Bifidobacterium属の系統分類学的研究 : 系統解析および生態に関する検討

Bifidobacterium属は1900年に乳児から初めて分離され、その後、1963年にBifidobacterium属の基準種となるB. bifidumが命名提案された。また、その分類・同定方法は表現形質に基づいて分類されてきたが、1969年からはDNA-DNA相同性試験による分類が主流になり、現在においても分類の基準となっている。ところが、Bifidobacterium属には、異なる菌種間で類似した表現型や高いDNA-DNA相同性を示す菌種が存在することが示唆され、これのみではBifidobacterium属の分類・同定に混乱を招くことから要因となることから、これらの代わる明確な分類・同定法の開発が必要とされてきた。また、16S rDNA塩基配列に基づいた系統解析が行われるようになり、表現型やDNA-DNA相同試験と同様に、Bifidobacterium属の菌種間で高い類似性を示す菌種が存在することが報告されている。このことから、これらの菌種については明確な分類法あるいは新たな分類体系が必要であると考えられる。また、Bifidobacterium属の分類学的な問題が指摘されてきた菌種のなかでも、B. infantis、B. longumおよびB. suisの3菌種は高いDNA-DNA相同性を示すことが知られている。そのことから、同一菌種であるかどうかが論じられているが、明らかにされていないのが実情であり、分類学的な検討を行う必要がある。一方で、ヒトおよび動物の腸管内に多種類の菌群が存在し、特に乳児の腸管内においてBifidobacterium属が優勢菌種として存在し、本菌が大腸内細菌叢の環境の改善や免疫調整または感染防御などとの関連性が重要視されている。また、腸管内には、高度嫌気生菌や特殊な培養法を必要とし、通常の培養法では培養が困難な菌種が存在することが知られている。しかしながら、Bifidobacterium属とそれら培養困難な菌種との拮抗関係、培養困難な菌種の構成または培養困難な菌種と乳児の健康との関連性は把握されていないのが実情である。そこで、上述のことから加味して、Bifidobacterium属における系統分類を明確するため、新しい分類・同定方法としてリボタピング解析を行い、16S rDNA塩基配列に基づく系統解析と比較を行った。また、B. infantis、B. longumおよびB. suisについては糖分解性状、DNA-DNA相同性試験、リボタピングおよびRAPD-PCRを用いて分類学的な検討を行った。さらに、Bifidobacterium属の生体に関する研究として、1ヶ月齢の乳児の糞便（母乳栄養、混合栄養、人工栄養）を対象とし、乳児の栄養法の違いに伴うBifidobacterium属と大腸内細菌叢の関連性を検討するために、従来の培養法とPCR法によるBifidobacterium属の検出、さらに分子生物学的手法の1つであるTerminal restriction fragment length polymorphism (T-RFLP) 解析および16S rRNAクローンライブラリーを用いた乳児の大腸内細菌叢の解析を行った。その成績は以下の通りである。　1) Bifidobacterium属28菌種2亜種の計97株を使用し、RiboPrinter^R System (TaKaRa) を用いたリボタイピング解析と16S rDNA塩基配列に基づいた系統解析を行った。今回用いたRiboPrinter^R SystemによるLactobacillusやStaphylococcusの分類学的検討では、類似度50～60%を基準とした型別でそれぞれの菌種を識別されている。そのことから、リボタイピング解析後、得られた各Bifidobacteriumのリボパターンを類似度60%で型別を行ったところ、クラスターIからIXの9つのクラスターに型別され、そのうちクラスターVには供試した28菌種のうち18菌種が含まれた。このことから、さらに詳細に検討するために、類似度90%を基準としてさらに細分類を行ったところ、各クラスターはさらに細分類された。なかでもクラスターVはa～wの23のサブクラスターに分けられた。これまでにB. indicumおよびB. coryneformeグループ、B. catenulatumおよびB. pseudocatenulatumグループ、B. saeculare、B. gallinarumおよびB. pullorumグループおよびB. infantis、B. longumおよびB. suisグループの各菌種間では高いDNA-DNA相同性を示すが、これらの各菌種はそれぞれ異なるサブクラスターに含まれ、分けることが可能であった。その一方で、2菌種以上が含まれるブクラスターや2つ以上のクラスターに分かれる菌種が認められた。リボタイピング解析において、異なる菌種間で類似したパターンを示す場合や1菌種が異なるパターンを示す場合、前者は同一菌種、後者は別種であることが推定される。そこで、各供試菌株の16S rDNA塩基配列を決定し比較したところ、各菌種は基準株に対し、類似度98%以上の類似度を示しそれぞれ同一菌種であることが認められた。このことから、Bifidobacterium属の場合リボパターンの多様性は種により異なることが考えられた。また、B. adolescentisの供試菌株はすべて異なるサブクラスターに分けられた。その16S rDNA塩基配列を比較したところ、V1およびV2領域に違いが認められ、このことからB. adolescentisは他の種よりもヘテロである事が認められた。さらに、B. pseudolongum subsp. globosumおよびB. pseudolongum subsp. pseudolongumはDNA-DNA相同性試験において基準株とは異なるグループ（中間型IおよびII）が存在することが報告されている。そこで、サブクラスターと比較したところ各中間型に相当する菌株は、基準菌株と異なるクラスターに含まれた。これらのことから、リボタピング解析はBifidobacterium属の分類・同定に有効であることが認められた。　2) B. infantis12株、B. longum11株およびB. suis2株の計25株について分類学的な検討を行ったところ、DNA-DNA相同性試験では、各菌種間のDNA-DNA相同値が42℃の条件下では67～81%、さらに52℃では63～85%の相同性を示し、同一菌種であることが認められた。