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Tohru Shimizu

Publications and source records attributed to Tohru Shimizu.

17 recordsLinked to original sources
PubMed2006

Detection of a group II intron without an open reading frame in the alpha-toxin gene of Clostridium perfringens isolated from a broiler chicken.

A DNA insertion of 834 bp, designated CPF-G2Im, was identified within the alpha toxin gene (cpa) of Clostridium perfringens strain CPBC16ML, isolated from a broiler chicken. Sequence analysis of CPF-G2Im indicated that it was integrated 340 nucleotides downstream of the start codon of cpa. However, the insertion did not abolish the phospholipase C and hemolytic activities of CPBC16ML. To investigate the expression of its alpha toxin, the intact copy of cpa was cloned into an expression vector and transformed into Escherichia coli M15 cells. Immunoblotting analysis showed that the protein expressed from the transformant as well as in the culture supernatant of C. perfringens strain CPBC16ML had the expected molecular weight detected in reference strains of C. perfringens. Northern hybridization and reverse transcriptase PCR (RT-PCR) analysis revealed that the entire CPF-G2Im insertion was completely spliced from the cpa precursor mRNA transcripts. The sequence of the insertion fragment has 95% and 97% identity to two noncoding regions corresponding to sequences that flank a predicted group II RT gene present in the pCPF4969 plasmid of C. perfringens. However, an RT was not encoded by the CPF-G2Im fragment. Based on the secondary structure prediction analysis, CPF-G2Im revealed typical features of group II introns. The present study shows that CPF-G2Im is capable of splicing in both C. perfringens and E. coli. To our knowledge, this is the first report that a group II intron without an open reading frame (ORF) is located in the cpa ORF of C. perfringens.

Animals↗
PubMed2005

Identification and characterization of a cell-wall anchored DNase gene in Clostridium perfringens.

Completion of the whole genome sequence of Clostridium perfringens strain 13 revealed the presence of an extracellular nuclease gene, cadA. Transcriptional analysis showed that the cadA gene is negatively regulated by the two-component VirR/VirS system and its secondary regulator VR-RNA. The CadA protein possesses an N-terminal signal sequence and a Gram-positive cell wall anchoring motif consisting of a sorting signal (LPXTG motif), a hydrophobic domain, and positively charged residues at the end of C-terminus. By comparing the DNase production between the wild type and the cadA mutant, and DNase activity assay with the recombinant truncated CadA protein, we confirmed that the cadA gene product is one of the DNases produced by C. perfringens.

Bacterial Proteins↗
PubMed2004

Organization and transcriptional regulation of myo-inositol operon in Clostridium perfringens.

myo-Inositol operon of Clostridium perfringens strain 13 consists of 13 genes with an upstream divergent regulator, iolR. Transcriptional analysis showed three separate transcripts for the operon of 15.6, 4.6 and 2.0 kb in length. iolR mutation studies showed that IolR is a negative regulator of the operon at transcriptional level. All the transcripts were induced by myo-Inositol in dose- and time-dependent manner. Glucose repressed the expression of all the transcripts of myo-Inositol operon. We also found that the operon was positively regulated by the two-component VirR/VirS system both in the presence and absence of myo-Inositol. This study shows that the global regulatory VirR/VirS system controls the expression of genes related to energy production (e.g. myo-Inositol operon) in addition to the virulence genes of C. perfringens strain 13.

Bacterial Proteins↗
PubMed2004

Staphylococcus aureus cell envelope antigen is a new candidate for the induction of IgA nephropathy.

