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Tetsuya Hayashi

Publications and source records attributed to Tetsuya Hayashi.

At least 19 recordsLinked to original sources

Large-scale genome analysis of bovine commensal Escherichia coli reveals that bovine-adapted E. coli lineages are serving as evolutionary sources of the emergence of human intestinal pathogenic strains.

How pathogens evolve their virulence to humans in nature is a scientific issue of great medical and biological importance. Shiga toxin (Stx)-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) are the major foodborne pathogens that can cause hemolytic uremic syndrome and infantile diarrhea, respectively. The locus of enterocyte effacement (LEE)-encoded type 3 secretion system (T3SS) is the major virulence determinant of EPEC and is also possessed by major STEC lineages. Cattle are thought to be the primary reservoir of STEC and EPEC. However, genome sequences of bovine commensal E. coli are limited, and the emerging process of STEC and EPEC is largely unknown. Here, we performed a large-scale genomic comparison of bovine commensal E. coli with human commensal and clinical strains, including EPEC and STEC, at a global level. The analyses identified two distinct lineages, in which bovine and human commensal strains are enriched, respectively, and revealed that STEC and EPEC strains have emerged in multiple sublineages of the bovine-associated lineage. In addition to the bovine-associated lineage-specific genes, including fimbriae, capsule, and nutrition utilization genes, specific virulence gene communities have been accumulated in stx- and LEE-positive strains, respectively, with notable overlaps of community members. Functional associations of these genes probably confer benefits to these E. coli strains in inhabiting and/or adapting to the bovine intestinal environment and drive their evolution to highly virulent human pathogens under the bovine-adapted genetic background. Our data highlight the importance of large-scale genome sequencing of animal strains in the studies of zoonotic pathogens.

Animals↗

TccP2 of O157:H7 and non-O157 enterohemorrhagic Escherichia coli (EHEC): challenging the dogma of EHEC-induced actin polymerization.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 and enteropathogenic E. coli (EPEC) trigger actin polymerization at the site of bacterial adhesion by inducing different signaling pathways. Actin assembly by EPEC requires tyrosine phosphorylation of Tir, which subsequently binds the host adaptor protein Nck. In contrast, Tir(EHEC O157) is not tyrosine phosphorylated and instead of Nck utilizes the bacterially encoded Tir-cytoskeleton coupling protein (TccP)/EspF(U), which mimics the function of Nck. tccP is carried on prophage CP-933U/Sp14 (TccP). Typical isolates of EHEC O157:H7 harbor a pseudo-tccP gene that is carried on prophage CP-933 M/Sp4 (tccP2). Here we report that atypical, beta-glucuronidase-positive and sorbitol-fermenting, strains of EHEC O157 harbor intact tccP and tccP2 genes, both of which are secreted by the LEE-encoded type III secretion system. Non-O157 EHEC strains, including O26, O103, O111, and O145, are typically tccP negative and translocate a Tir protein that encompasses an Nck binding site. Unexpectedly, we found that most clinical non-O157 EHEC isolates carry a functional tccP2 gene that encodes a secreted protein that can complement an EHEC O157:H7 DeltatccP mutant. Using discriminatory, allele-specific PCR, we have demonstrated that over 90% of tccP2-positive non-O157 EHEC strains contain a Tir protein that can be tyrosine phosphorylated. These results suggest that the TccP pathway can be used by both O157 and non-O157 EHEC and that non-O157 EHEC can also trigger actin polymerization via the Nck pathway.

Actins↗

Gender difference of atorvastatin's vasoprotective effect in balloon-injured rat carotid arteries.

