two independent basic vectors for Halomonas, named pUCpHAw and pHA1AT_32, consisting of ori regions from two plasmids isolated from Halomonas sp. A020, and chloramphenicol- and tetracycline-resistant genes as cloning markers, respectively. These vectors can independently transform and co-transform the Halomonas sp. KM-1 (KM-1). A protein that was highly and constitutively accumulated was identified as a hemolysin coregulated protein (Hcp) based on proteome analysis of KM-1. Using the hcp promoter, various genes, such as phaA and EGFP, were highly expressed. To establish a gene disruption system, the Streptococcus pyogenes cas9 gene and guide RNA for the pyrF gene, a yeast URA3 homologue, were expressed in pUCpHAw and pHA1AT_32, respectively. As a result, gene disruption mutants were isolated based on phenotypes, 5-fluoroorotic acid resistance, and uracil auxotrophy. A combination of KM-1 and these vectors could be a suitable platform for industrial chemical and protein production.

Halomonas species, which are aerobic, alkaliphilic, and moderately halophilic bacteria, produce diverse biochemicals. To identify food-related Halomonas strains for bioremediation and the industrial production of biochemicals, 20 strains were isolated from edible seashells, shrimp, and umeboshi (pickled Japanese plum) factory effluents. All isolates were phylogenetically classified into a large clade of Halomonas species. Most isolates, which grew in wide pH (6-13) and salt concentration (0-14%) ranges, exhibited the intracellular accumulation of poly(3-hydroxybutyrate) granules. The characteristics of these isolates varied. A020 isolated from umeboshi factory effluents exhibited enhanced stress tolerance and proliferation and comprised two plasmids. IMZ03 and A020 grew to more than 200 OD600, while IMZ03 produced 3.5% 3-hydroxybutyrate in inorganic medium supplemented with 10% sucrose. The mucus of TK1-1 cultured on agar medium comprised approximately 64‍ ‍mM of ectoine. Whole-genome sequencing of A020 was performed to elucidate its origin and genomic characteristics. The genome ana-lysis revealed a region exhibiting synteny with a large virus genome isolated from the ocean, but did not identify any predictable pathogenic genes. Therefore, saline foods and related materials may be suitable resources for isolating Halomonas strains exhibiting unique, useful, and innocuous features.

Four types of zinc (Zn)-doped hydroxyapatite (Zn-HAp) nanoparticles were prepared using calcium nitrate tetrahydrate as an anti-sintering agent during calcination at 600°C for 1 hr, to prevent calcination-induced aggregation. The Zn content of the nanopowders was determined at 0, 4.3, 9.2, and 14.7% [Zn/(Ca + Zn) × 100] using inductively coupled plasma atomic emission spectroscopic analysis. Based on X-ray diffraction analysis, the products were shown to possess an apatite structure without other crystalline impurities. The cell parameters of Zn-HAp nanoparticles decreased with increasing of Zn content in the HAp structures. This tendency implies that Zn ions substituted for Ca sites in the HAp crystal lattices. To investigate the biological effects of Zn-HAp nanoparticles, cell proliferation activity of MC3T3-E1 osteoblasts and antibacterial activity against Escherichia coli were evaluated in vitro. According to the results obtained, Zn-HAp nanoparticles containing of 14.7% Zn ions was noticeable shown shareability of the conflicting activities at 0.1 mg/mL.

Calcined and dispersible titanium-doped hydroxyapatite (Ti-HAp) nanoparticles at different [Ti/(Ca+Ti)] atomic ratios (0.3, 0.4, and 0.5) were prepared using an anti-sintering method. The Ti substitution ratios of the HAp structures in the feed of Ti-HAp preparation were approximately 80%. Ti-HAp nanoparticles were coated on polyethylene terephthalate (PET) sheets through polyacrylic acid graft-polymers. The PET substrate was almost completely covered with monolayer nanoparticles (over 95%). Antibacterial activity of coated Ti-HAp was calculated from the survival ratio of the bacteria, Staphylococcus aureus, after ultraviolet (UV) irradiation at 312 nm and 6.4 mW/cm² for 30 s. The number of S. aureus on the Ti-HAp coated substrate decreased by 43% compared to those on the original PET and normal HAp coatings as negative controls. The antibacterial activity of Ti-HAp coated substrate was, furthermore, no statistically difference with TiO₂ sheet as a positive control.

Thermotolerant strains are critical for low-cost high temperature fermentation. In this study, we carried out the thermal adaptation of A. pasteurianus IFO 3283-32 under acetic acid fermentation conditions using an experimental evolution approach from 37ºC to 40ºC. The adapted strain exhibited an increased growth and acetic acid fermentation ability at high temperatures, however, with the trade-off response of the opposite phenotype at low temperatures. Genome analysis followed by PCR sequencing showed that the most adapted strain had 11 mutations, a single 64-kb large deletion, and a single plasmid loss. Comparative phenotypic analysis showed that at least the large deletion (containing many ribosomal RNAs and tRNAs genes) and a mutation of DNA polymerase (one of the 11 mutations) critically contributed to this thermotolerance. The relationship between the phenotypic changes and the gene mutations are discussed, comparing with another thermally adapted A. pasteurianus strains obtained previously.

Chlamydia pneumoniae is an obligate intracellular pathogen responsible for respiratory diseases, including pneumonia and bronchitis, and is highly involved in chronic diseases, including atherosclerosis, asthma, and Alzheimer's disease. We previously showed that the host apoptotic factor caspase-9 played a crucial role for chlamydial multiplication and host apoptosis inhibition by chlamydial infection. To identify chlamydial genes interacting with human caspase-9, yeast two-hybrid screening was performed and 5 chlamydial genes, including Cpj0838 and pmpG were isolated from the C. pneumoniae genomic library. Pull-down experiments showed that caspase-9 physically bound to the Cpj0838 product and chlamydial cells, which contain PmpG proteins. This study could provide a clue to understanding host-Chlamydia interactions, especially the apoptosis repression by Chlamydia infection.

Although pancreatic enzyme replacement therapy (PERT) is effective in the alleviation of pancreatic exocrine insufficiency (PEI)-related symptoms in patients with chronic pancreatitis, its mechanism of action is poorly understood. Recent studies suggest that the intestinal microbiota is associated with the pathogenesis of chronic pancreatitis. Therefore, we hypothesized that PERT exerts its effect by modifying the intestinal microbiota in addition to its presumed role in promoting fat and protein absorption. To explore the mechanism of action of PERT, we analyzed the intestinal microbiotas of two groups of mice treated with either pancrelipase or tap water by using 16S rRNA amplicon sequencing. The results revealed that the bacterial compositions of the pancrelipase-treated mice were significantly different from those of the control samples. Akkermansia muciniphila, a key beneficial bacterium in the intestinal tract, showed a higher relative abundance in the pancrelipase-treated samples than in the control samples. Lactobacillus reuteri, a widely used probiotic bacterium known to relieve intestinal inflammation, also showed a higher relative abundance in the pancrelipase-treated samples. These results suggested that PERT induces the colonization of beneficial bacteria, thereby contributing to the attenuation of PEI-associated symptoms in addition to improvement of the nutritional state.