B. longumはB. infantisおよびB. longumに対し命名の優先性をもつことから、B. infantisとB. suisをB. longumとして統合することを提案した。ところが、これらの供試菌株の糖分解性状は多様であったが、リボタピングおよびPAPD-PCRによって3つに型別できることが認められた。このことから、3つの生物型；Infantis type、Longum typeおよびSuis typeとして分類することを提案した（以下、B. longumをB. longum longum type, B. infantisをB. longum infantis typeおよびB. suisをB. longum suis typeとする）。　3) Bifidobacterium属の生態学的な検討として、2002年2～8月に1ヶ月齢の栄養法の異なる健康乳児67例（母乳30例、混合26例および人工11例）から得られた新鮮排泄便を用いて、培養法によるBifidobacterium属の検出を試みたところ、その検出率は72.7%であったが、PCR法による検出では100%から検出された。PCR法による各菌種別の検出率では、B. longum longum typeが16例（62.7%）と最も高率に検出され、次いでB. longum infantis typeが22例（32.8%）、B. breveが16例（23.9%）から検出された。また、各栄養法からの各菌種の検出率は同じ傾向を示した。さらに、糞便中の総菌数と培養菌数の比較を行った結果、大腸内細菌叢の22～94%（平均70%）が培養困難な大腸内細菌であることが明らかになった。そこで、培養困難な大腸内細菌を含めた解析を行うために、T-RFLP解析を行ったところ、各検体は2クラスター（I；34例、II；33例）に分けられ、さらにa、b、c、dおよびeとして5つのサブクラスターに分けられた。クラスターと栄養法との関連性を調べたところ、各クラスターにそれぞれの栄養法が混在して含まれるため、栄養法ごとにその構成に特定の傾向がないことが明らかとなった。しかしながら、母乳栄養に比べ特徴的なクラスターは形成しなかった。このことから、母乳栄養の大腸内細菌叢は比較的類似した構成であるのに対し、混合栄養または人工栄養の大腸内細菌叢の構成は多様であることが明らかとなった。　4) PCR法により検出されたBifidobacterium属と各クラスターとの比較では、B. bifidumは7例（10.4%）から検出されて、サブクラスターa（4例）およびd（3例）のみから検出された。検出された16例のB. breveのうち13例がクラスターIに含まれ、その11例がサブクラスターaに含まれた。T-RFLPのクラスターによりBifidobacterium属の検出率が異なる傾向が見られた。さらに、T-RFLPの各ピークを解析する目的で母乳栄養児1例（baby 4：男児、生後一ヶ月齢）について16S rDNAクローンライブラリーを構築したところ、B. breve、Rothia dentcariosa、Staphylococcus epidermidis、Streptococcus indantis/Streptococcus mitisおよびStreptococcus salivariousの5菌種のクローンが検出された。このうちT-RFLP解析においてHha Iで全体のピーク面積の60.6%を占める579bpとMsp Iで58.5%を占める553bpのピークにはS. salivariusが該当した。一方、培養法で最優勢に分離されたB. breveのピーク面積はHha Iが4.4%およびMsp Iが3.6%であった。baby 4からは培養法ではBifidobacterium属が10.2log/g、Streptococcus属が9.2log/gであるのに対し、T-RFLPおよび16S rDNAクローンライブラリーは定量的には一致しない成績であった。このことは培養可能な菌種数が約30%であることから、培養では検出されにくいStreptococcus属が優勢に存在し、それが検出されていることが考えられた。その一方で、16S rDNAのコピー数やPCRバイアスによりStreptococcus属が優勢に検出された可能性も考えられ、さらに定量PCR法やFISH法を併用した定量的な検討を行う必要があると考えられた。The genus Bifidobacterium has recently been classified based on physiological features, DNA-DNA hybridization, and 16S rDNA sequence analysis. However, some species of Bifidobacterium have not been analyzed so thoroughly, because they showed a high similarity to each other. In this study, the genus Bifidobacterium was analyzed by ribotyping and 16S rDNA phylogenetic analysis and phylogenetic relations among different species of Bifidobacterium were investigated. Bifidobacterium infantis and Bifidobacterium longum isolated from human feces, and Bifidobacterium suis isolated from pig feces showed a high similarity in DNA-DNA hybridization and 16S rDNA sequence analysis, and may possibly represent a single species. To identify the taxonomic relationships between B. infantis, B. longum and B. suis, they were studied by carbohydrate fermentation, DNA-DNA