Biomedical subjects
Publications and source records attributed to A Okabe.
The mscL gene, which encodes the protein forming a large-conductance mechanosensitive channel (MscL) in Escherichia coli, has previously been cloned and sequenced by Sukharev et al. [Nature 368 (1994) 265-268]. We found a gene homologous to mscL in Clostridium perfringens which is located just downstream from the collagenase-encoding gene in the opposite direction.
The genesis and the significance of pacemaker-induced T-wave changes remain unclear. Changes in body surface potential mapping (BSM) were observed and compared with resting thallium-201 myocardial scintigraphy (T1-SC) findings before, during and after ventricular pacing (VP) in 10 patients with various bradyarrhythmias. All studies were performed with the patients taking no medication. In all patients, isoarea QRST maps showed a characteristic abnormal dipolar pattern with positive values distributed over the upper chest and negative values over the lower chest during VP at a physiological rate for 14 days or more. These abnormalities were preserved almost completely after pacing was terminated; and coincided with deep T-wave inversions in leads II, III, aVF and V4-6. In three patients, BSM performed before VP showed normal QRST isoarea maps with positive values distributed over the left lower chest. All patients in whom resting T1-SC was performed during chronic VP showed transient perfusion defects in the posteroinferior (seven cases) or inferolateral (one case) left ventricular wall. In three patients, T1-SC was performed before VP and showed a normal distribution. Both the pacing-induced perfusion defects and the T-wave abnormalities remained unchanged 2 h after ceasing VP, were attenuated 7 days later and disappeared within a month. These findings suggest that chronic ventricular pacing may produce myocardial ischaemia, and that it persists for a certain period after the cessation of pacing, resulting in post-pacing T-wave inversion.
A low-molecular-mass cytotoxin produced by Klebsiella oxytoca isolated previously from patients with antibiotic-associated haemorrhagic enterocolitis was purified, and its biological and chemical properties were elucidated. The toxin inhibited the syntheses of DNA and RNA by HEp-2 cells dose-dependently, whereas protein synthesis was only slightly inhibited, as measured by the incorporation of radioactive precursors. When synchronously cultured HEp-2 cells were examined in the presence of cytotoxin, inhibition of DNA synthesis occurred promptly within 5 h, but cell-rounding, the earliest visible morphological change, was not observed until 6 h after exposure. The intracellular levels of ATP decreased with an approximately similar time course. These results suggest that cytotoxicity toward HEp-2 cells is primarily due to the inhibitory effect of the cytotoxin on nucleic acid synthesis, possibly on DNA synthesis. Cell rounding and cell death were induced even in the absence of the cytotoxin after incubation with the cytotoxin for 6 h. The cytotoxin was heat-labile, cytotoxic activity decreasing to 50% of the initial level on heating at 70 degrees C for 20 min. Plasmids were extracted from three strains of K. oxytoca producing the cytotoxin and analysed by agarose gel electrophoresis. Two strains possessed plasmids of different sizes, but one strain possessed no plasmid, indicating that the cytotoxin is probably chromosomally encoded. Analysis by NMR and FAB-mass-spectrometry revealed that the molecular mass of the cytotoxin should be 217.1062 Da (exact mass), its molecular formula being C8H15O4N3.
The phospholipase C (alpha-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 were 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.
Adherence to a HEp-2 cell monolayer was tested for in four strains of Salmonella derby which were isolated from patients with diarrhea. One strain, SB1, was highly adherent and another strain, SB4, was nonadherent. The other two strains exhibited moderate adherence. Further in vitro study of invasion of HEp-2 cells by S. derby and its replication in murine peritoneal macrophages was carried out using SB1 and SB4. Thin section electron micrographs revealed that SB1 invaded HEp-2 cells but SB4 did not. The number of viable bacteria within macrophages was determined at intervals after inoculation of bacteria. The result indicates that SB1 can replicate in the macrophages but SB4 cannot. Flagella and fimbriae were compared by electron microscopy between SB1 and SB4, and their lipopolysaccharides and outer membrane proteins were also compared with each other by SDS-polyacrylamide gel electrophoresis. The presence of a 41 kDa protein in the outer membranes of SB1 was only the difference detected, suggesting that this protein could be a factor required for adherence of this serovar to epithelial cells.