Cry46Ab is a Cry toxin derived from Bacillus thuringiensis TK-E6. Cry46Ab is not significantly homologous to other mosquitocidal Cry or Cyt toxins and is classified as an aerolysin-type pore-forming toxin based on structural similarity. In this study, the potency of Cry46Ab was assessed for its potential application to mosquito control. A synthetic Cry46Ab gene, ciy46Ab-S1, was designed to produce recombinant Cry46Ab as a glutathione-S-transferase fusion in Escherichia coli. Recombinant Cry46Ab showed apparent toxicity to Culex pipiens larvae, with a 50% lethal dose of 1.02 mu g/ml. In an artificial lipid bilayer, Cry46Ab activated by trypsin caused typical current transitions between open and closed states, suggesting it functions as a pore-forming toxin similar to other Cry and Cyt toxins. The single-channel conductance was 103.3 +/- 4.1 pS in 150 mM KCl. Co-administration of recombinant Cry46Ab with other mosquitocidal Cry toxins, especially the combination of Cry4Aa and Cry46Ab, resulted in significant synergistic toxicity against C. pipiens larvae. Co-administration of multiple toxins exhibiting different modes of action is believed to prevent the onset of resistance in insects. Our data, taken in consideration with the differences in its structure, suggest that Cry46Ab could be useful in not only reducing resistance levels but also improving the insecticidal activity of Bt-based bio-insecticides. (C) 2017 Elsevier Ltd. All rights reserved.

type:Departmental Bulletin Paper [要旨] 梅干しは日本の伝統的な食品であり、医学的に有益な物質を多数含んでいる。しかし、伝統的な製法で作られた梅干しは酸っぱさと塩辛さが原因で忌避されるようになり、50年くらい前に登場した調味梅干しが梅干し市場で伝統的な梅干しに取って代わっている。この調味梅干しは、伝統的な梅干しを調味液につけることで塩濃度を下げ風味を加えた食べやすくした梅干しである。和歌山県は梅の一大産地で、全国の梅の生産量の65%を占めており、そのうちの70%が梅干しに加工され、多くが調味梅干しとして追加工される。梅干しの調味工程で発生する使用済み梅調味液は和歌山県内で年間18,000m3廃棄されるが、20%の糖や10%の食塩を含む使用済み梅調味液の処理には年間数億円のコストが発生していると推計される。本研究では使用済み梅調味液の再利用による最終的な処理コストの削減を目標として、梅調味廃液からピルビン酸や3－ヒドロキシ酪酸(3HB)、ポリヒドロキシ酪酸(PHB)などの有用物質を産生する好塩性ハロモナス菌の獲得を試みた。まず、梅調味廃液やその処理施設の廃液の沈殿から菌液を調製し、栄養が豊富な培地や無機塩類の培地、植物培養用培地などの培地を用いて菌を培養した。分離培養された65菌株のうち高塩濃度・高pHの培地で増殖した8菌株について16SrRNA遺伝子のDNA配列解析と分子系統解析を行った結果、それら8菌株がハロモナス(Halomonas)属に分類されること、そして多様なクレードに属する新規なハロモナス菌であることが分かった。さらに、好気条件下でこれら8菌株のうちA014とA024がピルビン酸を産生し、A020とA031、A064はPHBを高蓄積することが明らかになった。また、PHBを高蓄積していた3菌株に加え、A003とA063の5菌株は微好気培養によって3HBを産生した。これらの新規ハロモナス菌は梅調味廃液からの有用物質生産などホワイトバイオテクノロジーに有用であることが示された。 [Abstract] Umeboshi is a traditional, medically beneficial and extremely salty and sour pickled Japanese food made from fully ripened fruits of Japanese Ume tree (Prunus mume) without any fermentation processes. Recently the traditional umeboshi is less popular because of its saltiness and sourness. Instead, seasoned umeboshi was developed about 50 years ago and it has been replacing over the traditional one in the markets. A variety of seasoned umeboshi are made of the traditional umeboshi by re-pickling processes in different kinds of seasoning liquids. In Wakayama Prefecture, 65% of Japanese whole Ume are harvested and 70% of them are processed for the umeboshi. After the process for seasoning the umeboshi, 18,000 m3 of seasoning liquids are wasted a year in Wakayama Pref. Since it contains 20% saccharides, 10% salt, 3% citric acid, thus its pH is approximately 3, and amino acid-based seasonings, annual running costs for the disposal treatments are several hundred million yen and it is a big issue how to decrease the huge industrial waste and cost in Wakayama Pref. In this study, we have attempted to isolate halophilic Halomonas, which produces useful materials such as pyruvate, 3-hydroxybutyrate (3HB) and poly-3-hydroxybutyrate (PHB) from the wasted seasoning liquids. The precipitants were prepared from the waste liquids and spread on variety kinds of agar media, including a rich and middle pH medium for yeast and acetic acid bacteria, a high salt and high pH one for Spirulina, and a nutrient poor one for plant tissues. Out of 65 isolates, 8 isolates, which could grow on the high salt and high pH medium, were clarified as different and new strains of a genus Halomonas based on DNA sequences of whole 16S rRNA genes. Phylogenetic analysis using the DNA sequences illustrated that the 8 strains were allocated to different clades of Halomonas. Out of 8 isolates, A014 and A024 secreted pyruvate and A020, A031 and A064 highly accumulated PHB in the cells. Five isolates, A003, A063 and 3 of PHB highly accumulating isolates, secreted 3HB. Henceforth, we'll carry out the production tests of useful substances using the seasoning liquids of umeboshi, and continue to isolate more strains of Halomonas and other for industrial applications.

Background: Thirteen patients with chlorhexidine-silver sulfadiazine-impregnated catheters have experienced serious anaphylactic shock in Japan. These adverse reactions highlight the lack of commercially available catheters impregnated with strong antibacterial chemical agents. A system should be developed that can control both biocompatibility and antibacterial activity. Summary: Hydroxyapatite (HAp) is biocompatible with bone and skin tissues. To provide antibacterial activity by using an external physical stimulus, titanium (Ti) ions were doped into the HAp structure. Highly dispersible, Ti-doped HAp (Ti-HAp) nanoparticles suitable as a coating material were developed. In 3 kinds of Ti-HAp [Ti/(Ca + Ti) = 0.05, 0.1, 0.2], the Ti content in the HAp was approximately 70% of that used in the Ti-HAp preparation, as determined by inductively coupled plasma atomic emission spectroscopy (ICP-AES). ICP-AES and X-ray diffraction showed Ti ions were well substituted into the HAp lattice. The nanoparticles were almost uniformly coated on a polyethylene (PE) sheet in a near-monolayer with a surface coverage ratio > 95%. The antibacterial activity of the Ti-HAp nanoparticles containing 7.3% Ti ions and coating the sheet was evaluated by calculating the survival ratio of Pseudomonas aeruginosa on the coated sheet after ultraviolet (UV) irradiation. The Ti-HAp-coated sheet showed a 50% decrease in the number of P. aeruginosa compared with that on an uncoated control PE sheet after UV irradiation for 30 s. Key Messages: A system of biocompatibility and antibacterial activity with an on/off switch controlled by external UV stimulation was developed. The system is expected to be applicable in long-term implanted intravascular catheters.

Environmental adaptation is considered as one of the most challenging subjects in biology to understand evolutionary or ecological diversification processes and in biotechnology to obtain useful microbial strains. Temperature is one of the important environmental stresses; however, microbial adaptation to higher temperatures has not been studied extensively. For industrial purposes, the use of thermally adapted strains is important, not only to reduce the cooling expenses of the fermentation system, but also to protect fermentation production from accidental failure of thermal management. Recent progress in next-generation sequencing provides a powerful tool to track the genomic changes of the adapted strains and allows us to compare genomic DNA sequences of conventional strains with those of their closely related thermotolerant strains. In this article, we have attempted to summarize our recent approaches to produce thermotolerant strains by thermal adaptation and comparative genomic analyses of Acetobacter pasteurianus for high-temperature acetic acid fermentations, and Zymomonas mobilis and Kluyveromyces marxianus for high-temperature ethanol fermentations. Genomic analysis of the adapted strains has found a large number of mutations and/or disruptions in highly diversified genes, which could be categorized into groups related to cell surface functions, ion or amino acid transporters, and some transcriptional factors. Furthermore, several phenotypic and genetic analyses revealed that the thermal adaptation could lead to decreased ROS generation in cells that produce higher ROS levels at higher temperatures. Thus, it is suggested that the thermally adapted cells could become robust and resistant to many stressors, and thus could be useful for high-temperature fermentations.

