記述言語：日本語   出版者・発行元：日本細菌学会  

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1093/jtm/taae011

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Methicillin-resistant Staphylococcus spp. present challenges in clinical and veterinary settings because effective antimicrobial agents are limited. Phage-encoded peptidoglycan-degrading enzyme, endolysin, is expected to be a novel antimicrobial agent. The enzymatic activity has recently been shown to be influenced by the linker between functional domains in the enzyme. S6_ORF93 (ORF93) is one of the endolysins derived from previously isolated Staphylococcus giant phage S6. The ORF93 was speculated to have a catalytic and peptidoglycan-binding domain with a long linker. In this study, we examined the influence of linker shortening on the characteristics of ORF93. We produce wild-type ORF93 and the linker deletion mutants using an Escherichia coli expression system. These mutants were designated as ORF93-Δ05, ORF93-Δ10, ORF93-Δ15, and ORF93-Δ20, from which 5, 10, 15, and 20 amino acids were removed from the linker, respectively. Except for the ORF93-Δ20, ORF93 and its mutants were expressed as soluble proteins. Moreover, ORF93-Δ15 showed the highest yield and bacteriolytic activity, while the antimicrobial spectrum was homologous. The cold storage experiment showed a slight effect by the linker deletion. According to our results and other studies, linker investigations are crucial in endolysin development.

DOI： 10.1371/journal.pone.0310962

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記述言語：日本語   出版者・発行元：(一社)日本臨床微生物学会  

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Springer Science and Business Media LLC  

Abstract

Background

Subclinical infection with bovine leukemia virus (BLV) in cows can cause economic losses in milk and meat production in many countries, as BLV-related negative effects. The volatile fatty acids (VFAs) and microbiota present in the digestive tracts of cows can contribute to cow health. Here, we exploratorily investigated the VFAs and microbiota in the rumen and gut with respect to subclinical BLV infection using cows housed at a single farm.

Results

We analyzed a herd of 38 cows kept at one farm, which included 15 uninfected and 23 BLV-infected cows. First, the analysis of the VFAs in the rumen, gut, and blood revealed an absence of statistically significant differences between the uninfected and BLV-infected groups. Thus, BLV infection did not cause major changes in VFA levels in all tested specimens. Next, we analyzed the rumen and gut microbiota. The analysis of the microbial diversity revealed a modest difference between the uninfected and BLV-infected groups in the gut; by contrast, no differences were observed in the rumen. In addition, the investigation of the bacteria that were predominant in the uninfected and BLV-infected groups via a differential abundance analysis showed that no significant bacteria were present in either of the microbiota. Thus, BLV infection possibly affected the gut microbiota to a small extent. Moreover, bacterial associations were compared between the uninfected and BLV-infected groups. The results of this analysis suggested that BLV infection affected the equilibrium of the bacterial associations in both microbiota, which might be related to the BLV-related negative effects. Thus, BLV infection may negatively affect the equilibrium of bacterial associations in both microbiota.

Conclusions

Subclinical BLV infection is likely to affect the rumen and gut microbiota, which may partly explain the BLV-related negative effects.

DOI： 10.1186/s13213-023-01737-4

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その他リンク： https://link.springer.com/article/10.1186/s13213-023-01737-4/fulltext.html

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The deterioration in reproductive performance in association with low fertility leads to significant economic losses on dairy farms. The uterine microbiota has begun to attract attention as a possible cause of unexplained low fertility. We analyzed the uterine microbiota associated with fertility by 16S rRNA gene amplicon sequencing in dairy cows. First, the alpha (Chao1 and Shannon) and beta (unweighted and weighted UniFrac) diversities of 69 cows at four dairy farms that had passed the voluntary waiting period before the first artificial insemination (AI) were analyzed with respect to factors including farm, housing style, feeding management, parity, and AI frequency to conception. Significant differences were observed in the farm, housing style, and feeding management, except parity and AI frequency to conception. The other diversity metrics did not show significant differences in the tested factors. Similar results were obtained for the predicted functional profile. Next, the microbial diversity analysis of 31 cows at a single farm using weighted UniFrac distance matrices revealed a correlation with AI frequency to conception but not with parity. In correlation with AI frequency to conception, the predicted function profile appeared to be slightly modified and a single bacterial taxon, Arcobacter, was detected. The bacterial associations related to fertility were estimated. Considering these, the uterine microbiota in dairy cows can be varied depending on the farm management practices and may become one of the measures for low fertility. IMPORTANCE We examined the uterine microbiota associated with low fertility in dairy cows derived from four commercial farms via a metataxonomic approach using endometrial tissues prior to the first artificial insemination. The present study provided two new insights into the relevance of uterine microbiota with respect to fertility. First, the uterine microbiota varied depending on housing style and feeding management. Next, a subtle change was observed in functional profile analysis: a formation of uterine microbiota was detected to be different in correlation with fertility in one farm studied. Considering these insights, an examination system on bovine uterine microbiota is hopefully established based on continuous research on this topic.

DOI： 10.1128/spectrum.04764-22

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.18926/AMO/64355

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

BACKGROUND AND AIMS: Ulcerative colitis (UC) can develop colitis-associated colorectal neoplasm (CAN). Adenine-to-inosine RNA editing, which is regulated by adenosine deaminase acting on RNA (ADAR), induces the posttranscriptional modification of critical oncogenes, including antizyme inhibitor 1 (AZIN1), leading to colorectal carcinogenesis. Therefore, we hypothesized that ADAR1 might be involved in the development of CAN in UC. METHODS: We systematically analyzed a cohort of 139 UC cases (40 acute phase, 73 remission phase, 26 CAN). The degree of inflammation was evaluated using the Mayo endoscopic score (MES). RESULTS: The type 1 IFN-related inflammation pathway was upregulated in the rectum of active UC, rectum of UC-CAN, and tumor site of UC-CAN patients. ADAR1 expression was upregulated in the entire colon of CAN cases, while it was down-regulated in non-CAN MES0 cases. ADAR1 expression in the rectum predicted the development of CAN better than p53 or β-catenin, with an area under the curve of 0.93. The high expression of ADAR1 and high AZIN1 RNA editing in UC was triggered by type 1 IFN stimulation from UC-specific microbiomes, such as Fusobacterium in vitro analyses. The induction of AZIN1 RNA editing by ADAR1, whose expression is promoted by Fusobacterium, may induce carcinogenesis in UC. CONCLUSIONS: The risk of CAN can be evaluated by assessing ADAR1 expression in the rectum of MES0 UC patients, freeing UC patients from unnecessary colonoscopy and reducing their physical burden. RNA editing may be involved in UC carcinogenesis, and may be used to facilitate the prevention and treatment of CAN in UC.

DOI： 10.1093/ecco-jcc/jjac186

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Helicobacter pylori infection is an important risk factor for developing gastric cancer. However, only a few H. pylori-infected people develop gastric cancer. Thus, other risk factors aside from H. pylori infection may be involved in gastric cancer development. This study aimed to investigate whether the nitrate-reducing bacteria isolated from patients with atrophic gastritis caused by H. pylori infection are risk factors for developing atrophic gastritis and gastric neoplasia. Nitrate-reducing bacteria were isolated from patients with atrophic gastritis caused by H. pylori infection. Among the isolated bacteria, Actinomyces oris, Actinomyces odontolyticus, Rothia dentocariosa, and Rothia mucilaginosa were used in the subsequent experiments. Cytokine inducibility was evaluated in monocytic cells, and mitogen-activated protein kinase (MAPK) activity and cell cycle were assessed in the gastric epithelial cells. The cytotoxicities and neutrophil-inducing abilities of the Actinomyces and Rothia species were enhanced when cocultured with H. pylori. Th1/Th2-related cytokines were also expressed, but their expression levels differed depending on the bacterial species. Moreover, H. pylori and Actinomyces activated MAPK (ERK and p38) and affected cell cycle progression. Some nitrate-reducing bacteria cocultured with H. pylori may promote inflammation and atrophy by inducing cytokine production. In addition, the MAPK activation and cell cycle progression caused by these bacteria can contribute to gastric cancer development.

DOI： 10.3390/microorganisms10122495

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Amidst the global spread of antimicrobial resistance, New Delhi metallo-β-lactamase (NDM)-type carbapenemase-producing Enterobacterales (CPE) remain uncommon in Japan, and the detection of such highly drug-resistant organisms is limited to inbound cases. There is little evidence regarding the prevalence of NDM β-lactamase gene (blaNDM)-harboring CPE in the domestic community, especially in the provincial cities of Japan. Herein, we report the isolation of a blaNDM-1-harboring plasmid in Enterobacter cloacae complex strain isolated from an elderly woman without a history of traveling abroad who had resided in a long-term care facility in Okayama, Japan. The multidrug-resistant blaNDM-harboring CPE isolate was detected in a stool sample of the patient during routine screening at admission. We performed whole-genome sequencing analysis of the isolate using MiSeq (Illumina) and MinION (Oxford Nanopore Technologies) platforms. The isolate was identified as sequence type 171, which has predominantly been reported in the United States and China. The blaNDM-1 gene was encoded on the 46,161 bp IncX3 plasmid, with sequence similarity to plasmids of similar size isolated from individuals in China. Collectively, the genomic data suggest that an imported CPE isolate may have spread among healthy individuals in the regional area of Japan.

DOI： 10.1016/j.jiac.2022.08.019

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Bacteriophages (phages) are the most diverse and abundant life-form on Earth. Jumbophages are phages with double-stranded DNA genomes longer than 200 kbp. Among these, some jumbophages with uracil in place of thymine as a nucleic acid base, which we have tentatively termed "dU jumbophages" in this study, have been reported. Because the dU jumbophages are considered to be a living fossil from the RNA world, the evolutionary traits of dU jumbophages are of interest. In this study, we examined the phylogeny of dU jumbophages. First, tBLASTx analysis of newly sequenced dU jumbophages such as Bacillus phage PBS1 and previously isolated Staphylococcus phage S6 showed similarity to the other dU jumbophages. Second, we detected the two partial genome sequences of uncultured phages possibly relevant to dU jumbophages, scaffold_002 and scaffold_007, from wastewater metagenomics. Third, according to the gene-sharing network analysis, the dU jumbophages, including phages PBS1 and S6, and uncultured phage scaffold_002 formed a cluster, which suggested a new viral subfamily/family. Finally, analyses of the phylogenetic relationship with other phages showed that the dU jumbophage cluster, which had two clades of phages infecting Gram-negative and Gram-positive bacteria, diverged from the single ancestral phage. These findings together with previous reports may imply that dU jumbophages evolved from the same origin before divergence of Gram-negative and Gram-positive bacteria.

DOI： 10.1016/j.virusres.2022.198881

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Springer Science and Business Media LLC  

The emergence of high-level daptomycin (DAP)-resistant (HLDR) Corynebacterium striatum has been reported as a result of loss-of-function point mutations or premature stop codon mutations in a responsible gene, pgsA2. We herein describe the novel detection of an HLDR C. striatum clinical isolate, in which IS30-insertion was corroborated to cause destruction of pgsA2 gene. We isolated an HLDR C. striatum from a critically ill patient with underlying mycosis fungoides who had been treated with DAP for 10 days. With a sequence investigation, IS30-insertion was discovered to split pgsA2 in the HLDR C. striatum strain, which may cause disrupted phospholipid phosphatidylglycerol (PG) production. Future studies should survey the prevalence of IS-mediated gene inactivation among HLDR C. striatum clinical isolates.

DOI： 10.1007/s10096-021-04369-1

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その他リンク： https://link.springer.com/article/10.1007/s10096-021-04369-1/fulltext.html

記述言語：日本語   出版者・発行元：(一社)日本臨床微生物学会  

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記述言語：日本語   出版者・発行元：(一社)日本臨床微生物学会  

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Microbiology Society  

The options available for treating infections with carbapenemase-producing <italic>
<named-content content-type="family">
<ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://doi.org/10.1601/nm.3091" xlink:type="simple">Enterobacteriaceae</ext-link>
</named-content>
</italic> (CPE) are limited; with the increasing threat of these infections, new treatments are urgently needed. Biapenem (BIPM) is a carbapenem, and limited data confirming its <italic>in vitro</italic> killing effect against CPE are available. In this study, we examined the minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of BIPM for 14 IMP-1-producing <italic>
<named-content content-type="family">
<ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://doi.org/10.1601/nm.3091" xlink:type="simple">Enterobacteriaceae</ext-link>
</named-content>
</italic> strains isolated from the Okayama region in Japan. The MICs against almost all the isolates were lower than 0.5 µg ml⁻¹, indicating susceptibility to BIPM, while approximately half of the isolates were confirmed to be bacteriostatic to BIPM. However, initial killing to a 99.9 % reduction was observed in seven out of eight strains in a time–kill assay. Despite the small data set, we concluded that the <italic>in vitro</italic> efficacy of BIPM suggests that the drug could be a new therapeutic option against infection with IMP-producing CPE.

DOI： 10.1099/jmm.0.001430

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担当区分：最終著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：Wiley  

DOI： 10.1111/1348-0421.12888

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その他リンク： https://onlinelibrary.wiley.com/doi/full-xml/10.1111/1348-0421.12888

掲載種別：研究論文（学術雑誌）   出版者・発行元：MDPI AG  

The infection caused by Helicobacter pylori is associated with several diseases, including gastric cancer. Several methods for the diagnosis of H. pylori infection exist, including endoscopy, the urea breath test, and the fecal antigen test, which is the serum antibody titer test that is often used since it is a simple and highly sensitive test. In this context, this study aims to find the association between different antibody reactivities and the organization of bacterial genomes. Next-generation sequences were performed to determine the genome sequences of four strains of antigens with different reactivity. The search was performed on the common genes, with the homology analysis conducted using a genome ring and dot plot analysis. The two antigens of the highly reactive strains showed a high gene homology, and Western blots for CagA and VacA also showed high expression levels of proteins. In the poorly responsive antigen strains, it was found that the inversion occurred around the vacA gene in the genome. The structure of bacterial genomes might contribute to the poor reactivity exhibited by the antibodies of patients. In the future, an accurate serodiagnosis could be performed by using a strain with few gene mutations of the antigen used for the antibody titer test of H. pylori.