The effect of estrogen on neointimal formation in injured rat arteries has been reported to be a sexual dimorphic effect. Recently, it has been reported that 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) exhibit vasoprotective effects, which are independent of their cholesterol-lowering effects. In this study, we examined the gender differences of atorvastatin's effect on neointimal formation in balloon-injured rat arteries. Male and female Sprague Dawley rats underwent gonadectomy and balloon injury of the carotid artery. Ovariectomized female, as well as intact and castrated male, rats exhibited marked neointimal formation. Treatment with atorvastatin significantly reduced neointimal formation at day 14 (14 days after injury) and NADPH oxidase-dependent superoxide production at day 2 in ovariectomy, but not in intact and castrated males. In ovariectomized rats, 7 days of atorvastatin treatment from days -3 to 3 but not from days 7 to 14 suppressed neointimal formation at day 14. In this study, we showed that atorvastatin's effect on neointimal formation was female-specific and was more marked in ovariectomized female rats. NADPH oxidase-dependent superoxide production may be involved in the mechanism of the sexual dimorphic response seen in response to atorvastatin treatment. Furthermore, the results suggest the importance of treatment in the early phase after vascular injury.

Animals↗

An extensive repertoire of type III secretion effectors in Escherichia coli O157 and the role of lambdoid phages in their dissemination.

Several pathogenic strains of Escherichia coli exploit type III secretion to inject "effector proteins" into human cells, which then subvert eukaryotic cell biology to the bacterium's advantage. We have exploited bioinformatics and experimental approaches to establish that the effector repertoire in the Sakai strain of enterohemorrhagic E. coli (EHEC) O157:H7 is much larger than previously thought. Homology searches led to the identification of >60 putative effector genes. Thirteen of these were judged to be likely pseudogenes, whereas 49 were judged to be potentially functional. In total, 39 proteins were confirmed experimentally as effectors: 31 through proteomics and 28 through translocation assays. At the protein level, the EHEC effector sequences fall into >20 families. The largest family, the NleG family, contains 14 members in the Sakai strain alone. EHEC also harbors functional homologs of effectors from plant pathogens (HopPtoH, HopW, AvrA) and from Shigella (OspD, OspE, OspG), and two additional members of the Map/IpgB family. Genes encoding proven or predicted effectors occur in >20 exchangeable effector loci scattered throughout the chromosome. Crucially, the majority of functional effector genes are encoded by nine exchangeable effector loci that lie within lambdoid prophages. Thus, type III secretion in E. coli is linked to a vast phage "metagenome," acting as a crucible for the evolution of pathogenicity.

Bacteriophage lambda↗

Complexity of the genomic diversity in enterohemorrhagic Escherichia coli O157 revealed by the combinational use of the O157 Sakai OligoDNA microarray and the Whole Genome PCR scanning.

Escherichia coli O157, an etiological agent of hemorrhagic colitis and hemolytic uremic syndrome, is one of the leading worldwide public health threats. Genome sequencing of two O157 strains have revealed that the chromosome is comprised of a 4.1 Mb backbone shared by K-12 and a total of 1.4 Mb O157-specific sequences. Most of the large O157-specific sequences are prophages and prophage-like elements, which have carried many virulence genes into the O157 genome. This suggests that bacteriophages have played the key roles in the emergence of O157. The Whole Genome PCR Scanning (WGPScanning) analysis of O157 strains, on the other hand, revealed a high level of genomic diversity in O157. Variation of prophages has also been suggested as a major factor generating such diversity. In this study, we analyzed the gene content of O157 strains, by an oligoDNA microarray, using the same set of strains as examined by the WGPScanning method. Although most of the strains were typical O157 : H7, they differed remarkably in gene composition, particularly in those on prophages, and we identified more than 400 'variably absent or present' genes which included virulence-related genes. This confirms the role of prophages in generating the genomic diversity, and raises a possibility that some level of variation in potential virulence is present among O157 strains. Fine comparison of the two datasets obtained by microarray and WGPScanning provided much further details on the O157 genome diversity than illustrated by each method alone, indicating the usefulness of this combinational approach in the genomic comparison of closely related strains.

Combinatorial Chemistry Techniques↗

ppGpp with DksA controls gene expression in the locus of enterocyte effacement (LEE) pathogenicity island of enterohaemorrhagic Escherichia coli through activation of two virulence regulatory genes.

