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K Shimada

Publications and source records attributed to K Shimada.

At least 1,027 records · Page 57Linked to original sources
PubMed1991

Role of threonine 190 in modulating the catalytic function of malate dehydrogenase from a thermophile Thermus flavus.

Random mutagenesis of malate dehydrogenase from a thermophilic bacterium, Thermus flavus AT-62, had revealed that a Thr190----Ile replacement near the essential catalytic residue His187 caused marked modulation of the catalytic properties. For further exploration of a role of the residue at this position, this residue was substituted with each of the other amino acids by site-directed mutagenesis. Most of the mutations except for substitution with Ser caused increases in Km for oxaloacetate and increases Ki for oxaloacetate of 2-110 times. Substitution with His or Pro was characterized by the complete loss of substrate inhibition, along with a marked increase in Km for oxaloacetate. Kinetic analyses of the native and altered malate dehydrogenases at various pHs revealed that both Km and Ki for oxaloacetate decreased proportionally to the decrease in pH from 8.40 to 5.75, whereas kcat was nearly constant within the pH range. Apparent shifts of the optimum pH values toward acidity observed with most of the altered malate dehydrogenases were attributed to the increase in Ki, which facilitated the release from the substrate inhibition at a lower pH. Replacement of Thr310, a possible counterpart with which Thr190 forms a hydrogen bond, by Ile caused changes in the catalytic properties similar to those of the Thr190-substituted enzymes. These results suggest that not only the loss of the hydrogen bond between Thr190 and Thr310 but also properties of the residues introduced at position 190 cause modulation of the catalytic properties, probably through dislocation of the loop structure that contains the catalytic residue His187.

Base Sequence↗
PubMed1991

A sensitive nonisotopic method for the determination of intracellular azidothymidine 5'-mono-, 5'-di-, and 5'-triphosphate.

In order to analyze the efficacy of azidothymidine (AZT), it is important to know intracellular concentrations of AZT metabolites. However, it has been impossible to measure intracellular AZT 5'-monophosphate (AZT-MP), AZT 5'-diphosphate (AZT-DP), and AZT 5'-triphosphate (AZT-TP) without using isotopes. In the present study, we developed a new method to measure intracellular AZT metabolites without radiolabeled compounds. The method employed was a high-performance liquid chromatography (HPLC) system programmed for column switching technique, in which two columns were used: column 1 (TSK-G2000-SW, 300 x 7.5 mm) to preseparate AZT metabolites from major cell components, and column 2 (YMC-A-312-ODS, 150 x 6 mm) to determine the metabolites. The limit of detectability of this system was 3.3 pmol/injection. When MT-4 cells were incubated with various concentrations of AZT, intracellular concentrations of AZT-MP increased in parallel with extracellular AZT. Those of AZT-DP and AZT-TP, however, reached plateaus at 5 and 2 microM of AZT, respectively. In MT-4 and Molt-4 cells incubated with 5 microM AZT, concentrations of AZT-MP increased time dependently, while the AZT-DP/AZT-MP ratios decreased with time. These data suggest that high dose of AZT may not necessarily increase intracellular concentration of AZT-TP. The concentrations of AZT metabolites in peripheral blood mononuclear cells in a patient with AIDS and an asymptomatic carrier were measured; the concentrations were comparable to those in cultured cells. Quantitative analysis of intracellular AZT metabolites without the use of isotopes will increase safety and convenience of measurement, and take an effective step in studying pharmacokinetics of AZT in clinical materials.

Acquired Immunodeficiency Syndrome↗
PubMed1991

High-performance liquid chromatographic separation of bile acid pyrenacyl esters with cyclodextrin-containing mobile phase.

The high-performance liquid chromatographic separation of bile acid pyrenacyl esters with cyclodextrin-containing mobile phase is presented. Compared with conventional methods, inclusion chromatography gives much more satisfactory separation of derivatized bile acids in a short time. The application of this method to the separation of glycine-conjugated bile acids in human bile is also described.

