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Demand for banked bone allografts is increasing in Japan; however, there are too few bone banks and the bone bank network is not well-established. One reason for this was lack of funding for banks. Bone banks had to bear all material expenses of banked bone allografts themselves because this was not designated a covered expense. In December 2004, the Japanese government started a new "Advanced Medical Treatment" administration system which allowed an approved institution to charge the expense of authorized advanced medical treatments directly to patients. The treatment named "Cryopreserved allogenic bone and ligamentous tissue retrieved from cadaveric donor" was approved as an advanced medical treatment in March 2007. We present the calculation method and the expense per implantation of a banked bone allograft from a cadaveric donor under this treatment and raise issues which affect this advanced medical treatment and remain to be resolved in the Japanese orthopaedic field.

This study was undertaken to survey culturable heterotrophic bacteria associated with the marine ark shell Anadara broughtoni inhabiting in the Sea of Japan, and to test isolates for their antimicrobial, hemolytic and surface activities with an emphasis on low-molecular-weight metabolites search. A total of 149 strains were isolated and identified phenotypically. A total of 27 strains were selected to be investigated phylogenetically by 165 rRNA gene sequence analysis. The most bacteria were affiliated with members of the Gammaproteobacteria and Alphaproteobacteria, and Less with Firmicutes, Actinobacteria, and Cytophaga-Flavobacterium-Bacteroides (CFB) group. The isolates capable of hemolysis were numerically abundant in the genera Pseudoalteromonas, Aeromonas and Bacillus. The six Gram-positive isolates belonging to the genera Bacillus, Paenibacillus and Saccharothrix and two Gram-negative strains related to Pseudomonas and Sphingomonas, possessed antimicrobial activity against indicator strains and to each other. Antimicrobial, hemolytic and surface activities were revealed in butanot extracts of cells or cell-free supernatant of six active strains. This points to availability of active low-molecular-weight metabolites. Substances with hemolytic and surface activities were isolated from strain Bacillus pumilus An 112 and characterized as cyclic depsipeptides with molecular masses 1021, 1035, 1049, 1063 and 1077 Da. The recovery of strains producing antimicrobial and surface-active substances suggests that microorganisms associated with the marine bivalve are potential source of bioactive metabolites.

Tyrosinase inhibitor-producing fungus of Trichoderma sp. strain H1–7, which was isolated from marine sediment, was investigated by phylogenetic analysis and physiological characteristics. Phylogenetic analyses of the strain were similar to Trichoderma atroviride or T. viride. Physiological characteristics of the strain were similar to T. viride, and based on these results, it was identified as T. viride. Characterization of tyrosinase inhibitory activity of a culture supernatant of the strain was investigated. The inhibitory activity of the supernatant of the strain decreased after cultivation for more than 3 days. Furthermore, sea water was not essential for the production of the tyrosinase inhibitor (TI). When TI production of the strain was compared to T. viride, the strain showed higher activity than T. viride. From this result, it seemed that the strain had characteristic features comparable to T. viride, which was isolated from the terrestrial environment. As TI production of the strain showed higher potential than that of T. viride, it is necessary to elucidate the chemical structure of TI exemplified in the present study.

An aerobic, Gram-negative, motile, non-pigmented bacterium, strain KMM 3042(T), isolated from a deep-sea brittle star in the Fiji Sea, was subjected to a polyphasic taxonomic study. Phylogenetic analysis based on 16S rRNA gene sequences revealed marine Mn(II)-oxidizing isolate S185-2B as the closest neighbour of strain KMM 3042(T) (99.9 % sequence similarity). The two strains formed a distinct lineage within the genus Pseudomonas adjacent to the members of the Pseudomonas borbori cluster, sharing highest sequence similarity of 97.4 and 97.0 %, respectively, with P. borbori DSM 17834(T) and Pseudomonas flavescens DSM 12071(T). The DNA-DNA hybridization value (71 %) between strains KMM 3042(T) and S185-2B confirmed their assignment to the same species. On the basis of phylogenetic analysis, DNA-DNA hybridization and physiological and biochemical characterization, strains KMM 3042(T) and S185-2B should be assigned to a novel species of the genus Pseudomonas, for which the name Pseudomonas marincola sp. nov. is proposed. The type strain is KMM 3042(T) (=NRIC 0729(T) =JCM 14761(T)).

タイ産, ベトナム産および日本産の魚醤油66種を用いてそれらの化学組成を比較した。その結果, 魚醤油の性状が非常に多様であることを確認した。食塩濃度は14-28%であり, pHは5-8, 全窒素量1-6g/100mlと従来の報告と比べ, これらの値の範囲が広かった。また, 全窒素量が高い魚醤油はペプチド態窒素量が多かった。生体への機能が興味深いペプチドの含量は全窒素に対し全魚醤油の平均は32%であった。国別では日本産よりもタイ産, ベトナム産が高く, 亜熱帯から熱帯気候に位置するタイやベトナムは日本より高温で熟成させるため, プロテアーゼによる魚肉タンパク質の分解が高かったと考えられた。総遊離アミノ酸と代表的な旨味成分であるグルタミン酸との関係では総遊離アミノ酸量が多くなるに従ってグルタミン酸量は高い値を示し, その割合は平均で12%であった。一方, グルタミン酸の割合が20-30%と魚肉タンパク質の同組成値よりも高い値を示すものもあり, これらはグルタミン酸を添加している可能性が考えられた。