For a new pathogen to emerge, it must acquire both virulence genes and a system for responding to changes in environmental conditions. Starvation of nutrients or growth arrest induces the stringent response in Escherichia coli, via increased ppGpp. We found the adherence capacity of enterohaemorrhagic E. coli (EHEC) and gene expression in the locus of enterocyte effacement (LEE) were enhanced by a downshift in nutrients or by entry into the stationary growth phase, both of which increase the ppGpp concentration. The activation was dependent on relA and spoT, which encode enzymes for the synthesis and degradation of ppGpp, and on dksA, which encodes an RNA polymerase accessory protein required for the stringent response. Upon induction of RelA expression, LEE gene transcription was activated within 20 min, even without starvation. The expression of two LEE transcriptional regulators, Ler and Pch, was activated by ppGpp and essential for the enhancement of LEE gene expression. In addition, the ler and pch promoters were directly activated by ppGpp in an in vitro transcription system. These findings suggest that the regulation of virulence genes in EHEC is integrated with E. coli's stringent response system, through the regulation of virulence regulatory genes.

Adhesins, Bacterial↗

A case of renal cell carcinoma with multiple lung metastases refractory to interferon-alpha showing complete remission by interleukin-2 monotherapy.

We report a case in which a regimen of interleukin-2 (IL-2) achieved pathologically complete remission against renal cell carcinoma with multiple lung metastases. A 63-year-old man was admitted to the Osaka University Hospital with a right renal tumor and multiple lung metastases. Right radical nephrectomy was performed and the histological diagnosis was clear cell carcinoma, G3 > G2, INFbeta, pT3a, pN0. Postoperatively, despite treatment with interferon-alpha, the lung metastases progressed. Consequently, intravenous administration of IL-2 was started at a dose of 70 x 10(4) JRU/day five times per week. After 16 weeks of IL-2 therapy, most of the multiple lung metastases completely vanished and the largest metastatic lung mass was reduced in size. Resection of this residual lung mass was performed and pathological examination revealed no viable cancer cells.

Adenocarcinoma, Clear Cell↗

Angiotensin-II receptor blocker exerts cardioprotection in diabetic rats exposed to hypoxia.

BACKGROUND: Hypoxia caused by sleep apnea might be associated with an increased risk of cardiovascular events in subjects with metabolic syndrome. The aim of this study was to examine the effect of hypoxia on the left ventricular (LV) myocardium and evaluate the cardioprotective effect of an angiotensin-II receptor blocker (ARB) in diabetic rats. METHODS AND RESULTS: Male Otsuka Long-Evans Tokushima Fatty (OLETF) rats at 30 weeks of age (n=30) were divided into 2 groups that were treated with vehicle or candesartan 0.2 mg x kg(-1) x day (-1). The animals were housed in a hypoxic gas chamber (oxygen, 10.0+/-0.5%, mean +/- standard deviation) for 2 weeks. Hypoxia increased right ventricular (RV) systolic pressure (hypoxia; 78+/-14 mmHg vs control; 22+/-5, p<0.05), but did not increase LV systolic pressure (131+/-23 mmHg vs 121+/-10). Hypoxia exacerbated the degeneration of cardiomyocytes, and accelerated the expression of hypoxia inducible factor-1alpha (HIF-1 alpha) and vascular endothelial growth factor (VEGF) in the myocardium. Treatment with ARB decreased RV and LV pressures (46+/-7 and 100+/-18 mmHg, respectively), suppressed the expression of HIF-1alpha and VEGF, and preserved the fine structure of the LV myocardium. CONCLUSIONS: ARB exhibited cardioprotection under hypoxia, in part through the reduction of blood pressure and cytokine expression, in OLETF rats. Thus, ARB might be a potent agent for the treatment of diabetic patients with the complication of sleep apnea.

Angiotensin II Type 1 Receptor Blockers↗

[Synchronous ipsilateral renal cell carcinoma and transitional cell carcinoma: report of 2 cases].

Two cases of synchronous ipsilateral renal cell carcinoma and renal pelvic transitional cell carcinoma are presented, one in a 70-year-old man and another in a 54-year-old man. These two cases were diagnosed preoperatively as synchronous ipsilateral renal tumor and pelvic tumor from urine cytology, retrograde pyelography, computed tomography and magnetic resonance imaging, and in both two cases, nephroureterectomy was performed. Pathological diagnosis was renal cell carcinoma and renal pelvic transitional cell carcinoma, which existed incidentally in the same kidney. To our knowledge, these cases are the 34th and 35th reported cases of synchronous ipsilateral renal cell carcinoma and renal pelvic transitional cell carcinoma in Japan.