DOI： 10.3390/toxins13070467

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Informa UK Limited  

Background: Helicobacter pylori infection poses a risk of the occurrence of gastrointestinal diseases, such as gastric cancer. Its incidence rate is significantly reduced by eradication, and thereby, eradication therapy is generally performed. Disulfiram is an oral prescription drug mainly used for the treatment of alcohol dependence. In recent years, reports have been made on its anticancer and antibacterial effects, and thus, it has recently become an interesting subject. This study aimed to examine the antibacterial activity of disulfiram, investigate the presence or absence of its antibacterial activity on H. pylori, and determine whether it could be a new bactericidal drug against drug-resistant H. pylori. Materials and Methods: Drug-sensitive strains of H. pylori and amoxicillin-resistant, clarithromycin-resistant, and metronidazole-resistant strains were used, and a growth inhibition test of H. pylori using disulfiram was performed. Furthermore, the expression of urease, vacuolating cytotoxin A (VacA), and CagA, the virulence proteins of H. pylori, was quantitatively analyzed using the Western blotting method. In addition, for H. pylori used in this study, the 16SrDNA sequence, a ribosomal gene involved in protein production, was analyzed to examine the presence or absence of gene mutation. Results: Disulfiram suppressed the growth of 7 out of 12 H. pylori strains at 1 µg/mL, and no correlation was observed between their susceptibility/resistance to current eradication antimicrobial drugs and disulfiram resistance. Disulfiram reduced the expression levels of urease, VacA, and CagA proteins. H. pylori, which showed resistance to disulfiram, tended to have fewer gene deletions/insertions in the 16S rDNA sequence; however, no specific mutation was detected. Conclusion: Disulfiram has a bactericidal effect on H. pylori at low concentrations, suggesting that it can be used as a supplement for current H. pylori eradication drugs.

DOI： 10.2147/idr.s299177

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担当区分：最終著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

The genus Tsukamurella is a fastidious, environmental organism that potentially causes various infections in humans. Due to the morphological and biochemical similarities to others pathogens, such as Gordona, Rhodococcus, Corynebacterium, Nocardia, and Mycobacterium, a molecular-based approach is indispensable to correctly identify them. Herein, we describe a case of Tsukamurella inchonensis bacteremia complicated with septic pulmonary emboli (SPE), which is the first in the literature. A 44-year-old Japanese woman diagnosed with tongue cancer had undergone partial tongue resection. While receiving chemotherapy and radiotherapy, she developed high fever. Chest computed tomography suggested multiple emboli at the bilateral, peripheral lungs, indicating SPE. Blood culture detected Gram-positive rods, to which matrix-assisted laser desorption/ionization-time of flight mass spectrometry failed to identify. Then, we attempted to characterize it by 16S rRNA sequence, which suggested the organism to be Tsukamurella species but resulted in low resonance of the species-level identification. Additionally, we employed a confidence gene targeting groEL, leading to 100% matching (753/753 bps) with T. inchonensis NCTC 10741 (GenBank accession no. LR131273.1), which has been incorrectly registered as wrong species name Tsukamurella paurometabola in the database. Under the diagnosis of T. inchonensis-associated SPE, we successfully treated the patient with imipenem/cilastatin administration for 4 weeks. Sequencing analysis of groEL was of great use in identifying the organism in this case. More clinical cases based on molecular diagnosis of the fastidious pathogens need to be accumulated to further understand the characteristics and appropriate treatment regimen.

DOI： 10.1016/j.jiac.2020.09.024

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掲載種別：研究論文（学術雑誌）   出版者・発行元：The Society for Antibacterial and Antifungal Agents, Japan  

DOI： 10.4265/bio.26.137

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Springer Science and Business Media LLC  

<title>Abstract</title><sec>
<title>Objective</title>
<italic>Weissella confusa</italic> F213 (WCF213) and <italic>Lactobacillus rhamnosus</italic> FBB81 (LrFBB81) are two probiotic candidates isolated from humans in our previous study. Their functional activity on the mucosal barrier has not yet been adequately investigated. Therefore, the objective of this study was to investigate the effect of these strains on maintaining mucosal integrity in vitro. Caco-2 cell monolayers were pretreated with WCF213 and LrFBB81 before being exposed to hydrogen peroxide. The integrity of mucosal cells was evaluated by measuring the transepithelial resistance (TER), flux of FITC-labelled dextran, and ZO-1 protein distribution with the help of an immunofluorescence method.


</sec><sec>
<title>Results</title>
WCF213 was found to significantly maintain the TER better than the control hydrogen peroxide-treated cells (<italic>p </italic>&lt; 0.001), followed by the strain combination, and LrFBB81 alone (<italic>p </italic>&lt; 0.05). The permeability of mucosa was also successfully maintained by the WCF213 strain. This was illustrated by the significant reduction in the flux of FITC-labelled dextran (<italic>p </italic>&lt; 0.05), which was larger than that exhibited by the other groups. The ZO-1 distribution of strain-treated cells showed less disruption than hydrogen peroxide-treated cells, consistent with the TER and FITC experimental results. These findings indicate that WCF213 and LrFBB81 plays important roles in the maintenance of mucosal integrity in a strain-dependent manner.


</sec>

DOI： 10.1186/s13104-020-05338-1

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その他リンク： https://link.springer.com/article/10.1186/s13104-020-05338-1/fulltext.html

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Wiley  

Several nerve conduits have been investigated for their potential as alternative sources of autografts for bridging neural gaps. However, autologous nerve transplants remain the most effective for nerve repair. We examined clinically approved nerve conduits containing collagen and polyglycolic acid (PGA-c) combined with collagen-binding basic fibroblast growth factor (bFGF) containing a polycystic kidney disease (PKD) domain and collagen binding domain (CBD) (bFGF-PKD-CBD) in a rat 15-mm sciatic nerve critical-size defect model. The treatment groups were: PGA-c immersed in phosphate-buffered saline (PBS) (PGA-c/PBS group), bFGF (PGA-c/bFGF group), or bFGF-PKD-CBD (PGA-c/bFGF-PKD-CBD group), and no treatment (Defect group). Gait and histological analyses were performed. Four weeks after treatment, the recovery rate of the paw print area was significantly greater in the PGA-c/bFGFPKD-CBD group than the PGA-c/PBS and PGA-c/bFGF groups. Mean intensity of paw prints was significantly greater in the PGA-c/bFGF-PKD-CBD group than the PGA-c/PBS and Defect groups. Swing time was significantly greater in the PGA-c/PBS, PGA-c/bFGF, and PGA-c/bFGF-PKD-CBD groups than the Defect group. At 8 weeks, all three parameters were significantly greater in the PGA-c/PBS, PGA-c/bFGF, and PGA-c/bFGF-PKD-CBD groups than the Defect group. Regenerated myelinated fibers were observed in 7/8 (87.5%) rats in the PGA-c/bFGF-PKD-CBD group after 8 weeks, and in 1/8 (12.5%) and 3/8 (37.5%) rats in the PGA-c/PBS and PGA-c/bFGF groups, respectively. PGA-c/bFGF-PKD-CBD composites may be promising biomaterials for promoting functional recovery of long-distance peripheral nerve defects in clinical practice.

DOI： 10.1002/jbm.b.34391

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その他リンク： https://onlinelibrary.wiley.com/doi/full-xml/10.1002/jbm.b.34391

掲載種別：研究論文（学術雑誌）  

Background: Caco-2 cells monolayer is one of in vitro models to evaluate the translocation capacity of Lactobacillus spp probiotic strains. The translocation is influenced by mucosa permeability of enterocytes as shown by increasing transepithelial resistance (TER) and formation of tight junction proteins. The pore size of the supported permeable membrane used in in vitro assay was one of the crucial factors in performing bacterial translocation assay. Almost no study has been conducted using Caco-2 cells monolayer grown on 8-μm pore size polycarbonate membrane for evaluating probiotics translocation. Therefore this study aimed to determine whether the Caco-2 cells monolayer model was suitable as an in vitro translocation model. Methods: Caco-2 cells monolayer was seeded onto 8-μm collagen-coated polycarbonate membrane insert Transwell®. Differentiation of Caco-2 cells was detected by measuring the TER, while the ZO-1 protein (the tight junction proteins) was detected by immunofluorescence. H2 O2 was used as a tight junction disruptive agent. Data were analyzed using SPSS version 23 software to compare the mean of TER measurement between untreated and H2 O2-treated Caco-2 cells monolayer. Results: The result showed that the TER of Caco-2 cells monolayer was gradually increasing until day 14, reaching more than 800 ohm.cm2. Furthermore, the ZO-1 protein was successfully detected, indicated the tight junction formation. TER value of H2 O2-treated cells showed significantly lower than that of untreated cells (P<0.05), indicating a disturbance of cells monolayer integrity. Lactobacillus rhamnosus FBB81 was used for validating the translocation. There was no translocation observed; however, translocation was observed in H2 O2-treated cells. Conclusion: Altogether suggests that Caco-2 cells grown on 8 μm-pore size permeable filters could be considered as a suitable in vitro model for probiotics strains translocation.

DOI： 10.15562/bmj.v9i1.1633

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Springer Science and Business Media LLC  

DOI： 10.1186/s13027-019-0221-1

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その他リンク： http://link.springer.com/article/10.1186/s13027-019-0221-1/fulltext.html

掲載種別：研究論文（学術雑誌）   出版者・発行元：Wiley  

Background: Basic fibroblast growth factor (bFGF) has been applied for periodontal regeneration. However, the application depends on bone defect morphology because bFGF diffuses rapidly from defect sites. In a previous study, collagen-binding bFGF (CB-bFGF) has been shown to enhance bone formation by collagen-anchoring in the orthopedic field. The aim of this study is to demonstrate the efficacy of CB-bFGF with collagen scaffolds in bone regeneration of horizontal bone defect. Methods: Cell proliferation activity and collagen binding activity of CB-bFGF was confirmed by WST-8 assay and collagen binding assay, respectively. The retention of CB-bFGF in the collagen sheet (CS) was measured by fluorescence imaging. The rat horizontal alveolar bone defect model was employed to investigate the efficacy of CB-bFGF with collagen powder (CP). After 4 and 8 weeks, the regenerative efficacy was evaluated by microcomputed tomography, histological, and immunohistochemical analyses. Results: CB-bFGF had a comparable proliferation activity to bFGF and a collagen binding activity. CB-bFGF was retained in CS longer than bFGF. At 8 weeks postoperation, bone volume, bone mineral content, and new bone area in CB-bFGF/CP group were significantly increased compared with those in other groups. Furthermore, epithelial downgrowth was significantly suppressed in CB-bFGF/CP group. At 4 weeks, the numbers of osteocalcin, proliferating cell nuclear antigen, and osteopontin-positive cells at the regeneration site in CB-bFGF/CP group were greater than those in other groups. Conclusions: CB-bFGF/CP effectively promoted bone regeneration of horizontal bone defect possibly by sustained release of bFGF. The potential of CB-bFGF composite material for improved periodontal regeneration in vertical axis was shown.

DOI： 10.1002/jper.18-0674

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その他リンク： https://onlinelibrary.wiley.com/doi/full-xml/10.1002/JPER.18-0674

掲載種別：研究論文（学術雑誌）   出版者・発行元：Informa UK Limited  

DOI： 10.2147/idr.s196452

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Wiley  

DOI： 10.1111/febs.14611

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その他リンク： https://onlinelibrary.wiley.com/doi/full-xml/10.1111/febs.14611

担当区分：最終著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

The bacterium Vibrio alginolyticus, an opportunistic pathogen in humans, has a type III secretion system (T3SS) that is responsible for its cytotoxicity toward eukaryotic cells. The effector of T3SS that is responsible for the cytotoxicity had not been identified. Here we demonstrate that VepA, a homolog of the T3SS effector in V. parahaemolyticus, is required for cytotoxicity in V. alginolyticus. VepA induces lysosomal membrane permeabilization, and it allows the leakage of only small molecules into the cytosol. Our findings revealed that VepA induces cathepsin-independent cell death in mammalian cells. The ferrous ion, one of the small molecules in the lysosome contents, appears to be involved in the cell death caused by V. alginolyticus VepA.

DOI： 10.18926/AMO/56068

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Impact Journals, LLC  

Helicobacter pylori infections cause gastritis and affect systemic immune responses; however, no direct association between immune cells and stomach bacteria has yet been reported. The present study investigated DEC205-mediated phagocytosis of H. pylori and the role of DEC205-positive macrophages in the human gastric mucosa. DEC205 mediated phagocytosis of H. pylori was detected immunocytochemically in PMAstimulated macrophages differentiated from NOMO1 cells. Expression of DEC205 mRNA in peripheral blood mononuclear cells (PBMCs) from H. pylori-infected patients was analyzed following stimulation with H. pylori cell lysate. We found that anti-DEC205 antibodies inhibited phagocytosis of H. pylori. The number of cells double-positive for DEC205 and CD14 in human gastric mucosa was higher in H. pylori-infected patients. DEC205-positive macrophages invaded the extracellular space between epithelial cells within gastric pits. In addition, DEC205 mRNA expression was upregulated in human PBMCs stimulated with H. pylori lysate. These findings suggest DEC205-expressing macrophages are important for recognition of H. pylori in human gastric mucosa, which affects systemic immunity.

DOI： 10.18632/oncotarget.24574

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担当区分：最終著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：Springer Science and Business Media LLC  

Vibrio alginolyticus is an opportunistic pathogen in both humans and marine animals. Collagenase encoded by colA is considered to be one of the virulence factors. Expression of colA is regulated by multiple environmental factors, e.g., temperature, growth phase, and substrate. To elucidate the mechanism of regulation of colA expression, transposon mutagenesis was performed. VarS, a sensor histidine kinase of the two-component regulatory system, was demonstrated to regulate the expression of colA. VarA, a cognate response regulator of VarS, was also identified and shown to be involved in the regulation of colA expression. In vitro phosphorylation assays showed that phosphorylated VarS acted as a phosphoryl group donor to VarA. A site-directed mutagenesis study showed that the His300, Asp718 and His874 residues in VarS were essential for the phosphorylation of VarS, and the Asp54 residue in VarA was likely to receive the phosphoryl group from VarS. The results demonstrate that the VarS/VarA two-component regulatory system regulates the expression of collagenase in V. alginolyticus.