Bile Acids and Salts↗
PubMed1991

Magnetic shielding induces early developmental abnormalities in the newt, Cynops pyrrhogaster.

Developing larvae of the Japanese newt, Cynops pyrrhogaster, were subjected for 5 days to a shielded environment in which the static magnetic field was about 10,000 times weaker (5 nT) than the geomagnetic norm, which ranges between 30 and 60 microT at the earth's surface. Larvae from non-cleavage to neurula stages were exposed under shielded or normal (control) conditions and then examined for evidence of developmental abnormalities either 1 day or 20 days after treatment. The magnetic shielding was associated with an increased incidence of somatic defects, especially in larvae that were examined 20 days after shielding. Bi-headedness and intestinal protrusion were observed in magnetically shielded larvae but not in controls. Other abnormalities more frequently observed in shielded larvae were spinal curvature, malformed eyes, and retarded or blocked development. These data are among the first to illustrate the effects of magnetic-field deprivation on a developing animal.

Animals↗
PubMed1991

Role of bone marrow cells in autoantibody production and lymphoproliferation in the novel mutant strain of mice, CBA/KlJms-lprcg/lprcg.

The novel mutant gene, lprcg, is allelic with lpr, but complements the gld gene in induction of lymphoproliferation. Mice with this autosomal recessive mutation, CBA/KlJms-lprcg/lprcg (CBA-lprcg), served in this study to analyze the abnormalities of bone marrow (BM) stem cells responsible for autoantibody production and lymphoproliferation by BM transfer experiments. Transferred CBA-lprcg BM cells might have differentiated into so-called double-negative, anomalous lymphoid cells and caused production of autoantibodies such as anti-DNA antibodies in the environment of normal CBA/KlJms-(+)/+ (CBA-(+)) mice. Macroscopic graft-vs.-host-like disease as reported in lpr----non-lpr BM transfer was not observed in these recipients. In this BM chimera, however, lymphoproliferation did not ensue and the host's lymph nodes became atrophic. The lymphoproliferation required the coexistence of lprcg BM cells and lprcg lymph nodes in CBA-(+) mice. The results indicate that the functions of the lprcg gene are expressed at both BM and lymph node levels. Thus, this mutant strain of mice should provide an excellent model for analyzing aberrant lymphocyte differentiation from the BM cells leading to autoimmunity and lymphoproliferative disorder.

Animals↗
PubMed1991

A novel missense mutation in exon 8 of the ornithine transcarbamylase gene in two unrelated male patients with mild ornithine transcarbamylase deficiency.

We studied two unrelated male probands with mild ornithine transcarbamylase (OTC) (E.C.2.1.3.3) deficiency presenting a similar clinical course. Previous analyses of their liver OTCs also revealed similar properties. To identify the underlying molecular defects, we first cloned the entire coding region of the OTC gene from one proband and found a single base-substitution (C to T) leading to the substitution of tryptophan for arginine at amino acid position 277. Using a genomic amplification technique followed by allele specific oligonucleotide hybridization, we identified the same point mutation in the OTC gene of the other proband. We observed the presence of the mutation among family members in at least three generations, and in one asymptomatic hemizygous sibling in each family.

Alleles↗
PubMed1991

Evidence of effective multidisciplinary treatment for resectable pancreatic cancer from the viewpoint of the CA19-9 level.

Nineteen patients with resectable pancreatic cancer have been treated by a multidisciplinary approach at the National Cancer Center Hospital. This therapy included extended pancreatic resection, intraoperative irradiation by 30 Gy of electrons, and intraoperative hepatic arterial or portal infusion of mitomycin C. Furthermore, postoperative chemotherapy with mitomycin C, using Seldinger's method or iv administration, was added. The study consisted of 13 patients with carcinoma of the pancreatic head and six patients with carcinoma of the body and tail. The one- and three-year survival rates for these patients were 88 and 50%, respectively. The five-year survival rate was calculated as 28%, although there is only one five-year survivor. The effectiveness of this therapy was studied by changes in Carbohydrate Antigen 19-9 (CA19-9) level. Thirteen of the 15 patients with high CA19-9 levels showed a decrease to a level less than 50 U/mL after this therapy. In the postoperative course, the patients showing a tendency of elevation of the CA19-9 level could be suspected of having cancer recurrence at this time without any physical sign or other laboratory findings.

