Search PubMed⌕ Search

Biomedical subjects

M Watarai

Publications and source records attributed to M Watarai.

33 records · Page 2Linked to original sources
PubMed1996

Interaction of Ipa proteins of Shigella flexneri with alpha5beta1 integrin promotes entry of the bacteria into mammalian cells.

Shigella is a genus of highly adapted bacterial pathogens that cause bacillary dysentery in humans. Bacteria reaching the colon invade intestinal epithelial cells by a process of bacterial-directed endocytosis mediated by the Ipa proteins: IpaB, IpaC, and IpaD of Shigella. The invasion of epithelial cells is thought to be a receptor-mediated phenomenon, although the cellular components of the host that interact with the Ipa proteins have not yet been identified. We report here that in a Shigella flexneri invasive system and Chinese hamster ovary (CHO) cell monolayers, the Ipa proteins were capable of interacting directly with alpha5beta1 integrin. The invasive capacity of S. flexneri for CHO cells increased as levels of alpha5beta1 integrin were elevated. When CHO cells were infected with S. flexneri, the tyrosine phosphorylation both of pp 125FAK, an integrin-regulated 125 K focal adhesion kinase, and of paxillin was stimulated. In contrast, an isogenic strain of S. flexneri that was defective in invasion owing to a mutation in its spa32 gene failed to induce such phosphorylation. Under in vitro and in vivo conditions, the released IpaB, IpaC, and IpaD proteins bound to alpha 5 beta 1 integrin in a manner different from that of soluble fibronectin but similar to that of the tissue form of fibronectin. At the site of attachment of S. flexneri to CHO cells, alpha5beta1 integrin converged with polymerization of actin. These data thus suggest that the capacity of Ipa proteins to interact with alpha5beta1 integrin may be an important Shigella factor in triggering the reorganization of actin cytoskeletons.

Adhesins, Bacterial↗
PubMed1995

Contact of Shigella with host cells triggers release of Ipa invasins and is an essential function of invasiveness.

The invasion of colonic epithelial cells by Shigella, an early essential step for causing bacillary dysentery, is mediated by the IpaB, IpaC and IpaD proteins. Secretion of the Ipa proteins from Shigella requires functions encoded by the mxi and spa loci. In this study, we show that contact between the bacteria and epithelial cell triggers release of the Ipa proteins into the external medium, which results in a rapid decrease in levels of Ipa proteins presented on the cell surface. When the bacteria were used to infect polarized Caco-2 cells, release of Ipa proteins occurred efficiently from bacteria interacting with the basolateral surface rather than with the apical surface. Moreover, the interaction of bacteria with components of the extracellular matrix, such as fibronectin, laminin or collagen type IV, also stimulates the release of Ipa proteins. The release of Ipa proteins from Shigella required the surface-located Spa32 protein encoded by one of the spa genes on the large plasmid.

Adhesins, Bacterial↗
PubMed1995

Disulfide oxidoreductase activity of Shigella flexneri is required for release of Ipa proteins and invasion of epithelial cells.

Secretion of IpaB, IpaC, and IpaD proteins of Shigella flexneri, essential for the invasion of epithelial cells, requires a number of proteins encoded by the spa and mxi loci on the large plasmid. Introduction of dsbA::Tn5 into S.flexneri from Escherichia coli K-12 reduced invasiveness, which resulted from a decrease in the capacity to release IpaB, IpaC, and IpaD proteins into the external medium. Examination of the surface-presented Ipa proteins of the dsbA mutant, however, revealed Ipa proteins at levels similar to those on wild-type cells. Since the defective phenotype was similar to that of the spa32 mutant of S. flexneri and the Spa32 sequence possessed two Cys residues, the effect of dsbA mutation of the folding structure of Spa32 under reducing conditions and on the surface expression of Spa32 was investigated. The results indicated that Spa32 was a disulfide-containing protein whose correctly folded structure was required for its presentation on the outer membrane. Indeed, replacing either one of the two Cys residues in Spa32 with Ser by site-directed mutagenesis reduced its capacity to release Ipa proteins into the external medium and led to the accumulation of Spa32 protein in the periplasm. These results indicated that the DsbA protein performs an essential function during the invasion of mammalian cells, by facilitating transport of the Spa32 protein across the outer membrane.

Animals↗
PubMed1995

Identification of a novel virulence gene, virA, on the large plasmid of Shigella, involved in invasion and intercellular spreading.