BACKGROUND: IgA nephropathy is the most common form of glomerulonephritis worldwide. We previously reported a novel form of glomerulonephritis with glomerular IgA deposits following methicillin-resistant Staphylococcus aureus (S. aureus) infection. We investigated the role of S. aureus related antigens in the immunopathogenesis of IgA nephropathy by producing several monoclonal antibodies against S. aureus surface antigens and determining the epitopes of deposited antigens in patients with IgA nephropathy. METHODS: Cell membrane proteins were isolated from cultured S. aureus. Mouse monoclonal antibodies against these proteins were generated, and their target epitopes were determined by antibody affinity chromatography and amino acid sequence analysis, and by monoclonal antibody screening of Escherichia coli clones transfected with plasmids from the Lambda S. aureus Genomic Library. Renal biopsy specimens from 116 patients with IgA nephropathy and 122 patients with other forms of renal disease were examined for glomerular antigen depositions by immunofluorescence microscopy. RESULTS: . The major antigen recognized by monoclonal antibodies against S. aureus cell membrane was identified as the S. aureus cell envelope antigen designated 'probable adhesin' (ACCESSION AP003131-77, Protein ID; BAB41819.1). In 68.1% (79/116) of renal biopsy specimens from patients with IgA nephropathy, S. aureus cell envelope antigen was localized in the glomeruli, and the data confirmed that S. aureus cell envelope antigen was co-localized with IgA antibody in the glomeruli. No deposition of this antigen was detected in the glomeruli of patients with non-immune complex deposit forms of glomerulonephritis. CONCLUSION: S. aureus cell envelope antigen is a new candidate for the induction of IgA nephropathy.

Amino Acid Sequence↗
PubMed2003

The VirR/VirS regulatory cascade affects transcription of plasmid-encoded putative virulence genes in Clostridium perfringens strain 13.

We analyzed the transcriptional regulation of the putative virulence genes encoded on the plasmid pCP13 from Clostridium perfringens strain 13. The transcription of the beta2-toxin (cpb2) and possible collagen adhesin (cna) genes were regulated in both a positive and negative manner, respectively, by the two-component VirR/VirS system. The secondary regulator of the VirR/VirS system, VR-RNA, also affects the expression of both of these genes in the same fashion as the VirR/VirS system. This indicates that the global regulatory cascade of the VirR/VirS system controls the expression of virulence genes located on the plasmid, as well as those found chromosomally in C. perfringens strain 13.

Bacterial Proteins↗
PubMed2003

[Henoch-Schönlein purpura nephritis in a patient infected with the human immunodeficiency virus].

There are various forms of renal lesions in patients with human immunodeficiency virus(HIV), however reported cases of immune-complex glomerulonephritis are scarce. Here we describe an HIV-positive patient with Henoch-Schönlein purpura nephritis(HSPN), which presented as nephrotic syndrome. In addition to therapy combined with glucocorticosteroid and inhibition of the renin-angiotesin system(RAS), plasmapheresis and antiretroviral therapy produced a favorable outcome. A 26-year-old HIV positive man was admitted for purpura on both lower limbs. Despite glucocorticosteroid treatment, purpura recurred and urinary protein increased to 5-10 g daily. HSPN was diagnosed based on the skin and renal biopsies. During 2 months of treatment with combined glucocorticosteroid and RAS inhibition, nephrotic syndrome persisted. He received double filtration plasmapheresis(DFPP). Soon after, urine protein decreased to 2-3 g daily and macrohematuria decreased. The second renal biopsy showed a decrease in IgA deposition and improvement of acute inflammatory changes. In addition, highly active antiretroviral therapy was started to reduce the high viral load. After 3 weeks, HIV-1-RNA rapidly decreased and urine protein decreased to 1 g daily. After a year, urinary protein was negative, but mild microhematuria persisted. We speculate that the refractory nephrotic syndrome in this patient might be associated with the abnormal immunological condition due to HIV infection.

AIDS-Associated Nephropathy↗
PubMed2002

Identification of a novel locus that regulates expression of toxin genes in Clostridium perfringens.

