Biomedical subjects

H Yukawa

Publications and source records attributed to H Yukawa.

At least 37 records · Page 2Linked to original sources

PubMed2000

Sequence analysis of the cryptic plasmid pMG101 from Rhodopseudomonas palustris and construction of stable cloning vectors.

A 15-kb cryptic plasmid was obtained from a natural isolate of Rhodopseudomonas palustris. The plasmid, designated pMG101, was able to replicate in R. palustris and in closely related strains of Bradyrhizobium japonicum and phototrophic Bradyrhizobium species. However, it was unable to replicate in the purple nonsulfur bacterium Rhodobacter sphaeroides and in Rhizobium species. The replication region of pMG101 was localized to a 3.0-kb SalI-XhoI fragment, and this fragment was stably maintained in R. palustris for over 100 generations in the absence of selection. The complete nucleotide sequence of this fragment revealed two open reading frames (ORFs), ORF1 and ORF2. The deduced amino acid sequence of ORF1 is similar to sequences of Par proteins, which mediate plasmid stability from certain plasmids, while ORF2 was identified as a putative rep gene, coding for an initiator of plasmid replication, based on homology with the Rep proteins of several other plasmids. The function of these sequences was studied by deletion mapping and gene disruptions of ORF1 and ORF2. pMG101-based Escherichia coli-R. palustris shuttle cloning vectors pMG103 and pMG105 were constructed and were stably maintained in R. palustris growing under nonselective conditions. The ability of plasmid pMG101 to replicate in R. palustris and its close phylogenetic relatives should enable broad application of these vectors within this group of alpha-proteobacteria.

Bacterial Proteins↗

PubMed2000

Analysis of oxidation sensitivity of maleate cis-trans isomerase from Serratia marcescens.

The maleate cis-trans isomerase gene (maiA) from Serratia marcescens IFO3736 was cloned and sequenced. Serratia MaiA has 62.4% amino acid identity with Alcaligenes faecalis IFO13111 MaiA and 64.9% with Bacillus stearothermophilus MI-102 MaiA. All known ten amino acid sequences of MaiA had significant conserved regions containing cysteine residues, which were previously suggested to be involved in an active site of the enzyme. The maiA gene was expressed in Escherichia coli, and expressed products MaiA was purified and characterized. The purified enzyme of strain IFO3736 showed high activity at room temperature and high heat stability. It also showed higher activity in the presence of high concentration of aspartic acid than the enzyme of A. faecalis IFO13111, but it was also sensitive to chemical oxidation. By amino acid composition analysis, cysteine, methionine, and tyrosine residues were suggested to be oxidized to inactivate the enzyme by chemical oxidation. To investigate the mechanism of chemical oxidation of the enzyme, six methionine residues in the conserved regions of S. marcescens MaiA were replaced with cysteine residues by site-directed mutagenesis. The analysis of the constructed mutants suggested that the Met201 residue near the Cys198 residue is involved in the sensitivity of the enzyme to chemical oxidation.

Amino Acid Sequence↗

PubMed2000

Molecular analysis of maleate cis-trans isomerase from thermophilic bacteria.

Several strains of thermophilic bacteria containing maleate cis-trans isomerase were isolated from soil samples and identified as Bacillus stearothermophilus, Bacillus circulans, Bacillus brevis, and Deleya halophila. The maleate cis-trans isomerase was purified and characterized from one of the isolated strains, B. stearothermophilus MI-102. The purified enzyme of strain MI-102 showed higher thermal stability than the enzyme of a mesophile, Alcaligenes faecalis IFO13111. The seven maleate cis-trans isomerase genes (maiA) of thermophile were cloned and sequenced. B. stearothemophilus MI-102 MaiA has 67% amino acid identity with A. faecalis MaiA. All eight amino acid sequences of maiA gene products had significant conserved regions containing cysteine residues, which were previously suggested to be involved in an active site of the enzyme. To probe the catalytic mechanism, three cysteine residues in the conserved regions of A. faecalis MaiA were replaced with serine by site-directed mutagenesis. The results suggest that Cys80 and Cys198 play important roles in the enzyme activity.

