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Biomedical subjects

A Okabe

Publications and source records attributed to A Okabe.

At least 37 records · Page 2Linked to original sources
PubMed1999

The hydA gene encoding the H(2)-evolving hydrogenase of Clostridium perfringens: molecular characterization and expression of the gene.

A putative hydrogenase (hydA) gene of Clostridium perfringens encodes a protein with strong identity to Clostridium pasteurianum hydrogenase I. Disruption of the hydA gene abolished H(2) productivity, confirming its function. A putative butyrate kinase gene (buk) is adjacent to the hydA gene. When cultures were grown in medium with glucose, 1.8-kb hydA and 2.1-kb buk transcripts and a 3. 9-kb transcript hybridized with both hydA and buk-probe were detectable in all the exponential growth phases. In medium without glucose, these transcripts were decreased rapidly after the mid-exponential phase. These results suggest that the transcription of these two genes is probably regulated by a similar mechanism in response to glucose availability.

Bacterial Proteins↗
PubMed1999

Promoter upstream bent DNA activates the transcription of the Clostridium perfringens phospholipase C gene in a low temperature-dependent manner.

The phospholipase C gene (plc) of Clostridium perfringens possesses three phased A-tracts forming bent DNA upstream of the promoter. An in vitro transcription assay involving C.perfringens RNA polymerase (RNAP) showed that the phased A-tracts have a stimulatory effect on the plc promoter, and that the effect is proportional to the number of A-tracts, and more prominent at lower temperature. A gel retardation assay and hydroxyl radical footprinting revealed that the phased A-tracts facilitate the formation of the RNAP-plc promoter complex through extension of the contact region. The upstream (UP) element of the Escherichia coli rrnB P1 promoter stimulated the downstream promoter activity temperature independently, differing from the phased A-tracts. When the UP element was placed upstream of the plc promoter, low temperature-dependent stimulation was observed, although this effect was less prominent than that of the phased A-tracts. These results suggest that both the phased A-tracts and UP element cause low temperature-dependent activation of the plc promoter through a similar mechanism, and that the more efficient low temperature-dependent activation by the phased A-tracts may be due to an increase in the bending angle at a lower temperature.

Binding, Competitive↗
PubMed1999

Three distinct anti-allergic drugs, amlexanox, cromolyn and tranilast, bind to S100A12 and S100A13 of the S100 protein family.

To investigate the roles of calcium-binding proteins in degranulation, we used three anti-allergic drugs, amlexanox, cromolyn and tranilast, which inhibit IgE-mediated degranulation of mast cells, as molecular probes in affinity chromatography. All of these drugs, which have different structures but similar function, scarcely bound to calmodulin in bovine lung extract, but bound to the same kinds of calcium-binding proteins, such as the 10-kDa proteins isolated in this study, calcyphosine and annexins I-V. The 10-kDa proteins obtained on three drug-coupled resins and on phenyl-Sepharose were analysed by reversed-phase HPLC. It was found that two characteristic 10-kDa proteins, one polar and one less polar, were bound with all three drugs, although S100A2 (S100L), of the S100 family, was bound with phenyl-Sepharose. The cDNA and deduced amino acid sequence proved our major polar protein to be identical with the calcium-binding protein in bovine amniotic fluid (CAAF1, S100A12). The cDNA and deduced amino acid sequence of the less-polar protein shared 95% homology with human and mouse S100A13. In addition, it was demonstrated that the native S100A12 and recombinant S100A12 and S100A13 bind to immobilized amlexanox. On the basis of these findings, we speculate that the three anti-allergic drugs might inhibit degranulation by binding with S100A12 and S100A13.

Amino Acid Sequence↗
PubMed1999

Purification of bovine S100A12 from recombinant Escherichia coli.

S100A12, a member of the S100 family of EF-hand calcium-binding proteins, was purified from Escherichia coli cells expressing the corresponding cDNA. The procedure involved washing induced E. coli cells with EDTA-containing hypotonic solution, ion-exchange chromatography, and HPLC. Recombinant S100A12 was purified to homogeneity with the final yield around 6.7 mg per 20 ml of culture. The purified protein was identical to native S100A12 in the N-terminal amino acid sequence, lysylendopeptidase peptide mapping, mass spectrum, and Ca2+-dependent binding affinity to amlexanox, an antiallergy drug. However, the N-terminal methionine residue of the purified protein was not cleaved off as in the native protein. The method used in the present study permits the purification of recombinant S100A12 in large quantities and may also be applicable to preparation of other S100 family proteins.

Amino Acid Sequence↗
PubMed1999

Treatment of obstructive sleep apnea syndrome with a Kampo-formula, San'o-shashin-to: a case report.