The structural gene for perfringolysin O (pfoA), a thiol-activated hemolysin of Clostridium perfringens, was cloned into Escherichia coli JM109 on a 4.6-kilobase (kb) EcoRI-NdeI fragment which contained the 1.7-kb pfoA gene and an upstream 2.9-kb region. An E. coli strain transformed by this plasmid produced 20-fold more perfringolysin O than a strain containing only the 1.7-kb pfoA gene. The stimulatory effect of the upstream region on in vivo expression of the pfoA gene was further analyzed by using a set of deletion mutants. Stimulation was still observed with a 3.9-kb fragment, but stimulation was not observed with fragments that were 3.6 kb or less long, indicating that the upstream region between 3.9 and 1.7 kb was involved in activation of pfoA gene expression. Nucleotide sequencing showed that this region contained one open reading frame (pfoR) coding for 343 amino acids. The deduced amino acid sequence of pfoR possesses several motifs that are characteristic of DNA-binding proteins. When a region coding for a helix-turn-helix, one of the most important motifs of DNA-binding proteins, was deleted within pfoR, stimulation was completely abolished. These results indicate that pfoR positively controls expression of the pfoA gene.
The hepatitis B virus surface antigen containing the preS2 nine amino acid sequence produced by a recombinant Saccharomyces cerevisiae (yHBsAg) was purified and its physicochemical properties were determined. Ultrastructurally, the yHBsAg was found to be a homogeneous spherical particle with a diameter of 24 +/- 4 nm. The homogeneity of the yHBsAg particles was also demonstrated by analyses of their buoyant density and isoelectric point. They consisted of protein (53%), lipid (36%) and carbohydrate (11%), and the alpha-helix content was estimated to be 32%, differing from the reported values for human plasma-derived HBsAg (hHBsAg). Immunodiffusion analysis showed that the antigenic specificity of yHBsAg was identical to that of hHBsAg. Immunization of mice demonstrated that the immunogenicity of the yHBsAg was significantly higher than that of hHBsAg.
We constructed a plasmid, pBH103-ME5, in which the region encoding the 10 preS2 amino acid residues and the S domain of the hepatitis B surface antigen (HBsAg) were regulated by the promoter of the yeast repressible acid phosphatase gene. Saccharomyces cerevisiae carrying pBH103-ME5 produced the HBs antigen (yHBsAg), when it was cultured in a medium containing a low concentration of phosphate. The antigen was purified to homogeneity. Its molecular weight was determined by Western blotting to be 24,000, and its amino acid composition agreed well with that deduced from the nucleotide sequence. The C-terminal amino acid sequence of yHBsAg was exactly the same as that predicted from the nucleotide sequence, while the N-terminal amino acid acetylserine, which was followed by 8 amino acid residues coded by the preS2 region. These results indicate that the recombinant yeast produced a single polypeptide consisting of the preS2 region and the subsequent S domain after being processed at the N-terminus.
Monoclonal antibodies (McAbs) against a part of v-myb gene product were prepared for the detection of human c-myb gene product (p75c-myb). Western blotting analyses with these McAbs were performed on human leukemia-lymphoma cells. All T-cell lines were positive in p75c-myb expression. B-cell lines were variable, myeloid and erythroid cells were positive although the amount of expressed p75c-myb was less than the T-cell lines. Cells isolated from patients were positive in expression except for cells from acute myeloblastic leukemia with maturation (AML M2), acute hypergranular promyelocytic leukemia (AML M3) and erythroleukemia (AML M6) developed from myelodysplastic syndromes. Differences in p75c-myb expression seemed to depend upon the differentiation stage and distinctive lineage from which each cell line had been established. The p75c-myb expression in HL60 (acute promyelocytic leukemia cell line) showed remarkably high at logarithmic growth. When examined with HL60, p75c-myb expression significantly decreased during the differentiation induced by 12-O-tetradecanoylphorbol-13-acetate or retinoic acid. These results suggest that p75c-myb expression plays a crucial role in hematopoietic cell proliferation and differentiation and that multiple mechanisms including aberrant expression of p75c-myb is involved in leukemogenesis.