Alcaligenes↗

PubMed2000

Selenium and glutathione peroxidase mRNA in rat glioma.

The purpose of current study was to determine the step at which dietary selenium (Se) regulates the transcriptional expression of the gene for Se-glutathione peroxidase (Se-GPx) in rat brain and transplanted glioma tissue. Wistar rats were fed a Se-free diet or the same diet supplemented with 0.5, 2.0, and 4.0 mg Se/kg as sodium selenite for at least 3 wk. Then, the rats were transplanted with C6 rat glioma cells into the right frontal lobe parenchyma. All rats were observed for 30 d, then tumor and contralateral brain tissue were excised and divided into three portions for purification of selenium content, for the assay of Se concentration, Se-GPx activity, and for Se-GPx mRNA. Se concentration and Se-GPx activity are increased with Se supplementation both in tumor tissue and contralateral brain tissue, and Se concentration in tumor is higher than that in contralateral brain tissue at each dietary Se content. Se-GPx mRNA of brain and tumor were probed with fragments from a rat Se-GPx cDNA in Northern blot analysis. There was significant differences of Se-GPx mRNA transcription in brain tumor tissue among each dietary group of the Se content, and the steady-state level of Se-GPx mRNA was markedly reduced by Se deficiency. These results suggest that dietary Se exerts its augmenting effect on Se-GPx gene transcription.

Animals↗

PubMed2000

Cloning of genes participating in aerobic biodegradation of p-cumate from Rhodopseudomonas palustris.

Rhodopseudomonas palustris utilizes p-cumate as a carbon source both under anaerobic light and aerobic dark conditions. A gene cluster was isolated whose sequence showed high homology to genes which have been implicated the degradation of p-cumate in Pseudomonas pitida. Seven structural genes coding for dioxygenase-reductase, dihydroxy-dihydro dehydrogenase, and ring cleavage oxygenases were identified. A putative regulator and its possible recognition site was suggested on the basis of homology data. Mutant cells in which a kanamycin cassette was inserted into the dihydroxy-dihydro dehydrogenase gene could not grow aerobically on p-cumate. The mutation had no effect on growth using the para substituted benzoate derivatives 4-hydroxycinnamate, ferulate, protocatechuate, and 2,3,4-trihydroxybenzoate as sole carbon source. Moreover, mutant cells showed a growth pattern similar to wild type cells grown on these compounds under photoheterotrophic anaerobic conditions. These data suggest that genes of this operon are involved specifically in aerobic dissimilation of p-cumate. Intermediate products of p-cumate degradation could be detected from extracts of Escherichia coli heterologously expressing the first 5 genes responsible for the first two steps of p-cumate degradation in R. palustris. Primer extension analysis revealed the transcription regulation of the gene cluster which could be induced with para methyl-, ethyl- and isopropyl (cumate) benzoates. This is the first report on genes involved in aerobic degradation of these compounds in photosynthetic bacteria.

Aerobiosis↗

PubMed2000

Structure of the urease operon of Corynebacterium glutamicum.

Urease activity of Corynebacterium glutamicum results in a rapid pH increase upon addition of urea to the growth medium. The urease operon C. glutamicum was isolated of and sequenced. Seven open reading frames were identified; ureA, ureB, and ureC were homologues of other bacterial urease structural genes, ureE, ureF, ureG, and ureD exhibited homology to urease accessory genes. Disruption of ureC prevented the utilization of urea as a nitrogen source by C. glutamicum. Urease activity was induced by urea and appeared to be independent of the nitrogen regulatory system. Urease activity was not affected by pH. Heterologous expression of a truncated derivative of the urease gene cluster in Escherichia coli showed that ureD was necessary for active expression. Western-blot and primer extension analysis on C. glutamicum grown under different conditions confirmed that the operon was induced by urea. Transcriptional startpoint for the ureA gene was determined.