DOI： 10.1007/s00232-017-9991-9

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その他リンク： http://link.springer.com/content/pdf/10.1007/s00232-017-9991-9.pdf

記述言語：日本語   出版者・発行元：日本防菌防黴学会  

近年、環境汚染菌による院内感染が報告され、環境整備の重要性が認められつつある。今回、消毒剤および除菌剤の除染効果を比較検討するため、環境中の細菌の生存状態とATP測定の基礎的検討と各種消毒剤または除菌剤を含浸させたワイプによる拭き取り効果について検討した。乾燥による生菌数への影響に関する検討では、一定の濃度に調製した多剤耐性Acinetobacter baumanniiおよびBacillus cereusを乾燥状態で放置し、経時的に細菌数の指標としてATP値とコロニー数を計測した。シャーレの中で乾燥状態にしたB.cereusは1日目でATP値が有意に低下したが、A.baumanniiは有意な低下を認めるまでに7日間を要した。7日目のコロニー数を計測したところ、A.baumanniiおよびB.cereus共に生菌として検出された。拭き取りによる除菌効果は、ステンレス板とポリプロピレン板にA.baumanniiおよびB.cereusを塗布し、上記消毒剤または除菌剤含浸ワイプで拭き取り、その前後におけるATP量およびコロニー数を比較検討した。拭き取り効果は、材質による差は認められなかった。また、いずれの含浸ワイプによる拭き取りにおいても同様に有意な菌数の低下が認められ、いずれの細菌に対しても消毒剤または除菌剤は5log10CFU以上の減少を認めた。(著者抄録)

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Hindawi Limited  

Basic fibroblast growth factor 2 (bFGF) accelerates bone formation during fracture healing. Because the efficacy of bFGF decreases rapidly following its diffusion from fracture sites, however, repeated dosing is required to ensure a sustained therapeutic effect. We previously developed a fusion protein comprising bFGF, a polycystic kidney disease domain (PKD; s2b), and collagen-binding domain (CBD; s3) sourced from the<italic> Clostridium histolyticum</italic> class II collagenase, ColH, and reported that the combination of this fusion protein with a collagen-like peptide, poly(Pro-Hyp-Gly)₁₀, induced mesenchymal cell proliferation and callus formation at fracture sites. In addition,<italic> C. histolyticum</italic> produces class I collagenase (ColG) with tandem CBDs (s3a and s3b) at the C-terminus. We therefore hypothesized that a bFGF fusion protein containing ColG-derived tandem CBDs (s3a and s3b) would show enhanced collagen-binding activity, leading to improved bone formation. Here, we examined the binding affinity of four collagen anchors derived from the two clostridial collagenases to H-Gly-Pro-Arg-Gly-(Pro-Hyp-Gly)₁₂-NH₂, a collagenous peptide, by surface plasmon resonance and found that tandem CBDs (s3a-s3b) have the highest affinity for the collagenous peptide. We also constructed four fusion proteins consisting of bFGF and s3 (bFGF-s3), s2b-s3b (bFGF-s2b-s3), s3b (bFGF-s3b), and s3a-s3b (bFGF-s3a-s3b) and compared their biological activities to those of a previous fusion construct (bFGF-s2b-s3) using a cell proliferation assay in vitro and a mouse femoral fracture model in vivo. Among these CB-bFGFs, bFGF-s3a-s3b showed the highest capacity to induce mesenchymal cell proliferation and callus formation in the mice fracture model. The poly(Pro-Hyp-Gly)₁₀/bFGF-s3a-s3b construct may therefore have the potential to promote bone formation in clinical settings.

DOI： 10.1155/2018/8393194

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その他リンク： http://downloads.hindawi.com/journals/bmri/2018/8393194.xml

掲載種別：研究論文（学術雑誌）   出版者・発行元：Ovid Technologies (Wolters Kluwer Health)  

DOI： 10.1097/brs.0000000000002074

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Wiley  

DOI： 10.1002/term.2019

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Wiley  

We developed a new scaffold material-oriented collagen tubes (OCT)-and evaluated the potential of OCTs combined with basic fibroblast growth factor (bFGF) to repair of a 15 mm sciatic nerve defect in rats. The treatment groups consisted of OCT with adsorbed bFGF (OCT/bFGF group), OCT in phosphate-buffered saline (PBS) (OCT/PBS group), and a no-treatment group (Defect group). Functional evaluation of nerve regeneration was performed using the CatWalk system, and histological analyses of the defect sites were also performed. In rats treated with either OCT/bFGF or OCT/PBS, the walking function parameter of max contact area returned to normal levels by 4 weeks after grafting, and the regeneration of myelinated fibers was detected after 8 weeks. However, more regenerated myelinated fibers were observed in the OCT/bFGF group compared with the OCT/PBS group at 4 weeks. In addition, the max contact area and swing speed in the OCT/bFGF group were significantly recovered compared to the OCT/PBS and Defect groups at 8 weeks. Although the combination of bFGF and OCT was superior to OCT alone for nerve regeneration and functional recovery, the present findings demonstrate that OCT alone or in combination with bFGF accelerates nerve repair in a large peripheral nerve defect in rats. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 8-14, 2017.

DOI： 10.1002/jbm.a.35866

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Hindawi Limited  

Cell-based regenerative therapy has the potential to repair bone injuries or large defects that are recalcitrant to conventional treatment methods, including drugs and surgery. Here, we developed a multilayered cell-based bone formation system using cells coated with fibronectin-gelatin (FN-G) nanofilms. The multilayered mesenchymal cells (MLMCs) were formed after two days of culture and were shown to express higher levels of BMP-2 and VEGF compared to monolayer cultures of MCs. The MLMCs were used as a graft material in combination with a fusion protein consisting of basic fibroblast growth factor (bFGF), polycystic kidney disease (PKD) domain, and the collagen-binding domain (CBD) of<italic> Clostridium histolyticum</italic> collagenase. In femur sites grafted with the MLMCs, significantly higher levels of callus volume and bone mineral content were observed compared to the sham controls. The callus volume and bone mineral content were further increased in femur sites grafted with bFGF-PKD-CBD/MLMCs. Taken together, these results suggest that bFGF-PKD-CBD/MLMCs, which can be simply and rapidly generated in vitro, have the potential to promote bone repair when grafted into large defect sites.

DOI： 10.1155/2017/4371460

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その他リンク： http://downloads.hindawi.com/journals/bmri/2017/4371460.xml

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Wiley  

Growth factor delivered in combination with animal-derived collagen materials has been used to accelerate bone fracture healing in human patients. However, the introduction of bovine proteins into humans carries the risk of zoonotic and immunologic complications. Here, we developed a collagen-like polypeptide-based bone formation system consisting of poly(Pro-Hyp-Gly)10 , which mimics the triple helical conformation of collagen, and basic fibroblast growth factor (bFGF) fused to the polycystic kidney disease (PKD) domain and collagen-binding domain (CBD) of Clostridium histolyticum collagenase. Circular dichroism spectral analysis showed that when pepsin-soluble bovine type I collagen was treated at 50°C, a positive signal corresponding to the collagen triple helix at 220 nm was not detected. In contrast, poly(Pro-Hyp-Gly)10 retained the 220-nm positive peak, even when treated at 80°C. The combination of the collagen binding-bFGF fusion protein (bFGF-PKD-CBD) with poly(Pro-Hyp-Gly)10 induced greater bone formation compared to bFGF alone in mice bone fracture models. Taken together, these properties suggest that the bFGF-PKD-CBD/poly(Pro-Hyp-Gly)10 composite is a promising material for bone repair in the clinical setting. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1372-1378, 2016.

DOI： 10.1002/jbm.a.35670

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Springer Science and Business Media LLC  

BACKGROUND: To repair fractures with large bone defects or gaps, demineralized allogenic bone matrix (DBM) is often applied to the fracture site. However, studies have shown that the use of DBM alone has limited efficacy for repairing fractures. In the present study, we developed an allogenic demineralized bone powder (DBP) with basic fibroblast-derived growth factor containing a polycystic kidney disease (PKD) domain and collagen-binding domain (CBD) from Clostridium histolyticum collagenase (ColH) and investigated the stimulatory effects of bFGF-PKD-CBD combined with allogenic DBP on bone growth in a mouse femur fracture model. METHODS: DBP mixed with either phosphate-buffered saline (PBS) (DBP/PBS), 0.58 nmol basic fibroblast growth factor (bFGF) (0.58 nmol DBP/bFGF), 0.058 nmol bFGF-PKD-CBD (0.058 nmol DBP/bFGF-PKD-CBD), or 0.58 nmol bFGF-PKD-CBD (0.58 nmol DBP/bFGF-PKD-CBD) was grafted into fracture sites. RESULTS: bFGF-PKD-CBD/DBP composite accelerates callus formation in a bone fracture model in mice and clearly showed that the composite also increases bone mineral density at fracture sites compared to bFGF/DBP. In addition, bFGF-PKD-CBD/DBP increased callus volume and bone mineral content to similar levels in fractures treated with a tenfold higher amount of bFGF at 4 weeks. CONCLUSIONS: Our results suggest that bFGF-PKD-CBD/DBP may be useful for promoting fracture healing in the clinical setting.

DOI： 10.1186/s13018-015-0201-0

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その他リンク： http://link.springer.com/article/10.1186/s13018-015-0201-0/fulltext.html

掲載種別：研究論文（学術雑誌）   出版者・発行元：MDPI AG  

DOI： 10.3390/toxins7082906

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掲載種別：研究論文（学術雑誌）   出版者・発行元：International Union of Crystallography (IUCr)  

<italic>Clostridium histolyticum</italic>collagenases ColG and ColH are segmental enzymes that are thought to be activated by Ca²⁺-triggered domain reorientation to cause extensive tissue destruction. The collagenases consist of a collagenase module (s1), a variable number of polycystic kidney disease-like (PKD-like) domains (s2a and s2b in ColH and s2 in ColG) and a variable number of collagen-binding domains (s3 in ColH and s3a and s3b in ColG). The X-ray crystal structures of Ca²⁺-bound holo s2b (1.4 Å resolution,<italic>R</italic>= 15.0%,<italic>R</italic>_free= 19.1%) and holo s2a (1.9 Å resolution,<italic>R</italic>= 16.3%,<italic>R</italic>_free= 20.7%), as well as of Ca²⁺-free apo s2a (1.8 Å resolution,<italic>R</italic>= 20.7%,<italic>R</italic>_free= 27.2%) and two new forms of N-terminally truncated apo s2 (1.4 Å resolution,<italic>R</italic>= 16.9%,<italic>R</italic>_free= 21.2%; 1.6 Å resolution,<italic>R</italic>= 16.2%,<italic>R</italic>_free= 19.2%), are reported. The structurally similar PKD-like domains resemble the V-set Ig fold. In addition to a conserved β-bulge, the PKD-like domains feature a second bulge that also changes the allegiance of the subsequent β-strand. This β-bulge and the genesis of a Ca²⁺pocket in the archaeal PKD-like domain suggest a close kinship between bacterial and archaeal PKD-like domains. Different surface properties and indications of different dynamics suggest unique roles for the PKD-like domains in ColG and in ColH. Surface aromatic residues found on ColH s2a-s2b, but not on ColG s2, may provide the weak interaction in the biphasic collagen-binding mode previously found in s2b-s3.<italic>B</italic>-factor analyses suggest that in the presence of Ca²⁺the midsection of s2 becomes more flexible but the midsections of s2a and s2b stay rigid. The different surface properties and dynamics of the domains suggest that the PKD-like domains of M9B bacterial collagenase can be grouped into either a ColG subset or a ColH subset. The conserved properties of PKD-like domains in ColG and in ColH include Ca²⁺binding. Conserved residues not only interact with Ca²⁺, but also position the Ca²⁺-interacting water molecule. Ca²⁺aligns the N-terminal linker approximately parallel to the major axis of the domain. Ca²⁺binding also increases stability against heat and guanidine hydrochloride, and may improve the longevity in the extracellular matrix. The results of this study will further assist in developing collagen-targeting vehicles for various signal molecules.

DOI： 10.1107/s1399004714027722

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記述言語：日本語   掲載種別：研究論文（学術雑誌）  

Atrophic gastritis is caused by Helicobacter pylori infection, and is involved in gastric cancer. In this study, we investigated the association with total IgG and IgG subclass antibodies using several strains isolated from Japanese in H. pylori positive and negative individuals, and gastric atrophy using measuring pepsinogen I and II levels. We found that total IgG antibody measurement using typical Japanese genotype as an antigen was available for diagnosis of H. pylori infection, whereas IgG1 and IgG2 antibodies were not for diagnosis. Furthermore, the IgG1/G2 ratio was elevated in a patient with gastric cancer. The accuracy of serodiagnosis of H. pylori infection may increase when the optimal antigens are used, and measurement IgG subclass may provide additional prediction of gastric cancer.