A novel gene that regulates the alpha-toxin (plc), kappa-toxin (colA), and theta;-toxin (pfoA) genes was identified using toxin-negative mutant strains of Clostridium perfringens. The cloned 3.2-kb fragment contained the virX gene encoding a 51-amino acid polypeptide of unknown function that seemed to be responsible for the activation of toxin genes. The virX knock out mutant of wild-type strain 13 showed a reduced expression of the plc, colA, and pfoA genes, which was complemented by the transformation of the intact virX gene. Deletion and site-directed mutagenesis studies suggested that the virX gene acts as a regulatory RNA rather than as a peptide regulator. The virX locus found in this study might play a part in the signal transduction to regulate toxin production in C. perfringens.

Amino Acid Sequence↗
PubMed2002

Complete genome sequence of Clostridium perfringens, an anaerobic flesh-eater.

Clostridium perfringens is a Gram-positive anaerobic spore-forming bacterium that causes life-threatening gas gangrene and mild enterotoxaemia in humans, although it colonizes as normal intestinal flora of humans and animals. The organism is known to produce a variety of toxins and enzymes that are responsible for the severe myonecrotic lesions. Here we report the complete 3,031,430-bp sequence of C. perfringens strain 13 that comprises 2,660 protein coding regions and 10 rRNA genes, showing pronounced low overall G + C content (28.6%). The genome contains typical anaerobic fermentation enzymes leading to gas production but no enzymes for the tricarboxylic acid cycle or respiratory chain. Various saccharolytic enzymes were found, but many enzymes for amino acid biosynthesis were lacking in the genome. Twenty genes were newly identified as putative virulence factors of C. perfringens, and we found a total of five hyaluronidase genes that will also contribute to virulence. The genome analysis also proved an efficient method for finding four members of the two-component VirR/VirS regulon that coordinately regulates the pathogenicity of C. perfringens. Clearly, C. perfringens obtains various essential materials from the host by producing several degradative enzymes and toxins, resulting in massive destruction of the host tissues.

Amino Acids↗
PubMed2002

Gallbladder metastasis of renal cell carcinoma: report of two cases.

We report two extremely rare cases of metastasis to the gallbladder from renal cell carcinoma. In both men, aged 63 and 80 years, a pedunculated polypoid gallbladder tumor was incidentally found 27 and 8 years after surgery for renal cell carcinoma, respectively. The tumors showed hypervascularity on diagnostic imaging. A histopathological examination showed no tumor cells in the gallbladder mucosa. but clear cell carcinoma was predominantly observed below the mucosal layer. Furthermore, based on various specific and immunohistochemical studies as well as the electronmicroscopic findings, the patients were pathologically diagnosed to have gallbladder metastasis of renal cell carcinoma.

Aged↗
PubMed2002

Clostridial VirR/VirS regulon involves a regulatory RNA molecule for expression of toxins.

We analysed the region encoding VR-RNA (VirR-regulated RNA), which has been reported to be positively regulated by the two-component VirR/VirS system in Clostridium perfringens. The VR-RNA promoter identified by primer extension analysis was preceded by a probable VirR-binding site (CCAGTTNNNCAC), which resembled a repeated sequence motif present in the promoter region of the theta-toxin (pfoA) gene. A VR-RNA-null mutant, constructed by a homologous recombination, exhibited a reduced amount of transcription of the alpha- (plc) and kappa-toxin (colA) genes, which was restored by the complementation of intact VR-RNA, indicating that the VR-RNA region plays an important role in the regulation of the plc and colA genes in C. perfringens. It was found that the regulatory effect was observed even when the hyp7 gene encoded on VR-RNA was deleted or a nonsense mutation was introduced in the hyp7-coding region. We found that the small 3'-portion of VR-RNA was sufficient for the activation of toxin genes, which suggested that VR-RNA itself could act as an RNA regulatory molecule for the plc and colA genes mediating the regulatory information from the VirR/VirS system in C. perfringens.

Amino Acid Sequence↗
PubMed2002

The luxS gene is involved in cell-cell signalling for toxin production in Clostridium perfringens.