To develop a nanoscaled coating material for medical devices possessing weak antibacterial activity, dispersible and crystalline fluorinated hydroxyapatite (F-HAp) nanoparticles were prepared using antisintering agent to avoid calcination-induced sintering. The product was identical to fluorapatite, as determined by X-ray diffraction and Fourier transform infrared spectroscopy. The primary particles generally showed rod-shaped morphology with a length of 367 +/- 67 nm and a width of 223 +/- 21 nm measured by scanning electron microscopy (SEM). The dispersed average particle size (313 +/- 51 nm) in ethanol analyzed by dynamic light scattering was almost the same as that obtained from the SEM images. In the evaluation of solubility in acidic aqueous solution, F-HAp and original hydroxyapatite (HAp) nanoparticles started to dissolve at around pH 3.4 and 4.2, respectively. Thus, the stability of F-HAp in a living body increased compared with original HAp. The antibacterial activity of F-HAp nanoparticles was higher than that of fluoride in sodium fluoride alone or the original HAp nanoparticles. However, it was estimated that the effect of F-HAp was much lower compared with that of silver, one of the popular antibacterial materials. Thus, the dispersed F-HAp nanoparticles possessing weak antimicrobial activity can be useful without severe damage to the living tissue.

Asaia bogorensis, a member of acetic acid bacteria (AAB), is an aerobic bacterium isolated from flowers and fruits, as well as an opportunistic pathogen that causes human peritonitis and bacteraemia. Here, we determined the complete genomic sequence of the As. bogorensis type strain NBRC 16594, and conducted comparative analyses of gene expression under different conditions of co-culture with mammalian cells and standard AAB culture. The genome of As. bogorensis contained 2,758 protein-coding genes within a circular chromosome of 3,198,265 bp. There were two complete operons encoding cytochrome bo(3)-type ubiquinol terminal oxidases: cyoABCD-1 and cyoABCD-2. The cyoABCD-1 operon was phylogenetically common to AAB genomes, whereas the cyoABCD-2 operon belonged to a lineage distinctive from the cyoABCD-1 operon. Interestingly, cyoABCD-1 was less expressed under co-culture conditions than under the AAB culture conditions, whereas the converse was true for cyoABCD-2. Asaia bogorensis shared pathogenesis-related genes with another pathogenic AAB, Granulibacter bethesdensis, including a gene coding pathogen-specific large bacterial adhesin and additional genes for the inhibition of oxidation and antibiotic resistance. Expression alteration of the respiratory chain and unique hypothetical genes may be key traits that enable the bacterium to survive under the co-culture conditions.

Chlamydia is an obligate intracellular bacterial pathogen that replicates solely within a membrane-bound vacuole termed an inclusion. Chlamydia seems to perturb multiple cellular processes of the host, such as, rearrangement of the membrane trafficking system for its intracellular multiplication, and inhibition of host cell apoptosis for persistent infection. In an attempt to clarify host factor involvement in apoptosis regulation, we found that inhibition of Caspase-9 restricted, while Apaf-1 promoted, Chlamydia pneumoniae infection in HEp-2, HeLa, and mouse epithelial fibroblast (MEF) cells. These opposition contributions to the chlamydial infection were confirmed using caspase-9 (-/-) and apaf-1 (-/-) MEFs. Similar phenomena also appeared in the case of infection with Chlamydia trachomatis. Interestingly, caspase-9 in apaf-1 (-/-) MEFs was activated by chlamydial infection but during the infection caspase-3 was not activated. That is, caspase-9 was activated without support for multiplication and activation by Apaf-1, and the activated caspase-9 may be physically disconnected from the caspase cascade. This may be partially explained by the observation of caspase-9 accumulation within chlamydial inclusions. The sequestration of caspase-9 by chlamydia seems to result in apoptosis repression, which is crucial for the chlamydial development cycle. Because Apaf-1 shares domains with intracellular innate immune receptor NOD1, it may play a key role in the strategy to regulate chlamydial infection.

Gluconacetobacter xylinus (formerly Acetobacter xylinum and presently Komagataeibacter medellinensis) is known to produce cellulose as a stable pellicle. However, it is also well known to lose this ability very easily. We investigated the on and off mechanisms of cellulose producibility in two independent cellulose-producing strains, R1 and R2. Both these strains were isolated through a repetitive static culture of a non-cellulose-producing K. medellinensis NBRC 3288 parental strain. Two cellulose synthase operons, types I and II, of this strain are truncated by the frameshift mutation in the bcsBI gene and transposon insertion in the bcsCII gene, respectively. The draft genome sequencing of R1 and R2 strains revealed that in both strains the bcsBI gene was restored by deletion of a nucleotide in its C-rich region. This result suggests that the mutations in the bcsBI gene are responsible for the on and off mechanism of cellulose producibility. When we looked at the genomic DNA sequences of other Komagataeibacter species, several non-cellulose-producing strains were found to contain similar defects in the type I and/or type II cellulose synthase operons. Furthermore, the phylogenetic relationship among cellulose synthase genes conserved in other bacterial species was analyzed. We observed that the cellulose genes in the Komagataeibacter shared sequence similarities with the gamma-proteobacterial species but not with the alpha-proteobacteria and that the type I and type II operons could be diverged from a same ancestor in Komagataeibacter.

Purpose: Dispersible hydroxyapatite (HAp) nanoparticles are very useful for applying a monolayer to implantable medical devices using the nano-coating technique. To improve tolerance to infection on implanted medical devices, silver-doped HAp (Ag-HAp) nanoparticles with dispersiblity and crystallinity were synthesized, avoiding calcination-induced sintering, and evaluated for antibacterial activity. Methods: The Ca10-xAgx(PO4)(6)(OH)(2) with x = 0 and 0.2 were prepared by wet chemical processing at 100 degrees C. Before calcination at 700 degrees C for 2 h, two kinds of anti-sintering agents, namely a Ca(NO3)(2) (Ca salt) and a polyacrylic acid/Ca salt mixture (PAA-Ca), were used. Escherichia coli was used to evaluate the antibacterial activity of the nanopowder. Results: When PAA-Ca was used as an anti-sintering agent in calcination to prepare the dispersible nanoparticles, strong metallic Ag peaks were observed at 38.1 degrees and 44.3 degrees (2 theta) in the X-ray diffraction (XRD) profile. However, the Ag peak was barely observed when Ca salt was used alone as the anti-sintering agent. Thus, using Ca salt alone was more effective for preparation of dispersible Ag-HAp than PAA-Ca. The particle average size of Ag-HAp with 0.5 mol% of Ag content was found to be 325 +/- 70 nm when the formation of large particle-aggregations was prevented, as determined by dynamic light scattering instrument. The antibacterial activity of the Ag-HAp nanoparticles possessing 0.5 mol% against E. coli was greater than 90.0%. Conclusions: Dispersible and crystalline nano Ag-HAp can be obtained by using Ca salt alone as an anti-sintering agent. The nanoparticles showed antibacterial activity.