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Wiley  

DOI： 10.1002/jbm.a.34974

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

DOI： 10.1016/j.bbagen.2014.01.019

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Wiley  

DOI： 10.1002/jbm.a.34841

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その他リンク： https://onlinelibrary.wiley.com/doi/full-xml/10.1002/jbm.a.34841

掲載種別：研究論文（学術雑誌）   出版者・発行元：IOP Publishing  

DOI： 10.1088/1748-6041/9/3/035014

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その他リンク： https://iopscience.iop.org/article/10.1088/1748-6041/9/3/035014

掲載種別：研究論文（学術雑誌）   出版者・発行元：Ovid Technologies (Wolters Kluwer Health)  

DOI： 10.1097/cad.0b013e3283650bff

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Japanese Society of Allergology  

Background: In contrast to Staphylococcus aureus-derived superantigenic exotoxins, the role of non-superantigenic exotoxins in the pathogenesis of eosinophilic airway diseases remains obscure. We sought to characterize S. aureus alpha-toxin-induced cellular responses in chronic rhinosinusitis with nasal polyps (CRSwNP).<br> Methods: Dispersed nasal polyp cells and uncinate tissue cells were prepared from patients with CRS with and without nasal polyps, respectively. Cells were incubated with various concentrations of alpha-toxin or staphylococcal enterotoxin B and then the levels of IL-5, IL-13, IFN-γ, IL-17A, and IL-10 in the cell supernatants were determined. The pathophysiological significance of alpha-toxin-induced cytokine production was also determined including radiological severity of rhinosinusitis, tissue and blood eosinophilia, serum total IgE level, and 1-s forced expiratory volume/forced vital capacity ratio (FEV₁/FVC).<br> Results: Nasal polyp cells produced substantial amounts of IL-5, IL-13, IFN-γ, IL-17A, and IL-10 in response to alpha-toxin. Cytokine production was higher in nasal polyp cells than in uncinate tissue cells. The potency of alpha-toxin in stimulating IL-5, IL-13, and IL-10 production was comparable to that of enterotoxin. Alpha-toxin-induced IFN-γ, IL-17A, and IL-10 production significantly and negatively correlated with the degree of eosinophil infiltration into nasal polyps. Conversely, alpha-toxin-induced IFN-γ and IL-10 production significantly and positively correlated with FEV₁/FVC. IL-10 production was significantly lower in asthmatic patients compared to non-asthmatics<br> Conclusions: S. aureus-derived alpha-toxin can provoke cellular responses in nasal polyps. These responses, especially failure to synthesize IL-10, may play a role in the pathophysiology of CRSwNP.<br>

DOI： 10.2332/allergolint.14-oa-0703

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その他リンク： http://search.jamas.or.jp/link/ui/2015325519

記述言語：英語   出版者・発行元：Okayama University Medical School  

Atrophy of the gastric mucosa is a precursor of intestinal-type gastric cancer, and Helicobacter pylori infection causes atrophic gastritis. The aim of this study was to determine whether the genetic diversity of H. pylori virulence genes is associated with the development and progression of gastric atrophy in humans. We isolated and cultured H. pylori strains from patients with gastric ulcer and duodenal ulcer accompanied by atrophic gastritis in background mucosa. H. pylori strains were stored at －80℃ prior to the experiments being carried out. We analyzed iceA, babA, vacA, cagA, and cagE genes by PCR. The cagA gene was analyzed through sequencing of the C-terminal region containing the EPIYA motif, which is related to tyrosine phosphorylation. Severe atrophy was observed in patients with gastric ulcer. The major phenotype of the vacA gene was s1c/m1 (93ｵ). The cagA gene was detected in all strains. The cagE gene was not detected in 2 and 5 strains from the mild cases and severe cases, respectively. The major cagA EPIYA motif, which is amino acids repeat in the C terminus, was the A-B-D type (44 of 58 strains). The virulence genes were not statistically associated with the severity of atrophy in the background gastric mucosa in humans. Not only identification of bacterial virulence factors but also studies of the host response will be necessary to investigate the progression of gastric atrophy and subsequent cancer development in humans.

DOI： 10.18926/AMO/49667

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その他リンク： http://search.jamas.or.jp/link/ui/2014027956

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Ovid Technologies (Wolters Kluwer Health)  

BACKGROUND: Various carriers have been tested as drug delivery systems in an attempt to sustain the action of growth factors. Gene therapy has also been adopted to achieve lasting effects but without satisfactory results. Because the authors believe that the angiogenic effect of vascular endothelial growth factor (VEGF) can be enhanced by anchoring the fusion protein composed of the Clostridium-derived collagen-binding domain and recombinant VEGF-A164 (CB-VEGF-A) in the tissue, they examined the changes in blood flow of random pattern flaps following treatment of the dorsal region of the rat with the fusion proteins before skin flap elevation. METHODS: The authors administered CB-VEGF-A subcutaneously into the dorsal region of Sprague-Dawley rats 7 days before creation of skin flaps, and compared the necrosis rate observed on the seventh day after flap elevation with that of vehicle controls. The authors also performed comparison with a group treated by subcutaneous administration of non-collagen-binding domain-binding VEGF. The skin flaps were also examined histologically. RESULTS: The flap necrosis rate was lower in the CB-VEGF-A group (36.7 ± 7.4 percent) than in the control group (48.2 ± 5.4 percent). However, no improvement was observed in the non-collagen-binding domain-binding VEGF group. Moreover, histologic examination revealed an increase in the subcutaneous blood vessel counts. CONCLUSION: CB-VEGF-A has an angiogenic effect on rat dorsal skin flaps and improves flap survival.

DOI： 10.1097/prs.0b013e3182818b34

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記述言語：英語   出版者・発行元：Okayama University Medical School  

Clostridium botulinum type C and D strains recently have been found to produce PLC on egg yolk agar plates. To characterize the gene, enzymatic and biological activities of C. botulinum PLCs (Cb-PLCs), the cb-plc genes from 8 strains were sequenced, and 1 representative gene was cloned and expressed as a recombinant protein. The enzymatic and hemolytic activities of the recombinant Cb-PLC were measured and compared with those of the Clostridium perfringens alpha-toxin. Each of the eight cb-plc genes encoded a 399 amino acid residue protein preceded by a 27 residue signal peptide. The protein consists of 2 domains, the N- and C-domains, and the overall amino acid sequence identity between Cb-PLC and alpha-toxin was greater than 50%, suggesting that Cb-PLC is homologous to the alpha-toxin. The key residues in the N-domain were conserved, whereas those in the C-domain which are important in membrane interaction were different than in the alpha-toxin. As expected, Cb-PLC could hydrolyze egg yolk phospholipid, p-nitrophenylphosphorylcholine, and sphingomyelin, and also exhibited hemolytic activity;however, its activities were about 4- to over 200-fold lower than those of alpha-toxin. Although Cb-PLC showed weak enzymatic and biological activities, it is speculated that Cb-PLC might play a role in the pathogenicity of botulism or for bacterial survival.

DOI： 10.18926/AMO/49252

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その他リンク： http://search.jamas.or.jp/link/ui/2013249132

掲載種別：研究論文（学術雑誌）   出版者・発行元：American Society for Microbiology  

<title>ABSTRACT</title>

<named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">Clostridium histolyticum</named-content>
secretes collagenases, ColG and ColH, that cause extensive tissue destruction in myonecrosis. The C-terminal collagen-binding domain (CBD) of collagenase is required for insoluble collagen fibril binding and subsequent collagenolysis. The high-resolution crystal structures of ColG-CBD (s3b) and ColH-CBD (s3) are reported in this paper. The new X-ray structure of s3 was solved at 2.0-Å resolution (
<italic>R</italic>
= 17.4%;
<italic>R</italic>
_free
= 23.3%), while the resolution of the previously determined s3b was extended to 1.4 Å (
<italic>R</italic>
= 17.9%;
<italic>R</italic>
_free
= 21.0%). Despite sharing only 30% sequence identity, the molecules resemble one another closely (root mean square deviation [RMSD] C
_α
= 1.5 Å). All but one residue, whose side chain chelates with Ca
²⁺
, are conserved. The dual Ca
²⁺
binding site in s3 is completed by an unconserved aspartate. Differential scanning calorimetric measurements showed that s3 gains thermal stability, comparable to s3b, by binding to Ca
²⁺
(
<italic>holo</italic>
<italic>
T
_m
</italic>
= 94.1°C;
<italic>apo</italic>
<italic>
T
_m
</italic>
= 70.2°C).
<italic>holo</italic>
s3 is also stabilized against chemical denaturants urea and guanidine HCl. The three most critical residues for collagen interaction in s3b are conserved in s3. The general shape of the binding pocket is retained by altered loop structures and side chain positions. Small-angle X-ray scattering data revealed that s3 also binds asymmetrically to minicollagen. Besides the calcium-binding sites and the collagen-binding pocket, architecturally important hydrophobic residues and the hydrogen-bonding network around the
<italic>cis</italic>
-peptide bond are well conserved within the metallopeptidase subfamily M9B. CBDs were previously shown to bind to the extracellular matrix of various tissues. Compactness and extreme stability in physiological Ca
²⁺
concentration possibly make both CBDs suitable for targeted growth factor delivery.

DOI： 10.1128/jb.00010-12

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Wiley  

DOI： 10.1002/pro.2145

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Wiley  

DOI： 10.1002/ijc.27379

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Springer Science and Business Media LLC  

DOI： 10.1007/s00223-012-9626-1

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その他リンク： http://link.springer.com/article/10.1007/s00223-012-9626-1/fulltext.html

掲載種別：研究論文（学術雑誌）   出版者・発行元：American Chemical Society (ACS)  

DOI： 10.1007/s13361-011-0309-3

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その他リンク： http://link.springer.com/article/10.1007/s13361-011-0309-3/fulltext.html

掲載種別：研究論文（学術雑誌）  

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担当区分：最終著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

Two glucosylating toxins named toxins A and B play a role in the pathogenesis of Clostridium Difficile infection. The interaction of the toxins with host cell factors proceeds to downstream stages of cytotoxic effects in cells, in which involvement of other C. difficile factors remains unknown. We utilized culture filtrate of C. difficile with a low dilution to characterize the influence of putative minor proteins on the organization of the actin cytoskeleton in cultured epithelial cells and found a previously uncharacterized F-actin aggregated structure, termed "actin aggregate," at the juxtanuclear region. We reasoned that formation of actin aggregate was due to an additional factor(s) in the culture filtrate rather than the glucosylating toxins, because treatment of purified toxins rarely caused actin aggregate in cells. We focused on a previously uncharacterized hypothetical protein harboring a KDEL-like sequence as a candidate. The product of the candidate gene was detected in culture filtrate of C. difficile ATCC 9689 and was renamed Srl. Purified glutathione S-transferase-tagged Srl triggered formation of actin aggregate in the cells in the presence of either toxin A or B and enhanced cytotoxicity of each of the two toxins, including decreases in both cell viability and transepithelial resistance of cultured epithelial monolayer, although the recombinant Srl alone did not show detectable cytotoxicity. Srl-neutralized culture filtrate partially inhibited morphological changes of the cells in parallel with decreased actin aggregate formation in the cells. Thus, Srl might contribute to the modulation of toxin sensitivity of intestinal epithelial cells by enhancing cytotoxicity of C. difficile toxins.

DOI： 10.1128/IAI.00051-11

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Springer Science and Business Media LLC  

DOI： 10.1007/s00223-011-9485-1

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その他リンク： http://link.springer.com/article/10.1007/s00223-011-9485-1/fulltext.html

掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

DOI： 10.1016/j.ab.2009.07.017

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Wiley  

DOI： 10.1111/j.1742-4658.2009.07078.x

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

Histotoxic clostridia produce collagenases responsible for extensive tissue destruction in gas gangrene. The C-terminal collagen-binding domain (CBD) of these enzymes is the minimal segment required to bind to collagen fibril. Collagen binding efficiency of CBD is more pronounced in the presence of Ca2+. We have shown that CBD can be functional to anchor growth factors in local tissue. A H-1-N-15 HSQC NMR titration study with three different tropocollagen analogues ((POG)(10))(3), ((GPOG)(7)PRG)(3), and (GPRG(POG)(7)C-carbamidomethyl)(3), mapped a saddle-like binding cleft on CBD. NMR titrations with three nitroxide spin-labeled analogues of collagenous peptide, (PROXYL-G(POG)(7)PRG)(3), (PROXYL-G(POG)(7))(3), and (GPRG(POG)(7)C-PROXYL)(3) (where PROXYL represents 2,2,5,5-tetramethyl-L-pyrrolidinyloxy), unambiguously demonstrated unidirectional binding of CBD to the tropocollagen analogues. Small angle x-rays cattering data revealed that CBD binds closer to a terminus for each of the five different tropocollagen analogues, which in conjunction with NMR titration studies, implies a binding mode where CBD binds to the C terminus of the triple helix.

DOI： 10.1074/jbc.m807684200

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Springer Science and Business Media LLC  

DOI： 10.1007/s12104-008-9102-z

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その他リンク： http://link.springer.com/article/10.1007/s12104-008-9102-z/fulltext.html

担当区分：筆頭著者   記述言語：日本語   掲載種別：研究論文（研究会，シンポジウム資料等）  

Many hospitals have infection control education programs to facilitate the appropriate use of antimicrobial agents. Even with these efforts, however, it is not rare to encounter irregular prescriptions. In order to solve this discrepancy between knowledge and actual behavior, we chose an alternative approach to improve the decision making process. Recent advances in information technology have made it possible to not only instantly integrate various bacterial examination results using a computer, but to simultaneously carry out the statistical analyses at a much lower cost. We employed a client-server system to accomplish these tasks in Kagawa University Hospital. By connecting CCD camera-equipped microscopes to the system directly, image uploading has become a single-clicking job. Various microbial examination data were automatically transferred to the system once they became available in analytical devices such as BacT/ALERT 3D, VITEK, and an MIC analyzer. These data were presented to hospital doctors in well-designed web windows without delay. By removing psychological barriers to access laboratory examination data, statistics, and relevant information, more doctors seemed to independently follow scientific processes to choose antimicrobial agents. The daily behavior of hospital doctors has also been influenced by the system, e. g., pasting the microscopic images onto clinical records, or starting Gram staining in their own wards. These subtle but fundamental changes will eventually alter the way they make prescription decisions. The computer system was also useful for the infection control team to monitor and detect nosocomial infections, which has become essential to carry out its daily activities.

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Springer Nature  

Clostridium histolyticum collagenase is used to isolate cells from various organs and tissues for tissue engineering, and also to treat destructive fibrosis; thus, the demand for high-grade enzyme preparations is increasing. In this study, we constructed a plasmid encoding C. histolyticum type II collagenase (ColH) with a C-terminal hexahistidine tag (ColH-his) to facilitate the purification of the enzyme through immobilized metal affinity chromatography (IMAC). When ColH-his was expressed in a protease-deficient mutant of Clostridium perfringens, it was produced in the culture supernatant more efficiently than the untagged ColH. ColH-his exhibited the same hydrolytic activity as ColH against 4-phenylazobenzyloxy-carbonyl-Pro-Leu-Gly-Pro-D-Arg (Pz peptide), a synthetic collagenase substrate. From 100 ml of the culture supernatant, approximately 1 mg of ColH-his was purified by ammonium sulfate precipitation, IMAC, and high-performance liquid chromatography on a MonoQ column. When IMAC was performed on chelating Sepharose charged with Zn(2+) instead of Ni(2+), a potential carcinogenic metal, the specific activities against Pz peptide and type I collagen decreased slightly. However, they were comparable to those reported for other recombinant ColHs and a commercial C. histolyticum collagenase preparation, suggesting that this expression system is useful for large-scale preparation of high-grade clostridial collagenases.

