Biomedical subjects

K Numata

Publications and source records attributed to K Numata.

At least 19 recordsLinked to original sources

PubMed1996

A stable intermediate in the thermal unfolding process of a chimeric 3-isopropylmalate dehydrogenase between a thermophilic and a mesophilic enzymes.

The thermal unfolding process of a chimeric 3-isopropylmalate dehydrogenase made of parts from an extreme thermophile, Thermus thermophilus, and a mesophile, Bacillus subtilis, enzymes was studied by CD spectrophotometry and differential scanning calorimetry (DSC). The enzyme is a homodimer with a subunit containing two structural domains. The DSC melting profile of the chimeric enzyme in 20 mM NaHCO3, pH 10.4, showed two endothermic peaks, whereas that of the T. thermophilus wild-type enzyme had one peak. The CD melting profiles of the chimeric enzyme under the same conditions as the DSC measurement, also indicated biphasic unfolding transition. Concentration dependence of the unfolding profile revealed that the first phase was protein concentration-independent, whereas the second transition was protein concentration-dependent. When cooled after the first transition, the intermediate was isolated, which showed only the second transition upon heating. These results indicated the existence of a stable dimeric intermediate followed by the further unfolding and dissociation in the thermal unfolding of the chimeric enzyme at pH 10-11. Because the portion derived from the mesophilic isopropylmalate dehydrogenase in the chimeric enzyme is located in the hinge region between two domains of the enzyme, it is probably responsible for weakening of the interdomain interaction and causing the decooperativity of two domains. The dimeric form of the intermediate suggested that the first unfolding transition corresponds to the unfolding of domain 1 containing the N- and C-termini of the enzyme, and the second to that of domain 2 containing the subunit interface.

3-Isopropylmalate Dehydrogenase↗

PubMed1996

The epidemiology of human calicivirus/Sapporo/82/Japan.

Based on genome analysis of the RNA-dependent RNA polymerase region, it has been proposed that human caliciviruses (HuCV) can be classified into at least three genogroups: genogroup I is represented by Norwalk virus (NV), genogroup II by Snow Mountain agent (SMA) and genogroup III by HuCV/Sapporo/82/Japan (HuCV/Sa/82/J) virus. HuCV/Sa/82/J strain is genetically unique and more closely related to animal caliciviruses than are other known HuCVs, such as NV and SMA. HuCV/Sa/82/J strain was detected in four outbreaks of HuCV gastroenteritis occurring between 1977 and 1982 in an infant home in Sapporo. The HuCVs detected from these four outbreaks all showed a typical "Star of David" configuration by electron microscopy (EM), and they were identical antigenically and genetically. This strain has also been detected in other prefectures in Japan, as well as in the USA, UK, Saudi Arabia and Kenya. Seroepidemiological studies have shown a worldwide distribution of this virus, including Japan, USA, UK, Southeast Asia, Canada, China and Kenya. This virus has been circulating in Sapporo for at least 19 years (1977-1995). HuCV/Sa/82/J strain is thought to be one of the common causes of viral gastroenteritis worldwide. The HuCV/Sa/82/J strain has been detected mainly in infants. Age-related prevalence of antibody to this strain also shows that infections commonly occur in children less than 5 years old, although viruses in the NV and SMA genogroups commonly infect adults. The pattern of acquisition of antibodies to strain HuCV/Sa/82/J is similar to that of other common viral infections. HuCV/Sa/82/J strain is unique virologically and clinically among caliciviruses.

Age Factors↗

PubMed1996

Dot blot hybridization with a cDNA probe derived from the human calicivirus Sapporo 1982 strain.

A dot blot hybridization assay was developed for detection of human calicivirus/Sapporo/82/J (HuCV/Sa/82) or strains closely related to HuCV/Sa/82 in stool specimens. The cDNA derived from the RNA-dependent RNA polymerase (RDRP) region of HuCV/Sa/82 was used as a positive probe and the pBR322 DNA as a negative control probe. Both probes were labeled with digoxigenin and the products of hybridization reaction were detected with an anti-digoxigenin antibody-alkaline phosphatase conjugate. This assay was specific for HuCV/Sa/82 and for HuCV antigenically related to HuCV/Sa/82. The lower limit of sensitivity of this assay was estimated to be about 10(5) physical particles or 10 pg of cDNA, similar to that of the previously developed ELISA for HuCV. In 1273 stool specimens obtained from children with acute gastroenteritis in Sapporo, Japan, 110 (8.6%) contained small round structured viruses by EM and 23 (1.8%) were positive for HuCV antigenically related to HuCV/Sa/82 by either the hybridization assay or ELISA. A higher positive rate was obtained with the dot blot assay (21%) than by ELISA (10%), suggesting that the dot blot assay either detects HuCV more broadly than the ELISA or detects HuCV covered with fecal antibodies which interrupt antigen-antibody reactions in the ELISA. Negative results for detection of Norwalk virus (NV) cDNA and feline calicivirus (FCV) RNA by both this assay and the ELISA indicated that the HuCV/Sa/82 strain is distinct antigenically and genetically from NV and FCV.