Background: The purpose of this study was to evaluate contributing factors affecting deep infection and fracture healing of open tibia fractures treated with locked intramedullary nailing (IMN) by multivariate analysis. Materials and Methods: We examined 99 open tibial fractures (98 patients) treated with immediate or delayed locked IMN in static fashion from 1991 to 2002. Multivariate analyses following univariate analyses were derived to determine predictors of deep infection, nonunion, and healing time to union. The following predictive variables of deep infection were selected for analysis: age, sex, Gustilo type, fracture grade by AO type, fracture location, timing or method of IMN, reamed or unreamed nailing, debridement time (≤6 h or> 6 h), method of soft-tissue management, skin closure time (≤1 week or> 1 week), existence of polytrauma (ISS< 18 or ISS≥18), existence of floating knee injury, and existence of superficial/pin site infection. The predictive variables of nonunion selected for analysis was the same as those for deep infection, with the addition of deep infection for exchange of pin site infection. The predictive variables of union time selected for analysis was the same as those for nonunion, excluding of location, debridement time, and existence of floating knee and superficial infection. Results: Six (6.1%; type II Gustilo n=1, type IIIB Gustilo n=5) of the 99 open tibial fractures developed deep infections. Multivariate analysis revealed that timing or method of IMN, debridement time, method of soft-tissue management, and existence of superficial or pin site infection significantly correlated with the occurrence of deep infection ( P < 0.0001). In the immediate nailing group alone, the deep infection rate in type IIIB + IIIC was significantly higher than those in type I + II and IIIA ( P = 0.016). Nonunion occurred in 17 fractures (20.3%, 17/84). Multivariate analysis revealed that Gustilo type, skin closure time, and existence of deep infection significantly correlated with occurrence of nonunion ( P < 0.05). Gustilo type and existence of deep infection were significantly correlated with healing time to union on multivariate analysis (r 2 = 0.263, P = 0.0001). Conclusion: Multivariate analyses for open tibial fractures treated with IMN showed that IMN after EF (especially in existence of pin site infection) was at high risk of deep infection, and that debridement within 6 h and appropriate soft-tissue managements were also important factor in preventing deep infections. These analyses postulated that both the Gustilo type and the existence of deep infection is related with fracture healing in open fractures treated with IMN. In addition, immediate IMN for type IIIB and IIIC is potentially risky, and canal reaming did not increase the risk of complication for open tibial fractures treated with IMN.

It is important to predict the occurrence of deep infection in open fractures when treating such fractures. We tried to develop a new scoring system for predicting the occurrence of deep infection in open tibial fractures on the basis of the Hannover Fracture Scale '98 (HFS-98).A total of 202 open tibial fractures (195 patients) were retrospectively reviewed in the initial analysis. The relationship between Gustilo's grade and the eight items on the HFS in the open tibial fractures was first investigated by multivariate analysis. By this analysis, we selected significant items that correlated with Gustilo's grade. Among these cases, 179 patients with 185 open tibial fractures were used for the following infection analyses. The relationships between the incidence of deep infection and age (46 years, 46-60 years,60 years), grade of trauma (ISS18, 18or = ISSor = 30, ISS30), fracture location, or some significant items in the above initial analysis were further analyzed by multivariate analysis after univariate analysis. We devised a new scoring system of open tibial fractures based on p values in the above analysis. The discrimination of the newly devised scoring system was evaluated with receiver operating characteristic curves.The following were significant factors affecting the occurrence of deep infection on multivariate analysis: bone loss (p = 0.012); muscle injury (p = 0.012); wound contamination (p = 0.0001); and local circulation (p = 0.0001). We devised a new scoring system for open tibial fractures (bone loss: 0-20 points, muscle injury: 0-20 points, wound contamination: 0-30 points, and local circulation: 0-30 points). The cut-off point for occurrence of deep infection in these fractures was 60 by receiver operating characteristic analysis.This new scoring system was thought to be useful for predicting the occurrence of deep infection in open tibial fractures. However, further prospective study or extension of this scale for other open fractures would be needed.

An aerobic, Gram-negative, yellow-pigmented, non-motile bacterium, designated strain KMM 3882T, was isolated from a marine bivalve (Anadara broughtoni) collected from Peter the Great Bay, Sea of Japan, and was subjected to phenotypic and phylogenetic analyses. Strain KMM 3882T was found to exert a remarkable inhibitory activity against a number of Gram-positive micro-organisms. Phylogenetic analysis based on 16S rRNA gene sequences placed strain KMM 3882T within the genus Sphingomonas, as an independent lineage adjacent to Sphingomonas dokdonensis DS-4T and Sphingomonas panni DSM 15761T. Strain KMM 3882T showed the highest 16S rRNA gene sequence similarity to Sphingomonas dokdonensis DS-4T (97.3 %); similarities of 96.5–96.7 % were obtained with Sphingomonas pituitosa DSM 13101T, Sphingomonas azotifigens NBRC 15497T, Sphingomonas asaccharolytica NBRC 15499T, Sphingomonas trueperi DSM 7225T and Sphingomonas panni DSM 15761T. Chemotaxonomically, strain KMM 3882T contained sphingoglycolipid, C16 : 0 and C18 : 1 as predominant fatty acids and 2-OH C14 : 0 as a major 2-hydroxy fatty acid, confirming the affiliation of strain KMM 3882T with the genus Sphingomonas. On the basis of phylogenetic analysis, DNA–DNA hybridization and physiological and biochemical characterization, strain KMM 3882T should be classified as representing a novel species of the genus Sphingomonas, for which the name Sphingomonas molluscorum sp. nov. is proposed. The type strain is KMM 3882T (=An 18T=NRIC 0685T=JCM 14122T=CIP 109223T).

モチ米の性状と胚乳アミラーゼ活性について検討を行った。蒸米テスクチャーは, 試料米により硬さ, 付着性の強弱に差がみられた。また, 炊飯特性および粘度特性試験を用いた比較を検討し, 各試料米の差異を確認した。その結果, 膨張容積や糊化開始温度と蒸米テスクチャーとの間に相関がみられた。粘度特性試験中での生成還元糖の増加は, 胚乳アミラーゼ作用と考えられた。アミラーゼ活性染色より, 主要な4種のアミラーゼと3種の枝きり酵素活性バンドを確認した。試料米間で, バンドパターンと強弱で若干の差異がみられた。さらに調理条件であるpH7においてアミラーゼ活性を確認し, α-アミラーゼと枝きり酵素の活性量が試料間の差異としてみられた。それらの活性量と粘度の低下量との間に相関関係を認めた (p