DOI： 10.1007/s00253-008-1592-1

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記述言語：日本語   出版者・発行元：(一社)日本病院薬剤師会  

現在、眼科領域において難治性眼感染症の治療に注射用抗メチシリン耐性黄色ブドウ球菌(MRSA)薬を点眼液として院内で調製し使用している。これら院内製剤は安定性について不明であるにもかかわらず、慣例的に使用されているのが現状である。今回、これら院内製剤抗生物質点眼液の安定性を力価について試験し、それを基に最大投与日数の検討を行った。香川大学医学部附属病院において2006年の1年間に眼科病棟より依頼があり薬剤部で調製した抗生物質点眼液は、バンコマイシン67本、セフタジジム11本、イミペネム/シラスタチン3本であった。寒天平板法による阻止円の測定および液体培地中での生菌数の測定を行ったところ、バンコマイシンは180日以上、セフタジジムは14日間、イミペネム/シラスタチンは3日間、90%以上の力価を保つことが確認された。なお、これら点眼液の無菌試験を行ったところ、すべての点眼液で無菌であることが確認された。(著者抄録)

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その他リンク： https://search.jamas.or.jp/default/link?pub_year=2007&ichushi_jid=J04451&link_issn=&doc_id=20071005290003&doc_link_id=%2Fdg4hppha%2F2007%2F004310%2F001%2F1369-1372%26dl%3D0&url=https%3A%2F%2Fwww.medicalonline.jp%2Fjamas.php%3FGoodsID%3D%2Fdg4hppha%2F2007%2F004310%2F001%2F1369-1372%26dl%3D0&type=MedicalOnline&icon=https%3A%2F%2Fjk04.jamas.or.jp%2Ficon%2F00004_2.gif

記述言語：日本語   掲載種別：研究論文（学術雑誌）   出版者・発行元：公益社団法人日本薬学会  

Levocarnitine chloride is used for the therapeutic purpose of levocarnitine deficiency. For infants, however, levocarnitine chloride tablets must be crushed to avoid difficulties associated with swallowing, and also to administer an appropriately low dosage. Since the tablet is extremely hygroscopic and sour, it is dissolved in water containing simple syrup after crushing. In this study we investigated the stability of the drug after dissolution to optimize its preparation for clinical use. It was shown to be stable for at least 90 days after preparation, and microbes did not grow in 1—10% (w/v) solutions (pH 2.0—2.5) regardless of the presence or absence of simple syrup. Furthermore, the autoclaved levocarnitine chloride solution was as stable as the non-autoclaved one. In conclusion, the method employed in our hospital for the preparation of levocarnitine chloride for infants is appropriate and is recommended as a standard medicine supply method among different facilities.<br>

DOI： 10.1248/yakushi.126.805

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Wiley  

Epsilon-toxin (ET) of Clostridium perfringens, which causes fatal enterotoxemia in ungulates, was previously shown to bind to and form a heptameric pore within the detergent-resistant membranes (DRMs) of MDCK cells. Depletion of cholesterol has also been shown to decrease the cytotoxicity of ET and its heptamerization. In this study, we investigated the effects of changes in sphingolipids, other DRM components of MDCK cells, on the cells' susceptibility to ET. Treatment with fumonisin B1 and PDMP, inhibitors of sphingolipid and glycosphingolipid syntheses, respectively, increased the susceptibility, while D609, a sphingomyelin synthesis inhibitor, had the opposite effect. The exogenous addition of ganglioside G(M1) dramatically decreased the ET binding, heptamerization and cytotoxicity. These effects were shown not to be due to ET binding to G(M1) or to denaturation of ET. We also found that the ET cytotoxicity towards MDCK cells decreased with an increase in culture time. In accordance with the resistance observed for prolonged cultured cells, G(M3), a major ganglioside component, increased and sialidase treatment increased their susceptibility. These results suggest that membrane-anchored sialic acid of G(M3) within DRMs inhibits ET binding, leading to prevention of the heptamerization of ET and cell death. It is also suggested that sialidase produced by this organism aids the targeting of ET to MDCK cells.

DOI： 10.1111/j.1348-0421.2005.tb03726.x

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ACADEMIC PRESS INC ELSEVIER SCIENCE  

A "large" sialidase isozyme (NanI) from Clostridium perfringens is a representative microbial sialidase with broad substrate specificity, being used for the analysis of sialoglycoconjugates. It is also a possible virulence factor. However, purification of the native enzyme in a large quantity is not practical due to its low productivity. To obtain the enzyme in a satisfactory yield, a gene encoding the NanI was transcriptionally fused to the fdx gene promoter (P-fdx) in a shuttle-vector, pFF, and transformed into C perfringens 13. The resultant strain released the enzyme into the culture medium, as the original strain does. The enzyme activity increased during the first 6 h of culture and thereafter remained at maximal levels. The maximal activity was approximately 3000-fold compared with that of the original strain, and 15-fold compared with that of recombinant Escherichia coli, which possesses extra copies of the tRNA gene for selected rare codons. This suggests the usefulness of a P-fdx-based plasmid for expressing AT-rich genes in C perfringens. The enzyme was successfully purified by two-step procedure with a specific activity of 2860 U/mg using 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid and a yield of 1.69 mg of NanI per 100 ml of culture. The method described here can facilitate purification of NanI in enough quality and quantity to analyze the role of sialoglycoconjugates in cells and the pathogenic importance of NanI sialidase. (C) 2004 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.pep.2004.03.004

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

This study has revealed that a Clostridium perfringens ferredoxin gene (per-fdx) possesses a novel type of DNA curvature, which is formed by five phased A-tracts extending from upstream to downstream of the -35 region. The three A-tracts upstream of the promoter and the two within the promoter are located at the positions corresponding to A-tracts present in a C. perfringens phospholipase C gene (plc) and a Clostridium pasteurianum ferredoxin gene (pas-fdx), respectively. DNA fragments of the per-fdx, pas-fdx and plc genes (nucleotide positions -69 to +1 relative to the transcription initiation site) were fused to a chloramphenicol acetyltransferase reporter gene on a plasmid, pPSV, and their in vivo promoter activities were examined by assaying the chloramphenicol acetyltransferase activity of each C. perfringens transformant. Comparison of the three constructs showed that the order of promoter activity is, in descending order, per-fdx, pas-fdx and plc. Deletion of the three upstream A-tracts of the per-fdx gene drastically decreased the promoter activity, as demonstrated previously for the plc promoter. Substitution of the most downstream A-tract decreased the promoter activities of the per-fdx and pas-fdx genes. These results indicate that not only the phased A-tracts upstream of the promoter but also those within the promoter stimulate the promoter activity, and suggest that the high activity of the per-fdx promoter is due to the combined effects of these two types of A-tracts.

DOI： 10.1099/mic.0.26503-0

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

In this paper we show that Clostridium perfringens epsilon-toxin accumulates predominantly in the mouse kidney, where it is distributed mainly in glomeruli, capillaries, and collecting ducts. Although some pycnotic and exfoliated epithelial cells were observed in distal tubuli and collecting ducts, there were no findings indicative of severe renal injury. Bilateral nephrectomy increased the mouse lethality of the toxin, suggesting that the kidney contributes to the host defense against the lethal toxicity of epsilon-toxin.

DOI： 10.1128/IAI.71.9.5371-5375.2003

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Wiley  

DOI： 10.1093/emboj/cdg172

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掲載種別：研究論文（学術雑誌）   出版者・発行元：American Chemical Society (ACS)  

DOI： 10.1021/bi0206558

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

Clostridium perfringens epsilon-toxin, which is responsible for enterotoxaemia in ungulates, forms a heptamer in rat synaptosomal and Madin-Darby canine kidney (MDCK) cell membranes, leading to membrane permealization. Thus, the toxin may target the detergent-resistant membrane domains (DRMs) of these membranes, in analogy to aerolysin, a heptameric pore-forming toxin that associates with DRMs. To test this idea, we examined the distribution of radiolabeled epsilon-toxin in DRM and detergent-soluble membrane fractions of MDCK cells and rat synaptosomal membranes. When MDCK cells and synaptosomal membranes were incubated with the toxin and then fractionated by cold Triton X-100 extraction and flotation on sucrose gradients, the heptameric toxin was detected almost exclusively in DRMs. The results of a toxin overlay assay revealed that the toxin preferentially bound to and heptamerized in the isolated DRMs. Furthermore, cholesterol depletion by methyl-beta-cyclodextrin abrogated their association and lowered the cytotoxicity of the toxin toward MDCK cells. When epsilon-protoxin, an inactive precursor able to bind to but unable to heptamerize in the membrane, was incubated with MDCK cell membranes, it was detected mainly in their DRMs. These results suggest that the toxin is concentrated and induced to heptamerize on binding to a putative receptor located preferentially in DRMs, with all steps from initial binding through pore formation completed within the same DRMs.

DOI： 10.1074/jbc.m206731200

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Wiley  

DOI： 10.1016/s0014-5793(01)03148-9

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

DOI： 10.1074/jbc.m011527200

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

DOI： 10.1074/jbc.m003450200

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：GORDON BREACH PUBLISHING, TAYLOR & FRANCIS GROUP  

The substrate spectrum of the tandem collagen-binding domain (CBD) of Clostridium histolyticumclass I collagenase (ColG) was examined both in vitro and in vivo. CBD bound to insoluble type I. II, III and IV collagens in vitro, and to skin, aorta, tendon, kidney, trachea and corneal tissues containing various types of collagen fibrils or sheets. CBD bound to all kinds of collagen fibrils regardless of their diameters and also bound to sheet-forming collagen in the glomerular basal lamina or Descemet's membrane of the cornea. This wide substrate spectrum expands possible applications of the drug delivery system we proposed previously (PNAS 95:7018-7023, 1998). Therapeutic agents fused with CBD will bind not only to subcutaneous tissues, but also to other tissues containing non-type I collagen.

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

DOI： 10.1006/mpat.1999.0328

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Microbiology Society  

DOI： 10.1099/00221287-145-12-3377

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Oxford University Press (OUP)  

DOI： 10.1111/j.1574-6968.1999.tb08863.x

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Center for Academic Pub. Japan  

DOI： 10.1111/j.1348-0421.1999.tb03355.x

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Wiley  

DOI： 10.1093/emboj/18.12.3442

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

DOI： 10.1006/prep.1998.1026

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掲載種別：研究論文（学術雑誌）   出版者・発行元：American Society for Microbiology  

<title>ABSTRACT</title>

A
<italic>Clostridium histolyticum</italic>
116-kDa collagenase has an H
⁴¹⁵
EXXH motif but not the third zinc ligand, as found in already characterized zinc metalloproteinases. To identify its catalytic site, we mutated the codons corresponding to the three conserved residues in the motif to other amino acid residues. The mutation affecting His
⁴¹⁵
or His
⁴¹⁹
abolished catalytic activity and zinc binding, while that affecting Glu
⁴¹⁶
did the former but not the latter. These results suggest that the motif forms the catalytic site. We also mutated the codons corresponding to other amino acid residues that are likely zinc ligands. The mutation affecting Glu
⁴⁴⁷
decreased markedly both the enzymatic activity and the zinc content, while that affecting Glu
⁴⁴⁶
or Glu
⁴⁵¹
had smaller effects on activity and zinc binding. These mutations caused a decrease in
<italic>k</italic>
_cat
but no significant change in
<italic>
K
_m
</italic>
. These results are consistent with the hypothesis that Glu
⁴⁴⁷
is the third zinc ligand. The spacing of the three zinc ligands is the same in all known clostridial collagenases but not in other known gluzincins, indicating that they form a new gluzincin subfamily. The effects of mutations affecting Glu
⁴⁴⁶
and Glu
⁴⁵¹
suggest that the two residues are also involved in catalysis, possibly through an interaction with the two zinc-binding histidine residues.

DOI： 10.1128/jb.181.9.2816-2822.1999

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担当区分：筆頭著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：American Society for Microbiology  

<title>ABSTRACT</title>

<italic>Clostridium histolyticum</italic>
collagenase contains a number of different active components. Previously we have shown that
<italic>colH</italic>
encodes a 116-kDa collagenase (ColH) and a 98-kDa gelatinase. We purified a different 116-kDa collagenase (ColG) from the culture supernatant and sequenced its gene (
<italic>colG</italic>
). We also identified four other gelatinases (105, 82, 78, and 67 kDa) and determined their N-terminal amino acid sequences, all of which coincided with that of either ColG or ColH. Hybridization experiments showed that each gene is present in a single copy and each gene is transcribed into a single mRNA. These results suggest that all the gelatinases are produced from the respective full-length collagenase by the proteolytic removal of C-terminal fragments. The substrate specificities of the enzymes suggest that
<italic>colG</italic>
and
<italic>colH</italic>
encode class I and class II enzymes, respectively. Analysis of their DNA locations by pulsed-field gel electrophoresis and nucleotide sequencing of their surrounding regions revealed that the two genes are located in different sites on the chromosome.
<italic>C. histolyticum colG</italic>
is more similar to
<italic>C. perfringens colA</italic>
than to
<italic>colH</italic>
in terms of domain structure. Both
<italic>colG</italic>
and
<italic>colA</italic>
have a homologous gene,
<italic>mscL</italic>
, at their 3′ ends. These results suggest that gene duplication and segment duplication have occurred in an ancestor cell common to
<italic>C. histolyticum</italic>
and
<italic>C. perfringens</italic>
and that further divergence of the parent gene produced
<italic>colG</italic>
and
<italic>colA</italic>
.

DOI： 10.1128/jb.181.3.923-933.1999

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Proceedings of the National Academy of Sciences  

DOI： 10.1073/pnas.95.12.7018

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担当区分：筆頭著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

DOI： 10.1074/jbc.273.6.3643

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掲載種別：研究論文（学術雑誌）  

The extracellular matrix (ECM) is an attractive target for localizing exogenous growth factors and other peptide signaling molecules as therapeutic agents. We have produced fusion proteins consisting of growth factor moieties and a collagen-binding domain (CBD) derived from a bacterial collagenase which we expected to act as an anchor to the collagen fibrils in vivo. The fusion proteins carrying the epidermal growth factor (EGF) or basic fibroblast growth factor (bFGF) at the N-terminal of CBD (CBEGF and CBFGF) tightly bound to insoluble collagen and stimulated the growth of cultured fibroblasts as much as the unfused counterparts. CBEGF, when injected subcutaneously into mice, remained at the sites of injection for up to 10 days, but EGF was not detectable 24 h after injection. Although CBEGF did not exert a growth promoting effect in vivo, CBFGF, but not bFGF, strongly stimulated the DNA synthesis in stromal cells at 5 days and 7 days after injection. CBD may be used as an anchoring unit to produce fusion proteins which are nondiffusible and long-lasting in vivo.