The following describes a 76-year-old male with obstructive sleep apnea syndrome successfully treated with a Kampo-formula, San'o-shashin-to (Formula medicamentorum tres ad dispellendi cordis). Polysomnography, performed before and after administration of San'o-shashin-to, revealed that the apnea index decreased from 11.1 events/hour to 4.1 events/hour, and that the apnea plus hypopnea index decreased from 18.4 events/hour to 10.7 events/hour. The patient was normo-weight (body mass index: 20.4 kg/m2), and events of sleep apnea and hypopnea were mostly noted during a non-rapid eye movement sleep. It is possible that San'o-shashin-to has some alleviating effects on the upper airway resistance during sleep.

Aged↗
PubMed1999

A Clostridium perfringens hem gene cluster contains a cysG(B) homologue that is involved in cobalamin biosynthesis.

The hem gene cluster, which consists of hemA, cysG(B), hemC, hemD, hemB, and hemL genes, and encodes enzymes involved in the biosynthetic pathway from glutamyl-tRNA to uroporphyrinogen III, has been identified by the cloning and sequencing of two overlapping DNA fragments from Clostridium perfringens NCTC8237. The deduced amino acid sequence of the N-terminal region of C. perfringens HemD is homologous to those reported for the C-terminal region of Salmonella typhimurium CysG and Clostridium josui HemD. C. perfringens CysG(B) is a predicted 220-residue protein which shows homology to the N-terminal region of S. typhimurium CysG. Disruption of the cysG(B) gene in C. perfringens strain 13 by homologous recombination reduced cobalamin (vitamin B12) levels by a factor of 200. When grown in vitamin B12-deficient medium, the mutant strain showed a four-fold increase in its doubling time compared with that of the wild-type strain, and this effect was counteracted by supplementing the medium with vitamin B12. These results suggest that C. perfringens CysG(B) is involved in the chelation of cobalt to precorrin II as suggested for the CysG(B) domain of S. typhimurium CysG, enabling the synthesis of cobalamin.

Base Sequence↗
PubMed1999

Gene duplication and multiplicity of collagenases in Clostridium histolyticum.

Clostridium histolyticum collagenase contains a number of different active components. Previously we have shown that colH encodes a 116-kDa collagenase (ColH) and a 98-kDa gelatinase. We purified a different 116-kDa collagenase (ColG) from the culture supernatant and sequenced its gene (colG). We also identified four other gelatinases (105, 82, 78, and 67 kDa) and determined their N-terminal amino acid sequences, all of which coincided with that of either ColG or ColH. Hybridization experiments showed that each gene is present in a single copy and each gene is transcribed into a single mRNA. These results suggest that all the gelatinases are produced from the respective full-length collagenase by the proteolytic removal of C-terminal fragments. The substrate specificities of the enzymes suggest that colG and colH encode class I and class II enzymes, respectively. Analysis of their DNA locations by pulsed-field gel electrophoresis and nucleotide sequencing of their surrounding regions revealed that the two genes are located in different sites on the chromosome. C. histolyticum colG is more similar to C. perfringens colA than to colH in terms of domain structure. Both colG and colA have a homologous gene, mscL, at their 3' ends. These results suggest that gene duplication and segment duplication have occurred in an ancestor cell common to C. histolyticum and C. perfringens and that further divergence of the parent gene produced colG and colA.

Amino Acid Sequence↗
PubMed1999

Identification of metal ligands in the Clostridium histolyticum ColH collagenase.

A Clostridium histolyticum 116-kDa collagenase has an H415EXXH motif but not the third zinc ligand, as found in already characterized zinc metalloproteinases. To identify its catalytic site, we mutated the codons corresponding to the three conserved residues in the motif to other amino acid residues. The mutation affecting His415 or His419 abolished catalytic activity and zinc binding, while that affecting Glu416 did the former but not the latter. These results suggest that the motif forms the catalytic site. We also mutated the codons corresponding to other amino acid residues that are likely zinc ligands. The mutation affecting Glu447 decreased markedly both the enzymatic activity and the zinc content, while that affecting Glu446 or Glu451 had smaller effects on activity and zinc binding. These mutations caused a decrease in kcat but no significant change in Km. These results are consistent with the hypothesis that Glu447 is the third zinc ligand. The spacing of the three zinc ligands is the same in all known clostridial collagenases but not in other known gluzincins, indicating that they form a new gluzincin subfamily. The effects of mutations affecting Glu446 and Glu451 suggest that the two residues are also involved in catalysis, possibly through an interaction with the two zinc-binding histidine residues.