Amino Acid Sequence↗

PubMed2000

[Alcohol consumption and change of activity of daily living among the elderly living in an urban community].

Alcohol consumption has both adverse and beneficial effects on survival. We examined the baseline status of alcohol consumption and the changes of instrumental activity of daily living (I-ADL) by a prospective survey for the elderly living in an urban community who were participants in the "Longitudinal and Interdisciplinary Study on Aging by the Tokyo Metropolitan Institute of Gerontology (TMIG-LISA)". Detailed information on alcohol intake was collected during a structured interview. At a baseline survey in 1991, 1) the frequency of current drinkers decreased lineally by age. 2) the most popular alcohol beverage was beer for both sexes and average ethanol intake per day were 41 (10-45) g in men and 24 (8-28) g in women. Current drinkers had significantly stronger grip strength, higher velocity in maximum walking and higher score on the TMIG Index of Competence, designed to measure the higher-level competence in elderly community residents. After 4 years follow-up, in 1995, 73.3 percent of the participants completed the examination. They were classified into five categories as "current drinkers", "lifetime abstainers", "beginning to drink during follow-up period", "quit drinking during follow-up period" and "quit drinking at baseline". The decline of I-ADL was significantly greater among the elderly who "quit drinking during follow-up period" than "current drinkers". Further "current drinkers" had significantly lower decline of I-ADL than "lifetime abstainers". Our findings show beneficial effects of moderate alcohol intake in the maintenance of a higher level of competence in the elderly living in a community.

Activities of Daily Living↗

PubMed2000

[A case of left occipital lobe hemorrhage in a patient with progressive systemic sclerosis: evaluation of cerebral angiography and histology].

Involvement of the central nervous system is uncommon in progressive systemic sclerosis, with only 2 reported cases associated with intracerebral hemorrhage detected by neuroimaging. A 55-year-old woman with a 10-year history of scleroderma presented with left occipital lobe hemorrhage manifesting as headache and vomiting. She had no signs of hypertension, diabetes mellitus and hyperlipidemia. CT and MRI, on admission, showed left occipital lobe hemorrhage with ventricular rupture and acute left subdural hematoma. Serial cerebral angiography was performed on day 0, day 7 and day 14, and found no evidence of aneurysm, arteriovenous multiformation or tumor stain in the left occipital lobe. However, the bilateral anterior cerebral arteries showed increasing segmental narrowing suggestive of vasculitis. Histological examination of a section from the brain cortex adjacent to the hemorrhage revealed no evidence of vasculitis, fibrinoid degeneration or amyloid deposition. Focal vasculitis may have occurred secondary to the homorrhagic lesion.

Cerebral Angiography↗

PubMed1999

Characterization of a separate small domain derived from the 5' end of 23S rRNA of an alpha-proteobacterium.

We demonstrate the presence of a separate processed domain derived from the 5' end of 23S rRNA in ribosomes of Rhodopseudomonas palustris, a member of the alpha-++proteobacteria. Previous sequencing studies predicted intervening sequences (IVS) at homologous positions within the 23S rRNA genes of several alpha-proteobacteria, including R.palustris, and we find a processed 23S rRNA 5' domain in unfractionated RNA from several species. 5.8S rRNA from eukaryotic cytoplasmic large subunit ribosomes and the bacterial processed 23S rRNA 5' domain share homology, possess similar structures and are both derived by processing of large precursors. However, the internal transcribed spacer regions or IVSs separating them from the main large subunit rRNAs are evolutionarily unrelated. Consistent with the difference in sequence, we find that the site and mechanism of IVS processing also differs. Rhodopseudomonas palustris IVS-containing RNA precursors are cleaved in vitro by Escherichia coli RNase III or a similar activity present in R.palustris extracts at a processing site distinct from that found in eukaryotic systems and this results in only partial processing of the IVS. Surprisingly, in a reaction unlike characterized cases of eubacterial IVS processing, an RNA segment larger than the corresponding DNA insertion is removed which contains conserved sequences. These sequences, by analogy, serve to link the 23S rRNA 5' rRNA domains or 5.8S rRNAs to the main portion of other prokaryotic 23S rRNAs or to eukaryotic 28S rRNAs, respectively.