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Center for Academic Pub. Japan  

DOI： 10.1111/j.1348-0421.1997.tb01888.x

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Center For Academic Publications Japan  

The colH gene encoding 116-kDa collagenase of Clostridium histolyticum (cColH) was cloned into an Escherichia coli-Bacillus subtilis shuttle vector to develop a method for purification of recombinant collagenase (rColH). When plasmid pJCM310 containing the colH gene was introduced into B. subtilis DB104 and the transformant was grown in LB broth at 37C, stability of the plasmid was not maintained. However, stability was partly improved by growing the transformant in a modified LB broth containing 0.5M sodium succinate with gentle shaking at 35C. When the transformant was grown to an optical density of 0.4 at 600nm in this medium, pJCM310 was stable and rColH was produced in sufficient amounts. rColH was purified to homogeneity by ammonium sulfate precipitation, gel filtration and ion-exchange chromatography. The yield of rColH from an 800-ml culture was 0.53mg and its specific activity was estimated to be 1, 210U per mg of protein. The purified rColH was capable of degrading native type-I collagen fibril from bovine achilles tendon, as was demonstrated by zymography. A comparison of the N-terminal amino acid sequence between cColH and rColH revealed that rColH has 10 extra N-terminal amino acid residues. However, the peptide mapping of rColH with V8 protease was virtually identical to that of cColH. Furthermore, the molecular mass of rColH was estimated to be 112, 999 Da by mass spectrometry, coinciding with the value of 112, 977 Da, which was predicted from the nucleotide sequence of the colH gene. Therefore, the recombinant B. subtilis culture is capable of serving as a useful source for enzyme purification.

DOI： 10.1111/j.1348-0421.1996.tb01161.x

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Microbiology Society  

DOI： 10.1099/00221287-142-9-2561

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1111/j.1348-0421.1996.tb03344.x

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掲載種別：研究論文（学術雑誌）   出版者・発行元：American Society for Microbiology  

The lambda-toxin of Clostridium perfringens type B NCIB10691 was purified by ammonium sulfate precipitation, followed by size exclusion, anion-exchange, and hydrophobic interaction chromatography. The purified toxin had an apparent molecular mass of 36 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The toxin possessed casein-hydrolyzing activity, which was inhibited specifically by metal chelators, indicating that the toxin is a metalloprotease. The gene encoding the lambda-toxin (lam), which was shown by Southern analysis to be located on a 70-kb plasmid, was cloned into Escherichia coli cells. Nucleotide and N-terminal amino acid sequencing revealed that the lam gene encodes a 553-amino-acid protein, which is processed into a mature form, the molecular mass of which was calculated to be 35,722 Da. The deduced amino acid sequence of the mature enzyme contains an HEXXH motif characteristic of zinc metalloproteases and is homologous to other known enzymes belonging to the thermolysin family. The purified toxin degraded various biologically important substances, such as collagen, fibronectin, fibrinogen, immunoglobulin A, and the complement C3 component. It caused an increase in vascular permeability and hemorrhagic edema on injection into the dorsal skin of mice. These results suggest that the toxin contributes to the pathogenesis of histolytic infection by lambda-toxin-producing C. perfringens.

DOI： 10.1128/iai.64.1.230-237.1996

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掲載種別：研究論文（学術雑誌）   出版者・発行元：American Society for Microbiology  

The phylogenetic interrelationships between strains of 5 toxin types (A to E) of Clostridium perfringens were examined by analysis of differences in the nucleotide sequences of phospholipase C genes (plc genes) among 10 strains, including 3 strains for which the plc gene sequences have been previously reported. A plc gene was also cloned from a Clostridium novyi type A strain and sequenced to analyze the interspecies diversity of plc genes. Phylogenetic trees constructed by the neighbor-joining method revealed that the phylogeny of C. perfringens strains is not related to toxin typing, in agreement with the results of a comparative genome mapping study by Canard et al. (B. Canard, B. Saint-Joanis, and S. T. Cole, Mol. Microbiol. 6:1421-1429, 1992). Various C. perfringens phospholipase C enzymes were purified from cultures of Escherichia coli cells into which the encoding plc genes had been cloned. All of the enzymes showed the same specific activity. On the other hand, the level of plc transcripts differed greatly (up to 40-fold) from one C. perfringens strain to another. No significant difference in the nucleotide sequence of the plc promoter region was observed for any of the plc genes. These results suggest that the variation in phospholipase C activity among different strains is not due to mutation in the plc coding region but to that in an extragenic region. The evolution of C. perfringens phospholipase C is discussed on the basis of similarities and differences between clostridial plc genes.

DOI： 10.1128/jb.177.24.7164-7170.1995

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掲載種別：研究論文（学術雑誌）   出版者・発行元：American Society for Microbiology  

We developed a method to purify Clostridium perfringens phospholipase C from a culture of recombinant Bacillus subtilis cells. This method consists of three purification steps, and it allowed us to obtain 6.2 mg of pure phospholipase C from 800 ml of culture.

DOI： 10.1128/aem.61.11.4114-4115.1995

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掲載種別：研究論文（学術雑誌）  

DOI： 10.1099/00207713-45-4-804

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担当区分：筆頭著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

DOI： 10.1016/0378-1119(95)00490-w

PubMed

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掲載種別：研究論文（学術雑誌）   出版者・発行元：American Society for Microbiology  

Clostridium perfringens type A strains which differed in alpha-toxin (phospholipase C [PLC]) productivity were inoculated intraperitoneally or intravenously into mice, and then their 50% mouse lethal doses (LD50) were determined. Strain NCTC 8237 produced ninefold higher PLC activity than strain 13. The mean LD50 for the former was 1 log unit lower than that for the latter. Two isogenic strains were constructed from strain 13: strain 13(pJIR418 alpha) (pJIR418 alpha contains the plc gene), which produced ninefold higher PLC activity than strain 13; and strain 13 PLC-, which showed no PLC productivity at all because of transformation-mediated gene disruption. The mean LD50 for strain 13(pJIR418 alpha) was 1 log unit lower than those for strain 13 PLC- and strain 13. These results indicate that PLC functions as a virulence-determining factor when it is produced in a sufficient amount. Such a difference in LD50 was also observed between Bacillus subtilis with and without the cloned plc gene. Inoculation of B. subtilis PLC+ intravenously into mice caused marked thrombocytopenia and leukocytosis. Mice inoculated with B. subtilis at 2 LD50 died because of circulatory collapse. Histological examination revealed that intravascular coagulation and vascular congestion occurred most prominently in the lungs. These results suggest that PLC plays a key role in the systemic intoxication of clostridial myonecrosis, probably by affecting the functions of platelets and phagocytes.

DOI： 10.1128/iai.62.11.5032-5039.1994

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掲載種別：研究論文（学術雑誌）   出版者・発行元：American Society for Microbiology  

The colH gene encoding a collagenase was cloned from Clostridium histolyticum JCM 1403. Nucleotide sequencing showed a major open reading frame encoding a 116-kDa protein of 1,021 amino acid residues. The deduced amino acid sequence contains a putative signal sequence and a zinc metalloprotease consensus sequence, HEXXH. A 116-kDa collagenase and a 98-kDa gelatinase were copurified from culture supernatants of C. histolyticum. While the former degraded both native and denatured collagen, the latter degraded only denatured collagen. Peptide mapping with V8 protease showed that all peptide fragments, except a few minor ones, liberated from the two enzymes coincided with each other. Analysis of the N-terminal amino acid sequence of the two enzymes revealed that their first 24 amino acid residues were identical and coincided with those deduced from the nucleotide sequence. These results indicate that the 98-kDa gelatinase is generated from the 116-kDa collagenase by cleaving off the C-terminal region, which could be responsible for binding or increasing the accessibility of the collagenase to native collagen fibers. The role of the C-terminal region in the functional and evolutional aspects of the collagenase was further studied by comparing the amino acid sequence of the C. histolyticum collagenase with those of three homologous enzymes: the collagenases from Clostridium perfringens and Vibrio alginolyticus and Achromobacter lyticus protease I.

DOI： 10.1128/jb.176.21.6489-6496.1994

PubMed

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Center For Academic Publications Japan  

The virulence of methicillin-resistant Staphylococcus aureus (MRSA) was compared with that of methicillin-sensitive S. aureus (MSSA), using 13 MRSA and 7 MSSA strains isolated from clinical specimens. The infectivity and lethality of the two groups were examined as to the inoculum required to infect 50% of guinea pigs (ID₅₀) and to kill 50% of mice (LD₅₀), respectively. The mean ID₅₀ [log10 colony forming units (CFU)] for MRSA strains was 7.1±0.60 standard deviation, which was 1.5 higher than that for MSSA strains (P<0.001). The mean LD₅₀ (log10 CFU) for MRSA strains was 9.0±0.42, being 1.1 higher than that for MSSA strains (P=0.001). Pretreatment of mice with cyclophosphamide decreased the mean LD₅₀ for MRSA strains more than that for MSSA strains, resulting in the difference in the mean LD₅₀ being insignificant (P=0.502). These results indicate that MRSA is less virulent than MSSA in normal hosts, but that they are equally virulent in immunocompromised hosts. The growth of MRSA strains was much slower than that of MSSA strains in the lag phase, although their growth rates were almost the same in the exponential growth phase, suggesting that the difference in virulence between them may be at least partly due to such a difference in growth.

DOI： 10.1111/j.1348-0421.1994.tb01829.x

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

DOI： 10.1006/plas.1994.1035

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掲載種別：研究論文（学術雑誌）   出版者・発行元：American Society for Microbiology  

We examined the enterotoxicity of a Klebsiella oxytoca cytotoxin which is produced by K. oxytoca OK-1, a strain from a patient with antibiotic-associated hemorrhagic colitis. Injection of the cytotoxin into ligated ileal and colonic loops in rabbits caused the accumulation of fluid in the loops. The fluid was bloody in the ileal loops but not in the colonic ones. Histological examination revealed intense mucosal hemorrhage with erosion in the ileum, whereas no microscopic change was noted in the colon. The fluid accumulation was shown to be a dose-dependent response in both ileal and colonic loops. The amounts of the cytotoxin required for maximal fluid accumulation in ileal and colonic loops were 60 and 10 micrograms, respectively. Fluid accumulation was first noticeable in ileal loops 12 h and in colonic ones 5 h after the injection of these doses of the cytotoxin and then proceeded with time. When K. oxytoca OK-1, a cytotoxin-producing strain, was inoculated into the loops at doses of 1 x 10(8) and 5 x 10(9) CFU, similar fluid accumulation was observed. However, inoculation of K. oxytoca ATCC 13182, a non-cytotoxin-producing strain, at the same doses did not cause any change. These results suggest that the cytotoxin-producing strain of K. oxytoca is the causative organism of antibiotic-associated hemorrhagic colitis and that the toxin is the factor responsible for pathogenesis.

DOI： 10.1128/iai.62.1.172-177.1994

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担当区分：筆頭著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：American Society for Microbiology  

Clostridium perfringens type C NCIB 10662 produced various gelatinolytic enzymes with molecular masses ranging from approximately 120 to approximately 80 kDa. A 120-kDa gelatinolytic enzyme was present in the largest quantity in the culture supernatant, and this enzyme was purified to homogeneity on the basis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme was identified as the major collagenase of the organism, and it cleaved typical collagenase substrates such as azocoll, a synthetic substrate (4-phenylazobenzyloxy-carbonyl-Pro-Leu-Gly-Pro-D-Arg [Pz peptide]), and a type I collagen fibril. In addition, a gene (colA) encoding a 120-kDa collagenase was cloned in Escherichia coli. Nested deletions were used to define the coding region of colA, and this region was sequenced; from the nucleotide sequence, this gene encodes a protein of 1,104 amino acids (M(r), 125,966). Furthermore, from the N-terminal amino acid sequence of the purified enzyme which was found in this reading frame, the molecular mass of the mature enzyme was calculated to be 116,339 Da. Analysis of the primary structure of the gene product showed that the enzyme was produced with a stretch of 86 amino acids containing a putative signal sequence. Within this stretch was found PLGP, the amino acid sequence constituting the Pz peptide. This sequence may be implicated in self-processing of the collagenase. A consensus zinc-binding sequence (HEXXH) suggested for vertebrate Zn collagenases is present in this bacterial collagenase. Vibrio alginolyticus collagenase and Achromobacter lyticus protease I showed significant homology with the 120-kDa collagenase of C. perfringens, suggesting that these three enzymes are evolutionarily related.

DOI： 10.1128/jb.176.1.149-156.1994

PubMed

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掲載種別：研究論文（学術雑誌）   出版者・発行元：American Society for Microbiology  

The Clostridium perfringens plc gene encoding phospholipase C (alpha-toxin) was cloned from type C NCIB 10662, a strain which produces low levels of phospholipase C activity. The nucleotide sequence of a cloned 3.1-kb HindIII fragment was determined. The same fragment was also cloned from type A NCTC 8237, a phospholipase C-overproducing strain. In this case, an open reading frame (ORF2) truncated in the previously cloned 2-kb fragment was also sequenced. Comparison of the nucleotide sequence between the 3.1-kb fragments of the two type strains shows some differences both in the plc gene and in ORF2. However, when the 3.1-kb fragment was cloned into plasmid pUC19 and expressed in Escherichia coli, the plc genes from both type strains were similarly expressed and the toxins produced showed similar levels of activity. Northern blot analysis revealed that the type A strain produced 16 to 23 times more plc mRNA than the type C strain. These results indicate that in C. perfringens the two plc genes are transcribed at different rates, probably because of a difference in a locus lying outside of the cloned fragments. Gel retardation analysis showed that the type A strain possessed two different proteins that bound different regions of the plc gene. However, one of these proteins, which binds within the plc coding region, was not found in the type C strain, suggesting that it plays a role in the regulation of the plc gene expression.