A Gram-positive anaerobic pathogen, Clostridium perfringens, causes clostridial myonecrosis or gas gangrene in humans by producing numerous extracellular toxins and enzymes that act in concert to degrade host tissues. C. perfringens possesses a homologue of the luxS gene that is reported to be responsible for the production of autoinducer 2 (AI-2), which participates in quorum sensing in bacteria. The luxS mutant was constructed using C. perfringens strain 13, and the role of the luxS gene in toxin production was examined. The cell-free culture supernatant from wild-type strain 13 greatly stimulated the luminescence of Vibrio harveyi BB170, whereas that from the luxS mutant caused no significant stimulation, indicating that the luxS gene is necessary for AI-2 production in C. perfringens. The luxS mutant showed a reduced level of production of alpha-, kappa- and theta-toxins. In the luxS mutant, the transcription of the theta-toxin gene (pfoA) was lower at mid-exponential growth phase, whereas alpha- and kappa-toxin gene transcription was not significantly affected. The production of toxins in the luxS mutant was stimulated by the addition of the culture supernatant from the wild-type cells, possibly because of the presence of AI-2. Moreover, the expression of the pfoA gene in the luxS mutant was apparently activated when the mutant cells were cultured in the presence of culture supernatants from the wild-type C. perfringens, Escherichia coli DH5alpha carrying the luxS gene of C. perfringens. A deletion analysis of the luxS operon showed that the luxS gene alone is responsible for cell-cell signalling, and that the metB or cysK genes located upstream of luxS are not involved in regulating toxin production. Our results indicate that cell-cell signalling by AI-2 plays an important role in the regulation of toxin production in C. perfringens.

Bacterial Proteins↗
PubMed2002

Hybrid carcinomas of the salivary glands: report of nine cases with a clinicopathologic, immunohistochemical, and p53 gene alteration analysis.

Hybrid carcinomas of the salivary gland are a recently defined and rare tumor entity, consisting of two histologically distinct types of carcinoma within the same topographic area. In this study, we examined nine such cases, which mainly arose in the parotid gland (seven cases), with an additional one each from submandibular and lacrimal glands, and analyzed their clinicopathologic profiles, including immunohistochemical features and p53 gene alterations. The prevalence of hybrid carcinomas was 0.4% among the 1863 cases of parotid gland tumors in our series. The nine patients comprised five men and four women, ranging in age from 40 to 81 years (mean, 62 y). Tumor size ranged from 2 to 10 cm (mean, 4.2 cm). Of the seven patients who were followed up, two were alive with disease and five were alive with no evidence of disease, although the follow-up period was short. Three cases had cervical lymph nodal metastases. The combinations of carcinoma components in our hybrid carcinomas were as follows: epithelial-myoepithelial carcinoma and basal cell adenocarcinoma in two cases, epithelial-myoepithelial carcinoma and squamous cell carcinoma in one case, salivary duct carcinoma and adenoid cystic carcinoma in two cases, myoepithelial carcinoma and salivary duct carcinoma in one, acinic cell carcinoma and salivary duct carcinoma in one, and squamous cell carcinoma and salivary duct carcinoma in two. Although the proportion of each carcinoma component in a tumor mass varied from case to case, the minor component always represented >or= 10% of the area. Differences in cellular composition were studied by immunohistochemistry and electron microscopy. The Ki-67-labeling index apparently differed between the two carcinoma elements in five cases. Diffusely positive p53 immunoreactivity was observed in four cases, restricted to the more aggressive component in each pair. Furthermore, p53 gene alteration analysis of these p53-positive cases revealed that all and three cases demonstrated loss of heterozygosity at p53 microsatellite loci and p53 gene point mutations, respectively, which were detected only in the p53-immunoreactive carcinoma component. Therefore, there is the possibility that such molecular-genetic events take an integral part for inducing the transformation from histologically lower to higher grade tumor during the hybrid carcinoma genesis of the salivary glands.