Acute Disease↗

PubMed1996

Outbreaks of nosocomial rotavirus gastro-enteritis in a paediatric ward.

UNLABELLED: Faecal samples were collected from patients with gastro-enteritis during two winter seasons on a paediatric ward. Three outbreaks of nosocomial rotavirus gastro-enteritis were identified by latex agglutination and the virus strains were characterized by polyacrylamide gel electrophoresis of the genome nucleic acid and by subgrouping and serotyping enzyme-linked immunosorbent assays (ELISA). One outbreak was caused by serotype 1 rotavirus, one by serotype 2 and the remaining outbreak was caused by a mixture of serotypes 1 and 4. Identical electrophoretic patterns of the rotavirus genome in each outbreak combined with the ELISA results indicate that these three outbreaks were hospital-acquired cases. The index cases in the three outbreaks were community-acquired and one of two index cases in the second outbreak was hospital-acquired. On each occasion, susceptible roommates were easily infected from the index cases and then cross-infection occurred in the paediatric ward. Possible vehicles were the medical staff, especially doctors, parents of infected patients and infected patients who were moved to other rooms. One patient who had been treated with a series of antitumour therapies excreted rotaviruses in faeces for a long time period and probably played a role as a source of the outbreak. Moreover, some patients still excreted rotaviruses in their normal stool 1 week after recovery from gastro-enteritis. These findings indicate that continual examination of stool samples for rotaviruses until they are negative may be important to prevent the spread of rotavirus infection. CONCLUSION: Nucleic acid analysis and serotyping ELISA are useful tools for analyzing nosocomial rotavirus gastro-enteritis and important to prevent the spread of rotavirus infection in institutions.

Child↗

PubMed1996

Ventromedial hypothalamic lesion-induced vagal hyperactivity stimulates rat pancreatic cell proliferation.

BACKGROUND & AIMS: Ventromedial hypothalamic (VMH) lesions cause an increase in DNA content in the rat pancreas. This study examined the role of cell proliferation in the mitotic response of the rat pancreas after VMH lesion formation. METHODS: Alterations in rat pancreatic DNA content, DNA synthesis, and labeling indices using antiproliferation cell nuclear antigen molecular antibody were measured 0,1,3, and 7 days after VMH lesion formation. Additionally, the effects of vagotomy, atropine, or anti-insulin antibody on VMH lesion-induced alterations in DNA synthesis were examined. Pancreatic samples were also treated with double immunostaining: first for PCNA and then for insulin, glucagon, and somatostatin. RESULTS: Pancreatic weight, DNA content, and DNA synthesis increased in animals receiving VMH lesions. Proliferation was primarily observed in islet B and acinar cells beginning 1 day after VMH lesion formation, reaching a maximum rate after 3 days. VMH lesion-induced stimulation of DNA synthesis was completely inhibited by vagotomy or atropine administration but not by anti-insulin antibody. CONCLUSIONS: Vagal hyperactivity produced by VMH lesions stimulated cell proliferation of rat pancreatic islet B and acinar cells primarily through a cholinergic receptor mechanism.

Analysis of Variance↗

PubMed1995

Molecular characterization of a human calicivirus with sequence relationships closer to animal caliciviruses than other known human caliciviruses.

cDNA clones were produced from a morphologically typical human calicivirus (HuCV) in stool specimens collected in 1982 during an outbreak of gastroenteritis in Sapporo, Japan. The cDNA clones were generated separately in two laboratories by reverse transcriptase-polymerase chain reaction (RT-PCR) using primers 35 and 36 derived from Norwalk virus. The RT-PCR product from six specimens was of the predicted size, had a continuous protein encoding frame on the positive strand, and contained GLPS and YGDD amino acid motifs at the predicted distance from the primers. RT-PCR amplification with primer 35 and a HuCV/Sapporo-specific primer 36 of four HuCV/Sapporo-positive stool specimens from a 1986 Houston day care center outbreak yielded products with 93% nucleotide and 99% predicted amino acid sequence identity with the HuCV/Sapporo strain from the 1982 outbreak. The HuCV/Sapporo strains are genetically distinct from previously characterized HuCVs and more closely related to known animal CVs than other known HuCVs.