DOI： 10.1128/iai.61.2.457-463.1993

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Center For Academic Publications Japan  

Lactobacillus plantarum ATCC 8014 grew on melibiose at 30C, but not at 37C, although it grew on galactose or lactose at either temperature. ATCC 8014 grown on lactose at 30 or 37C accumulated melibiose slowly, suggesting that melibiose may partly be transported by a lactose transport system. A lactose-negative mutant, NTG 21, derived from ATCC 8014 was isolated. The mutant was totally deficient in lactose transport, but retained normal melibiose transport activity. In NTG 21, the melibiose transport activity was induced by melibiose at 30C, but not at 37C. The transport activity itself was found to be stable for at least 3hr at 37C, suggesting that the induction process in the cytoplasm rather than the inducer entrance is temperature-sensitive in the organism. The organism also failed to form α-galactosidase at 37C when grown on melibiose. The enzyme synthesis, however, was induced by galactose in NTG 21 (and also by lactose in ATCC 8014) even at 37C, indicating that the induction of the enzyme is essentially not temperature-sensitive. In NTG 21, melibiose transport system and α-galactosidase were induced by galactose, melibiose and o-nitrophenyl-α-D-galactopyranoside when the strain was grown at 30C. Raffinose induced melibiose transport system only a little, while it was a good inducer for α-galactosidase. Inhibition studies revealed that galactose may be a weak substrate of the melibiose transport system; no inhibition was demonstrated with lactose and raffinose.

DOI： 10.1111/j.1348-0421.1992.tb02116.x

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掲載種別：研究論文（学術雑誌）  

DOI： 10.1128/aem.58.11.3593-3597.1992

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Center For Academic Publications Japan  

The phospholipase C (α-toxin) gene (plc) of Clostridium perfringens was cloned into pUC19 and the effects of the upstream regions on expression of the plc gene were examined in Escherichia coli JM109. When the 0.7-kb region just upstream of the putative -35 site of the gene was deleted, production of phospholipase C increased approximately 10-fold. Northern blot hybridization analysis of the plc transcript showed that the upstream region inhibited transcription from the plc promoter. Nucleotide sequencing of this upstream region revealed that there are three periodically repeated (dA)_5-6 tracts between positions -66 and -40 of the plc gene. A fragment containing this sequence showed anomalously slow electrophoretic mobility at low temperature, indicating that the region immediately upstream of the plc promoter is a locus of sequence directed DNA-bending. Nested deletions of the upstream region were created from its 5' end by exonuclease III and the effects of deletions on the expression of the plc gene were examined. When the 77-bp fragment containing the two (dA)_5-6 tracts was deleted, phospholipase C production increased markedly. These results indicate that the intrinsic DNA curvature upstream of the plc promoter is involved in the negative regulation of the plc gene transcription.

DOI： 10.1111/j.1348-0421.1992.tb02060.x

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Center For Academic Publications Japan  

Yersinia enterocolitica is capable of growing in a broad range of temperatures from 4 to 45C. How this organism alters its membrane lipids in response to the change of growth temperature is very interesting. The fatty acids of membrane lipids of cells cultured at 5, 15, 25 and 37C were analyzed and the physical states of these membrane lipids were characterized. The major phospholipids of this bacterium were phosphatidylethanolamine, phosphatidylglycerol, cardiolipin, lysophosphatidylglycerol and lysophosphatidylethanolamine. No significant difference in phospholipid composition in response to culture temperatures was observed. It was reported in our previous paper that the major fatty acids of membrane phospholipids of Y. enterocolitica were C_15:0, C_16:0, C_16:1, cyclopropane C_17:0 and C_18:0. Some differences in the fatty acid composition were, however, observed with the change of culture temperature. When the culture temperature was raised, the saturated and cyclopropane fatty acids substantially increased and the unsaturated ones decreased. A reverse phenomenon was observed when culture temperature was lowered. From the viewpoints of membrane physical state, adaptational changes were analyzed using a nylon microcapsule method. Phase transition in membrane lipids of cells grown at each culture temperature took place in the range of about 5C below and about 10C above the culture temperature. It is, therefore, considered that Y. enterocolitica maintains its membrane rigidity and fluidity in response to growth temperature by changing the membrane fatty acid composition.

DOI： 10.1111/j.1348-0421.1991.tb01630.x

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担当区分：筆頭著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：American Society for Microbiology  

Escherichia coli transformed with a plasmid containing a Bacteroides ruminicola endoglucanase (carboxymethyl cellulase [CMCase]) gene produced three immunologically cross-reacting CMCases which had molecular weights of 40,500, 84,000, and 88,000, while B. ruminicola produced CMCases with molecular weights of 82,000 and 88,000. The two B. ruminicola enzymes (purified from culture supernatants) had different N-terminal amino acid sequences, but each enzyme was encoded by the same gene (three independent clones had the same DNA sequence). The 88,000-molecular-weight CMCase (88K CMCase) gene appeared to contain two open reading frames which overlapped for 18 bp and were -1 out of frame, and each open reading frame contained several stop codons near the overlap region. The two 88K CMCase open reading frames had enough DNA to produce a protein of 106K, but the mobility of the enzyme in sodium dodecyl sulfate gels gave a value which was 20% lower. On the basis of the -1 frame shift and the large deviation in theoretical versus actual size, it appears that an unusual event (e.g., ribosomal hopping or RNA splicing) is involved in either the translation or the transcription of the 88K B. ruminicola CMCase gene. The 82K CMCase was completely encoded in the second reading frame, and its size was in agreement with the DNA sequence.

DOI： 10.1128/jb.173.21.6919-6926.1991

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Center For Academic Publications Japan  

We developed a novel method for the detection of Mycoplasma hominis from vaginal swabs using an indirect immunofluorescence technique. It is a rapid and simple method that can be finished in only 5hr and is more sensitive than the usual culture isolation method. The indirect immunofluorescence method was applied to vaginal smears from 193 healthy women and 33.7% gave a positive test. This value was much higher than that (11.4%) obtained from the same specimens by the culture method. When vaginal smears were subjected to Papanicolaou staining after the indirect immunofluorescence method, the specific immunofluorescence of the epithelial cells was located exactly at the sites of granular aggregates stained with Papanicolaou stain. A histological examination by Papanicolaou staining showed that the incidence of inflammation seems to be slightly higher in M. hominis-carriers than in non-carriers.

DOI： 10.1111/j.1348-0421.1991.tb02023.x

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担当区分：筆頭著者   掲載種別：研究論文（学術雑誌）  

DOI： 10.1128/jb.172.7.3620-3630.1990

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担当区分：筆頭著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：American Society for Microbiology  

Escherichia coli K-12 strains isolates carrying plasmid pBR322 were grown in the presence of subinhibitory concentrations of lincomycin, which stimulated beta-lactamase synthesis about 2.5-fold, and the effects of the drug on the synthesis and degradation of bla mRNA were studied. The bla mRNA levels determined by 1-min pulse-labeling with [3H]uridine were significantly higher in a lincomycin-containing culture than in the control culture, indicating that stimulation of beta-lactamase synthesis is caused by an increase in the amount of bla mRNA. The enhancing effect of lincomycin was observed in strains harboring pBR322 delta P1 and pBR322 delta P3, which lacked the P1 or P3 promoter, respectively, as well as in the strain harboring pBR322. S1 nuclease analysis showed that the half-life of bla mRNA increased about 2.7-fold when lincomycin was present. These results indicate that the increase in beta-lactamase synthesis caused by lincomycin is due to an increase in the stability of bla mRNA rather than activation of its synthesis.

DOI： 10.1128/aac.33.6.805

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：JAPAN ANTIBIOTICS RESEARCH ASSOCIATION  

Sub-inhibitory concentrations of lincomycin slightly inhibit growth of Escherichia coli carrying plasmid RP4 and cause a 2-fold increase in TEM-2 β-lactamase. To analyze this effect, cultures were pulse-labeled with [³H]leucine, chased with non-radioactive leucine and immunoprecipitated with anti-β-lactamase antiserum. The synthesis rate of β-lactamase was two times higher in inhibited cultures than in control cultures. No significant decrease of labeled enzyme occurred during the 30 minutes chase, indicating no degradation of β-lactamase. The rate of maturation of pre-β-lactamase was determined by measuring the decrease in the amount of pre-β-lactamase after a 1-minute labeling interval. There was no significant difference between the control and lincomycin-treated cultures, indicating that posttranslational translocation is not involved in the stimulation. Both plasmid encoded and chromosomally encoded TEM-1 β-lactamase increased in the presence of lincomycin. The effects of other protein synthesis inhibitors on the synthesis of TEM-1 β-lactamase were examined. The stimulation of β-lactamase synthesis by lincomycin appears to be specific for macrolide and related antibiotics and is not a general phenomenon resulting from partial inhibition of protein synthesis.

DOI： 10.7164/antibiotics.41.667

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掲載種別：研究論文（学術雑誌）   出版者・発行元：American Society for Microbiology  

Lincomycin increased the TEM-2 beta-lactamase activity of Escherichia coli K-12 cells carrying plasmid RP4 at a concentration which slightly inhibited cell growth. In a control culture beta-lactamase activity reached its maximal level in late log phase, whereas when lincomycin was present beta-lactamase activity continued to increase into the stationary phase. Lincomycin (100 micrograms/ml) inhibited both cell growth and protein synthesis by about 35% but stimulated beta-lactamase activity 2.5-fold per ml of culture and about 4-fold per cell after 20 h of growth. The amount of beta-lactamase produced in each culture was also compared by densitophotometry of a stained sodium dodecyl sulfate-polyacrylamide gel. The relative values were in good agreement with the relative enzyme activities, indicating that the stimulatory effect of lincomycin was due to an increase in the amount of beta-lactamase protein. Inactivation of beta-lactamase appeared to be faster when lincomycin was present. This was determined by measuring the decrease in beta-lactamase activity when phenethyl alcohol was present to prevent maturation of the enzyme. There was no significant difference in plasmid copy number between the cells grown in the presence or absence of lincomycin. These results indicate that lincomycin stimulates transcription, translation, or translocation of beta-lactamase.

DOI： 10.1128/aac.30.1.82

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記述言語：英語   出版者・発行元：日本細菌学会  

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記述言語：英語   出版者・発行元：日本細菌学会  

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記述言語：日本語   出版者・発行元：日本細菌学会  

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J-GLOBAL

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J-GLOBAL

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記述言語：英語   出版者・発行元：日本細菌学会  

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記述言語：日本語   出版者・発行元：日本細菌学会  

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J-GLOBAL

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記述言語：日本語   出版者・発行元：日本細菌学会  

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記述言語：英語   出版者・発行元：日本結合組織学会  

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記述言語：英語   出版者・発行元：日本細菌学会  

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J-GLOBAL

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記述言語：日本語   出版者・発行元：(NPO)日本歯周病学会  

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記述言語：英語   出版者・発行元：日本細菌学会  

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記述言語：日本語   出版者・発行元：日本防菌防黴学会  

近年、環境汚染菌による院内感染が報告され、環境整備の重要性が認められつつある。今回、消毒剤および除菌剤の除染効果を比較検討するため、環境中の細菌の生存状態とATP測定の基礎的検討と各種消毒剤または除菌剤を含浸させたワイプによる拭き取り効果について検討した。乾燥による生菌数への影響に関する検討では、一定の濃度に調製した多剤耐性Acinetobacter baumanniiおよびBacillus cereusを乾燥状態で放置し、経時的に細菌数の指標としてATP値とコロニー数を計測した。シャーレの中で乾燥状態にしたB.cereusは1日目でATP値が有意に低下したが、A.baumanniiは有意な低下を認めるまでに7日間を要した。7日目のコロニー数を計測したところ、A.baumanniiおよびB.cereus共に生菌として検出された。拭き取りによる除菌効果は、ステンレス板とポリプロピレン板にA.baumanniiおよびB.cereusを塗布し、上記消毒剤または除菌剤含浸ワイプで拭き取り、その前後におけるATP量およびコロニー数を比較検討した。拭き取り効果は、材質による差は認められなかった。また、いずれの含浸ワイプによる拭き取りにおいても同様に有意な菌数の低下が認められ、いずれの細菌に対しても消毒剤または除菌剤は5log10CFU以上の減少を認めた。(著者抄録)

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J-GLOBAL

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記述言語：英語   出版者・発行元：日本細菌学会  

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記述言語：英語   出版者・発行元：日本細菌学会  

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記述言語：英語   出版者・発行元：日本細菌学会  

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記述言語：日本語   出版者・発行元：(公社)日本整形外科学会  

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記述言語：日本語   出版者・発行元：日本細菌学会  

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記述言語：日本語   出版者・発行元：日本細菌学会  

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記述言語：英語   出版者・発行元：日本細菌学会  

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記述言語：日本語   出版者・発行元：(一社)日本移植学会  

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記述言語：日本語   出版者・発行元：(公社)日本整形外科学会  

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記述言語：日本語   出版者・発行元：(公社)日本整形外科学会  

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記述言語：日本語  

PubMed

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記述言語：日本語   出版者・発行元：(公社)日本整形外科学会  

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記述言語：日本語   出版者・発行元：(公社)日本整形外科学会  

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記述言語：日本語   出版者・発行元：(公社)日本整形外科学会  

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記述言語：日本語   出版者・発行元：(公社)日本整形外科学会  

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記述言語：日本語   出版者・発行元：(公社)日本整形外科学会  

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記述言語：日本語   出版者・発行元：(一社)日本耳科学会  

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記述言語：日本語  

CiNii Article

CiNii Books

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記述言語：日本語  

CiNii Article

CiNii Books

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担当区分：筆頭著者   出版者・発行元：Elsevier BV  

DOI： 10.1016/s0041-0101(01)00163-5

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担当区分：筆頭著者,　最終著者,　責任著者   記述言語：日本語   出版者・発行元：Japanese Society for Bacteriology  