Amino Acid Sequence↗
PubMed1998

Collagen-binding growth factors: production and characterization of functional fusion proteins having a collagen-binding domain.

The autocrine/paracrine peptide signaling molecules such as growth factors have many promising biologic activities for clinical applications. However, one cannot expect specific therapeutic effects of the factors administered by ordinary drug delivery systems as they have limited target specificity and short half-lives in vivo. To overcome the difficulties in using growth factors as therapeutic agents, we have produced fusion proteins consisting of growth factor moieties and a collagen-binding domain (CBD) derived from Clostridium histolyticum collagenase. The fusion proteins carrying the epidermal growth factor (EGF) or basic fibroblast growth factor (bFGF) at the N terminal of CBD (CBEGF/CBFGF) tightly bound to insoluble collagen and stimulated the growth of BALB/c 3T3 fibroblasts as much as the unfused counterparts. CBEGF, when injected subcutaneously into nude mice, remained at the sites of injection for up to 10 days, whereas EGF was not detectable 24 h after injection. Although CBEGF did not exert a growth-promoting effect in vivo, CBFGF, but not bFGF, strongly stimulated the DNA synthesis in stromal cells at 5 days and 7 days after injection. These results indicate that CBD may be used as an anchoring unit to produce fusion proteins nondiffusible and long-lasting in vivo.

3T3 Cells↗
PubMed1998

A study of the collagen-binding domain of a 116-kDa Clostridium histolyticum collagenase.

The Clostridium histolyticum 116-kDa collagenase consists of four segments, S1, S2a, S2b, and S3. A 98-kDa gelatinase, which can degrade denatured but not native collagen, lacks the C-terminal fragment containing a part of S2b and S3. In this paper we have investigated the function of the C-terminal segments using recombinant proteins. Full-length collagenase degraded both native type I collagen and a synthetic substrate, Pz-peptide, while an 88-kDa protein containing only S1 and S2a (S1S2a) degraded only Pz-peptide. Unlike the full-length enzyme, S1S2a did not bind to insoluble type I collagen. To determine the molecular determinant of collagen binding activity, various C-terminal regions were fused to the C terminus of glutathione S-transferase. S3 as well as S2bS3 conferred collagen binding. However, a glutathione S-transferase fusion protein with a region shorter than S3 exhibited reduced collagen binding activity. S3 liberated from the fusion protein also showed collagen binding activity, but not S2aS2b or S2b. S1 had 100% of the Pz-peptidase activity but only 5% of the collagenolytic activity of the full-length collagenase. These results indicate that S1 and S3 are the catalytic and binding domains, respectively, and that S2a and S2b form an interdomain structure.

Catalysis↗
PubMed1998

The Pasteurella haemolytica 35 kDa iron-regulated protein is an FbpA homologue.

In a previous investigation, a 35 kDa iron-regulated protein was identified from total cellular proteins of Pasteurella haemolytica grown under iron-depleted conditions. This study reports identification of the gene (fbpA) encoding the 35 kDa protein based on complementation of an entA Escherichia coli strain transformed with a plasmid derived from a P. haemolytica lambda ZAP II library. Cross-reactivity was demonstrated between an anti-35 kDa mAb and a 35 kDa protein expressed in this strain. Furthermore, a translated ORF identified on the recombinant plasmid corresponded with the N-terminal amino acid sequence of the intact and a CNBr-cleaved fragment of the 35 kDa iron-regulated protein. Nucleotide sequence analysis of the gene encoding the 35 kDa protein demonstrated homology with the cluster 1 group of extracellular solute-binding proteins, especially to the iron-binding proteins of this family. Complete sequence analysis of the recombinant plasmid insert identified three other predominant ORFs, two of which appeared to be in an operonic organization with fbpA. These latter components (fbpB and fbpC) showed homology to the transmembrane and ATPase components of ATP-binding cassette (ABC)-type uptake systems, respectively. Based on amino acid/DNA sequencing, citrate competition assay of iron affinity and visible wavelength spectra, it was concluded that the P. haemolytica 35 kDa protein functions as an FbpA homologue (referred to as PFbpA) and that the gene encoding this protein is part of an operon comprising a member of the FbpABC family of iron uptake systems. Primary sequence analysis revealed rather surprisingly that PFbpA is more closely related to the intracellular Mn/Fe-binding protein IdiA found in cyanobacteria than to any of the homologous FbpA proteins currently known in commensal or pathogenic members of the Pasteurellaceae or Neisseriaceae.

Amino Acid Sequence↗
PubMed1998

A case of subwakefulness syndrome.