Animals↗

PubMed1995

Ventromedial hypothalamic lesions induce the proliferation of gastrointestinal mucosal cells in the rat.

We reported recently that ventromedial hypothalamic (VMH) lesions increased the synthesis of DNA in the gastrointestinal tract of rats by the firing of vagus nerve activity, mainly via cholinergic receptor mechanisms. In the present study, we examined whether the mitotic response is due to proliferation of a cell population--mucosal, submucosal, or muscular layer. A monoclonal antibody to proliferating cell nuclear antigen (PCNA) has previously been shown to be capable of identifying proliferating cells. Samples of formalin-fixed gastrointestinal epithelium, taken before and after VMH lesioning, were immunostained with the anti-PCNA monoclonal antibody, and the labeling index (LI) was determined. To discriminate the effect of hyperphagia in VMH lesioned rats, we utilized the method of pair-feeding. Cell proliferation was examined by the PCNA-labeling technique 0, 1, 3, and 7 days after VMH lesioning. The increase in proliferation was confined to cells in the mucosa and did not involve the muscularis and serosa. Studies in control animals showed that the LI was higher in the small intestine than in other gut segments, and higher in the large intestine than in the stomach. The mean PCNA-LI began to increase at 1 day and continued to increase for 3 days, then decreased 7 days following the lesioning. Results indicate that the gastrointestinal mucosa is in a state of hyperproliferation after VMH lesioning.

Animals↗

PubMed1995

Thermal stability of chimeric isopropylmalate dehydrogenase genes constructed from a thermophile and a mesophile.

Chimeric isopropylmalate dehydrogenases were constructed by connecting the genes isolated from an extreme thermophile, Thermus thermophilus, and a mesophile, Bacillus subtilis. These genes were expressed in Escherichia coli. The enzymes were purified and analysed. Enzymes of T.thermophilus and B.subtilis and chimeric enzymes showed similar enzymological characteristics except for thermal stability. The stability of each enzyme was approximately proportional to the content of the amino acid sequence from the T.thermophilus enzyme. The results suggested that amino acid residues contributing the thermal stability distribute themselves, in general, evenly at least in the N-terminal half of the amino acid sequence of T.thermophilus isopropylmalate dehydrogenase.

3-Isopropylmalate Dehydrogenase↗

PubMed1995

The crystal structure of thermostable mutants of chimeric 3-isopropylmalate dehydrogenase, 2T2M6T.

A chimeric 3-isopropylmalate dehydrogenase (IPMDH), 2T2M6T, was produced by replacing the amino acid sequences of the Thermus thermophilus enzyme with those of the Bacillus subtilis enzyme from residues 75 to 113. Decreased thermostability of the chimeric enzyme was recovered by either evolutionary engineering (I93L) or site-directed mutagenesis (S82R). The 3-D structures of the mutants have been determined by X-ray diffraction at 2.1 A resolution. Although S82R was refined routinely, I93L required the preliminary rigid-body refinement of each domain. The R-factors were reduced to 0.18 for both mutants. Removal of the unfavorable torsion angle at isoleucine 93 may have made I93L more thermostable than 2T2M6T. In the case of S82R, the replaced arginine residue contributed to the extra hydrogen bond with water molecules. The large replaced residue decreased the entropy of the solvent, which may have caused the improvement in enzyme thermostability. Denaturation by heating may be interpreted from these structural results.

3-Isopropylmalate Dehydrogenase↗

PubMed1995

Growth impairment resulting from expression of influenza virus M2 protein in Saccharomyces cerevisiae: identification of a novel inhibitor of influenza virus.

The gene encoding M2, the ion channel-forming protein of influenza virus A, was expressed under the control of an inducible promoter in Saccharomyces cerevisiae. By using single and multicopy plasmids containing GAL promoter-M2 fusions, a correlation was observed between plasmid copy number and growth in medium inducing M2 expression. Cells expressing M2 from multicopy plasmids have reduced growth rates, suggesting that high levels of M2 are toxic to growth. The addition of amantadine, a compound known to block the ion channel activity of certain M2 alleles, restores the growth rates to wild-type levels in cells expressing an amantadine-susceptible allele of M2 but not an amantadine-resistant allele of M2, suggesting that M2 expression in S. cerevisiae results in the formation of functional M2 ion channels. Measurements of extracellular acidification by microphysiometry suggest that proton efflux in M2-expressing cells is altered and that the addition of amantadine permits the reestablishment of the proton gradient. The growth impairment phenotype resulting from M2 expression was used to develop a high-capacity screening assay which identified a novel inhibitor possessing an antiviral profile similar to that of amantadine.