食品衛生法で表示義務を定められている小麦について，PCR法における新しいプライマーセットの開発を行った．その結果，UNI2fとVIErプライマーの組合せで，約800bpと500bpに小麦および小麦アレルゲンを含有するライ小麦に特徴的なバンドを示した．特徴バンド以外にもバンドが存在するため，検出時の判定の簡便化のために両バンドをクローニング，シークエンスを行った．決定した配列を基にSTSプライマーを設計した．構築したプライマーのうち，K500は加工食品由来DNAで試験した際にバンドが明瞭であった．以上，本研究では新しい小麦検出用プライマーセットK500を開発した．

In this study, we report the primary structure of fructose 1, 6-bisphosphate aldolase encoded by Cystofilobasidium capitatum FBAl (CcFBAl) and the regulation of its gene expression. CcFBAl consists of a 1, 080 bp ORF corresponding to a protein of 360 amino acid residues, and the calculated molecular weight of CcFbalp is 39, 626 Da. The CcFbalp has both aldolase class-II signatures 1 and 2. CcFBAl is expressed at the same level during growth on several different carbon sources, although expression level may be slightly higher on glucose. Transcription of CcFBAl is not influenced by temperature or existence of oxygen, although it was reduced during the stationary phase. We show that CcFbalp is a constitutive enzyme in C. capitatum, and suggest that FBAl could be used as a marker for the detection of pectinolytic psychrophilic yeasts using the PCR method.

In this study, we attempted to classify the methylotrophic yeasts based on diversities of alcohol oxidase (AOD), i.e. zymogram patterns and partial amino acid sequences. According to zymogram patterns for AOD, members of the methylotrophic yeasts separate into two major lineages, one group involving strains having a single AOD and the other group, including Pichia methanolica, Candida pignaliae and C. sonorensis, showing nine AOD isozymes. Based on partial amino acid sequences of AOD, the methylotrophic yeasts could be divided into five groups, and this classification agrees mostly with grouping based on 26S domain D1/D2 rDNA nucleotide sequences, except for some strains. Moreover, the strains having AOD isozymes constitute one group with P. trehalophila, P. glucozyma and Pichia sp. strain BZ159, although these strains are divided into two types, based on amino acid sequences of second AODs. On the other hand, these AOD isozymes consist of two subunits; the first subunits are induced not only by methanol but also by glycerol and pectin, although the second subunits are mainly induced by methanol. These data indicate that AOD isozymes and second AOD genes distribute widely in several methylotrophic yeasts in the natural environment, and second AOD genes may have evolved as methylotrophic genes that can adapt to the environmental conditions of higher methanol concentrations.

The treatment of type IIIB open tibial fractures remains a challenge for orthopedic surgeons, particularly with respect to the soft-tissue and subsequent bony reconstruction. The primary shortening and limb lengthening (PSLL) simplifies wound closure for severe open injuries without requiring microsurgical procedures as a main advantage. This method is thought to be also useful for type IIIB patients with polytrauma and other life-threatening injuries because it helps to control both wound sepsis and their general state. In the present study, we attempted to assess the problems, long-term functional outcome, and quality of life (QOL) of patients who were treated by PSLL for Gustilo type IIIB open tibial fractures in our facility.Six patients with type IIIB open tibial fractures treated with PSLL were retrospectively reviewed. The mean shortening length was 7.4 cm (range, 4.5-10.3 cm). The mean percent shortening of the entire bone was 18.7% (range, 12.3-29.7%). Limb lengthening started at a mean interval of 10.3 months (range, 3-18 months) after the original injury. The mean healing index was 56.5 days/cm (range, 31.3-86.7 days/cm). The complications, functional outcome, and quality of life were evaluated for all cases.One superficial infection at the initial corticotomy, one deep infection around the shortening site, one refracture at the healed docking site, several wire breaks in external frames in two cases, and two severe equinovarus deformities occurred as complications of these procedures. Regarding functional outcome, three patients showed good outcome, two showed fair outcome, and one showed poor outcome. The percent shortening of the entire bone in the two fair cases were more than 25%. The median scale of physical health summary, mental health summary, and total general health summary in Short Form-36 (QOL) were lower than the standard scale in age-matched individuals.This PSLL treatment was thought to be a useful option for severe open fracture of the tibia, which had bony defect in more than 4.5 cm in length after serial debridement, although several complications occurred in this regimen. However, it is difficult to achieve an excellent function and QOL using these techniques. In addition, it is difficult for patients who underwent limb lengthening after shortening more than 25% of the total length of bone to gain good function.

Risk factors for deep infection in secondary intramedullary nailing (IMN) after external fixation (EF) for open tibial fractures were investigated by multivariate analysis following univariate analyses.Forty-two open tibial fractures were treated with secondary IMN after EF. The open tibial fractures were classified according to the criteria proposed by Gustilo et al.: type II, 11; type IIIA, 8; type IIIB, 22 and type IIIC, 1. Locked IMNs with limited reaming were performed in 27 patients, and locked IMNs without reaming in 15 patients. The following factors contributing to deep infection were selected for analysis: age, gender, Gustilo type (II or III), fracture grade by AO type (A or B+C), fracture site, existence of multiple trauma (Injury Severity Score, ISS18 or ISSor = 18), existence of floating knee injury, debridement time (or = 6 h or6 h), reamed (R) versus unreamed (UR) nailing, duration of external fixation (or = 3 weeks or3 weeks), interval between removal of EF and IMN (or = 2 weeks or2 weeks), skin closure time (or = 1 week or1 week), existence of superficial infection (+ or -) and existence of pin tract infection (+ or -). The relationship between deep infection and the above factors was evaluated by univariate analyses.Seven (16.7%) of the 42 open tibia fractures developed deep infections. All deep infections occurred in Gustilo type III (22.6%, 7/31). Only the skin closure time was a significant factor affecting the occurrence of deep infection on the present analysis (p = 0.006).The present evaluation showed that early skin closure within 1 week is the most important factor in preventing deep infections when treating open tibial fractures with secondary IMN after EF.