Aged↗

Differential effects of different statins on endothelin-1 gene expression and endothelial NOS phosphorylation in porcine aortic endothelial cells.

It has been reported that 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase inhibitors (statins) produce a variety of cardiovascular protective effects independent of their ability to lower total and low-density lipoprotein cholesterol. Recent studies have also reported that statins produce pleiotropic effects through improved endothelial function, enhanced fibrinolysis, and antithrombotic actions. In the present study, we examined the effects of pitavastatin, pravastatin, atorvastatin, and cerivastatin on endothelin (ET)-1 production in cultured porcine aortic endothelial cells (PAECs). Treatment with cerivastatin but not pitavastatin, pravastatin, or atorvastatin decreased basal and TNF-alpha-stimulated ET-1 release from PAECs in a dose-dependent manner (1-10 microM). Northern blot analysis showed that cerivastatin markedly suppressed prepro ET-1 mRNA expression in both conditions. In addition, these inhibitory effects of cerivastatin on ET-1 release and prepro ET-1 mRNA expression were completely abolished by simultaneous treatment with 200 microM mevalonate. Furthermore, cerivastatin did not have any effects on endothelial nitric oxide synthase (eNOS) protein levels, but induced eNOS phosphorylation at Ser1177. From these findings, it is most likely that cerivastatin suppresses ET-1 production, possibly through an increase in eNOS activity and the subsequent nitric oxide production in PAECs. These findings also suggest that cerivastatin may have beneficial effects on ET-1-related diseases.

Animals↗

The genome sequence of Clostridium botulinum type C neurotoxin-converting phage and the molecular mechanisms of unstable lysogeny.

Botulinum neurotoxins (BoNTXs) produced by Clostridium botulinum are among the most poisonous substances known. Of the seven types of BoNTXs, genes for type C1 and D toxins (BoNTX/C1 and D) are carried by bacteriophages. The gene for exoenzyme C3 also resides on these phages. Here, we present the complete genome sequence of c-st, a representative of BoNTX/C1-converting phages. The genome is a linear double-stranded DNA of 185,682 bp with 404-bp terminal direct repeats, the largest known temperate phage genome. We identified 198 potential protein-coding regions, including the genes for production of BoNTX/C1 and exoenzyme C3. Very exceptionally, as a viable bacteriophage, a number of insertion sequences were found on the c-st genome. By analyzing the molecular structure of the c-st genome in lysogens, we also found that it exists as a circular plasmid prophage. These features account for the unstable lysogeny of BoNTX phages, which has historically been called "pseudolysogeny." The PCR scanning analysis of other BoNTX/C1 and D phages based on the c-st sequence further revealed that BoNTX phages comprise a divergent phage family, probably generated by exchanging genomic segments among BoNTX phages and their relatives.

Bacteriophages↗

Active HSF1 significantly suppresses polyglutamine aggregate formation in cellular and mouse models.

Polyglutamine diseases are inherited neurodegenerative diseases characterized by misfolding and aggregation of proteins possessing expanded polyglutamine repeats. As overexpression of some heat shock protein (Hsp) suppresses polyglutamine aggregates and cell death, it is assumed that combined overexpression of Hsps will suppress that more effectively. Here, we examined the impact of active forms of heat shock transcription factor 1 (HSF1), which induces a set of Hsps, on polyglutamine inclusion formation and disease progression. We found that active HSF1 suppressed polyglutamine inclusion formation more significantly than any combination of Hsps in culture cells, possibly by regulating expression of unknown genes, as well as major Hsps. We crossed R6/2 Huntington disease mice with transgenic mice expressing an active HSF1 (HSF1Tg). Analysis of the skeletal muscle revealed that the polyglutamine inclusion formation and its weight loss were improved in R6/2/HSF1Tg mice. Unexpectedly, the life span of R6/2/HSF1Tg mice was significantly improved, although active HSF1 is not expressed in the brain. These results indicated that active HSF1 has a strong inhibitory effect on polyglutamine aggregate formation in vivo and in vitro.