Amantadine↗

PubMed1995

Intracameral ascorbic acid, glutathione and protein levels in albino and pigmented rabbits.

We performed a comparative investigation into differences between albino and pigmented rabbits in respect of intracameral concentrations of ascorbic acid, glutathione and total protein with high-performance liquid chromatography. The intracameral concentrations of reduced ascorbic acid, total ascorbic acid, reduced glutathione, total glutathione and protein in albino rabbits were similar (p > 0.05) to those in pigmented rabbits.

Animals↗

PubMed1994

Facilitation of liver regeneration after partial hepatectomy by ventromedial hypothalamic lesions in rats.

Whether or not the hypothalamus is involved in initiating hepatic DNA synthesis after partial hepatectomy is unclear. To determine the role of the ventromedial hypothalamic nuclei in liver regeneration after partial hepatectomy, we studied hepatic DNA synthesis during liver regeneration in rats with bilateral lesions of these nuclei. Lesions of the ventromedial hypothalamus accelerated the increase in hepatic DNA synthesis and raised the peak level of thymidine incorporation after partial hepatectomy. These effects of hypothalamic lesions were completely inhibited by hepatic vagotomy. Thus, lesions of the ventromedial hypothalamus appear to promote hepatic regeneration by increasing vagal stimulation of the liver.

Animals↗

PubMed1994

Three-dimensional structures of chimeric enzymes between Bacillus subtilis and Thermus thermophilus 3-isopropylmalate dehydrogenases.

The 3-D structures of two chimeric enzymes (4M6T and 2T2M6T) between the Bacillus subtilis and Thermus thermophilus 3-isopropylmalate dehydrogenases were analysed by X-ray diffraction in order to investigate their different thermostabilities. The structure of 2T2M6T was determined by the difference Fourier method and that of 4M6T by rigid body refinement, as based on the structure of the T. thermophilus enzyme. These structures were refined stereochemically to an R-factor of 0.193 at 2.5 A resolution for 4M6T and to an R-factor of 0.195 at 2.2 A resolution for 2T2M6T. The 3-D structures of 4M6T and 2T2M6T were very close to the structure of the T. thermophilus enzyme, conspicuous differences being at the molecular surface. In particular, 2T2M6T having a larger reduction in thermostability was more closely related to the T. thermophilus enzyme. However, their correlations between C alpha-atom displacements and the root squares of the temperature factors were significantly different from each other.

3-Isopropylmalate Dehydrogenase↗

PubMed1994

Epidemiological study of Norwalk virus infections in Japan and Southeast Asia by enzyme-linked immunosorbent assays with Norwalk virus capsid protein produced by the baculovirus expression system.

In this study, we investigated Norwalk virus (NV) antigen and antibody to recombinant NV (rNV) in human populations in Japan and Southeast Asia by enzyme-linked immunosorbent assays (ELISAs). Baculovirus-expressed recombinant NV (rNV) capsid protein was used for preparing antisera to rNV or used as an antigen for detecting antibody to rNV. The ELISAs were specific for NV and had sensitivities equivalent to or higher than those of the previously developed radioimmunoassays. In 159 stool samples obtained from children, mainly younger than 10 years old, with acute gastroenteritis due to small round structured viruses in Japan, only 1 was positive for NV antigen. The pattern of acquisition of antibody to rNV was quite different from those of antibodies to group A rotavirus and human calicivirus Sapporo (HuCV-Sa) strain. The prevalence of antibody to rNV remained at a low level throughout childhood and then showed a steep rise during school age and early adulthood in Japan. A high prevalence of antibody was observed in samples collected from healthy adults in Japan and Southeast Asia. These results suggested that NV infection is common in adults in Japan and Southeast Asia but may be rare in infants in Japan. The HuCV-Sa strain was negative by the ELISA, and no serological relationship between NV and the HuCV-Sa strain was found. NV may be quite different from the HuCV-Sa strain, although both viruses are classified in the family Caliciviridae.

Adolescent↗

PubMed1994

[Effects of hyperventilation upon the spinal pain modulating system (third report)].