hybridization, ribotyping and random amplified polymorphic DNA-PCR (RAPD-PCR). Twenty-eight species and two subspecies (total 97 strais) were analyzed by ribotyping. The species that showed high similarity (DNA-DNA hybridization 50-75%, 16S rDNA sequences above 99%) with each other, clearly fell into distinct clusters. A species was contained in one cluster at 90% similarity. In contrast, each species were fallen into two or more clusters by ribotyping. Of the species, all strains of Bifidobacterium adolescentis were divided into distinct clusters. Moreover, the analysis of 16S rDNA from the strains of B. adolescentis showed different sequences in the V1 and V2 region of 16S rDNA. B. adolescentis seem to be rich in diversity within the species compared with other species. The relationships among Bifidobacterium infantis, B. longum and B. suis were examined by means of carbohydrate fermentation, DNA-DNA hybridization, ribotyping and random amplified polymorphic DNA-PCR (RAPD-PCR). The levels of DNA-DNA hybridization among the strains of B. infantis, B. longum and B. suis used in this study were 67 to 81% under optimal conditions (42℃) and 63 to 85% under stringent conditions (52℃). Although the strains showed varied carbohydrate fermentation patterns, three species were divided into three types, namely the infantis type, the longum type and the suis type, respectively, by ribotyping and RAPD-PCR. Because of these results, strains of B. infantis, B. longum and B. suis were recognized as distinct groups within a single species. It was conclude that B. infantis and B. suis should be unified as B. longum and divided them into three biotypes, the infantis type, the longum type and the suis type by molecular methods. (The species name is shown as follows: B. longum is classified as B. longum longum type, B. infantis is classified as B. longum infantis type and B. suis is classified as B. longum suis type) The genus Bifidobacteirum exists in infants intestinal tract and is an important bacteria in infant intestinal tract involved in the proper balance of normal intestinal tract the formation of barriers against the proliferation of exogenous pathogens, and in the reinforcement of immune functions. Therefor, the relevance of Bifidobacterium and other organisms and the differences in infant intestinal microbiota by diet were investigated. Sample feces from a total of 67 infants consisting of 30 breast-fed, 27 mix-fed and 11 bottle-fed infants were analyzed. Bifidobacterium was detected by culture method and PCR with species-specific primers, and clonal organisms in the infants feces were detected by Terminal restriction fragment length polymorphism (T-RFLP) and 16S rDNA sequence library. Based on the culture method, the breast-fed, mix-fed