Adenoviridae↗

Vascular endothelial growth factor-expressing mesenchymal stem cell transplantation for the treatment of acute myocardial infarction.

OBJECTIVE: Vascular endothelial growth factor (VEGF) plays an important role in inducing angiogenesis. Mesenchymal stem cells (MSCs) may have potential for differentiation to several types of cells, including myocytes. We hypothesized that transplantation of VEGF-expressing MSCs could effectively treat acute myocardial infarction (MI) by providing enhanced cardioprotection, followed by angiogenic effects in salvaging ischemic myocardium. METHODS AND RESULTS: The human VEGF165 gene was transfected to cultured MSCs of Lewis rats using an adenoviral vector. Six million VEGF-transfected and LacZ-transfected MSCs (VEGF group), LacZ-transfected MSCs (control group), or serum-free medium only (medium group) were injected into syngeneic rat hearts 1 hour after left coronary artery occlusion. At 1 week after MI, MSCs were detected by X-gal staining in infarcted region. High expression of VEGF was immunostained in the VEGF group. At 28 days after MI, infarct size, left ventricular dimensions, ejection fraction, E wave/A wave ratio and capillary density of the infarcted region were most improved in the VEGF group, compared with the medium group. Immunohistochemically, alpha-smooth muscle actin-positive cells were most increased in the VEGF group. CONCLUSIONS: This combined strategy of cell transplantation with gene therapy could be a useful therapy for the treatment of acute MI.

Actin Cytoskeleton↗

ATP regulation of ciliary beat frequency in rat tracheal and distal airway epithelium.

Ciliary beat frequency (CBF) was measured by video-optical microscopy in rat tracheal and distal airway ciliary cells using a slice preparation. In tracheal ciliary cells (tracheal slice), ATP or 2-methylthio ATP (MeSATP) increased CBF, which was inhibited by suramin (100 microm, an inhibitor of purinergic receptor). Ionomycin (5 microm) or thapsigargin (2 microm) increased CBF similarly. Ca2+-free solution or addition of Ni2+ (1 mm) decreased CBF gradually by approximately 25% and subsequent stimulation with ATP (10 microm) increased CBF transiently. The purinergic agonist experiments demonstrated that ATP increases CBF in tracheal ciliary cells via both P2X and P2Y receptors. ATP increased the intracellular calcium concentration ([Ca2+]i) in tracheal ciliary cells. However, in distal airway ciliary cells (lung slice), ATP did not increase CBF and [Ca2+]i, although a Ca2+-free solution decreased CBF, and ionomycin (5 microm) or thapsigargin (2 microm) increased it. Moreover, acetylcholine (100 microm) did not increase CBF in distal airway ciliary cells, although it increased CBF in tracheal ciliary cells. Terbutaline (10 microm), a selective beta2-adrenergic agonist, increased CBF in both tracheal and distal airway ciliary cells. These observations suggest that the Ca2+-mobilization mechanisms via purinergic or muscarinic receptors of the distal airway ciliary cell may be different from those of the tracheal ciliary cell. In conclusion, the CBF increase is differently regulated in the tracheal and distal airway epithelia of the rat.

Acetylcholine↗

Dual regulatory pathways integrating the RcsC-RcsD-RcsB signalling system control enterohaemorrhagic Escherichia coli pathogenicity.