A novel virulence gene (virA) was identified upstream of the virG gene on the large plasmid of Shigella flexneri 2a YSH6000. Characterization of virA mutants infecting MK2 epithelial cell monolayers revealed that their invasive capacity was decreased to less than one fifth of the wild-type level. Nevertheless, the bacteria were capable of expressing and secreting IpaB, IpaC and IpaD proteins. The virA mutants were also impaired in their ability to spread intercellularly, since the bacteria gave rise to a small number of foci in a focus-plaque-forming test with MK2 cells. Although virG expression was slightly decreased in the virA mutants, introduction of a cloned virG gene into a virA mutant, N1945, failed to restore spreading ability. Although, introduction of a cloned virA gene into N1945 restored invasiveness and spreading ability, the reduced virG transcription level was not affected, indicating that the reduced virG expression in virA mutants does not play a major role in defective intercellular spreading. The nucleotide sequence of the virA region revealed that the virA gene was located 528 bp upstream of the virG gene, in the opposite orientation. The deduced amino acid sequence of the VirA protein indicated a 44.7 kDa protein with no homology to known proteins. The VirA protein was secreted into the culture supernatant, a process that required the Mxi and Spa loci. The expression of virA was under the control of the virB gene, the positive regulator of the ipa, mxi and spa operons. These results indicate that virA is a new member of the invasion regulon directed by virB and that the VirA function is involved in invasion and intercellular spreading.

Amino Acid Sequence↗
PubMed1994

Long-term follow-up of coronary narrowing with spasm.

In order to clarify the fate of coronary narrowing with spasm, repeat angiograms of coronary narrowing with and without spasm were compared. The mean interval between the first and second angiograms was 3.6 years (range, 1.1-8.5 years). Improvement of narrowing was more frequent in the vasospastic group (23%) than in the group without spasm (3%, P < 0.005). The cause of this improvement in the vasospastic group may have been the resolution of the spasm in the first angiogram, but the presence of intravascular thrombus or bleeding or edema of the coronary arterial wall may have resolved in the second angiogram.

Angina Pectoris, Variant↗
PubMed1994

vacC, a virulence-associated chromosomal locus of Shigella flexneri, is homologous to tgt, a gene encoding tRNA-guanine transglycosylase (Tgt) of Escherichia coli K-12.

The genetic determinants required for invasion of epithelial cells by Shigella flexneri and for the subsequent bacterial spreading are encoded by the large virulence plasmid. Expression of the virulence genes is under the control of various genes on the large plasmid as well as on the chromosome. We previously identified one of the virulence-associated loci near phoBR in the NotI-C fragment of the chromosome of S. flexneri 2a YSH6000 and designated the locus vacC. The vacC mutant showed decreased levels of IpaC, and IpaD proteins as well as transcription of ipa, an operon essential for bacterial invasion (N. Okada, C. Sasakawa, T. Tobe, M. Yamada, S. Nagai, K. A. Talukder, K. Komatsu, S. Kanegasaki, and M. Yoshikawa, Mol. Microbiol. 5:187-195, 1991). To elucidate the molecular nature of the vacC locus, we cloned the vacC region from YSH6000 on a 1.8-kb SalI-BamHI DNA fragment. The nucleotide sequence of the 1,822-bp vacC clone was highly (> 98%) homologous to the tgt region of Escherichia coli K-12, which is located at 9.3 min on the linkage map. Complementation tests indicated that the vacC function was encoded by an open reading frame expressing a 42.5-kDa protein, which corresponded to the tgt gene of E. coli K-12, coding for tRNA-guanine transglycosylase (Tgt) (K. Reuter, R. Slany, F. Ullrich, and H. Kersten, J. Bacteriol. 173:2256-2264, 1991). The cloned tgt gene from E. coli K-12 restored the virulence phenotype to the vacC mutant of YSH6000. Characterization of the vacC mutant indicated that levels of VirG, a protein essential for bacterial spreading, and VirF, the positive regulator for the expression of the virG and ipaBCD operons, decreased significantly compared with those of the wild type. Similar phenotypic changes occurred in vacC mutants constructed by insertion of a neomycin resistance gene in shigellae and enteroinvasive E. coli strains, consistent with the hypothesis that the vacC (tgt) gene contributes to the pathogenicity of Shigella flexneri.

Amino Acid Sequence↗
PubMed1993

Comparative study of QRST values from body surface potential mapping, 12-lead ECGs, VCGs in detecting inferior myocardial infarction, and evaluating the severity of left ventricular wall motion abnormalities in simulated left bundle branch block.