and bottle-fed infants had Bifidobacterium as the predominant intestinal microbiota. The existence of Bifidobacterium in all infants was confirmed by PCR methods, whereas Bifidobacterium was isolated from 72.7% of infants by culture method. Using species-specific PCR, B. longum longum type was detected in 62.7%, B. longum infantis type was detected in 32.8% and B. breve was detected in 23.9% of infants from feces. Following that, B. adolescentis, B. bifidum and B. catenulatum group were detected from 10.5 to 13.4%. The incidence of species of Bifidobacterium in each diet groups was not significantly different. In comparing the total count of bacteria with the count of cultivable bacteria in infant intestinal microbiota, the cultivable bacteria accounted for only 30% of the total count and the other 70% were uncultured bacteria. After T-RFLP analysis of intestinal microbiota and cluster analysis, the T-RFLP of samples were divided into cluster I and II and subcluster from a to e. Those from breast-fed infants tended to be contained in subcluster a, and mix-fed and bottle-fed had no specific cluster. It seemed that intestinal microbiota from breast-fed infant were simple and had similar composition of bacteria with each other, and mix-fed and bottle-fed types showed great variety on the contrary. In comparison of T-RFLP clusters with the constitution of each Bifidobacterium, B. breve tended to be contained in subcluster a. This result showed a correlation between B. breve and breast-fed. Moreover, to analyze each T-RFLP peak of samples from breast-fed infants, the 16S rDNA clone library of one male one-month-old breast-fed infant (baby4) was employed. B. breve, Rothia dentcariosa, Staphylococcus epidermidis, Streptococcus indantis/Streptococcus mitis and Streptococcus salivarious clones were detected. In analysis, 579 bp of T-RF peak which shared 60.6% of total peak area after digestion of Hha I and 553 bp of T-RF peak which shared 58.5% of total peak area after digestion of Msp I corresponded to S. salivarius. On the other hand, B. breve, which was predominantly isolated from infant feces by culture method, had only 4.4% (Hha I) and 3.6% (Msp I). 10.5 log/g of Bifidobacterium and 9.15 log/g of Streptocuccus were isolated from this subject by the 10.2 log/g culture method. The results from culture and T-RFLP analysis were not quantitatively in agreement. Since Streptococcus might be the predominant species of uncultured bacteria, the peak associated with Streptococcus was predominantly detected. On the other hand, PCR bias such as primer suitability of differentiation of 16S rDNA copy number might make Streptococcus predominant. Further quantitative work using FISH and real time PCR for intestinal microflora will be needed.博士(学術)麻布大学甲第12