Bacterial pathogenesis is strictly regulated in response to changes in environmental conditions. A His-Asp phosphorelay system consisting of a sensor kinase and response regulator is used by Gram-positive and Gram-negative bacteria to control gene expression in response to environmental stimuli. We screened His-Asp phosphorelay systems for their effect on virulence expression in enterohaemorrhagic Escherichia coli (EHEC), and found rcsD or rcsB overexpression enhanced locus for enterocyte effacement (LEE) gene transcription and adherence to Caco-2 cells through transcriptional activation of the ler regulatory gene. An EHEC-specific regulator GrvA, encoded by ECs1274, was required for ler transcription activation by RcsB. Furthermore, GrvA activated ler transcription in E. coli K12. Stimulation of the RcsDCB regulatory system by RcsF overexpression slightly increased EspB expression in the wild type but not the ECs1274 mutant. However, EspB expression in an rcsB deletion mutant increased compared with wild type, suggesting that RcsB negatively regulates LEE gene expression and that active RcsB protein is present under normal growth conditions. Deletion of pchA, which encodes a positive regulator for ler, abolished the effect of the rcsB deletion, suggesting that pchA mediated the negative RcsB effect. pchA transcript levels decreased when RcsB expression increased. Thus, LEE gene transcription may be regulated by RcsB through two oppositely regulated O157-specific regulators, PchA and GrvA.

Adaptation, Physiological↗

Selective endothelin ET(B) receptor antagonist improves left ventricular function but exaggerates degeneration of cardiomyocytes in J2N-k hamsters.

BACKGROUND: Endothelin-1 (ET-1) receptor antagonist is expected to improve the prognosis of patients with heart failure, but the role of the ET(B) receptor in cardiac function and structure is complicated. In the present study the NADPH diaphorase activity and ET-1 content in the failing heart treated with ET(A) or ET(B) receptor antagonist were evaluated in a model of dilated cardiomyopathy. METHODS AND RESULTS: Selective ET(A) receptor antagonist, ABT-627 (10 mg/kg per day), or selective ET(B) antagonist, A-192621 (30 mg/kg per day), was given to 22-week-old J2N-k cardiomyopathic hamsters for 8 weeks. The effects of ABT-627 and A-192621 on cardiac function, left ventricular (LV) histology, ET-1 content and NADPH diaphorase activity in the LV were evaluated. Treatment with ABT-627, but not A-192621, significantly decreased ET-1 content and NADPH diaphorase activity. Although the improvement of LV function was modest, ABT-627 prevented tissue damage in J2N-k hamsters. In contrast, A-192621 worsened the degeneration of cardiomyocytes despite improving hemodynamic parameters. CONCLUSIONS: Selective ET(A) antagonist, but not ET(B) antagonist, reduced the ET-1 content as well as the NADPH diaphorase activity, and preserved the fine structure of LV myocardium in cardiomyopathic hamsters. Long-term blockade of ET(B) receptor might worsen the degeneration of cardiomyocytes through the ET-1/ET(A) system even if LV function could be improved.

Animals↗

Mechanisms of combined treatment with celiprolol and candesartan for ventricular remodeling in experimental heart failure.

BACKGROUND: Both beta-adrenergic blockers and angiotensin-II receptor blockers were reported to improve the prognosis of patients with heart failure, but the efficacy of combination therapy with these agents has not been fully elucidated. Also the efficacy of celiprolol, a beta1-selective adrenoceptor antagonist with partial beta2-agonist properties, for heart failure treatment is still controversial. We examined the cardioprotective effects and mechanisms of the therapy with celiprolol or candesartan, an angiotensin-II receptor blockers and their combination in heart failure induced by isoproterenol (ISO). METHODS AND RESULTS: ISO 300 mg/kg was injected in rats to produce heart failure. Two months after the injection, the ISO-injected rats were divided into 4 groups (8 rats each) and treated for 4 weeks as follows: (a) vehicle; (b) celiprolol 10 mg/kg per day (BB); (c) candesartan 0.2 mg/kg per day (ARB); and (d) their combination BB+ARB. ISO significantly elevated left ventricular (LV) end-diastolic pressure, decreased peak-negative dP/dt and LV ejection fraction. BB and ARB similarly ameliorated cardiac dysfunction due to ISO, but BB+ARB were more potent than the individual therapies. Separately, ARB preserved the histological structure in LV myocardium. In contrast, BB ameliorated calcium handling, as shown by the increased ratio of SERCA2 to phospholamban protein, despite having little effect on the histology. CONCLUSION: Both celiprolol and candesartan showed cardioprotective effects in this heart failure model. The potential use of the combination treatment in heart failure might result in a synergistic effect through the different cardioprotective mechanisms of celiprolol and candesartan.

Animals↗