Alphaproteobacteria↗

PubMed1999

Adenovirus-mediated gene transfer of Bcl-xL prevents cell death in primary neuronal culture of the rat.

Bcl-xL is a Bcl-2-related gene that regulates programmed cell death in a bcl-2-independent fashion. It is expressed in tissues containing long-surviving postmitotic cells, such as neurons in adult brains. To investigate the possibility of gene therapy for transferring this anti-apoptotic gene into the neuron for the treatment of vascular occlusive or neurodegenerative diseases, we examined the effect of a replication-deficient recombinant adenovirus vector coding human Bcl-xL gene on the augmentation of the survival of primarily-cultured rat neuronal cells in vitro. Immunoblot analysis revealed that primarily-cultured neuronal cells were successfully infected and transferred with this gene by recombinant adenovirus vector with high transduction efficiency. Bcl-xL gene transfer to the primarily-cultured neurons could prevent these cells from cell death.

Adenoviridae↗

PubMed1999

Effects of diadenosine tetraphosphate on systemic and regional hemodynamics in dogs.

PURPOSE: Diadenosine tetraphosphate (AP(4)A) produces vasodilation and hypotension. If AP(4)A is to be employed clinically, its influence on systemic and regional hemodynamics needs to be investigated. In this study, we observed systemic and regional hemodynamics during reduction of mean arterial pressure (MAP) induced by AP(4)A in dogs. METHODS: Nineteen mongrel dogs were allocated to three groups: those given physiological saline (vehicle group) and dogs in which MAP was decreased either by 8% (8% group) or by 30% (30% group) by infusion of AP(4)A. Systemic hemodynamics and microsphere-determined regional blood flow to vital organs were assessed before and during AP(4)A infusion. RESULTS: In the 8% group, cardiac output (CO) increased, and systemic vascular resistance (SVR) decreased during AP(4)A infusion. Although regional blood flow to myocardium and portal organs increased, hepatic blood flow decreased. In the 30% group, heart rate and SVR decreased, and stroke volume index increased without change in CO. Regional blood flow to myocardium, kidneys, and portal organs increased. In both groups, cerebral blood flow remained unchanged. CONCLUSION: During the decrease in MAP induced by AP(4)A, there were increases in regional blood flow distributed to the myocardium, kidneys, and portal organs, without change in the blood supply to the brain. This finding suggests that AP(4)A may be clinically useful for reducing blood pressure without compromising blood flow to vital organs.

Journal Article↗

PubMed1999

Cloning and nucleotide sequencing of the secA gene from coryneform bacteria.

Taking advantage of highly conserved domains present in the secA gene from Escherichia coli and Bacillus subtilis, we designed degenerate oligonucleotides (oligos) corresponding to these regions. These oligos were used as primers in PCR in order to amplify DNA sequences from Brevibacterium flavum MJ233 chromosomal DNA. The PCR product was used as a probe to recover genomic fragments from a lambda library of Br. flavum MJ233. The complete nucleotide sequence (nt) of the cloned 5.3-kb EcoR1 fragment containing the secA homolog from Br. flavum MJ233 indicated that the deduced gene product of the Br. flavum secA homolog is composed of 845 amino acids (aa) with a deduced molecular weight (MW) of 95429. Comparison of this aa sequence to the corresponding sequences from E. coli and B. subtilis revealed a high degree of conservation and suggested that the Br. flavum secA homolog has putative ATP binding regions.

Adenosine Triphosphatases↗

PubMed1999

Adenovirus-mediated gene transfer of a truncated form of fibroblast growth factor receptor inhibits growth of glioma cells both in vitro and in vivo.