地球温暖化やその影響が顕在化し，CO₂排出抑制につながる新たなエネルギー削減技術の開発が求められている．発酵産業においても同様で，耐熱性微生物を用いることによって省エネ型の高温発酵が可能になり，これが次世代型の発酵技術として期待される．その実現のため，安定な高温発酵系の構築に向けた基盤的情報として耐熱性発酵微生物のもつ耐熱性分子機構の把握は不可欠となる．

type:Departmental Bulletin Paper [要旨] 発酵産業はビールやヨーグルト, 醤油や食酢といった食品生産だけではなく, 医薬品や工業製品の原材料生産も含む巨大産業である. その近代的な生産現場においては高い生産性を維持するために, 発酵熱による発酵槽の過高温を抑制する大規模な冷却装置が導入され, 徹底した温度管理が行われている. 一方, 伝統的発酵産業では, その過高温が原因となり醸造菌の増殖停止や死滅が発生することもあり, 夏期には醸造を休止するところも多い. 本研究では, 発酵生産時の冷却コスト(つまりはCO_2の排出)の削減と高温発酵による生産効率の向上を目的として, 食酢醸造における優占種であるAcetohacter pasteurianusを利用し, 育種法と細胞融合法を用いて食酢合成能を有する耐熱性酢酸菌株の開発とその耐熱性発酵菌の社会実装にむけた無冷却高温発酵実験を実施した. 育種法にはA.pasteurianus NBRC 3283から分離されたAp32株(食酢合成能あり, 常温性)を用いて, 生育限界温度を超えて増殖が可能な耐熱性育種株MU株を樹立した. さらにMU株を高濃度エタノール存在下で繰り返し静置培養することにより, 高い食酢合成能を有する耐熱性育種株(JK株)の獲得に成功した. 細胞融合法ではAp32株と耐熱性育種株Ap01/42C株(食酢合成能なし)を細胞融合し, 食酢合成能を有する耐熱性細胞融合株(mika株)の樹立に成功した. 酢酸菌を用いた育種法では, 生育限界温度という多因子性形質に対する酢酸菌の高い柔軟性(ゲノム易変異性)が再確認された. 今後はゲノム解析による耐熱性形質を付与する遺伝子群の同定を進める. また, 細胞融合法では遺伝子組換えに頼らない簡便な形質導入法の開発に繋がる可能性を示すことができた. 一細胞ゲノム解析などの技術を利用し, 細胞融合及びゲノム融合の動的解析を進めたい. 発酵槽に冷却装置を設置しない伝統的な食酢醸造を行う田村造酢(株)では, 発酵熱により発酵槽が過高温となるため猛暑期には食酢醸造は行われない. そこで耐熱性発酵菌の社会実装にむけた無冷却高温発酵実験として, 2014年の8月と9月にJK株とmika株を用いた250L大型発酵試験とJK株を用いた日本酒を原料とする320L食酢醸造試験を行った. いずれの発酵実験においても酢酸度は目標となる数値まで上昇した. しかし, 2014年の夏は異常な冷夏であり, 田村造酢(株)の種菌を用いた場合でさえも発酵槽の過高温は発生せず, 耐熱性菌の耐熱性形質が発揮される条件は得られなかった. 耐熱性発酵菌の社会実装にむけて, 耐熱性形質の有効性を確認するための無冷却高温発酵実験を引き続き計画している. [Abstract] In order to realize the continual and steady reduction of greenhouse gas emissions in the mid and long term, we are working to obtain fruitful research leading to green innovation as a team for "Genome-based research and development of thermo-tolerant microbes aiming at low-cost fermentation" (a JST-ALCA project). Microbial fermentation is important in bio-industry such as not only brewing or food production but also a variety and abundant of productions of medicinal and industrial materials. Facing with a global warming and also an electric power crisis, it becomes more requested to keep the process at low temperature to make sure the stable fermentation or high productivities. We aim at developing methods to provide useful thermo-tolerant microbes able to ferment stably above 40℃ through genome engineering on the basis of breeding with adaptive evolution, cell-cell fusion (or bacterial crossbreeding) and genetic manipulation using genes related to this thermo-tolerance.Traditional breeding is suitable to modify microbes regarding to multi-gene phenotypes and most acceptable for the society based on its verified safety and reliability. Compared to the method, artificial genetic manipulation is more efficient to provide aimed functions to microbes and appropriate for maintenance of microbial diversity in many companies, but it is not welcome to the people in many countries. Thus a new method for systematic genomeengineering with advantages of traditional breeding and artificial genetic manipulation is requested. However no systems have been established yet, cell-cell fusion is seemed as a prospected candidate to develop such systems. Here we chose Acetobacter pasteurianus, which is widely used for vinegar production in the world, as a model organism for development of methods to generate thermo-tolerant fermentation microbes. As results, we successfully established two strains with two phenotypes, thermo-tolerant (growing at up to 42℃) and pellicle formation. One of the two strains was established from a strain with pellicle formation and thermo-labile (growing at 39℃ or low) phenotypes based on breeding with adaptive evolution. The other one was generated by cell-cell fusion with a pellicle formation but thermo-labile strain and a non-pellicle formation but thermo-tolerant strain. Using these strains, we attempted to brew vinegar in 250 and 320 liter scales without any cooling system in the middle of this summer as implementation experiments prier to utilization in fermentation companies. Both strains could form stable pellicles and produce acetic acid up to the expected concentrations under a procedure employed during cool seasons in Tamura Zousu Company. Using this procedure, the temperature of fermentation tanks generally rise above one which a thermo-labile strain can survive and ferment vinegar. Unfortunately it was abnormally cold in this summer and the temperatures of tanks did not reach even at 35℃, and thus the test point of thermo-tolerant phenotype was not validated.

Acidomonas methanolica (former name: Acetobacter methanolicus) is a unique acetic acid bacterium capable of growing on methanol as a sole carbon source. We reported the draft genome sequencing of A.methanolica type strain MB58, showing that it contains 3270 protein-coding genes, including the genes involved in oxidation of methanol, such as mxaFJGIRSACKL and hxlAB, and oxidation of ethanol, such as adhAB and adhS.

type:Departmental Bulletin Paper [要旨] クラミジアはヒトをはじめとする動物細胞中でのみ増殖可能な偏性細胞内寄生性細菌で, ヒトにおける多様な疾患の原因・増悪要因となる. たとえば, 肺炎クラミジアは世界的に成人の60％以上が罹患歴を持ち, その持続感染は喘息や動脈硬化症の発症・増悪の原因となる. また, クラミジア・トラコマティスは先進国において成人の数％が性器感染しており, その慢性感染は子宮外妊娠や不妊症の原因となる. さらに, トラコマティスは眼結膜炎を引き起こし, その慢性化が原因となり発展途上国では年間数百万人が失明している. このようにクラミジアは宿主動物の免疫系によって駆逐されず, また宿主を直接的に死に至らしめることもなく感染と増殖を繰り返している. この巧みな感染機構の解明をめざし, 生化学解析やゲノム解析が進められている. ここでは, クラミジアの持続感染と, その持続感染に重要な意味を持つ宿主アポトーシスの制御に関する研究を紹介する. [Abstract] Chlamydiae are Gram-negative and obligate intracellular parasitic bacteria responsible for a wide range of diseasescausing severe clinical and public health problems. Chlamydia pneumoniae is known as a leading cause of respiratory tract infections and its persistent infection has been shown to relate to atherosclerosis in the last two decades. Chlamydia trachomatis is the most common sexually transmitted bacterial pathogen worldwide, and its chronic infection increased risk of ectopic pregnancy, chronic pelvic pain and infertility. Chlamydial ways of killing host cells tenderly must be attributed to molecular mechanisms, by which Chlamydia modifies intracellular environments to scrape off nutrients but maintain the integrity of the host cells. One of the most focused topics is the prevention of the host cells from undergoing apoptosis induced by intracellular stress for its long-term multiplication or persistence. The main aim of this review is to summarize the data illustrating persistent infection and host apoptosis regulation by Chlamydia.