The authors compared the ability of QRST time-integral values (QRST values) from body surface potential maps (BSPM), 12-lead electrocardiograms (ECGs), and Frank lead vectorcardiograms (VCGs) in diagnosing a prior inferior myocardial infarction (MI) in simulated left bundle branch block (LBBB). The study included 32 patients whose digitized ECGs were recorded simultaneously for BSPM, ECGs, and VCGs during normal sinus rhythm and during right ventricular pacing simulating LBBB (18 with and 14 without an inferior MI). QRST values were calculated in each lead point of ECGs. Data on 608 normal subjects were used as controls; mean +/- 2 SD was regarded as the normal range. The following parameters were derived: sigma DM, sigma DE, sigma DV, the sum of the differences between the normal mean QRST value, and the QRST value of a given patient in leads where the QRST value was less than the normal range ("-2 SD area") in BSPM, ECGs, and VCGs (Y lead). The correlation coefficients for sigma DM, sigma DE, and sigma DV between the two activation sequences were highly significant. Sensitivity and specificity were as follows: 89% and 93% for sigma DM > 100 mV.ms, 89% and 93% for sigma DE > 50 mV.ms, and 56% and 100% for sigma DV > 10 mV.ms, respectively. Although sigma DM, sigma DE, and sigma DV were significantly (P < .01) correlated with the asynergy index calculated from left ventriculograms, sigma DM showed the best correlation. QRST values from BSPM, ECGs, and VCGs provide information that is useful in detecting an inferior MI and in estimating the severity of left ventricular wall motion abnormalities in the setting of LBBB. Of the three parameters, BSPM showed the best correlation with the severity of left ventricular wall motion abnormalities.

Aged↗
PubMed1993

Effects of right ventricular pacing on QRST isointegral maps in patients with and without myocardial infarction: body surface distribution of significant changes in QRST area compared to supraventricular complex.

To assess the effects of right ventricular (RV) pacing on body surface QRST distributions, we recorded QRST isointegral maps (I-maps) during sinus rhythm and RV pacing in 25 patients with anterior myocardial infarction (MI), 19 with inferior MI, and 14 without MI. The QRST values at each lead point recorded during sinus rhythm and RV pacing with an 87-lead system were analyzed with a paired t-test in each patient. An abnormal decrease in the QRST value of the I-map was assessed by the difference map, which indicated a "-2SD area," where the QRST integral value was less than the normal range (mean - 2SD) calculated from 608 normal individuals. The I-maps were similar during the two activation sequences in patients with and without MI. However, during RV pacing, QRST values significantly decreased over the upper right anterior chest and increased over the lower left anterior chest and back. The sigma DMs (sum of QRST integral values below the normal range) for both activation sequences were strongly correlated in patients with anterior MI and with inferior MI (r = 0.91 and r = 0.92, respectively; P < 0.001). Although small but significant changes in QRST values were detected, the distribution of the "-2SD area" and the sigma DM were similar during both activation sequences in patients with prior MI. Thus, these findings demonstrate that an altered activation sequence produces small but significant changes in QRST values but that I-maps still provide information that is useful for the diagnosis of MI during RV pacing.

Adult↗
PubMed1993

Comparison of etiological and immunological characteristics of two attenuated Erysipelothrix rhusiopathiae strains of serotypes 1a and 2.

Two acriflavine-fast attenuated Erysipelothrix rhusiopathiae strains Koganei 65-0.15 of serotype 1a (strain Kg-1a) and 2 (strain Kg-2) were comparatively characterized. Biochemical characterization showed the similar reactions with slight variation between the strains. Strain Kg-2 was more resistant to acriflavine dye than strain Kg-1a. Pathogenicity of strain Kg-2 was higher than strain Kg-1a in mice of strains ddY. C3H/He and A/J. Significant differences of clinical signs between strains Kg-1a and Kg-2 were observed in occurrence of arthritis (P < 0.05) and systemic signs (P < 0.01) of only ddY mice. C3H/He mice was more resistant than ddY and A/J mice to the infection of strains Kg-1a and Kg-2. Three culture fractions, whole culture: WC, culture filtrate: CF and killed cells: KC, of strain Kg-2 were more protective than those of strain Kg-1a in ddY mice. CF of strain Kg-2 was most protective in all fractions. Heating at 56 degrees C and 100 degrees C or treatment with trypsin completely reduced the protective activity of WC of the two strains, indicating that major protective antigens of WC were protein. The present results demonstrated that immunogenicity and pathogenicity for mice were different between the two attenuated strains.

Acriflavine↗
PubMed1992

Long-term study of recurrent vasospastic angina using coronary angiograms during ergonovine provocation tests.

Chronologic changes of coronary spasm were examined by repeated ergonovine provocation tests during angiography. A total of 322 patients who had variant angina without severe atherosclerosis demonstrated a positive response to the first test. Ninety of these patients had recurrent variant anginal symptoms after an angina-free period of 38 +/- 12 months (mean +/- SD). Of these 90 patients, 76 (84%) had symptoms or electrocardiographic (ECG) findings similar to those of the first test. The initial 9 of these 76 patients underwent a second provocation test and showed coronary responses analogous to those on the first test. Of the 90 patients, 14 (16%) had different symptoms or ECG findings from those elicited at the first episode. All 14 patients again had a positive response to a second ergonovine test and the following angiographic changes were observed in the three major vessels between the two tests. Of the 21 vessels that had spasm on the first test, eight vessels (19%) did not have spasm on the second test. Of the 21 vessels that did not demonstrate spasm on the first test, 10 (24%) demonstrated spasm on the second test. In the present study it is concluded that the majority of patients with recurrent angina seemed to have consistency in the location of coronary spasm, while in some patients the fluctuation of coronary spasm was confirmed by two ergonovine provocation tests.