The conversion method from external fixation (EF) to intramedullary nailing (IMN) for open tibia fractures, especially to Gustilo type IIIB open tibia fractures, have potentially high risk of infections. We document a report of a more progressive approach in four consecutive cases of type IIIB open tibial fractures successfully managed with early unreamed IMN without a safety interval and simultaneous flap coverage following EF. The mean patients age at the time of injury was 43.8 years (range 23-64 years), and three patients were male. The timing from EF to IMN without safety interval combined with well-vascularised flap (free latissimus dorsi flaps in two patients and pedicled soleus flaps in two patients) ranged 48 to 72 hours. Average time to union was 14 months (range 9-21 months). There was one nonunion patient whose fracture healing was gained by reamed IMN without bone grafting. However, there were no infections. The functions in all patients were satisfactory. This early unreamed IMN without a safety interval and with simultaneous flap coverage following EF is a useful and effective option for treating type IIIB open tibial fractures.

Two Gram-negative, non-fermentative, non-denitrifying, non-pigmented, rod-shaped bacteria that were motile by means of polar flagella, designated strains KMM 330T and KMM 331, were isolated from a deep-sea sponge specimen and subjected to a polyphasic taxonomic study. The new isolates exhibited 16S rRNA gene sequence similarity of 99·9 %, and their mean level of DNA–DNA relatedness was 82 %. Phylogenetic analysis based on their 16S rRNA gene sequences placed the strains within the genus Pseudomonas as an independent deep clade. Strain KMM 330T shared highest sequence similarity (96·3 %) with each of Pseudomonas fulva NRIC 0180T, Pseudomonas parafulva AJ 2129T and Pseudomonas luteola IAM 13000T; sequence similarity to other recognized species of the genus Pseudomonas was below 95·7 %. The marine sponge isolates KMM 330T and KMM 331 could be distinguished from the other recognized Pseudomonas species based on a unique combination of their phenotypic characteristics, including growth in 8 or 10 % NaCl, the absence of pigments, the inability to denitrify and lack of carbohydrate utilization. On the basis of phylogenetic analysis, physiological and biochemical characterization, strains KMM 330T and KMM 331 should be classified as a novel species of the genus Pseudomonas, for which the name Pseudomonas pachastrellae sp. nov. is proposed. The type strain is KMM 330T (=JCM 12285T=NRIC 0583T=CCUG 46540T).

Two Pseudomonas-like yellow-orange-pigmented non-fluorescent denitrifying strains KMM 235 and KMM 1447T were isolated from marine ascidian specimens and investigated by a polyphasic approach to clarify their taxonomic status. On the basis of 16S rDNA gene sequence data the new isolates clustered with the Pseudomonas stutzeri species group with sequence similarities of >98%. The results of DNA-DNA hybridization and biochemical characterization showed genetic and phenotypic distinction between strains KMM 235 and KMM 1447T and from the other validly described Pseudomonas species. Strain KMM 235 was found to be closely related to the type strain of Pseudomonas stutzeri in their phenotypic and genetic characteristics and represented, probably, a new P. stutzeri genomovar. It is proposed that strain KMM 1447T be classified as a new species of the genus Pseudomonas, Pseudomonas xanthomarina sp. nov., with the type strain KMM 1447T (=JCM 12468T=NRIC 0617T=CCUG 46543T).

市販食肉試料より一般細菌を分離し, 菌数を測定した。また, 分離菌の中で低温性Pseudomonas属細菌の同定を行った。その結果, 一般生菌数は102-106cfu/g確認され, 食肉のすべてにPseudomonas属細菌が存在した。また, 分離Pseudomonas属細菌の大半は4℃で生育し, 脂質やタンパク質を分解するPseudomonas fluorescensと同定された。菌数, 菌種共に20年前の状況と変わらないことが示唆された。

Five strains of Gram-positive, endospore-forming, moderately halotolerant bacteria were studied taxonomically. Four were isolated from Vietnamese fish sauce and one from the Gulf of Mexico. Phylogenetic analysis based on 16S rRNA gene sequences showed that these strains clustered within the radiation of the genus Bacillus but separately from recognized Bacillus species. DNA G+C composition of the isolates ranged from 43 to 44 mol%. Strains 15-1T and NRRL B-14850 showed high levels of DNA–DNA relatedness (82–100 %) to each other and to the other strains isolated here; they displayed low levels of DNA–DNA relatedness (Bacillus species. They grew in 15 % NaCl and optimally in 1 % NaCl, which is characteristic of moderately halotolerant bacteria. The isolates grew at pH 6·5 to 10·0 but not at pH 6·0. Their cell walls contained meso-diaminopimelic acid. The major isoprenoid quinone was MK-7 and the principal cellular fatty acids were anteiso-C15 : 0, iso-C15 : 0 and anteiso-C17 : 0. Based on these results, the strains tested were regarded as members of a novel Bacillus species for which the name Bacillus vietnamensis sp. nov. is proposed. The type strain is 15-1T (=JCM 11124T=NRIC 0531T=NRRL 23890T).

Cervical spinal cord injury is one of the most frequent causes of sudden death in blunt trauma. However, upper airway obstruction due to retropharyngeal haematoma also can be life-threatening even when the patient is neurologically intact. We report the case of a 67-year-old man who developed fatal massive retropharyngeal haematoma secondary to minor blunt trauma sustained in a motor vehicle accident.

To assess the stability of DNA from heated food products, the PCR of the 18S rDNA gene was utilized. The results show as follows; 1. DNA extracted from heated wheat flour was available for PCR amplification when heat temperature was less than 130°C. 2. DNA was directly decomposed by heat over 130°C. 3. DNA in food products was stable for heat compare with DNA or DNA of wheat flour. As a result, the thermal history of the center of the food product is important for DNA thermostability.