We report a patient, a 30-year-old male Japanese-Brazilian migrant construction worker, suffering from excessive daytime sleepiness for at least 6 months. Electroencephalogram recordings during his waking states showed that 10-Hz and 60-microV alpha activity was present prominently in the occipital regions. From the multiple sleep latency test, it was found that stages 1-2 NREM sleep episodes appeared repetitively without any REM episodes, and that the mean sleep latency was 10.2 min. These findings support the diagnosis that this patient suffers from subwakefulness syndrome.

Adult↗
PubMed1998

Neurotoxicity of Clostridium perfringens epsilon-toxin for the rat hippocampus via the glutamatergic system.

The neurotoxicity of epsilon-toxin, one of the major lethal toxins produced by Clostridium perfringens type B, was studied by histological examination of the rat brain. When the toxin was injected intravenously at a lethal dose (100 ng/kg), neuronal damage was observed in many areas of the brain. Injection of the toxin at a sublethal dose (50 ng/kg) caused neuronal damage predominantly in the hippocampus: pyramidal cells in the hippocampus showed marked shrinkage and karyopyknosis, or so-called dark cells. The dark cells lost the immunoreactivity to microtubule-associated protein-2, a postsynaptic somal and dendric marker, while acetylcholinesterase-positive fibers were not affected. Timm's zinc staining revealed that zinc ions were depleted in the mossy layers of the CA3 subfield containing glutamate as a synaptic transmitter. The cerebral blood flow in the hippocampus was not altered significantly before or after administration of the toxin, as measured by laser-Doppler flowmetry, excluding the possibility that the observed histological change was due to a secondary effect of ischemia in the hippocampus. Prior injection of either a glutamate release inhibitor or a glutamate receptor antagonist protected the hippocampus from the neuronal damage caused by epsilon-toxin. These results suggest that epsilon-toxin acts on the glutamatergic system and evokes excessive release of glutamate, leading to neuronal damage.

Animals↗
PubMed1998

NMDA receptor-mediated differential laminar susceptibility to the intracellular Ca2+ accumulation induced by oxygen-glucose deprivation in rat neocortical slices.

Slices of somatosensory cortex taken from immature rats on postnatal day (P)7-14 were labeled with fura-2. Intracellular Ca2+ concentration ([Ca2+]i) was monitored in identified pyramidal cells as the ratio of fluorescence intensities (RF340/F380) during oxygen-glucose deprivation. The RF340/F380 ([Ca2+]i) of individual pyramidal cells was monitored in each of the cortical layers II-VI simultaneously. Neurons in all neocortical layers exhibited significant increases in [Ca2+]i that varied with the duration of oxygen-glucose deprivation. Individual neurons responded to oxygen-glucose deprivation with abrupt increases in [Ca2+]i after various latencies. The ceiling level of the [Ca2+]i increase differed from cell to cell. Neurons in layer II/III showed significantly greater increases in [Ca2+]i than those in layers IV, V, or VI. Kynurenic acid, a nonselective glutamate receptor antagonist, and bicuculline, a selective gamma-aminobutyric acid (GABA)A receptor antagonist, suppressed the intracellular Ca2+ accumulation induced by oxygen-glucose deprivation in all neocortical layers examined. After kynurenic acid, but not after bicuculline, there was no longer a differential [Ca2+]i increases in layer II/III. Both 2-amino-5-phosphonopentanoic acid (AP5), a selective N-methyl-D-aspartate (NMDA) receptor antagonist, and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a non-NMDA receptor antagonist, strongly suppressed the intracellular Ca2+ accumulation induced by oxygen-glucose deprivation in all layers. The laminar difference in terms of the [Ca2+]i increases was abolished by AP5, but not by CNQX. These results indicate that layer II/III cells are the most prone to oxygen-glucose deprivation-induced intracellular Ca2+ accumulation, and that this is primarily mediated by NMDA receptors. Thus, layer II/III neurons would be more likely to suffer cellular Ca2+ overload and excitotoxicity during ischemia than layer IV-VI cells. Such a differential laminar vulnerability might play an important role in determining the pathological characteristics of the immature cortex and its sequelae later in life.

2-Amino-5-phosphonovalerate↗
PubMed1998

Changes in intracellular Ca2+ induced by GABAA receptor activation and reduction in Cl- gradient in neonatal rat neocortex.