Aged↗
PubMed1991

Developmental expression of the prolactin gene in the chicken.

Steady-state levels of prolactin (PRL) mRNA in the pituitary gland during embryonic development were determined by dot blot analyses to relate the changes with those of pituitary and plasma levels of PRL. Steady-state levels of the 1.38-kb mRNA encoding the PRL prohormone remained low until Day 18 of incubation, increased on Day 19 of incubation, and reached maximum levels on the day of hatch but decreased 1 day after hatch. Changes in both pituitary and plasma concentrations of PRL closely mimicked those changes in PRL mRNA levels. Subsequently, both the levels of the pituitary PRL mRNA and PRL remained unchanged whereas those of plasma PRL increased 7 days after hatch. The results indicate that a progressive expression of PRL gene in the pituitary gland occurs 1-2 days before the hatch and concomitant increases in plasma concentrations of prolactin may be associated with physiologic changes in pulmonary respiration and hatching.

Amniotic Fluid↗
PubMed1991

Vascular smooth muscle cells from genetically hyperlipidemic rabbit (WHHL rabbit) exhibit decreased growth response.

The Watanabe Heritable Hyperlipidemic (WHHL) rabbit is a widely studied animal model for the human genetic disorder familial hypercholesterolemia, and spontaneously develops atherosclerotic disease. We studied the growth characteristics of cultured vascular smooth muscle cells (VSMC) from WHHL rabbits compared with VSMC from Japanese white rabbits. We measured cell proliferation, DNA synthesis, and c-myc proto-oncogene expression, in response to growth stimuli such as fetal bovine serum (FBS) and platelet-derived growth factor (PDGF). VSMC from Japanese white rabbits exhibited a 4-fold increase in cell numbers during a 5-day incubation period compared with those from WHHL rabbits. FBS and PDGF stimulated DNA synthesis, as measured by thymidine incorporation into VSMC, in both Japanese white rabbits and WHHL rabbits, however the response was significantly higher in the former strain. The intracellular pH value of VSMC determined using the pH-sensitive fluorescence dye 2',7'-bis-carboxyethyl-carboxyfluorescein was significantly higher in WHHL rabbits than in Japanese white rabbits. Proto-oncogene c-myc was induced by exposure of VSMC to FBS, however there was no significant difference in c-myc mRNA levels between the two strains. These results suggest that VSMC from WHHL rabbits are not genetically growth accelerated, but show decreased growth response to growth stimuli.

Animals↗
PubMed1991

Quantitative estimation of human immunodeficiency virus type-1 provirus in CD4+ T lymphocytes using the polymerase chain reaction.

The proviral burden of peripheral blood lymphocytes in 29 patients infected with human immunodeficiency virus type-1 (HIV-1) was estimated using a polymerase chain reaction method which we recently refined. The mean numbers of HIV-1 provirus in eight patients with AIDS and AIDS-related complex treated with zidovudine (AZT), in 10 asymptomatic patients treated with AZT, and in 11 asymptomatic untreated patients were 892, 436, and 406 copies in 1 x 10(5) CD4- T lymphocytes, respectively. These results demonstrate that patients may have more HIV-1 provirus copies in an advanced than early stage of disease, and that the fraction of the infected lymphocytes is much higher than previously thought, especially in asymptomatic patients. There was no difference in the viral burden in CD4+ T lymphocytes regardless of whether AZT had been administered or not. These findings validate the method used here to quantitate HIV-1 provirus DNA and confirm that AZT is not effective in reducing the amount of provirus DNA in lymphocytes.

AIDS-Related Complex↗
PubMed1991

Post-mortem diagnosis of human T lymphotrophic virus type-1 (HTLV-1) associated myelopathy by detection of HTLV-1 DNA in the spinal cord of a patient with post-transfusional myelopathy.