Carboxyl group-donated silver (Ag) nanoparticles for coating on medical devices were prepared by a two-phase reduction system in situ. AgNO3 was the Ag ion source, tetraoctylammonium bromide [N(C8H17)(4)Br] the phase-transfer agent, sodium tetrahydroborate (NaBH4) the reducing agent and 10-carboxy-1-decanthiol (C11H22O2S, CDT) the capping agent. The characterizations of the Ag nanoparticles were conducted by diffuse reflectance Fourier transform infrared (FT-IR) spectroscopy, thermogravimetric differential thermal analysis (TG/DTA) and transmission electron microscope. With CDT capped on Ag nanoparticles, we found that the band around 3,100 cm(-1) was attributed to COO-H stretching vibration, two adsorptions at 2,928 and 2,856 cm(-1) to C-H symmetric/anti-symmetric stretching vibration, and at 1,718 cm(-1) to C=O stretching vibration in the FT-IR spectra. The organic components of the carboxylated Ag nanoparticles were 5.8-25.9 wt%, determined by TG/DTA. The particle sizes of the carboxylated Ag nanoparticles were well controlled by the addition of the capping agent, CDT, into the reaction system. The antimicrobial activity of the Ag nanoparticles covered with different contents of CDT against E. coli was evaluated. Smaller-size Ag nanoparticles showed higher antibacterial activity, which depended on a surface area that attached easily to a microorganism cell membrane.

Rickettsia japonica is an obligate intracellular alphaproteobacteria that causes tick-borne Japanese spotted fever, which has spread throughout East Asia. We determined the complete genomic DNA sequence of R. japonica type strain YH (VR-1363), which consists of 1,283,087 base pairs (bp) and 971 protein-coding genes. Comparison of the genomic DNA sequence of R. japonica with other rickettsiae in the public databases showed that 2 regions (4,323 and 216 bp) were conserved in a very narrow range of Rickettsia species, and the shorter one was inserted in, and disrupted, a preexisting open reading frame (ORF). While it is unknown how the DNA sequences were acquired in R. japonica genomes, it may be a useful signature for the diagnosis of Rickettsia species. Instead of the species-specific inserted DNA sequences, rickettsial genomes contain Rickettsia-specific palindromic elements (RPEs), which are also capable of locating in preexisting ORFs. Precise alignments of protein and DNA sequences involving RPEs showed that when a gene contains an inserted DNA sequence, each rickettsial ortholog carried an inserted DNA sequence at the same locus. The sequence, ATGAC, was shown to be highly frequent and thus characteristic in certain RPEs (RPE-4, RPE-6, and RPE-7). This finding implies that RPE-4, RPE-6, and RPE-7 were derived from a common inserted DNA sequence.

Gluconacetobacter xylinus is involved in the industrial production of cellulose. We have determined the genome sequence of G. xylinus NBRC 3288, a cellulose-nonproducing strain. Comparative analysis of genomes of G. xylinus NBRC 3288 with those of the cellulose-producing strains clarified the genes important for cellulose production in Gluconacetobacter.

We developed a specific and rapid detection system for Rickettsia japonica and R. heilongjiangensis, the causative agents of spotted fever, using a TaqMan minor groove binder probe for a particular open reading frame (ORF) identified by the R. japonica genome project. The target ORF was present only in R. japonica-related strains.

Acetobacter species have been used for brewing traditional vinegar and are known to have genetic instability. To clarify the mutability, Acetobacter pasteurianus NBRC 3283, which forms a multiphenotype cell complex, was subjected to genome DNA sequencing. The genome analysis revealed that there are more than 280 transposons and five genes with hyper-mutable tandem repeats as common features in the genome consisting of a 2.9-Mb chromosome and six plasmids. There were three single nucleotide mutations and five transposon insertions in 32 isolates from the cell complex. The A. pasteurianus hyper-mutability was applied for breeding a temperature-resistant strain grown at an unviable high-temperature (42 degrees C). The genomic DNA sequence of a heritable mutant showing temperature resistance was analyzed by mutation mapping, illustrating that a 92-kappa b deletion and three single nucleotide mutations occurred in the genome during the adaptation. Alpha-proteobacteria including A. pasteurianus consists of many intracellular symbionts and parasites, and their genomes show increased evolution rates and intensive genome reduction. However, A. pasteurianus is assumed to be a free-living bacterium, it may have the potentiality to evolve to fit in natural niches of seasonal fruits and flowers with other organisms, such as yeasts and lactic acid bacteria.

Rac GTPases consist of Rac1, 2 and 3, and each of them have redundant and differential functions. Rac1 is the most ubiquitously and abundantly expressed of the three and has been shown to work as a "molecular switch" in various signal transduction pathways. Although Rac1 and Rac2 are both activated by TCR ligation, little is known about the function of Rac GTPases in the development and activation of T cells. In order to investigate the precise function of Rac GTPases in T cells in vivo, we established dominant negative Rac1 transgenic (dnRac1-Tg) mice controlled by the human CD2 promoter. Total numbers of thymocytes of dnRac1-Tg mice were significantly decreased because of impaired transition from the CD4CD8 double negative stage to the CD4CD8 double positive (DP) stage. Although positive selection of CD4 single positive (SP) was not altered, positive selection of CD8-SP was slightly increased. On the contrary, the number of mature CD4-SP and CD8-SP cells in the spleen, mesenteric lymph nodes and peripheral blood was severely decreased in dnRac1-Tg mice. Proliferation of splenic CD4-SP cells upon TCR stimulation in vitro was unaltered, however, homeostatic proliferation of dnRac1-Tg splenic CD4-SP cells in lymphopenic mice was severely reduced. Finally, we found increased spontaneous apoptosis of DP thymocytes and mature T cells in dnRac1-Tg mice, possibly because of reduced phosphorylation of Akt with or without TCR stimulation. Collectively, the current results indicate that Rac GTPases are important in survival of DP thymocytes and mature T cells in vivo by regulating Akt activation. (c) 2009 Elsevier B.V. All rights reserved.

RhoH is an atypical small G protein with defective GTPase activity that is specifically expressed in hematopoietic lineage cells. RhoH has been implicated in regulation of several physiological processes including hematopoiesis, integrin activation, and T cell differentiation and activation. In the present study, we investigated the role of RhoH in mast cells by generating RhoH knockout mice. Despite observing normal development of mast cells in vivo, passive systemic anaphylaxis and histamine release were impaired in these mice. We also observed defective degranulation and cytokine production upon Fc epsilon RI ligation in RhoH-deficient bone marrow-derived mast cells. Furthermore, Fc epsilon RI-dependent activation of Syk and phosphorylation of its downstream targets, including LAT, SLP76, PIC gamma 1, and PLC gamma 2 were impaired, however phosphorylation of the gamma-subunit of Fc epsilon RI remained intact. We also found RhoH-Syk association that was greatly enhanced by active Fyn. Our results indicate that RhoH regulates Fc epsilon RI signaling in mast cells by facilitating Syk activation, possibly as an adaptor molecule for Syk. The Journal of Immunology, 2009, 182: 957-962.