Recombinant plasmids containing v-myb' (803 bp fragment of the 3' end of v-myb) were constructed to induce sense or antisense v-myb' RNA expression with dexamethasone in human cells. These plasmids were used as a tool for the investigation of the role of c-myb gene in human leukemia cell proliferation and differentiation. They were transfected by electroporation into the K562 human leukemia cell line derived from a patient with chronic myelogenous leukemia in blastic crisis. After induction of transcription by dexamethasone, the plasmid with antisense v-myb' repressed the expression of p75c-myb from the endogenous c-myb gene of K562 cells. It also reduced the proliferation rate of K562 cells to 50% of the control level, and induced these K562 cells to express the myelomonocytic differentiation cell surface marker CD13 and increased NBT reducing activity. The plasmid with sense v-myb' did not have an effect on p75c-myb expression, the proliferation of K562 cells or the expression of myelomonocytic differentiation phenotypes. These observations suggest that antisense v-myb' RNA represses p75c-myb expression and that a decrease of p75c-myb suppresses K562 cell proliferation and induces its differentiation towards the myelomonocytic lineage.
The production of heat-labile enterotoxin (LT) in 76 strains of human enterotoxigenic Escherichia coli (ETEC) varied by a factor of 100. Three ETEC strains that differ in the levels of LT production were chosen for the cloning of LT genes (toxAB) into plasmid pBR322, and the gene structure and expression were compared in E. coli HB101. The recombinant of the low LT-producing strain produced LT at the same level as that of the moderate LT-producing strain, but that of the high-level producer continued to produce at a level 14-21 times higher than the others. The restriction maps of the coding regions of the cloned LT genes (toxAB) were identical, but the flanking regions were dissimilar. The content of LT mRNA per cell, examined by Northern blot analysis, was higher in the high producer than the others by 6 times. The promoter strengths of the recombinants were all alike. LT mRNA of the high producer was more stable than that of the moderate one by 1.3 times, but the difference was not large enough to explain the difference of the content of LT mRNA. It was shown that LT production can be controlled at a transcriptional step, and DNA structure of the flanking regions may be involved in the control of the LT gene expression.
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The gene encoding phospholipase C (alpha-toxin) of Clostridium perfringens was cloned into lambda gt10. The maximal size of the coding region was 1.4 kb and the minimum was 1.1 kb as determined by subcloning into the vector pBR322 and testing for activity. The nucleotide sequence of this region contained a single open reading frame of 1194 bp corresponding to a protein of Mr 45473 with a possible N-terminal signal sequence of 28 amino acids which when removed, would give a mature protein of Mr 42521. This is in good agreement with the reported size of 43 kDa. The coding region has a dG + dC content of 33.7%, and the codon usage displays a pronounced preference for codons with the lowest dG + dC content.