Human T lymphotrophic virus type-1(HTLV-1) is thought to be an etiological agent of HTLV-1 associated myelopathy (HAM). Therefore, a definitive diagnosis of HAM can not be made if information on the presence of antibody against HTLV-1 is not available, even though clinical and histopathological features strongly indicate the disease. In this study, HTLV-1 DNA was detected in the formalin-fixed, paraffin-embedded spinal cord of an autopsy case with post-transfusional myelopathy using the polymerase chain reaction (PCR). Ante-mortem HAM was not suspected of and the antibody has not been examined. Neuropathological examination of the sequential sections revealed a loss of axons and myelin sheaths in the white matter, resembling the histopathology of HAM. The post-mortem detection of HTLV-1 DNA by PCR may be useful for the confirmation of HAM in cases which were not provided with appropriate clinical data.

Autopsy↗
PubMed1991

Immune response of mice infected with recombinant vaccinia viruses carrying the HIV gag gene.

We examined mouse immune response to 4 kinds of recombinant vaccinia viruses carrying the HIV gag gene, including vac-gag/pol, which produces HIV-like particles with processed gag proteins; vac-gag, which also produces HIV-like particles but with unprocessed gag protein; and vac-gag-pol-fuse and vac-es-gag/pol, neither of which produces such particles but releases reverse transcriptase and gag protein, respectively, from infected cells. Although infection of mice with recombinant vaccinia viruses induced production of the anti-p24 antibody in all mice, vac-gag/pol and vac-es-pol induced higher production than the other two recombinants. Increase in [3H]thymidine uptake by splenic lymphocytes following p24 antigen stimulation was most evident in mice infected with vac-gag/pol. Thus, the highest immune reaction, both humoral and cellular, was elicited by vac-gag/pol, indicating that among those tested, this recombinant vaccinia virus is the best candidate for a vaccine that induces anti-HIV gag immunity.

Animals↗
PubMed1991

Factors associated with silent multiple lacunar lesions on magnetic resonance imaging in asymptomatic elderly hypertensive patients.

1. Factors associated with advanced cerebrovascular damage incidentally seen on brain magnetic resonance imaging (MRI) were investigated in a population of 34 normotensive and 54 hypertensive asymptomatic elderly individuals (69 +/- 5 years). 2. Four or more 'lacunar' lesions per subject were considered to be advanced changes, based on the distribution in normotensive subjects and were found in 11 (20%) out of 54 hypertensive subjects. 3. Compared with the remaining 43 hypertensive patients with fewer or no lacunae, the proportion of subjects over 70 years of age who had electrocardiographic (ECG) evidence of left ventricular hypertrophy (LVH) was significantly higher in this group of hypertensive patients. Patients with multi-lacunar lesions also showed significantly higher 'office' and average ambulatory, in particular, blood pressure during sleep and less nocturnal blood pressure fall, than those with fewer or no lacunae. Furthermore, they showed a higher predicted whole blood viscosity and a lower high density lipoprotein (HDL)-cholesterol. The predictive values of LVH on ECG for the presence of multi-lacunar lesions in hypertensive patients were substantially high (70-90%). 4. The data suggest that silent advanced cerebrovascular damages on MRI are not rare among asymptomatic elderly hypertensive patients and are associated with various cerebrovascular risk factors. A useful marker for their identification appears to be ECG-LVH.

Aged↗
PubMed1991

[Bacteriological examination of infections in the field of oral surgery].