Adult↗
PubMed2002

Keratocystoma of the parotid gland: a report of two cases of an unusual pathologic entity.

Benign salivary gland tumors composed of purely squamous cells are quite unusual and are not included in the World Health Organization classification. We have seen two benign parotid gland tumors characterized by multicystic spaces with stratified squamous linings and focal solid epithelial nests. Seifert et al. recently described such a case as a choristoma; we, however, herein propose a new designation, keratocystoma, for this unique tumor group, because of its distinctive histological features. These tumors occurred in men aged 18 and 38 years with enlarging parotid gland tumors. Both had largely similar gross and histological features, with some variations. The epithelium lining of the cysts showed apparent keratinization through a parakeratotic or orthokeratotic pathway without forming a granular cell layer. Stratification of the epithelium was always regularly oriented from the outer basal to the inner keratotic cell layer. Focally, the outer layer had bud-like protrusions. In some areas, solid squamous cell islands surrounded by basement membrane were enclosed within the collagenous stroma. These cystic and solid structures were randomly distributed, showing no definite lobular architecture. All of the tumor cells had uniform, bland nuclei and abundant eosinophilic cytoplasm. Scattered mitotic figures were observed, limited to the outer epithelial layer, and showed no abnormal patterns. Transformation from the parotid ductal epithelium to the tumor cells is evident. Foci of foreign-body reactions against keratin materials were present. Immunoreactivities for cytokeratins reconfirmed the nature of squamous differentiation of the tumor cells. Ki-67-positive cells were confined along the outer basal layer of the tumor epithelium. Tests for alpha-smooth muscle actin and S-100 protein were completely negative. Both patients had no evidence of recurrence 3 and 2 years after subtotal parotidectomy, respectively, without any additional therapy. We believe that this lesion represents a benign cystic neoplasm rather than a malignant tumor or pseudoneoplastic metaplastic condition. It is important to recognize that this peculiar benign tumor does originate from the salivary gland.

Adult↗
PubMed2002

Proteome and transcriptome analysis of the virulence genes regulated by the VirR/VirS system in Clostridium perfringens.

The proteins under the control of the two-component system VirR/VirS in Clostridium perfringens were analyzed by using two-dimensional gel electrophoresis of the culture supernatant from the wild type and the virR mutant. Based on matrix-assisted laser desorption ionization-time of flight/mass spectrometry, seven positively regulated proteins and eight negatively regulated proteins were identified. Transcriptome analysis confirmed that 7 of the 15 proteins were regulated by the VirR/VirS system at the transcriptional level, but the remaining proteins were modified with a VirR/VirS-directed protease at the posttranslation and secretion levels. We purified and characterized the VirR/VirS-directed protease from the culture supernatant and identified it as a kind of clostripain. Because this proteolytic activity was strongly inhibited by leupeptin and antipain, it was concluded that this protease was a member of the family of cysteine proteases of C. perfringens.

Bacterial Proteins↗
PubMedDate not supplied

Mucinous tubular and spindle cell carcinoma of the kidney: Report of a case with imprint cytologic features.

BACKGROUND: Mucinous tubular and spindle cell carcinoma of the kidney is a newly established subtype in the World Health Organization classification. The tumor has a good prognosis, and its diagnosis is clinically important. However, there are no reports of its cytologic features. Here we report the cytologic findings of this rare tumor. CASE: A 68-year-old female had left-sided abdominal pain, fatigue and hematuria. A large mass in the left kidney was removed. Histologically, the tumor was compatible with mucinous tubular and spindle cell carcinoma. Imprint cytologic examination revealed a large amount of tumor cells arranged in tubular, sheetlike and spindle structures. Some tumor cells showed clear cytoplasm. Nuclear atypia was mild, with occasional distinctive nucleoli. CONCLUSION: The cytologic features of mucinous tubular and spindle cell carcinoma have a varied appearance. It should be included in the differential diagnosis to avoid overtreatment.

Adenocarcinoma, Mucinous↗