The purpose of this study is to investigate the mechanisms of the effect of hyperventilation on the spinal pain modulating system by using phentolamine. Under enflurane anaesthesia, cats received mid-collicular decerebration and lumbar laminectomy. The spinal cord was transected at T12-L1. WDR cells, responding primarily to noxious peripheral stimuli, were sampled with a microelectrode at the depth of 2,000 microns from the cord dorsum. Following the control period, ventilation was changed to induce hypocapnia of PCO2 20-25 mmHg. After activities were well suppressed, phentolamine 0.5 mg with normal saline 1.0 ml was injected on the spinal cord. Changes of firings were investigated. When normocapnia was resumed, recovery followed. Hypocapnia of PCO2 20-25 mmHg significantly suppressed the activities of WDR cells. Phentolamine significantly antagonized the suppressive effects of hyperventilation upon the activities of WDR cells. Our results suggest that the hyperventilation has suppressive effects on single-unit activity of WDR cell and the mechanisms of those suppressive effects are related to adrenergic pain modulating system.

Action Potentials↗

PubMed1994

[Clinical evaluation of controlled hypotensive anesthesia with MR7S1].

Efficacy, safety and the optimal dose of MR7S1, an injectable preparation of sodium nitroprusside, were studied in 37 patients (ASA class I and II) under nitrous oxide-oxygen-enflurane anesthesia. MR7S1 was administered by intravenous infusion. The dose of MR7S1 was increased gradually starting from 0.25 micrograms.kg-1.min-1 to the dose which could achieve the target value of systolic blood pressure (80-100 mmHg). Thereafter this dose level was maintained. During the period in which the dose was increased, the blood pressure was reduced in proportion to the rate of administration. With the rate of administration of 1.0 to 3.0 micrograms.kg-1.min-1, a significant decrease in systolic blood pressure (SBP) was observed compared with the pretreatment level of SBP. During the maintenance period, the SBP was maintained around 80 to 100 mmHg at a rate of administration of 0.25 to 3.5 micrograms.kg-1.min-1. Two out of 37 patients showed a slight decrease in PaO2, but these values returned to normal without any treatment. These findings suggest that MR7S1 is a useful agent to control blood pressure for the hypotensive anesthesia.

Adult↗

PubMed1994

[The effects of hyperventilation upon the spinal pain modulating system (second report)].

The purpose of this study is to investigate the effect of hyperventilation on the spinal pain modulating system by using naloxone. Under enflurane anaesthesia, cats were prepared with midcollicular decerebration and lumbar laminectomy. The spinal cord was transected at T12-L1. WDR cells, responding primarily to noxious peripheral stimuli, were sampled with a microelectrode at the depth of 2,000 microns from the cord dorsum. Following the control period, ventilation was adjusted to produce hypocapnia of PCO2 20-25 mmHg. After activities of WDR cells were well suppressed, naloxone 0.1 mg.kg-1 was given intravenously. Changes of firings of WDR cells were investigated. Returning to normocapnia, recovery of firings was followed. Hypocapnia of PCO2 20-25 mmHg significantly suppressed the activities of WDR cells. Naloxone significantly antagonized the suppressive effects of hyperventilation upon the activity of WDR cell. Our results suggest that the hyperventilation has suppressive effects on single-unit activity of WDR cell and the mechanisms of those suppressive effects are related to pain modulating system by endogenous opiates.

Action Potentials↗

PubMed1993

Comparison of DNA synthesis in white and brown adipose tissue in rats with ventromedial hypothalamic lesions.

Ventromedial hypothalamic (VMH) lesions cause excessive fat accumulation in white adipose tissue (WAT), and brown adipose tissue (BAT); however, little information is available on whether or not cell proliferation occurs in WAT and BAT after VMH lesioning. In this study, we determined the DNA content and thymidine incorporation in unilateral parametrial WAT and interscapular BAT 0, 1, 3, and 7 days after VMH lesioning, and examined the mechanism of increased DNA content in WAT. In rats with VMH lesions, the weight of WAT and BAT had increased significantly at 7 days, and the DNA content and thymidine incorporation of WAT had increased significantly at 3 days and continued to increase for up to 7 days, while those of BAT did not increase for as long as 7 days after VMH lesioning. Restricted food intake according to the pair-feeding method partially inhibited the increased DNA content in WAT. The increased DNA content in WAT was mostly restored but not completely by the administration of anti-insulin antibody, and by administration of propranolol, a beta-adrenergic blocker. The results demonstrated that VMH lesions induced DNA synthesis in WAT early after VMH lesioning, but did not induce DNA synthesis in BAT, and suggested that either hyperinsulinemia or a beta-adrenergic receptor mechanism or both may be responsible for the increased DNA content in WAT.

Adipose Tissue↗