Certain strains of Klebsiella oxytoca isolated from patients with hemorrhagic enterocolitis produced a unique cytotoxin. The cytotoxin induced rounding of tissue culture cells, such as HEp-2, Vero, CHO and HeLa cells. The induced morphologic changes were indistinguishable between cell types. Seventy to 80% of the rounded cells died in 48 h incubation. The cytotoxin was purified 1000-fold from culture supernatant by Sephadex G-25 and Bio-Gel P-2 gel filtration followed by reversed-phase high-performance liquid chromatography. The molecular weight of the purified cytotoxin was estimated to be less than or equal to 651 by mass spectrometry. The minimum concentrations of the purified cytotoxin required to cause 50% of rounding of cells were 0.6 micrograms/ml for HEp-2, 0.8 micrograms/ml for Vero, 0.8 micrograms/ml for CHO and 1.4 micrograms/ml for HeLa cells. The type strain of K. oxytoca, ATCC 13182, did not produce the cytotoxin and only the clinically isolated strains did, suggesting that the cytotoxin may play a role in the pathogenesis of the organisms.
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.
Sub-inhibitory concentrations of lincomycin slightly inhibit growth of Escherichia coli carrying plasmid RP4 and cause a 2-fold increase in TEM-2 beta-lactamase. To analyze this effect, cultures were pulse-labeled with [3H]leucine, chased with non-radioactive leucine and immunoprecipitated with anti-beta-lactamase antiserum. The synthesis rate of beta-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 beta-lactamase. The rate of maturation of pre-beta-lactamase was determined by measuring the decrease in the amount of pre-beta-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 beta-lactamase increased in the presence of lincomycin. The effects of other protein synthesis inhibitors on the synthesis of TEM-1 beta-lactamase were examined. The stimulation of beta-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.
To detect significant coronary lesions based on exercise-induced reversible asynergy, two-dimensional echocardiograms were recorded before and immediately after treadmill exercise test in 15 patients with angina pectoris (AP) and six patients with neurocirculatory asthenia (NCA). Short-axis views of the left ventricle were analyzed quantitatively, using the following indices: segmental area change (%A); segmental wall thickness change (%Th); and relative curvature (rC) which was the product of curvature of each segment multiplied by end-diastolic circumference. The results were compared with those of coronary angiography, T1-201 myocardial emission computed tomography, and exercise electrocardiography. 1. The results of comparisons of quantitative analysis by %A, %Th, and rC with qualitative (visual) analysis were as follows: the sensitivities were 57%, 74% and 91%; the specificities were 75%, 75% and 90%; and the accuracies were 68%, 75% and 90%, respectively. 2. To detect coronary stenosis of more than 75%, visual assessments and assessments by rC were superior to assessments by %Th or %A. The accuracies were 93%, 91%, 73% and 61%, respectively. 3. In five cases with three-vessel disease, all diseased coronary arteries were detected by treadmill exercise echocardiography using rC as an index. However, by treadmill exercise T1-201 myocardial emission computed tomography, the diagnosis of three-vessel disease was possible in only one case. 4. The results of treadmill exercise electrocardiography were positive in 11 of 15 the AP patients and in all six NCA patients. The results of exercise echocardiography using rC as an index were normal in all NCA patients and abnormal in 14 of the 15 AP patients.(ABSTRACT TRUNCATED AT 250 WORDS)
We established a new method for detecting enteropathogenic Escherichia coli adhering to HEp-2 cells. An essential part of the method is an assay of beta-galactosidase activity of adhered bacterial cells. It consisted of the following steps: (1) culture of bacterial cells in a medium containing isopropyl-thio-beta-D-galactoside, an inducer of beta-galactosidase; (2) incubation of a bacterial culture with monolayered HEp-2 cells in a 96-well culture plate; (3) washing wells to remove bacterial cells which did not adhere to HEp-2 cells, and (4) enzymic reaction for beta-galactosidase activities. However, a calibration curve for the enzyme activity, obtained from each bacterial sample, showed that 10(5) bacteria per well permitted an accurate estimation. The enzyme activity of adhered bacteria to the monolayered cells showed that 10(7) bacteria were appropriate for the adherence assay. The number of adhered bacteria thus obtained was in good agreement with a viable cell count. The result indicates that the new method is more reliable than a widely used method, counting the number of bacteria under a microscope. The present method also makes it easy to detect adherent strains of E. coli in large numbers of specimens.