Adult↗
PubMed1992

Long-term changes of infarct-related lesions. Comparison of angiograms in recent and remote phases.

Coronary angiograms recorded in the recent phase of an acute myocardial infarction in 73 patients were compared with those taken in the remote phase at least 12 months from the onset of the infarction. The infarct-related lesions of 23 patients (32%) showed a regression of 20% or more. Analysis of recent-phase angiograms proved that any one of several features (long-segment narrowing, intraluminal thrombus, flap-like structure or atheromatous ulceration) was present more frequently in the lesions showing a marked regression (18/23, 78%) than in the lesions without regression (15/40, 38%) (p less than 0.01). Thus, the narrowings of infarct-related lesions seen on the recent-phase angiograms regress frequently. The possibility of regression can be predicted from the angiographic features.

Coronary Angiography↗
PubMed1992

Clinical significance of residual collaterals immediately after successful coronary angioplasty.

The clinical significance of collaterals visible on angiography immediately after successful percutaneous transluminal coronary angioplasty (PTCA) was analyzed in 221 patients who underwent successful PTCA for coronary arteries receiving collaterals. Filling of the collaterals was classified as good; filling the entire epicardial segment of the stenosed site, fair; partially filling the epicardial segments distal to the stenosed site, and faint; visible but not filling the epicardial segments of the diseased vessel. Fifteen of 41 good collaterals remained good or fair on angiography immediately after PTCA. Among the 114 fair collaterals, 26 remained fair and 20 of 66 faint collaterals remained visible on the angiogram immediately after PTCA. There was no relationship between the degree of residual stenosis after PTCA and the degree of residual collaterals. Repeat coronary angiography was obtained in 156 patients. There was no correlation between the presence, absence or degree of collaterals observed on angiography immediately after successful PTCA and the rate of restenosis. Thus, collaterals to the vessels dilated by PTCA often remain on the angiogram immediately after PTCA and are dependent primarily on their degree before dilation. They do not indicate inadequate dilation or predict restenosis.

Aged↗
PubMed1991

[Ecological behavior of plasmids and resistance to lead of Enterobacter agglomerans isolated from soil].

Soil samples taken monthly in 1990 from five parks outside Tokyo were examined for Enterobacter agglomerans. A total of 348 strains were isolated and 250 of them were tested for the presence of plasmids by DNA agarose gel electrophoresis. All the isolates carried at least two kinds of plasmids. Those isolated from July to September showed four or five kinds of plasmids (group I) and those isolated from January to June and from October to December showed two or three kinds of plasmids (group II). A majority of the plasmids detected were of 1,500 or fewer base pairs. The isolates were tested for the Pb2+ resistance; group I strains were more resistant to lead than group II strains. It is presumed that bacterial plasmids are related with the ecosystem of soil and the resistance to lead in E. agglomerans.

Drug Resistance, Microbial↗
PubMed1989

Vectorcardiographic criteria for diagnosis of high lateral infarction--supplement for Chou's criteria.

The concept of high lateral myocardial infarction (HLMI) has not been clearly defined, so criteria for its vectorcardiographic (VCG) diagnosis have had no firm basis. However, we have reported that HLMI, expressed as abnormal Q waves in lead aVL on the electrocardiogram, corresponds to necrosis of the area usually supplied by the diagonal branches of the left anterior descending coronary artery. Here, we evaluated conventional VCG criteria for the diagnosis of HLMI on the basis of angiographic findings, and selected the criteria of Chou as typical. The frontal plane VCG was analyzed in 46 patients with HLMI on angiograms; other subjects were 233 healthy controls and 194 patients with left ventricular hypertrophy. Chou's criteria had good specificity, but sensitivity was unsatisfactory (32/46, or 70%). Accordingly, we examined various parameters of the QRS and T loops and found that the addition of four new criteria to Chou's improved sensitivity (46/46, or 100%) without impairment of specificity. The additional criteria are; 1) initial counterclockwise rotation of the QRS loop, 2) the ratio of the maximal QRS magnitude to the maximal T magnitude less than 4.5, 3) direction of the maximal T between +60 degrees and +180 degrees, 4) the QRS-T angle between 40 degrees and 135 degrees, all in the frontal plane.

Adult↗