Chlamydophila pneumoniae, an obligate intracellullar eubacterium, changes its form from a vegetative reticulate body into an infectious elementary body during the late stage of its infection cycle. Comprehension of the molecular events in the morphological change is important to understand the switching mechanism between acute and chronic infection, which is deemed to relate to the pathogenesis of atherosclerosis. Herein, we have attempted to screen genes expressed in the late stage with a genome-wide DNA microarray, resulting in nomination of 17 genes as the late-stage genes. Fourteen of the 17 genes and six other genes predicted as late-stage genes were confirmed to be up-regulated in the late stage with a quantitative reverse transcriptase-pollymerase chain reaction. These 20 late-stage genes were classified into two groups by clustering analysis: 'drastically induced' and 'moderately induced' genes. Out of eight drastically induced genes, four contain sigma(28) promoter-like sequences and the other four contain an upstream common sequence. It suggests that besides sigma(28) there are certain up-regulatory mechanisms at the late stage, which may be involved in the chlamydial morphological change and thus pathogenesis.

A group of genes thought to encode members of the unique chlamydial polymorphic membrane protein (pmp) family were recently described in the Chlamydophila felis genome. This study aimed to commence characterisation of a subset of 12 of these putative pmp genes by developing and using gene-specific real-time (Q)PCR assays to confirm their presence in a wide range of C. felis field isolates and laboratory strains, and to look for pmp mRNA expression during in vitro infection. Sequencing of 525698 base pair regions of pmp genes 7,9-11, 13-20 for two laboratory strains of C.felis and alignment with the published Fe/C-56 sequence found only a single nucleotide polymorphism present in pmp9. Following the development of gene-specific (Q)PCR assays, analysis of genomic DNA extracted from 40 C. felis field isolates and 4 laboratory strains found that all 12 pmp genes were represented in all cases. Reverse transcription (RT)-QPCR analysis of RNA extracted from cell cultures at 24 and 48 h post inoculation with I of 5 different strains of C. felis detected transcripts for all 12 pmp genes at both time points. Analysis of the relative levels of pmp gene transcription suggested that down-regulation of the expression of multiple C. felis pmp genes occurs between 24 and 48 h post inoculation. This study provides the first evidence that 12 of the putative pmp C. felis genes are transcribed during in vitro infection, and shows that these genes are present in a large range of C felis field isolates and multiple passage laboratory-grown strains. (c) 2007 Elsevier B.V. All rights reserved.

Rac1, one of the Rho family small guanosine triphosphatases, has been shown to work as a "molecular switch" in various signal transduction pathways. To assess the function of Rac1 in the differentiation process of CD4 single-positive (CD4-SP) T cells from CD4CD8 double-positive (DP) cells, we used a DP cell line DPK, which can differentiate into CD4-SP cells upon TCR stimulation in vitro. DPK expressing dominant-negative (dn)Rac1 underwent massive apoptosis upon TCR stimulation and resulted in defective differentiation of CD4-SP cells. Conversely, overexpression of dnRac2 did not affect differentiation. TCR-dependent actin polymerization was inhibited, whereas early ERK activation was unaltered in duRac2-expressing DPK. We found that TCR-dependent induction of Bcl-2 was suppressed greatly in dnRac1-expressing DPK, and this suppression was independent of actin rearrangement. Furthermore, introduction of exogenous Bcl-2 inhibited TCR-dependent induction of apoptosis and restored CD4-SP generation in dnRac1-expressing DPK without restoring TCR-induced actin polymerization. Collectively, these data indicate that Rac1 is critical in differentiation of CD4-SP from the DP cell line by preventing TCR-induced apoptosis via Bcl-2 up-regulation.

SET domain genes have been identified in numbers of bacterial genomes based on similarity to SET domains of eukaryotic histone methyltransferases. Herein, a Chlamydophila pneumoniae SET domain gene was clarified to be coincidently expressed with hctA and hctB genes encoding chlamydial histone H1-like proteins, Hc1 and Hc2, respectively. The SET domain protein (cpnSET) is localized in chlamydial cells and interacts with Hc1 and Hc2 through the C-terminal SET domain. As expected from conservation of catalytic sites in cpnSET, it functions as a protein methyltransferase to murine histone H3 and Hc1. However, little is known about protein methylation in the molecular pathogenesis of chlamydial infection. cpnSET may play an important role in chlamydial cell maturation due to modification of chlamydial histone H1-like proteins.

Phosphoinositide 3-kinase (PI3K) has important functions in various biological systems, including immune response. Although the role of PI3K in signaling by antigen-specific receptors of the adaptive immune system has been extensively studied, less is known about the function of PI3K in innate immunity. In the present study, we demonstrate that macrophages deficient for PI3K (p85 alpha regulatory subunit) are impaired in nitric oxide ( NO) production upon lipopolysaccharide and interferon-gamma stimulation and thus vulnerable for intracellular bacterial infection such as Chlamydophila pneumoniae. Although expression of inducible nitric-oxide synthase (iNOS) is induced normally in PI3K-deficient macrophages, dimer formation of iNOS protein is significantly impaired. The amount of intracellular tetrahydrobiopterin, a critical stabilizing cofactor for iNOS dimerization, is decreased in the absence of PI3K. In addition, induction of GTP cyclohydrolase 1, a rate-limiting enzyme for biosynthesis of tetrahydrobiopterin, is greatly reduced. Our current results demonstrate a critical role of class IA type PI3K in the bactericidal activity of macrophages by regulating their NO production through GTP cyclohydrolase 1 induction.

Chlamydophila felis (Chlamydia psittaci feline pneumonitis agent) is a worldwide spread pathogen for pneumonia and conjunctivitis in cats. Herein, we determined the entire genomic DNA sequence of the Japanese C. felis strain Fe/C-56 to understand the mechanism of diseases caused by this pathogen. The C. felis genome is composed of a circular 1 166 239 bp chromosome encoding 1005 protein-coding genes and a 7552 bp circular plasmid. Comparison of C. felis gene contents with other Chlamydia species shows that 795 genes are common in the family Chlamydiaceae species and 47 genes are specific to C. felis. Phylogenetic analysis of the common genes reveals that most of the orthologue sets exhibit a similar divergent pattern but 14 C. felis genes accumulate more mutations, implicating that these genes may be involved in the evolutional adaptation to the C. felis-specific niche. Gene distribution and orthologue analyses reveal that two distinctive regions, i.e. the plasticity zone and frequently gene-translocated regions (FGRs), may play important but different roles for chlamydial genome evolution. The genomic DNA sequence of C. felis provides information for comprehension of diseases and elucidation of the chlamydial evolution.

Objectives: Chlamydiae are obligate intracellular bacteria, causing a variety of diseases, i.e. pneumonia, sexually transmitted disease, conjunctivitis and zoonosis. Tryptophan depletion by interferon-gamma (IFN-gamma) is the most important host defence system against chlamydial infection. Thus chlamydial tryptophan metabolism is thought to play key roles for IFN-gamma resistance, persistent infection and host/tissue tropisms. We tested tryptophan derivatives for activity against chlamydia-infected cells. Methods: Rates of chlamydial infection and sizes of the inclusions were evaluated by in vitro infection using three Chlamydiaceae species, Chlamydia trachomatis, Chlamydophila pneumoniae and Chlamydophila felis, which show significant divergence of tryptophan synthesis genes and different susceptibilities to IFN-gamma. Results: Melatonin and serotonin, which are recognized as neural hormones for maintenance of organism homeostasis, reduced chlamydial infection but not other bacterial growth tested here. Unlike IFN-gamma, melatonin limited infection of all three chlamydiae and the effects were not recovered by tryptophan supplementation. Melatonin treatment only of host cells could diminish infection and the infection reduction was neutralized by a pertussis toxin, an inhibitor of G proteins. Ligands of melatonin and serotonin receptors also hampered infection. Conclusions: Inhibition mechanisms of chlamydial infection by melatonin and serotonin appear to be different from those of IFN-gamma and involve specific G-protein-coupled receptors. Melatonin is deemed to inhibit early progression of the chlamydial development cycle, such as establishment of intracellular infection and/or conversion from elementary body to reticulate body. Utilization of melatonin, serotonin or their derivatives may be advantageous for harmless prevention of chlamydial infection.