Two bacterial strains (KMM 3633(T) and KMM 3634) were isolated from marine coastal sea-ice and characterized by using phenotypic and molecular methods. The isolates were aerobic, Gram-negative, psychrophilic, halophilic and motile by means of a single polar flagellum. The DNA G + C content was 45.3-45.6 mol%. The major cellular fatty acids were C(16 :0), C(16 : 1)omega9c and C(18 : 1)omega7c. Comparison of almost-complete 16S rDNA sequences demonstrated that the strains were phylogenetically closely related to each other (99.5% sequence identity), and related to Marinomonas species (94.4-96.4% identity). DNA-DNA reassociation between KMM 3633(T) and KMM 3634 occurred at a level of 92%. Based on phenotypic, chemotaxonomic and phylogenetic properties, the name Marinomonas primoryensis sp. nov. is proposed for strains KMM 3633(T) and KMM 3634; the type strain is KMM 3633(T) (= JCM 11775(T) = NRIC 523(T)).

The objective of this study was to evaluate bony healing and predict factors affecting bony healing of femoral fractures treated with interlocking nailing (ILN) in static or dynamic conditions, and in reamed or unreamed procedures. Seventy-four femoral fractures (69 patients) were initially stabilized with ILNs in static condition. Among these fractures, ten static ILNs were dynamized after approximately 6 (median 6.4, range 1–13) months because of poor fracture healing. Reamed ILNs were performed for 55 fractures and unreamed ILNs for 19 fractures. Clinical and roentgenograhic processes were analyzed with emphasis on whether or not ILNs were dynamized. To evaluate any significant contributing factors affecting the nonunion of femoral shaft fractures treated with ILNs, logistic regression analysis was done. The union rates of static ILNs and dynamized ILNs were 92% (59/64) and 70% (7/10) respectively, but there was no significant difference between them. Five nonunions were seen in reamed ILNs and three in unreamed ILNs. The predictive logistic regression equation for nonunion was as follows: Log 1-p/p=1.05 −1.20 × AO/ASIF fracture grade in the femur −3.07 × existence of multiskeletal trauma in lower extremity + 0.06 × age −1.11 × smoking history −0.3 × existence of polytrauma −0.626 × the severity of soft tissue injury (p=0.002; each variable in the above equation was arranged according to the significant order). Among the variables, AO fracture grade (type C) in the femur and existence of multiskeletal trauma in lower extremity were significantly related to nonunion. Static ILN in most femoral shaft fractures does not inhibit the process of fracture healing. The following associated skeletal lesions were our concerns for nonunion and broken nail in static or dynamic ILNs: (1) C-type femoral fracture; and (2) existence of multiskeletal trauma, such as double lesions in the ipsilateral femur, floating knee injury, and bilateral femoral fractures.

食品の材料やその種類をDNA情報から特定する方法が進められている。多くの方法はその過程でPCRを行うが, PCRによる増幅がみられない場合は食品中に対象物が含まれていないのか, 抽出DNAの量や質が不十分であるかの判断が困難であるものが多かった。そこで, 本研究では抽出DNAのPCR増幅性をあらかじめ確認することで, 抽出DNAの質的量的評価を行い, PCR増幅時に起こりうる問題の原因の特定を目指すことを目的とした。 PCR増幅性の対象遺伝子領域には16S rDNA遺伝子領域, 18S rDNA遺伝子領域, ITS領域の各領域を選択し, それぞれのユニバーサルプライマーにてPCR増幅を行った。各生物より抽出したDNAを用いた結果, 増幅産物の単一性などから18S rDNA遺伝子領域が優れていることが明らかとなった。また, アニーリング温度の検討結果からも同遺伝子領域の増幅性が最も優れていることが明らかとなった。以上のことから, 食品よりDNAを抽出した際には, 目的遺伝子領域を増幅する前に18S rDNA遺伝子領域のPCR増幅を行い, あらかじめ抽出DNAの評価をするほうが望ましいと考えた。

In this study, we examined identification of glutinous rice breed by RAPD method. Seven polished rice, Hiyokumochi, Hakutyoumochi, Himenomoti, Koganemoti, Miyakoganemoti, Wataboushi, and Chinese rice were used in this study. In addition, these rices were produced in 2000. DNA was extracted and purified by CTAB method. DNA was amplified with OPB, OPM, and OPT primers (Operon Inc.) by PCR. Molecular weight of amplified DNA was determined by electrophoresis. As a result, all breeds of rice were determined by RAPD method.

Multiple-injury patients absorb a large amount of trauma energy in their trunk, resulting in multiple organ injuries and often long-bone and axial skeletal fractures. Severity of these injuries can be quantified using the Trauma Index (TI) and Injury Severity Score (ISS). These systems allow not only a comparison of mortality and morbidity among institutions, but also serve as criteria for triage, emergency, and subsequent management of patients. We reviewed 229 multiple-trauma patients with orthopedic injuries who were treated in our trauma center. Based on the aim of orthopedic intervention performed within 1 week of injury, patients were divided into four groups: group A, surgery for supporting patient's life (n=8); group B, surgery for salvaging limbs and limb function (n=81); group C, surgery for preventing the development of serious complications (n=92); and group D, minor surgery performed according to the general situation (n=48). Both the TI and ISS were compared among these four groups. Furthermore, both the TI and ISS were compared between patients who died within 24 h and those who survived for longer. The ISS was significantly different between groups B and C (P

A protein transiently expressed in the neural precursors of developing tissues (TENP) was found to be present in emu (Dromaius novaehollandiae) egg white as one of the major proteins. Nucleotide analysis of its encoding cDNA revealed a sequence of 452 amino acids including a 19 amino acid peptide signal. Phylogenetic analysis determined that emu TENP was clustered within the bactericidal/permeability-increasing protein (BPI) superfamily together with other avian TENPs. RT-PCR analysis revealed that the emu TENP gene was highly expressed in the magnum of the oviduct, indicating that TENP is a major egg white component. Emu TENP was purified by anion exchange chromatography and ammonium sulfate fractionation. Unlike BPI, emu TENP exhibited antibacterial activity against Gram-positive bacteria, including Micrococcus luteus and Bacillus subtilis, but not against Gram-negative bacteria such as Escherichia coli and Salmonella Typhimurium. The results suggest that emu TENP is a potent novel antibacterial protein with a spectrum distinct from that of BPI.