Controlling oxygen coordination and valence of network forming cations

Understanding the structure-property relationship of glass material is still challenging due to a lack of periodicity in disordered materials. Here, we report the properties and atomic structure of vanadium phosphate glasses characterized by reverse Monte Carlo modelling based on neutron/synchrotron X-ray diffraction and EXAFS data, supplemented by Raman and NMR spectroscopy. In vanadium-rich glass, the water durability, thermal stability and hardness improve as the amount of P2O5 increases, and the network former of the glass changes from VOx polyhedra to the interplay between VOx polyhedra and PO4 tetrahedra. We find for the first time that the coordination number of oxygen atoms around a V4+ is four, which is an unusually small coordination number, and plays an important role for water durability, thermal stability and hardness. Furthermore, we show that the similarity between glass and crystal beyond the nearest neighbour distance is important for glass properties. These results demonstrate that controlling the oxygen coordination and valence of the network-forming cation is necessary for designing the properties of glass

Structural properties and thermodynamic stability of Ba-doped silicon type-I clathrates synthesized under high pressure

We present a joint experimental and theoretical study of the stability and structural properties of Ba-doped silicon type-I clathrates Ba8Si46 synthesized under high pressures. The thermodynamic stability of Ba8Si46 under high pressure has been discussed from the total energy calculations of some barium silicides within the local density approximation (LDA). We have theoretically found that pressure favors the formation of the clathrate phase as experimentally observed. We have also performed a synchrotron x-ray-diffraction experiment of Ba8Si46 prepared under high pressures. Some of the missing endohedral Ba elements in the small cage of Si(20) have been observed by x-ray crystallography, while big cages of Si(24) are found to be completely occupied by Ba elements. The stabilization energies of Ba atoms in the endohedral sites estimated within the present LDA calculation suggest that this is presumably attributed to the energetical site preference of Ba atoms between d(6) and a(2) sites. In addition, the isothermal parameter of Ba in the big cage of Si(24) has been found to be larger than that in the small Si(20) unit, which is consistent with some theoretical predictions in earlier works

Rice Annotation Database (RAD): a contig-oriented database for map-based rice genomics

A contig-oriented database for annotation of the rice genome has been constructed to facilitate map-based rice genomics. The Rice Annotation Database has the following functional features: (i) extensive effort of manual annotations of P1-derived artificial chromosome/bacterial artificial chromosome clones can be merged at chromosome and contig-level; (ii) concise visualization of the annotation information such as the predicted genes, results of various prediction programs (RiceHMM, Genscan, Genscan+, Fgenesh, GeneMark, etc.), homology to expressed sequence tag, full-length cDNA and protein; (iii) user-friendly clone / gene query system; (iv) download functions for nucleotide, amino acid and coding sequences; (v) analysis of various features of the genome (GC-content, average value, etc.); and (vi) genome-wide homology search (BLAST) of contig- and chromosome-level genome sequence to allow comparative analysis with the genome sequence of other organisms. As of October 2004, the database contains a total of 215 Mb sequence with relevant annotation results including 30 000 manually curated genes. The database can provide the latest information on manual annotation as well as a comprehensive structural analysis of various features of the rice genome. The database can be accessed at http://rad.dna.affrc.go.jp/

Effects of loading a magnetic field longitudinal to the linear particle-beam track on yields of reactive oxygen species in water

The effects of a magnetic field longitudinal to the ion beam track on the generation of hydroxyl radicals (•OH) and hydrogen peroxide (H2O2) in water were investigated. A longitudinal magnetic field was reported to enhance the biological effects of the ion beam. However, the mechanism of the increased cell death by a longitudinal magnetic field has not been clarified. The local density of •OH generation was estimated by a method based on the EPR spin-trapping. A series of reaction mixtures containing varying concentrations (0.76‒2278 mM) of DMPO was irradiated by 16 Gy of carbon- or iron-ion beams at the Heavy-Ion Medical Accelerator in Chiba (HIMAC, NIRS/QST, Chiba, Japan) with or without a longitudinal magnetic field (0.0, 0.3, or 0.6 T). The DMPO-OH yield in the sample solutions was measured by X-band EPR and plotted versus DMPO density. O2-dependent and O2-independent H2O2 yields were measured. An aliquot of ultra-pure water was irradiated by carbon-ion beams with or without a longitudinal magnetic field. Irradiation experiments were performed under air or hypoxic conditions. H2O2 generation in irradiated water samples was quantified by an EPR spin-trapping, which measures •OH synthesized from H2O2 by UVB irradiation. Relatively sparse •OH generation caused by particle beams in water were not affected by loading a magnetic field on the beam track. O2-dependent H2O2 generation decreased and oxygen-independent H2O2 generation increased after loading a magnetic field parallel to the beam track. Loading a magnetic field to the beam track made •OH generation denser or made dense •OH more reactive