二種のガス壊疸起因菌Clostridium perfringens, C. histolyticumから計三種類のコラゲナーゼを精製した。それらの構造遺伝子を解析したところ,予想一次配列にセグメント構造(S1, S2, S3)が認められた。N末端側のS1には金属プロテアーゼに共通なモチーフ(HEXXH)が存在していた。C末端側のS2, S3には重複が認められ,酵素により重複パターンが異なっていた。C. histolyticumの酵素の一つColHを用いて構造活性相関の解析を試みた。N末端側のS1のみからなる組換え酵素が水解活性を示したので,S1は触媒ドメインを形成すると考えられた。単離C末端領域が不溶性コラーゲンに結合することから,この領域はコラーゲン結合ドメイン(CBD)を形成すると考えられた。CBDを用いて細胞成長因子をコラーゲンにアンカーリングし,局所で長時間作用させることができた。CBDの構造が新しい薬物送達システム(drug delivery system, DDS)の開発に応用できる可能性が示された。

DOI： 10.3412/jsb.54.753

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その他リンク： https://jlc.jst.go.jp/DN/JALC/00085759732?from=CiNii

記述言語：日本語   出版者・発行元：Japanese Society for Bacteriology  

亜鉛結合性メタロプロテアーゼの研究は近年めざましい進歩を遂げた。構造遺伝子の塩基配列の情報からメタロプロテアーゼが予測され,メタロプロテアーゼとしての機能が浮き彫りにされてきたことが飛躍的な発展の原動力となっている。本酵素は原核生物,真核生物いずれにも幅広く存在している。構成タンパク,機能タンパクを特異的に分解する真核生物のメタロプロテアーゼはそれぞれの個体において重要な生理的役割を果たしている。一方細菌の産生するメタロプロテアーゼは宿主の構成タンパク,あるいは感染防御上重要なタンパクを分解し,病原因子として働くものが多い。その基質特異性は非常に厳密なものもあるが,真核生物のものに比べると一般にかなり幅広い。このことは病原因子としてはむしろ適していると考えられる。しかし,細菌自身の生命現象にとって重要な触媒作用を有する酵素が存在している可能性もある。今後の細菌性メタロプロテアーゼ研究の進展にとって多面的な視点とアプローチが必要と考えられる。

DOI： 10.3412/jsb.50.971

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その他リンク： https://jlc.jst.go.jp/DN/JALC/00082000653?from=CiNii

出願人：ザ ボード オブ トラスティーズ オブ ザ ユニバーシティ オブ アーカンソー

出願番号：特願2018-038513  出願日：2018年3月5日

公開番号：特開2018-104461  公開日：2018年7月5日

特許番号/登録番号：特許第6697019号  登録日：2020年4月27日 

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出願人：ザ ボード オブ トラスティーズ オブ ザ ユニバーシティ オブ アーカンソー; 学校法人北里研究所

出願番号：特願2019-543282  出願日：2018年2月9日

公表番号：特表2020-508984  公表日：2020年3月26日

特許番号/登録番号：特許第7397440号  登録日：2023年12月5日  発行日：2023年12月13日

権利者：ザ ボード オブ トラスティーズ オブ ザ ユニバーシティ オブ アーカンソー; 学校法人北里研究所

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出願人：The Board of Trustees of the University of Arkansas

出願番号：US 15/407,589  出願日：2017年1月17日

公開番号：US 2017/0204390 A1  公開日：2017年7月20日

特許番号/登録番号：US 11,001,820 B2  登録日：2021年5月11日 

権利者：The Board of Trustees of the University of Arkansas;Montefiore Medical CenterThe Kitasato Institute;National University Corporation Kagawa University

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出願人：THE BOARD OF TRUSTEES OF THE UNIVERSITY OF ARKANSAS

出願番号：US 15/387,817  出願日：2016年12月22日

公開番号：US 2017/0101457 A1  公開日：2017年4月13日

特許番号/登録番号：US 10,202,434 B2  登録日：2019年2月12日 

権利者：THE BOARD OF TRUSTEES OF THE UNIVERSITY OF ARKANSAS;OCHSNER CLINIC FOUNDATION;NATIONAL UNIVERSITY CORPORATION KAGAWA UNIVERSITY

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出願人：The Board of Trustes of the University of Arkansas

出願番号：US 15/386,626  出願日：2016年12月21日

公開番号：US 2017/0106093 A1  公開日：2017年4月20日

特許番号/登録番号：US 10,213,488 B2  登録日：2019年2月26日 

権利者：The Board of Trustes of the University of Arkansas;The Kitasato Institute;Montefiore Medical Center

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出願人：ザ ボード オブ トラスティーズ オブ ザ ユニバーシティ オブ アーカンソー

出願番号：特願2016-157345  出願日：2016年8月10日

公開番号：特開2017-25069  公開日：2017年2月2日

特許番号/登録番号：特許第6366653号  登録日：2018年7月13日  発行日：2018年8月1日

権利者：ザ ボード オブ トラスティーズ オブ ザ ユニバーシティ オブ アーカンソー;国立大学法人 香川大学;オクスナー クリニック ファウンデイション

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出願人：学校法人北里研究所

出願番号：特願2016-554137  出願日：2015年10月16日

公表番号：WO2016/060252  公表日：2016年4月21日

特許番号/登録番号：特許第6699821号  登録日：2020年5月7日  発行日：2020年5月27日

権利者：学校法人北里研究所;株式会社アトリー;国立大学法人 岡山大学;国立大学法人 香川大学;株式会社ニッピ

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出願人：ザ ボード オブ トラスティーズ オブ ザ ユニバーシティ オブ アーカンソー

出願番号：特願2015-173678  出願日：2015年9月3日

公開番号：特開2016-094385  公開日：2016年5月26日

特許番号/登録番号：特許第5989876号  登録日：2016年8月19日 

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出願人：Ochsner Clinic Foundation

出願番号：US 14/743,629  出願日：2015年6月18日

公開番号：US 2015/0284701 A1  公開日：2015年10月8日

特許番号/登録番号：US 9,528,099 B2  登録日：2016年12月27日 

権利者：Ochsner Clinic Foundation;National University Corporation Kagawa University;The Board of Trustees of the University of Arkansas

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出願人：ザ ボード オブ トラスティーズ オブ ザ ユニバーシティ オブ アーカンソー

出願番号：特願2013-194484  出願日：2013年9月19日

公開番号：特開2014-040431  公開日：2014年3月6日

特許番号/登録番号：特許第5821066号  登録日：2015年10月16日 

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出願人：National University Corporation Kagawa University

出願番号：US 13/898,058  出願日：2013年5月20日

公開番号：US 2013/0337017 A1  公開日：2013年12月19日

特許番号/登録番号：US 9,062,300 B2  登録日：2015年5月23日 

権利者：National University Corporation Kagawa University;Ochsner Clinic Foundation;The Board of Trustees of the University of Arkansas

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出願人：The Kitasato Institute

出願番号：US 14/378,067  出願日：2013年2月11日

公開番号：US 2015/0038423 A1  公開日：2015年2月5日

公表番号：WO2013/120060  公表日：2013年8月15日

特許番号/登録番号：US 9,526,765 B2  登録日：2016年12月27日 

権利者：The Kitasato Institute;Montefiore Medical Center;Board of Trustees of the University of Arkansas

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出願人：ザ ボード オブ トラスティーズ オブ ザ ユニバーシティ オブ アーカンソー

出願番号：特願2014-547503  出願日：2012年12月14日

公表番号：特表2015-513312  公表日：2015年5月7日

特許番号/登録番号：特許第6366507号  登録日：2018年7月13日  発行日：2018年8月1日

権利者：ザ ボード オブ トラスティーズ オブ ザ ユニバーシティ オブ アーカンソー;学校法人 北里研究所;モンテフィオーレ メディカル センター;国立大学法人 香川大学

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出願人：The Board of Trustees of the University of Arkansas

出願番号：EP 18173596.0 A  出願日：2012年12月14日

公開番号：EP 3391899 A1  公開日：2018年10月24日

公表番号：12857691.5/2 790 717  公表日：2018年10月24日

特許番号/登録番号：EP 3 391 899 B1  登録日：2020年7月15日 

権利者：The Board of Trustees of the University of Arkansas;The Kitasato Institute;Montefiore Medical Center;National University Corporation Kagawa University

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出願人：The Kitasato Institute

出願番号：US 14/365,226  出願日：2012年12月14日

公開番号：US 2014/0377215 A1  公開日：2014年12月25日

公表番号：WO013/090770  公表日：2013年6月20日

特許番号/登録番号：US 9,579.273 B2  登録日：2017年2月28日 

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出願人：The Board of Trustees of the University of Arkansas

出願番号：EP 12857691.5 A  出願日：2012年12月14日

公開番号：EP 2790717 A4  公開日：2016年3月9日

公表番号：WO2013/090770  公表日：2013年6月20日

特許番号/登録番号：EP 2 790 717 B1  登録日：2018年5月30日 

権利者：The Board of Trustees of the University of Arkansas;The Kitasato Institute;Montefiore Medical Center;National University Corporation Kagawa University

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出願人：THE BOARD OF TRUSTEES OF THE UNIVERSITY OF ARKANSAS

出願番号：US 2012/069831  出願日：2012年12月14日

公表番号：WO2013/090770  公表日：2013年6月20日

特許番号/登録番号：CA 2 859 412 C  登録日：2021年5月25日 

権利者：THE BOARD OF TRUSTEES OF THE UNIVERSITY OF ARKANSAS;MONTEFIORE MEDICAL CENTER;THE KITASATO INSTITUTE

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出願人：学校法人北里研究所

出願番号：特願2013-515034  出願日：2012年3月26日

特許番号/登録番号：特許第5512887号  登録日：2014年4月4日 

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出願人：SCHOOL JURIDICAL PERSON KITASATO INSTITUTE

出願番号：US 14/117,599  出願日：2012年3月26日

公開番号：US 2014/0335146 A1  公開日：2014年11月13日

公表番号：WO2012/157339  公表日：2012年11月22日

特許番号/登録番号：US 9,248,164 B2  登録日：2016年2月2日 

権利者：SCHOOL JURIDICAL PERSON KITASATO INSTITUTE;NATIONAL UNIVERSITY CORPORATION KAGAWA UNIVERSITY

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出願人：School Juridical Person Kitasato Institute

出願番号：EP 12785014.7 A  出願日：2012年3月26日

公開番号：EP 2708246 A4  公開日：2014年11月19日

公表番号：WO2012/157339  公表日：2012年11月22日

特許番号/登録番号：EP 2 708 246 B1  登録日：2017年11月1日 

権利者：School Juridical Person Kitasato Institute;National University Corporation Kagawa University

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出願人：ザ ボード オブ トラスティーズ オブ ザ ユニバーシティ オブ アーカンソー

出願番号：特願2010-503048  出願日：2008年4月9日

特許番号/登録番号：特許第5520811号  登録日：2014年4月11日 

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出願人：The Board of Trustees of The University of Arkansas

出願番号：EP 16169069.8 A  出願日：2008年4月9日

公開番号：EP 3091075 A1  公開日：2016年11月9日

特許番号/登録番号：EP 3 091 075 B1  登録日：2018年6月13日 

権利者：THE BOARD OF TRUSTEES OF THE UNIVERSITY OF ARKANSAS;National University Corporation Kagawa University;Ochsner Clinic Foundation

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出願人：The Board of Trustees of the University of Arkansas

出願番号：US 12/594,547  出願日：2008年4月9日

公開番号：US 2010/0129341 A1  公開日：2010年5月27日

特許番号/登録番号：US 8.450,273 B2  登録日：2013年5月28日 

権利者：The Board of Trustees of the University of Arkansas;Ochsner Clinic Foundation;National University Corporation Kagawa University

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出願人：THE BOARD OF TRUSTEES OF THE UNIVERSITY OF ARKANSAS

出願番号：EP 08742686.2 A  出願日：2008年4月9日

公開番号：EP 2155874 A4  公開日：2010年9月1日

公表番号：WO2008/124166  公表日：2008年10月16日

特許番号/登録番号：EP 2 155 874 B1  登録日：2016年5月11日 

権利者：THE BOARD OF TRUSTEES OF THE UNIVERSITY OF ARKANSAS;National University Corporation Kagawa University;Ochsner Clinic Foundation

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出願人：The Board of Trustees of the University of Arkansas; National University Corporation Kagawa University; Ochsner Clinic Foundation

出願番号：2 683 862  出願日：2008年4月9日

公開日：2008年10月16日

特許番号/登録番号：CA 2 930 681 C  登録日：2019年10月15日 

権利者：The Board of Trustees of the University of Arkansas;Ochsner Clinic Foundation;National University Corporation Kagawa University

出願人：THE BOARD OF TRUSTEES OF THE UNIVERSITY OF ARKANSAS

出願番号：2 683 862  出願日：2008年4月9日

公開日：2008年10月16日

特許番号/登録番号：CA 2 930 681 C  登録日：2019年10月15日 

権利者：THE BOARD OF TRUSTEES OF THE UNIVERSITY OF ARKANSAS;NATIONAL UNIVERSITY CORPORATION KAGAWA UNIVERSITY;OCHSNER CLINIC FOUNDATION

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出願人：THE BOARD OF TRUSTEES OF THE UNIVERSITY OF ARKANSAS

出願番号：US2008/004589  出願日：2008年4月9日

公表番号：WO2008/124166 A3  公表日：2008年10月16日

特許番号/登録番号：CA 2 683 862 C  登録日：2016年8月2日 

権利者：THE BOARD OF TRUSTEES OF THE UNIVERSITY OF ARKANSAS;NATIONAL UNIVERSITY CORPORATION KAGAWA UNIVERSITY;OCHSNER CLINIC FOUNDATION

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出願人：The Board of Trustees of the University of Arkansas; National University Corporation Kagawa University; Ochsner Clinic Foundation

出願番号：US2008/004589  出願日：2008年4月9日

公表番号：WO2008/124166 A3  公表日：2008年10月16日

特許番号/登録番号：CA 2 683 862 C  登録日：2016年8月2日 

権利者：The Board of Trustees of the University of Arkansas;National University Corporation Kagawa University;Ochsner Clinic Foundation

出願人：生化学工業株式会社

出願番号：特願平9-310887  出願日：1997年11月12日

公開番号：特開平11-137256  公開日：1999年5月25日

特許番号/登録番号：特許第4484971号  登録日：2010年4月2日 

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