The description of Pseudomonas straminae lizuka and Komagata 1963 was emended with data newly obtained. The spelling of the name of this taxon is also corrected as Pseudomonas straminea. Strains that were previously named 'Pseudomonas ochracea' were identified as P. straminea.

The proteolytic activity of Pseudomonas strains including P. fragi, P. taetrolens, P. aeruginosa, P. chlororaphis, P. fluorescens, P. putida, and P. synxantha was determined at various temperatures from 4°C to 37°C. Out of 43 strains tested, 33 strains grew well at 10°C within 3 days, and all strains except for a P. aeruginosa strain grew at 4°C within 7days. Four P. fragi strains, one P. taetrolens strain, one P. chlororaphis strain, two P. fluorescens strains, and one P. synxantha strain decomposed skimmilk at 10°C within 7 days. Ten P. fragi strains, one P. taetrolens strain, one P. chlororaphis strain, three P. fluorescens strains, and one P. synxantha strain did so at 4°C within 3 weeks. Strains of P. putida did not decompose skimmilk within 3 weeks. The proteolytic activity of P. fragi, P. taetrolens and their related species at low temperatures was discussed from the viewpoint of food hygiene

Survival of microorganisms in the food is interesting from the viewpoint of food preservation. However, it is difficult to apply data from one kind of the food to another kind of the food because of different conditions of foods. A total of 61 strains, 30 species, and 10 genera, including lactic acid bacteria and natto-bacilli, were used in this study. Various bacteria were preserved in pure water at low temperature (4°C) and room temperature (25°C), and the numbers of surviving cells were determined after one and four years' preservation. Most strains tested survived in pure water though the numbers of the cells were reduced from 106-108/ml to 104-106/ml. Moreover, survival of bacteria was correlated with the kinds of bacteria and temperatures for preservation. Various bacteria including putrefactive bacteria were considered to be alive in the food. The survived bacteria which survied on the food would be concerned with food putrefaction.

Low-Intensity Pulsed Ultrasound (LIPUS) provided a mechanical stimulus, and was thought to promote fracture healing by signal transduction through integrin, a cytoskeletal protein. Meanwhile, teriparatide, a drug for osteoporosis treatment, showed efficacy in promoting bone metabolism. This drug also appeared to prevent fractures in patients with serious osteoporosis by improving bone mineral density and bone quality, which in turn resulted from promoting action for bone metabolism. Further, clinical trials and fundamental research reported that teriparatide demonstrated the effect of promoting fracture healing. Mechanical stimulus by LIPUS had a topical effect on fractures; on the other hand, teriparatide (peptide hormone) had both topical and systemic effects. Both LIPUS and teriparatide had the effect of fracture healing, but it was supposed that the characteristics of each effect were different because of the different mechanism of action. Moreover, the combination therapy of LIPUS and teriparatide was expected to produce synergies. We used elderly rats as models for the femoral fracture to examine the effects of LIPUS and teriparatide on promoting fracture healing for treatment delay by aging. We observed the fracture healing process in 40-week-old rats as an elderly model using simple radiographs, and recognized a delay in fracture healing compared with that of 8-week-old rats. As discussed in histomorphology, it was demonstrated that the period of endochondral ossification, from chondrogenesis to teleost cross-linked callus, was prolonged and the fracture healing process was delayed by aging. Next, we treated the elderly fracture models with LIPUS for 20 minutes a day from the first day after the fracture, and compared them with non-treated models. The bone unions of the treated models were observed earlier than those of non-treated models in the simple radiographs. LIPUS shortened the period of endochondral ossification. Further, we gave the elderly fracture models teriparatide subcutaneously 5 μg/kg three times a week from the first day after the fracture. Bone unions of the treated models were observed earlier than those of non-treated models in simple radiographs as well. In micro CT analysis, it was demonstrated that lamellar bone transforming and bone remodeling of the trabecular structure of external callus were especially accelerated. The results of these trials showed that both LIPUS and teriparatide demonstrated the effect of promoting fracture healing, and each had unique characteristics.

Lysozyme (LZ), a bacteriolytic enzyme, is found in the egg white of many avian eggs and plays an important role in host defense; however, LZ activity in emu (Dromaius novaehollandiae) egg white is exceptionally undetectable. We cloned and characterized emu goose-type LZ (LZG) and chicken-type LZ (LZC) genes. RT-PCR analysis revealed very low LZG gene expression levels and absence of LZC gene expression in the emu oviduct. Sequencing of full-length LZG and LZC cDNAs indicated that their amino acid sequences show high similarities to ostrich LZG and LZC, respectively, with conserved catalytic residues for enzymatic activities. Whereas recombinant emu LZG prepared using Pichia pastoris exhibited similar enzyme activity as ostrich LZG, recombinant emu LZC exhibited significantly higher lytic activity than chicken LZC. We concluded that emus have functional genes for both LZG and LZC like many other avians, and the LZG gene is expressed in oviduct probably as in other ratite, however, its expression levels in egg white were low to be detected.

The aim of the study was to survey cultivable heterotrophic bacteria from sea ice samples collected from Peter the Great Bay of the Sea of Japan, and to test the isolates for antagonistic activity. A total of 195 strains were isolated and investigated by a phenotypic approach. The 16S rRNA gene sequence analysis of thirty eight strains revealed primarily representatives of the class Gammaproteobacteria, phylum Bacteroidetes, and phylum Actinobacteria. Members of the class Alphaproteobacteria were a minor component. Most strains shared 98-99% sequence similarity to recognized species, including those recovered previously from Antarctic or Arctic sea ice or polar environments, Shewanella frigidimarina, Psychrobacter urativorans, Psychrobacter glacincola, Psychrobacter fozii, Pseudomonas veronii, or Pseudomonas proteolytica. At least seven bacterial groups may represent novel species within known genera. Five isolates have been previously described as the novel species Marinomonas primoryensis and Psychrobacter maritimus. Antagonistic activity assays revealed a number of strains-antagonists of the Pseudomonas fluorescens group, Bacillus and Nocardioides displaying remarkable antifungal and antibacterial activities. For the first time our findings show that sea ice offshore in the Sea of Japan represents an untapped source of bacterial biodiversity and microorganisms capable of antibacterial and antifungal metabolite production.