We have studied the effects of gamma-aminobutyric acid (GABA) and of reducing the Cl- gradient on the [Ca2+]i in pyramidal neurons of rat somatosensory cortex. The Cl- gradient was reduced either with furosemide or by oxygen-glucose deprivation. Immature slices taken at postnatal day (P)7-14 were labeled with fura-2, and [Ca2+]i was monitored in identified pyramidal cells in layer II/III as the ratio of fluorescence intensities (RF340/F380). The magnitude of the [Ca2+]i increases induced by oxygen-glucose deprivation was significantly reduced (by 44%) by bicuculline (10 microM), a GABAA receptor antagonist. Under normal conditions, GABA generally did not raise [Ca2+]i, although in some neurons a small and transient [Ca2+]i increase was observed. These transient [Ca2+]i increases were blocked by Ni2+ (1 mM), a blocker of voltage-dependent Ca2+ channels (VDCCs). Continuous perfusion with GABA did not cause a sustained elevation of [Ca2+]i but bicuculline caused [Ca2+]i oscillations. After inhibition of Cl- extrusion with furosemide (1.5 mM), GABA induced a large [Ca2+]i increase consisting of an initial peak followed by a sustained phase. Both the initial and the sustained phases were eliminated by bicuculline (10 microM). The initial but not the sustained phase was abolished by Ni2+. In the presence of Ni2+, the remaining sustained response was inhibited by the addition of 2-amino-5-phosphonopentanoic acid (AP5, 20 microM), a selective N-methyl-D-aspartate (NMDA) receptor antagonist. Thus the initial peak and the sustained phase of the GABA-evoked [Ca2+]i increase were mediated by Ca2+ influx through VDCCs and NMDA receptor channels, respectively, and both phases were initiated via the GABAA receptor. These results indicate that, in neocortical pyramidal neurons, a reduction in the Cl- gradient converts the GABAA receptor-mediated action from nothing or virtually nothing to a large and sustained accumulation of cellular Ca2+. This accumulation is the result of Ca2+ influx mainly through the NMDA receptor channel. Thus GABA, normally an inhibitory transmitter, may play an aggravating role in excitotoxicity if a shift in the Cl- equilibrium potential occurs, as reported previously, during cerebral ischemia.

2-Amino-5-phosphonovalerate↗
PubMed1997

Lambda-toxin of Clostridium perfringens activates the precursor of epsilon-toxin by releasing its N- and C-terminal peptides.

The effect of lambda-toxin, a thermolysin-like metalloprotease of Clostridium perfringens, on the inactive epsilon-prototoxin produced by the same organism was examined. When the purified epsilon-prototoxin was incubated with the purified lambda-toxin at 37 C for 2 hr, the 32.5-kDa epsilon-prototoxin was processed into a 30.5-kDa polypeptide, as determined by SDS-polyacrylamide gel electrophoresis. A mouse lethality test showed that the treatment activated the prototoxin: the 50% lethal doses (LD50) of the prototoxin with and without lambda-toxin treatment were 110 and 70,000 ng/kg of body weight, respectively. The lethal activity of the prototoxin activated by lambda-toxin was comparable to that with trypsin plus chymotrypsin and higher than that with trypsin alone: LD50 of the prototoxin treated with trypsin and trypsin plus chymotrypsin were 320 and 65 ng/kg of body weight, respectively. The epsilon-toxin gene was cloned and sequenced. Determination of the N-terminal amino acid sequence of each activated epsilon-prototoxin revealed that lambda-toxin cleaved between the 10th and 11th amino acid residues from the N-terminus of the prototoxin, while trypsin and trypsin plus chymotrypsin cleaved between the 13th and 14th amino acid residues. The molecular weight of each activated epsilon-prototoxin was also determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The C-terminus deduced from the molecular weight is located at the 23rd or 30th amino acid residue from the C-terminus of the prototoxin, suggesting that removal of not only N-terminal but also C-terminal peptide is responsible for activation of the prototoxin.

Amino Acid Sequence↗
PubMed1996

Kindling induces neuropsin mRNA in the mouse brain.

Neuropsin mRNA expression was analyzed and mapped in the mouse brains after kindling epileptogenesis by using in situ hybridization histochemistry. Dynamic increases of the neuropsin mRNA were observed in the layer II of prelimbic, somatosensory, auditory, perirhinal, entorhinal, and piriform cortices in an activity-dependent manner, though no neuropsin gene was expressed in these areas in control mice. In addition to the confirmation of our previous studies showing increases of mRNA in the hippocampus and amygdaloid complex, there were also remarkable increases of the neuropsin mRNA in the limbic areas, such as the accessory olfactory nucleus, the medial and lateral septal nucleus, the nucleus of diagonal band, the substantia innominata and the zona incerta. The dynamic activity-dependent changes of the gene expression and the site-specificity of neuropsin localization are suggesting that this molecule is implicated in cortical- and limbic-specific neuronal reorganization.

Animals↗