Basic fibroblast growth factor (FGF-2) and high affinity FGF receptor (FGFR) have been detected in the nucleus as well as the cytoplasm of many human gliomas, and are known to stimulate cellular proliferation and angiogenesis in the tumors. To investigate the effects of inactivation of FGFR on the growth of malignant gliomas, we constructed a replication-deficient recombinant adenovirus vector encoding a truncated form of chicken FGFR1 (AxCA delta FR). AxCA delta FR-infected cells were confirmed to express truncated FGFR protein by immunoblotting and FGF-2-dependent clonogenicity of NIH3T3 cells was suppressed by infection with this virus vector. Then human malignant glioma cell lines U-251MG and T98G, both of which have been reported to express FGF-2 and FGFR, were infected with AxCA delta FR. These infected cells showed nuclear as well as cytoplasmic expression of a truncated FGFR protein. Proliferation rate and the ability to form colonies in soft agar of the cells infected with this virus vector were significantly suppressed compared with those of uninfected and lacZ-expressing adenovirus-infected cells. Moreover, intratumoral injection of AxCA delta FR significantly suppressed the subcutaneous tumor growth of the glioma cells in nude mice. We concluded that inactivation of the cytoplasmic and nuclear FGFR using this truncated FGFR-expressing adenovirus vector can inhibit the growth of malignant gliomas both in vitro and in vivo.

3T3 Cells↗

PubMed1999

Re-evaluation of appropriate size of the laryngeal mask airway.

We have assessed 32 males and 31 females in a randomized, crossover study to see if there was any difference in the correct positioning of the laryngeal mask, optimal ventilation (defined as no gas leak around the mask at an airway pressure of 18 cm H2O) and cuff visibility between sizes 4 and 5 masks in males and sizes 3 and 4 in females. The position of the mask in relation to the glottis was assessed using a fibreoptic bronchoscope. There was no significant difference in correct positioning between the two sizes in either sex. Gas leak was significantly less frequent for a larger than a smaller mask (P < 0.01 for both sexes), whereas the cuff was more often seen in the mouth with larger masks (P < 0.02 for males and P < 0.01 for females). Therefore, larger masks (size 4 in females and size 5 in males) provided a better seal than smaller sizes without worsening the relative position of the mask to the glottis; however, the larger mask came up within the mouth more often, which could interfere with tonsillectomy and could increase the risk of sore throat or lingual nerve damage.

Adult↗

PubMed1999

Molecular and functional characterization of the Rhodopseudomonas palustris no. 7 phosphoenolpyruvate carboxykinase gene.

The pckA gene, encoding the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK), was cloned by PCR amplification from the purple nonsulfur bacterium Rhodopseudomonas palustris No. 7. Sequencing of a 2.5-kb chromosomal SmaI-PstI fragment containing the structural gene revealed an open reading frame encoding 537 amino acids, homologous to known pckA genes. Primer extension analysis identified a transcriptional start site 72 bp upstream of the pckA initiation codon and an upstream sequence similar to sigma70 promoters. Studies of a pckA-lacZ gene fusion indicated that when cells were grown in minimal media with various carbon sources, such as succinate, malate, pyruvate, lactate, or ethanol, under both anaerobic light and aerobic dark conditions, the pckA gene was induced in log phase, irrespective of the carbon source. A R. palustris No. 7 PEPCK-deficient strain showed growth characteristics identical to those of the wild-type strain either anaerobically in the light or aerobically in the dark when a C4-dicarboxylic acid, such as succinate or malate, was used as a carbon source. These results indicate that in R. palustris No. 7, an alternative gluconeogenic pathway may exist in addition to PEPCK.

Amino Acid Sequence↗

PubMed1999

Inhibition of rat vascular smooth muscle cell proliferation in vitro and in vivo by recombinant replication-competent herpes simplex virus.