We previously isolated Streptomyces racemochromogenes strain 10-3, which produces a phospholipase D (PLD) with high transphosphatidylation activity. Here, we purified and cloned the PLD (PLD103) from the strain. PLD103 exerted the highest hydrolytic activity at a slightly alkaline pH, which is in contrast to the majority of known Streptomyces PLDs that have a slightly acidic optimum pH. PLD103 shares only 71-76% amino acid sequence identity with other Streptomyces PLDs that have a slightly acidic optimum pH; thus, the diversity in the primary structure might explain the discrepancy observed in the optimum pH. The purified PLD displayed high transphosphatidylation activity in the presence of glycerol, L: -serine, and 2-aminoethanol hydrochloride with a conversion rate of 82-97% in a simple one-phase system, which was comparable to the rate of other Streptomyces PLDs in a complicated biphasic system.

Previously we isolated six actinomycetes strains, 9-4, 10-1, 10-2, 10-3, 10-6, and 21-4, that produce phospholipase D (PLD) with high transphosphatidylation activity. In this study, we identified these strains, and the PLD activities were compared with those of reference strains. 16S rDNA sequences and DNA-DNA hybridization tests indicated taxonomic affiliations of strain 9-6 with Streptomyces senoensis, strains 10-1 and 10-6 with S. vinaceus, and strains 10-2 and 10-3 with S. racemochromogenes. Strain 21-4, though identified as a Streptomyces sp., could not be identified with any known species. Meanwhile, most of the culture supernatants of reference strains demonstrated no or very weak PLD activity, while those of our strains exhibited significantly higher activity. All of the strains in this study were identified as Streptomyces species. The PLD activity of our strains exceeded most of the reference Streptomyces strains. The findings in this study imply that the Streptomyces strains, although they are members of the same species, can produce different quantities of PLD enzyme.

Emu riboflavin-binding protein (RBP) was purified from egg white and yolk, and its N-terminal amino acid sequence was determined. The molecular mass of emu RBP was estimated at approximately 48 and 45 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, i.e., 10 kDa larger than chicken RBP. The molecular mass of deglycosylated RBPs indicated that the content of oligosaccharide chain in emu RBP was approximately 3 times greater than that in chicken RBP. The gene encoding the RBP precursor was cloned from emu oviduct cDNA by PCR and found also in the liver and ovary cDNAs as well as oviduct cDNA. The complete cDNA consisted of an open reading frame of 714 bp encoding a protein of 238 amino acids. The amino acid sequence deduced from the cDNA sequence revealed that many essential structural features were conserved in emu RBP including 18 cysteine residues, 2 N-glycosylation sites, a clustered phosphorylation region, and riboflavin-binding sites. Two additional potential N-glycosylation sites were found in the amino acid sequences of RBPs from the emu and other sources such as the turtle and frog, which might in part account for the greater content of oligosaccharide chain of emu RBP as compared to chicken RBP.

An aerobic, Gram-negative bacterium, strain KMM 329(T), was isolated from a deep-sea sponge specimen from the Philippine Sea and subjected to a polyphasic taxonomic investigation. Comparative 16S rRNA gene sequence analysis showed that strain KMM 329(T) clustered with the species of the genus Lysobacter. The highest level of 16S rRNA gene sequence similarity (97.0 %) was found with respect to Lysobacter concretionis KCTC 12205(T); lower values (96.4-95.2 %) were obtained with respect to the other recognized Lysobacter species. The value for DNA-DNA relatedness between strain KMM 329(T) and L. concretionis KCTC 12205(T) was 47 %. Branched fatty acids 16 : 0 iso, 15 : 0 iso, 11 : 0 iso 3-OH and 17 : 1 iso were found to be predominant. Strain KMM 329(T) had a DNA G+C content of 69.0 mol%. On the basis of the phenotypic, chemotaxonomic, DNA-DNA hybridization and phylogenetic data, strain KMM 329(T) represents a novel species of the genus Lysobacter, for which the name Lysobacter spongiicola sp. nov. is proposed. The type strain is KMM 329(T) (=NRIC 0728(T) =JCM 14760(T)).

It is important to predict the occurrence of deep infection in open fractures when treating such fractures. We tried to develop a new scoring system for predicting the occurrence of deep infection in open upper and lower extremity fractures on the basis of the Hannover Fracture Scale'98 (HFS-98).A total of 394 open upper and lower extremity fractures (351 patients) were retrospectively reviewed in the initial analysis. The relationship between Gustilo's grade and the eight items on HFS-98 in the open extremity fractures was first investigated by multivariate analysis. By this analysis, we selected significant items that correlated with Gustilo's grade. Among these cases, 318 patients with 352 open extremity fractures (humerus = 27, forearm = 62, femur = 76, tibia = 187) were used for the following infection analyses. The relationships between the incidence of deep infection and sex (male or female), age (30, 30-50,50 years), grade of polytrauma (ISS18, 18or = ISSor = 30, ISS30), site of fracture (humerus, forearm, femur, tibia), existence of fracture line around joint (+ or -) or some significant items in the above initial analysis were further analyzed by multivariate analysis after univariate analysis. We devised a new scoring system of open extremity fractures based on P values in the above analysis. The discrimination of the newly devised scoring system was evaluated with receiver operating characteristic (ROC) curves.The following factors: muscle injury (MI, P = 0.0001); wound contamination (WC, P = 0.0001); and local circulation (LC, P = 0.0001) were significant factors affecting the occurrence of deep infection on multivariate analysis. We devised a new scoring system for open extremity fractures (MI: 0-20 points, WC: 0-20 points, and LC: 0-20 points). The cut-off point for occurrence of deep infection in these fractures was 35 by ROC analysis.This new scoring system was thought to be useful for predicting the occurrence of deep infection in open extremity fractures. However, further prospective study or multicenter study would be needed to clarify the validity of this scale.