Characterization of the novel mutant A78T-HERG from a long QT syndrome type 2 patient: Instability of the mutant protein and stabilization by heat shock factor 1

Background:The human ether-a-go-go-related gene (HERG) encodes the α-subunit of rapidly activating delayed-rectifier potassium channels. Mutations in this gene cause long QT syndrome type 2 (LQT2). In most cases, mutations reduce the stability of the channel protein, which can be restored by heat shock (HS). Methods: We identified the novel mutant A78T-HERG in a patient with LQT2. The purpose of the current study was to characterize this mutant protein and test whether HS and heat shock factors (HSFs) could stabilize the mutant protein. A78T-HERG and wild-type HERG (WT-HERG) were expressed in HEK293 cells and analyzed by immunoblotting, immunoprecipitation, immunofluorescence, and whole-cell patch clamping. Results: When expressed in HEK293 cells, WT-HERG gave rise to immature and mature forms of the protein at 135 and 155 kDa, respectively. A78T-HERG gave rise only to the immature form, which was heavily ubiquitinated. The proteasome inhibitor MG132 increased the expression of immature A78T-HERG and increased both the immature and mature forms of WT-HERG. WT-HERG, but not A78T-HERG, was expressed on the plasma membrane. In whole-cell patch clamping experiments, depolarizing pulses evoked E4031-sensitive HERG channel currents in cells transfected with WT-HERG, but not in cells transfected with A78T-HERG. The A78V mutant, but not A78G mutant, remained in the immature form similarly to A78T. Maturation of the A78T-HERG protein was facilitated by HS, expression of HSF-1, or exposure to geranyl geranyl acetone. Conclusions: A78T-HERG was characterized by protein instability and reduced expression on the plasma membrane. The stability of the mutant was partially restored by HSF-1, indicating that HSF-1 is a target for the treatment for LQT2 caused by the A78T mutation in HERG

A unique actual experience for recognizing the various roles of healthcare professionals while enhancing pharmacy students\u27 motivation

薬学生に医療従事者の役割や医療チームの重要性を認識させることは極めて重要である。そこで我々は、病院診療科の視察体験が薬学生にどのような効果をもたらすか検討した。本学薬学4年生15名に宮崎大学医学部附属病院放射線科への視察の機会を与え、他の15名には、対照群として視察の機会を与えなかった。視察体験をした学生は、医療従事者が行う診断や患者への治療を、説明を受けながら観察し、さらに医療従事者と質疑応答の時間を持った。視察体験が学生に及ぼす効果は、学生への質問調査によって評価した。視察体験をした学生は医師及びコメディカル職員とのコミュニケーションに大きな興味を示した。また、視察体験をした全ての学生は医療チームの重要性を認識し、医療における知識の修得が必要と考えていた。それ故に診療科の視察体験は、高学年の薬学生において医療従事者の役割の重要性を理解する有意な実地演習であり、教育方略になると考えた。This study aimed to have pharmacy students understand the medical care team and recognize the importance of each healthcare professional\u27s role in medical practice through the implementation of a oneday visitation experience. Thirty fourth-year pharmacy students participated in the study. Half were given an opportunity to visit the Department of Radiology at University of Miyazaki Hospital; the other half were not, and served as the control group. The students observed the medical team diagnose and provide medical treatment to patients and received explanations from various healthcare professionals. They then had a question-and-answer session with healthcare professionals. The students were subsequently given questionnaires to evaluate the impact the visitation experience had on them. Students evinced huge enthusiasm in communicating with physicians and allied healthcare professionals in the one-day experience. The experience increased their motivation to study pharmacy and helped them recognize the importance of the medical care team. Students showed a positive attitude toward the one-day experience. This experiential visit was therefore very meaningful for senior-year pharmacy students. A visitation experience in a department providing diagnosis and treatment for patients affords a unique practical training and educational strategy for understanding the role of each healthcare professional