BACKGROUND AND PURPOSE: The proliferation of vascular smooth muscle cells (VSMCs) is a common feature associated with vascular proliferative disorders such as atherosclerosis and restenosis after balloon angioplasty. We examined the antiproliferative effects of recombinant replication-competent herpes simplex virus (HSV), hrR3, to proliferative VSMCs both in vitro and in vivo. METHODS: Early passages of Sprague-Dawley rat VSMCs were infected with hrR3 at a low multiplicity of infection (0.01 to 1.0) to examine the in vitro cytotoxic activity of this recombinant HSV to VSMCs in a proliferative state. Sprague-Dawley rats underwent balloon dilatation injury of the left carotid artery to induce neointimal formation. The injured carotid arteries were infected with hrR3 five days after balloon injury. Two weeks after injury, the left carotid arteries were fixed, and the areas of the neointimal and medial layers were analyzed microscopically. Because the reporter Escherichia coli lacZ gene in hrR3 is expressed only in infected cells in which the virus is actively replicating, virus replication was confirmed by X-gal staining. RESULTS: A morphometric analysis revealed that there were significant differences in the intima/media ratio between the HSV-treated group and mock-infected group (0. 354+/-0.068 and 1.08+/-0.055, respectively). In the histological study (X-gal staining), positive X-gal staining was observed chiefly in the VSMCs in the medial layer just beneath the internal elastic lamina, indicating active viral replication. CONCLUSIONS: Virus-mediated cytocidal therapy using recombinant HSV vector is a promising modality for the treatment of the restenosis after balloon angioplasty.

Angioplasty, Balloon↗

PubMed1999

[Compliance concerning external protectors for hip fractures among the institutionalized elderly in Japan].

An external protector (EHP) which was developed in Europe to protect the femoral neck from direct impact on falling is available but has not been examined sufficiently in Japan. In order to explore the compliance of use of the EHP among the institutionalized elderly with in case of hip fracture who are at high risk of falling, we conducted the four-week intervention study using two types of a EHP. The subjects of a study consisted of 10 elderly people (2 men, 8 women) with a mean age 85.7 living in nursing home in a village. Informed consent was obtained from all participants. At the end of the study, rate of subjects who wore EHP was relatively high for the Finnish EHP (Safety Pants) group compared to the Danish EHP (Hip Protector) group. There were no significant differences between variables in age, sex, fall experience during the previous year, history of diseases etc. The reasons for dropout were first: difficult in wearing EHP and accompanying delay in toilet, secondly: taking much time to wear, thirdly: a sense of incongruity, too small or too tight'. If the EHP is redesigned to suit Japanese elderly, the compliance might increase. Thorough explanation to institutionalized elderly who may have cognitive impairment, physical problems, or both, is required.

Aged↗

PubMed1999

[Compliance in use of external protectors for hip fractures among the community elderly in Japan].

To protect femoral neck fractures which are the most serious complication of osteoporosis and are increasing in frequency in Japan, an external hip protector (EHP) fixed in special underwear has been proven to absorb a direct impact to the greater trochanter during a fall from standing height. In this study, we investigated compliance concerning the use of EHPs, for six months using two types of EHP, i.e., hard pad type (Hip Protector, Sahvatex) from Denmark (Fig. 1) and soft pad type (Safety Pants, Rounomo Oy) from Finland. The subjects were 20 elderly women aged 70 years or more who had at least one experience of falling within the year preceding the baseline survey in September of 1997. The compliance rate is shown in Fig. 2. Though the soft type EHP had relatively better compliance than the hard type EHP, there was no significant difference between them. The main reason for early dropout (one or two weeks after base-line) was "difficulty to remove especially with regard to using the toilet". The main reason for later dropout was "too tight to wear in winter". There were no significant differences with regard to anthropometric measurements, physical activity, ADL, and rate of falls between compliers and dropouts except age (73.6 vs 78.5 yrs). Sufficient explanation at baseline and generatively good motivation for wearing the EHP will maintain a high compliance which may result in the effective prevention of hip fractures among the community elderly.

Aged↗