In patients with unstable pelvic ring fractures, the factors related to poor outcome are still controversial. The purpose of our study was to evaluate the long-term functional outcome of patients with unstable pelvic ring fractures and correlate it with various other factors.Fifty-seven patients who had a minimal follow-up of 2 years completed the three-view plain radiographs, physical examination, and functional assessment with questionnaire. There were 28 male and 29 female patients with an average age of 42.4 years and Injury Severity Score of 24.6 points. The mean time of follow-up was 47.2 months. Thirty-nine patients were Tile type B, and 18 were type C. Twenty-three patients were treated conservatively, 22 with external fixation, and 12 with internal fixation. The results were scored with the Majeed score, the Iowa Pelvic Score, and the Medical Outcomes Study Short-Form 36-item Health Survey (SF-36). Statistical analysis was performed by use of the Pearson correlation coefficient test and multiple regression analysis.The average Majeed score was 79.7, the average IPS was 80.7, and the average physical component summary of the SF-36 was 13.4 points worse than that of the population norm. These scores correlate significantly with each other. The average residual displacement was 7.3 mm anteriorly and 5.2 mm posteriorly. Multiple regression analysis revealed that the Majeed score and the physical component summary of the SF-36 correlated with the presence of neurologic injury, and the Iowa Pelvic Score correlated with the presence of a mental disorder, posterior displacement, and neurologic injury.The long-term functional outcome after unstable pelvic ring fracture was not associated with Injury Severity Score, fracture location or fracture type. We discovered a close correlation between neurologic injury and functional outcome.

An aerobic, Gram-negative, non-motile, yellow-pigmented bacterium, strain KMM 3046T, was isolated from a deep-sea brittle star from the Fiji Sea and was subjected to a polyphasic taxonomic analysis. Strain KMM 3046T grew at 5–32 °C and in the presence of 1–12 % (w/v) NaCl. It contained MK-6 as the predominant menaquinone and 3-OH i16 : 0, 3-OH i17 : 0 and 3-OH a17 : 0 as the major fatty acids. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain KMM 3046T forms a distinct evolutionary lineage within the family Flavobacteriaceae (phylum Bacteroidetes), displaying 92.3–91.9 % sequence similarity with respect to Salegentibacter species. On the basis of the phenotypic and phylogenetic data, strain KMM 3046T represents a novel genus and species of the family Flavobacteriaceae, for which the name Marixanthomonas ophiurae gen. nov., sp. nov. is proposed. The type strain of Marixanthomonas ophiurae is KMM 3046T (=NRIC 0684T=JCM 14121T).

Background There are a number of different methods for generation of trees and algorithms for phylogenetic analysis in the study of bacterial taxonomy. Genotypic information, such as SSU rRNA gene sequences, now plays a more prominent role in microbial systematics than does phenotypic information. However, the integration of genotypic and phenotypic information for polyphasic studies is necessary for the classification and identification of microbes. Thus, we devised an algorithm that objectively identifies discriminative characteristics for focused clusters on generated trees from a dataset composed of coded data, such as phenotypic information. Moreover, this algorithm has been integrated into the polyphasic analysis software, InforBIO. Results We developed a differential-character-finding algorithm based on information measures and used this algorithm to identify the characteristic that best discriminates operational taxonomic unit clusters. For all characteristics in a dataset, the algorithm estimates commonality in focused clusters and diversity among clusters by scoring based on Shannon's and relative entropies. All the characteristics selected for scoring are equally weighted. Thresholds for the scores are defined to identify discriminative characteristics for clusters efficiently from a database. The unique feature of the algorithm, which is implemented in the InforBIO software, is that it can identify the phenotypic characteristics that discriminate and are associated with the clusters of a phylogenetic tree. We successfully applied this algorithm to the study of phylogenetic clusters of Pseudomonas species. Conclusion The algorithm in the InforBIO software is a novel and useful approach for microbial polyphasic studies. The algorithm can also be applied to diverse cluster analyses. The InforBIO software is available from the download site http://wdcm.nig.ac.jp/inforbio/. This software is free for personal but not commercial use.

The present study was conducted to screen microorganisms that produce phospholipase D (PLD), and we especially focused on the strains having high transphosphatidylation activity. Eighty bacterial strains were isolated from soil samples by a screening method utilizing a preliminary selection medium with phosphatidylcholine (PC) as the sole carbon source. The culture supernatants were then assayed for PLD activity. The finding of dual PLD activities in cultures revealed that the hydrolytic and transphosphatidylation activities were correlated. Consequently, six strains were selected as stably producing PLD enzyme(s) during continuous subcultures. The culture supernatants of selected strains synthesized phosphatidylglycerol, phosphatidylserine and phosphatidylethanolamine from PC with high conversion rates. These isolated strains will be made available to carry out phospholipid modification through the efficient transphosphatidylation activity of the PLD that they produce.

We purified phospholipase D (PLD) enzyme from peanut seeds, and the PLD enzyme eluted as two distinct peak fractions on Mono-Q chromatography, the first of which was characterized. N-terminal sequencing indicated that the N-terminus was blocked. The molecular mass of the purified enzyme was estimated to be 92 kDa by SDS-PAGE. The pH optimum of the enzyme was 5.0, and the Km value against its substrate phosphatidylcholine (PC), in the presence of 10 mM CaCl2 and 4 mM deoxycholate, was estimated to be 0.072 mM. The enzyme catalyzed two reactions, i.e., hydrolysis of PC generating phosphatidic acid (PA) and choline, and transphosphatidylation of the PA-moiety in the PC molecule to the acceptor glycerol, generating phosphatidylglycerol. Furthermore, we cloned two types of full-length cDNA, Ahpld1 and Ahpld2, each encoding distinct PLD molecules having 794 and 807 residues, respectively. The partial amino acid sequence of the purified PLD was consistent with the deduced sequence of AhPLD2.