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A Okabe

Publications and source records attributed to A Okabe.

At least 55 records · Page 3Linked to original sources
PubMed1996

Tissue extract from dopamine-depleted striatum enhances differentiation of cultured striatal type-1 astrocytes.

The effects of tissue extract from dopamine (DA)-depleted striatum (lesion extract, L-ext) on morphological and electrophysiological natures of cultured striatal astrocytes were investigated. L-ext treatment suppressed the proliferation of type-1 astrocytes. They became fibrous in a concentration-dependent manner. These changes were not observed in type-2 astrocytes. By whole cell patch-clamp recording, two kinetically and pharmacologically distinct voltage-activated potassium currents, A current and delayed rectifier, were identified. L-ext treatment enhanced both currents in type-1 astrocytes, but only A current in type-2. Data suggest that in tissue extract from DA-depleted striatum, there are increased trophic activities that promote the differentiation of type-1 astrocytes.

Animals↗
PubMed1996

An upstream activating sequence containing curved DNA involved in activation of the Clostridium perfringens plc promoter.

The plc gene, which encodes phospholipase C (alpha-toxin) of Clostridium perfringens, possesses three poly(A) tracts forming an intrinsically curved DNA region immediately upstream of the promoter. The in vivo transcriptional activity of the plasmid-borne plc gene was stimulated by this curved-DNA-containing sequence, depending on its proper linear and rotational orientation. The in vitro transcriptional activity of the plc gene was also stimulated by the upstream sequence. In addition, the stimulatory effect of the sequence and the degree of DNA bending were greater at lower temperature, as was demonstrated by both in vitro and in vivo transcription assays, and a gel-mobility assay, respectively. A similar temperature effect was also observed with the chromosomal plc gene. These observations suggest that the upstream DNA curvature per se stimulates the initiation of transcription of the plc gene, possibly through direct contact with RNA polymerase.

Base Sequence↗
PubMed1996

Expression of the colH gene encoding Clostridium histolyticum collagenase in Bacillus subtilis and its application to enzyme purification.

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 37 C, stability of the plasmid was not maintained. However, stability was partly improved by growing the transformant in a modified LB broth containing 0.5 M sodium succinate with gentle shaking at 35 C. When the transformant was grown to an optical density of 0.4 at 600 nm 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 0.53 mg and its specific activity was estimated to be 1,210 U 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.

Amino Acid Sequence↗
PubMed1996

Analysis of the phospholipase C gene of Clostridium perfringens KZ1340 isolated from Antarctic soil.

Clostridium perfringens KZ1340 isolated from Antarctic soil was first classified as Clostridium plagarum and later as a lecithinase-negative variant of C. perfringens. Although the strain produced no detectable lecithinase (phospholipase C, PLC) activity in the culture supernatant, it was shown by Southern blot hybridization to possess a PLC-encoding gene (plc). To determine the cause of the PLC deficiency, we cloned and sequenced the plc gene from KZ1340. The deduced amino acid sequence consists of 398 amino acid residues, coinciding with those of the plc genes previously determined. Tyrosine was substituted for histidine at amino acid position 148, which is thought to bind a zinc ion essential for PLC activity. Northern blot analysis revealed that KZ1340 expressed the plc gene at an extremely low level. Furthermore, the plc gene cloned from C. perfringens strain 13 into a plasmid was expressed weakly in KZ1340, compared to that in strain 13. This indicates that the former strain represses plc gene expression in trans. When a phylogenetic tree of plc genes was constructed, the KZ1340 plc gene formed a monophyletic branch along with those of various other C. perfringens strains, supporting the classification of the strain as a variant of C. perfringens.

Amino Acid Sequence↗
PubMed1996

Purification, characterization, and primary structure of Clostridium perfringens lambda-toxin, a thermolysin-like metalloprotease.

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.

Amino Acid Sequence↗
PubMed1995

Identification of the gene encoding a mechanosensitive channel MscL homologue in Clostridium perfringens.

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.

Amino Acid Sequence↗
PubMed1995

Phylogenetic analysis of phospholipase C genes from Clostridium perfringens types A to E and Clostridium novyi.

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.

Amino Acid Sequence↗
PubMed1994

A Clostridium perfringens vector for the selection of promoters.

A promoter selection vector for Clostridium perfringens genes was constructed from a C. perfringens-Escherichia coli shuttle vector, pJIR418. The plasmid carries a promoterless chloramphenicol acetyltransferase gene (catP), derived from pIP401, downstream of the multiple cloning sites of pUC18. When a promoter region of the phospholipase C gene was inserted into one of the cloning sites, derivatives of C. perfringens strain 13 carrying the resultant plasmid acquired resistance to chloramphenicol. This plasmid should be useful reporter system for C. perfringens genes.

Base Sequence↗
PubMed1994

Comparison of the virulence of methicillin-resistant and methicillin-sensitive Staphylococcus aureus.

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.

Animals↗
PubMed1994

Enterotoxic activity of Klebsiella oxytoca cytotoxin in rabbit intestinal loops.

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.

Animals↗
PubMed1994

Role of alpha-toxin in Clostridium perfringens infection determined by using recombinants of C. perfringens and Bacillus subtilis.

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.

Animals↗
PubMed1994

Purification and characterization of Clostridium perfringens 120-kilodalton collagenase and nucleotide sequence of the corresponding gene.

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.

Amino Acid Sequence↗
PubMed1994

Cloning and nucleotide sequence analysis of the colH gene from Clostridium histolyticum encoding a collagenase and a gelatinase.

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.

Amino Acid Sequence↗
PubMed1993

Comparison of the effect of zopiclone and brotizolam on sleep EEG by quantitative evaluation in healthy young women.

We studied the effect of brotizolam (BRO) (0.25 mg) and zopiclone (ZPC) (7.5 mg) on the sleep electroencephalograms (EEG) of seven healthy young women (ages 20-21 years). In addition to conventional sleep scoring, we performed spectral analysis of EEG wave forms using the Fast Fourier Transformation (FFT) method. Four weeks following two consecutive polysomnographic recordings with placebo administration (for baseline data), polysomnography with BRO or ZPC administration was performed on the subjects who were then crossed over to polysomnography with ZPC or BRO, respectively, 1 week later. Total rapid eye movement (REM) time was decreased on the ZPC and BRO nights and REM latency was prolonged on the ZPC night compared with baseline (BL) night. Other parameters, however, were not significantly different between the drug nights and the BL night. There were also no differences in standard scoring results between both drug nights. FFT analysis revealed significant differences in total power density percentage and mean power density percentage in both nonrapid eye movement (NREM) and REM sleep between ZPC and BRO nights and between both drug nights compared to the BL night. Total power density and mean power density per epoch showed significant decreases in the theta band (6.0-8.0 Hz) and sigma band (12.0-14.0 Hz) on the ZPC night versus the BRO night or BL night during specific NREM periods. The results of this study corroborate previous reports that suggest computer-aided quantitative electroencephalography aids in differentiating specific effects of hypnotics and other central-nervous-system-acting agents on the sleep EEG.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1993

Comparison of the alpha-toxin genes of Clostridium perfringens type A and C strains: evidence for extragenic regulation of transcription.

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.

Amino Acid Sequence↗
PubMed1992

A possible mechanism for pacemaker-induced T-wave changes.

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.

Adult↗