By using the algorithm for identifying transcription units-i.e. independently regulated ORFs and operons, and pseudo-genes, that was described in our earlier paper (Suckow et al., FEES Letters, in press), the 1,738,505 base sequence that covers the whole genome of Pyrococcus sp. OT3 has been analyzed. ORFs of the number, 1,819, and 51 RNA genes have been identified. For 42% of the identified ORFs significant homology has been found to the known protein genes of known function, and thus these were identified as protein genes, while for 31% homology has been found to the known ORFs of unknown function. For the rest 27% no significant homology has been found.

Srp1p, the protein encoded by SRP1 of the yeast Saccharomyces cerevisiae, is a yeast nuclear localization signal (NLS) receptor protein. We have previously reported isolation of a protein kinase from yeast extracts that phosphorylates Srp1p complexed with NLS peptides/proteins. From partial amino acid sequences of the four subunits of the purified kinase, we have now identified this protein kinase to be identical to yeast casein kinase II (CKII). It was previously thought that autophosphorylation of the 36 kDa subunit of the yeast enzyme was stimulated by the substrate, GST-Srp1p. However, with the use of a more refined system, no stimulation of autophosphorylation of the 36 kDa subunit of yeast CKII was observed. Biochemical and mutational analyses localized the in vitro phosphorylation site of Srp1p by CKII to serine 67. It was shown that, in the absence of NLS peptides/proteins, phosphorylation of the intact Srp1p protein is very weak, but deletion of the C-terminal end causes great stimulation of phosphorylation without NLS peptides/proteins. Thus, the CKII phosphorylation site is apparently masked in the intact protein structure by the presence of a C-terminal region, probably between amino acids 403 and 516. Binding of NLS peptides/proteins most likely causes a change in protein conformation, exposing the CKII phosphorylation site. Mutational alterations of serine 67, the CKII phosphorylation site, to valine (S67V) and aspartic acid (S67D) were not found to cause any significant deleterious effects on cell growth. Analysis of in vivo phosphorylation showed that at least 30% of the wild type Srp1p molecules are phosphorylated in growing cells, and that the phosphorylation is mostly at the serine 67 CKII site. The ability of Srp1p purified from E coli and treated with calf intestinal phosphatase to bind a SV40 T-antigen NLS peptide was compared with that of Srp1p which was almost fully phosphorylated by CKII. No significant difference was observed. It appears that NLS binding does not require any phosphorylation of Srp1p, either by CKII or by some other protein kinase.

Subunit alpha of prokaryotic RNA polymerases plays key roles in protein-protein contacts for both subunit assembly and transcription activation, To gain an insight into the roles of subunit 3, the eukaryotic homologue of alpha, temperature-sensitive mutants of the fission yeast, Schizosaccharomyces pombe, have been isolated after transformation of the mutagenized rpb3 gene encoding mutant subunit 3 of RNA polymerase II, A total of 68 ts mutants were classified into two groups: mutants comprising one group ceased growing immediately after a temperature up-shift, while mutants comprising the other group exhibited delayed growth arrest at high temperatures, RNA polymerase II partially purified from Ts54, one of the group 2 mutants, was thermolabile in vitro, as measured by a non-specific transcription assay, This mutant carries double mutations in domain A of subunit 3, and thus can be used as a reference mutant of RNA polymerase II.

Srp1p, the protein encoded by SRP1 of Saccharomyces cerevisiae, is a nuclear-pore-associated protein. Its Xenopus homolog, importin, was recently shown to be an essential component required for nuclear localization signal (NLS)-dependent binding of karyophilic proteins to the nuclear envelope [Gorlich, D., Prehn, S., Laskey, R. A. & Hartman, E. (1994) Cell 79, 767-778]. We have discovered a protein kinase whose activity is stimulated by Srp1p (Srp1p fused to glutathione S-transferase and expressed in Escherichia coli) and is detected by phosphorylation of Srp1p and of a 36-kDa protein, a component of the protein kinase complex. The enzyme, called Srp1p kinase, is a protein-serine kinase and was found in extracts in two related complexes of approximate to 180 kDa and 220 kDa. The second complex, when purified, contained four protein components including the 36-kDa protein. We observed that, upon purification of the kinase, phosphorylation of Srp1p became very weak, while activation of phosphorylation of the 36-kDa protein by Srp1p remained unaltered. Significantly, NLS peptides and the nuclear proteins we have tested greatly stimulated phosphorylation of Srp1p, suggesting that Srp1p, complexed with karyophilic proteins carrying an NLS, is the in vivo substrate of this protein kinase.

Influenza virus nucleoprotein (NP) is associated with the genome RNA, forming ribonucleoprotein cores. To identify the amino acid sequence involved in RNA binding, we performed Northwestern blot analysis with a set of N- and C-terminal deletion mutants of NP produced in Escherichia coli. The RNA binding region has been mapped between amino acid residues 91 and 188, a stretch of residues that contains a sequence that is not only highly conserved among NPs from A-, B-, and C-type influenza viruses but also similar to the RNA binding domain of a plant virus movement protein.

To improve our understanding of the structure and function of eukaryotic RNA polymerase II, we purified the enzyme from the fission yeast Schizosaccharomyces pombe. The highly purified RNA polymerase II contained more than eleven polypeptides. The sizes of the largest, the second-, and the third-largest polypeptides as measured by SDS-polyacrylamide gel electrophoresis were about 210, 150, and 40 kilodaltons (kDa), respectively, and are similar to those of RPB1, 2, and 3 subunits of Saccharomyces cerevisiae RNA polymerase II. Using the degenerated primers designed after amino acid micro-sequencing of the 40 kDa third-largest polypeptide (subunit 3), we cloned the subunit 3 gene (rpb3) and determined its DNA sequence. Taken together with the sequence of parts of PCR-amplified cDNA, the predicted coding sequence of rpb3, interrupted by two introns, was found to encode a polypeptide of 297 amino acid residues in length with a molecular weight of 34 kDa. The S.pombe subunit 3 contains four structural domains conserved for the alpha-subunit family of RNA polymerase from both eukaryotes and prokaryotes. A putative leucine zipper motif was found to exist in the C-terminal proximal conserved region (domain D). Possible functions of the conserved domains are discussed.

A human MDR1 cDNA was introduced into yeast cells. Immunoblot analysis and indirect immunostaining showed that some of the P-glycoprotein produced was situated in its native orientation in the yeast plasma membrane. Drug-binding activities of the recombinant P-glycoproteins were markedly decreased compared to that of the authentic P-glycoprotein. To identify the bases of decreased binding we studied the effects of membrane component sterols on the azidopine binding and found that ergosterol, which is the main sterol in the yeast membrane, and calciferol, which is produced from ergosterol by UV irradiation, inhibited azidopine binding. These sterols in yeast membrane probably inhibit the function of human P-glycoprotein as a multidrug transporter in yeast cells, because expression of P-glycoprotein in yeast cells did not confer resistance to doxorubicin.

The gene, rpb1, encoding the largest subunit of RNA polymerase II has been cloned from Schizosaccharomyces pombe using the corresponding gene, RPB1, of Saccharomyces cerevisiae as a cross-hybridization probe. We have determined the complete sequence of this gene, and parts of PCR-amplified rpb1 cDNA. The predicted coding sequence, interrupted by six introns, encodes a polypeptide of 1,752 amino acid residues in length with a molecular weight of 194 kilodaltons. This polypeptide contains eight conserved structural domains characteristic of the largest subunit of RNA polymerases from other eukaryotes and, in addition, 29 repetitions of the C-terminal heptapeptide found in all the eukaryotic RNA polymerase II largest subunits so far examined.

Zinc (Zn) ions, a biological essential trace element, was doped hydroxyapatite (Zn-HAp) was developed in this study. Evaluation of material properties, fabrication of an inorganic-polymer composite, examination of anaphylactoid reactions, and elucidation of antibacterial mechanisms were conducted. In details, establishment of crystalline and dispersible Zn-HAp nanoparticles, establishment of a coating of nanoparticles on polymer substrate, manifesting of sustainable antibacterial activities, and elucidation of mechanisms of antibacterial effects (phenomena of ion-exchange and accumulation of ROS in bacteria) were done. Based on these results, a novel nanomaterial possessing sustainable antibacterial activities and poor at inducing an allergic-like reactions in vitro was developed.

Zika virus (ZIKV) causes congenital abnormality, and has an aspect of sexually transmitted disease agent. Its major protective immunity - inducing antigen is the envelope (E) protein. Mucosal epithelium is the contact interface to infectious agents, and mucosal immunity consists of systemic immune network. In order to confirm whether effective protective immunity against ZIKV infection can be induced to mucosa of sexual organ by oral immunization with Saccharomyces or lactobacilli on which Zika virus E protein is expressed, we tried to establish the Saccharomyces and the lactobacillus strains which stably express Zika virus E protein on cell surface for the purpose of using as oral mucosal vaccine antigen, and those which express GFP on cell surface to use as control antigen. As results, we have succeeded in establishing the Saccharomyces strains which stably express Zika virus E protein or GFP on their cell surface. Establishing of the lactobacillus strains is however still under way.

私達は熱帯環境から耐熱性微生物を分離するとともに、実験室進化によって「適応育種」耐熱化微生物を取得し、これら耐熱性・耐熱化発酵微生物を用いた「高温発酵系の開発」と、その比較ゲノム解析・発現解析・生理学的解析を通じた「耐熱性・耐熱化機構の解明」を目指しています。発酵の省エネルギーとロバスト化を可能にする高温発酵系の確立は、ホワイトバイオテクノロジーの活性化を通じて、「低炭素化」社会の実現に貢献します。

私達は熱帯環境から耐熱性微生物を分離するとともに、実験室進化によって「適応育種」耐熱化微生物を取得し、これら耐熱性・耐熱化発酵微生物を用いた「高温発酵系の開発」と、その比較ゲノム解析・発現解析・生理学的解析を通じた「耐熱性・耐熱化機構の解明」を目指しています。発酵の省エネルギーとロバスト化を可能にする高温発酵系の確立は、ホワイトバイオテクノロジーの活性化を通じて、「低炭素化」社会の実現に貢献します。

Acetic acid bacteria (AAB) are characterized by their ability to transform alcohols and sugar-alcohols into their corresponding organic acids. To acquire new metabolic systems practicable in high sugar concentration and to achieve a model system to comprehend environmental responses of AAB, we have performed comparative genome, transcriptome and proteome analyses First, to determine draft genome DNA sequences of 25 AAB, we carried out DNA sequencing, assembling and gene extraction by using a next generation sequencer. We will present our recent data for the metabolic and gene regulations by the stress of high glucose concentration.

肺炎クラミジアの冠状動脈硬化部における感染状況を調べたところ、軽度、高度狭心症計50人の冠動脈硬化部の平滑筋細胞組織の100%で動脈壁平滑筋細胞に肺炎クラミジアの高密度感染を検出し、局所炎症の惹起に関与していた。肺炎クラミジアの約100遺伝子は真核生物の遺伝子に類似する。これら主要な病原因子と相互作用するヒト大動脈遺伝子を2ハイブリッド法を用いてcDNAライブラリーから検出した。肺炎クラミジアset遺伝子(ヒトクロマチン関連因子ホモローグ)やmpg1遺伝子は肺炎クラミジア自身か宿主のクロマチン構造に関連すると考えられ、ある種の細胞外マトリックス蛋白質(大動脈壁平滑筋層で弾性を制御している細胞外マトリックスタンパクファミリーフィブリンのEGF様Ca結合ドメイが相互作用ドメインであった)との相互作用が同定された(クラミジア感染患部で、大動脈弾性繊維の断裂病変形成に関与?)。この肺炎クラミジアのキネシン/ミオシン類似因子であるKhc蛋白質や封入体膜構成蛋白質であるIncA2をベイトにすると、それぞれミトコンドリア膜に局在する蛋白質ATP6,NADH dehydrogenaseとamine oxidaseが同定された(大動脈平滑筋細胞の増殖や脱分化制御に関与?)。この封入体膜遺伝子をHeLa細胞に導入・発現させたところ、発現タンパク質の局在はミトコンドリアに一致しており同膜電位を変化させて透過性に関与することがわかった。同遺伝子導入細胞はアポトーシスを起こしやすいことを解明した。これら相互作用因子の結合ドメインも解明した。それらの相互作用による宿主細胞変化をさらに解析中である。その他約80の全く新しい肺炎クラミジア菌?宿主間タンパク相互作用を検出している。50種程度の薬物スクリーニングの結果、アスピリンなど数種の薬物が肺炎クラミジア感染排除に働くことも判明した。 肺炎クラミジア近縁種のクラミジア・フェリスの全ゲノム構造を決定したので両菌遺伝子の比較解析により動脈硬化関連因子探索の手がかりとなることが期待される。ヒト正常大動脈由来平滑筋細胞とHEp2細胞(対照)それぞれに肺炎クラミジア、クラミジア・フェリス菌を感染させて、ヒト3万遺伝子発現をマイクロアレイ解析して動脈硬化・心筋梗塞関連遺伝子データベースと比較することにより、肺炎クラミジア感染大動脈由来平滑筋にユニークな動脈硬化症関連遺伝子16コと新たな動脈硬化関連候補遺伝子合計224個を検出した)。

動脈硬化や心筋梗塞の冠状動脈硬化巣の80%以上で検出される肺炎クラミジアの感染細胞では、動脈壁傷害の修復への関与が示唆される一群の細胞外マトリックスタンパクファミリー遺伝子の発現が感染後期に低下していたが、大動脈壁に由来するcDNAライブラリーと肺炎クラミジアの病原因子との間で、two-hybrid studyによる相互作用分子のヒトゲノム網羅的スクリーニングを実施したしたところ、肺炎クラミジアset遺伝子産物(一般にはクロマチン因子とされる遺伝子のホモローグ)がこれらマトリックスタンパクと相互作用することを見い出した。SETタンパクは菌体が宿主細胞外に放出された後でこれらマトリックスタンパクに結合し、平滑筋細胞表面のインテグリンと弾性繊維との結合を阻害することによって弾性板や平滑筋層などの動脈壁の構造を変容し、脆弱なものにしている可能性が考えられる。今後さらに肺炎クラミジアSETタンパクと動脈壁平滑筋細胞表面のインテグリンと弾性繊維との関係を詳しく解析することが必要である。一方、我々は日本学術振興会未来開拓学術研究推進事業の成果として肺炎クラミジアの全ゲノム解析に続いて、肺炎クラミジアに最も近似したゲノムを持つにもかかわらず、全く異なる疾病を起こす(動脈硬化VS結膜炎)ネコクラミジア菌(C. felis)の全ゲノムDNA配列を決定し得たので(投稿中)、両菌の病原性の比較解析で、動脈硬化発生・病態の解明や感染排除という難解な問題を解明する手がかりをつかめることを目指している。

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