Etiology of bacterial infections in the field of oral surgery was studied. A total of 270 samples collected from patients with encapsulated abscess in their oral cavities was examined and bacteria were isolated from the 244 samples (90.4%). The following results were found; 1) Organisms more than one from one sample were frequently isolated from cases with parodontitis, pericoronitis and gnathitis. Isolation of anaerobic bacteria was common (54.2%). 2) Streptococcus milleri and Streptococcus sanguis and Capnocytophaga species were the most common isolates among aerobic gram-positive and gram-negative bacteria, respectively. 3) Peptostreptococcus micros and Eubacterium lentum were most frequent isolates among gram-negative anaerobic bacteria. Among gram-negative bacteria, Oral Group Bacteroides, especially Bacteroides gingivalis, Bacteroides intermedius, Bacteroides buccae and Bacteroides oralis were most prominent. 4) Isolation frequency of bacteria (both species and strains) was high from samples obtained from patients before antibiotic chemotherapy. 5) Most strains were sensitive to Midecamycin acetate and Josamycin. Minimum inhibitory concentration of 80% isolates (MIC80) against these antibiotics was 0.39 microgram/ml.

Bacteria↗
PubMed1991

[Ultrasonic nebulised pentamidine for Pneumocystis carinii pneumonia].

Our retrospective study for ultrasonic nebulised pentamidine in 13 patients shows that 7 of 8 patients with Pneumocystis carinii pneumonia (PCP), proven by biopsy examination, were cured (recovery rate, 88%). Two of these 8 patients were mainly treated by nebulised pentamidine and the other 5 patients were administered parenteral pentamidine before the nebulised pentamidine therapy and the final 1 patient died on day 9 without clinical improvement with nebulised pentamidine as well as conventional therapy (trimethoprim-sulphamethoxazole, parenteral pentamidine). No relapse occurred in the 7 cured patients. The inhalation increased the cough in 4 of 13 cases and caused vomiting in 1 patient. Two patients discontinued the nebulisation because of the side-effect. No extra-pulmonary side-effects of nebulised pentamidine were seen. These results show that an efficacy of nebulised pentamidine is at least equal to conventional therapy with fewer side-effects and that only patients with mild PCP without severe hypoxaemia are eligible for the inhalation therapy.

Aerosols↗
PubMed1991

[A case of human-immunodeficiency virus infection related Mycobacterium tuberculosis with atypical clinical features].

Increasing rates of human immunodeficiency virus (HIV) related tuberculosis have been noted and recently the clinical importance of the disease has been mentioned. The diagnosis of tuberculosis is more difficult in those patients with HIV seropositive than those with seronegative, because those with seropositive have atypical clinical features. A 29-year-old male, who was infected with HIV heterosexually in Central Africa in 1986, was admitted to our hospital with a history of general malaise and weight loss in April, 1989. Laboratory and physical examinations revealed anemia, thrombocytopenia, the elevation of LDH, and giant intraabdominal lymphadenopathies, suspecting malignant lymphoma. Mycobacterium was isolated from the sputa in April and was confirmed as M. tuberculosis using a DNA probe in May, 1989. Clinical symptoms including giant lymphadenopathies and laboratory abnormalities improved with antituberculosis therapy. Development of a rapid method for the diagnosis of tuberculosis was warranted in this case.

AIDS-Related Complex↗
PubMed1991

Evaluation of acridinium-ester-labeled DNA probes for identification of Mycobacterium tuberculosis and Mycobacterium avium-Mycobacterium intracellulare complex in culture.

The detectability of mycobacteria in culture by the use of nonisotopic, chemiluminescent DNA probes for Mycobacterium tuberculosis and the M. avium-M. intracellulare complex (MAC) was evaluated and compared with that by the use of 125I-labeled DNA probes for the same mycobacteria. In the assay, rRNA-directed DNA probes labeled with acridinium ester (AE-DNA probes) were used. Unhybridized probes were chemically degraded, and the esterified acridinium on the hybridized probes was hydrolyzed by the addition of alkaline hydrogen peroxide solution, resulting in the production of visible light which was measured with a luminometer. The detection limits of the AE-DNA probes were almost the same as those of the 125I-labeled DNA probes. A total of 107 clinical isolates of mycobacteria (47 isolates of M. tuberculosis, 36 MAC, and 24 atypical mycobacteria other than MAC) were tested. The sensitivity and specificity of the AE-DNA probes for M. tuberculosis were 100% both for the conventional method and with the 125I-labeled DNA probe. The sensitivity and specificity of the AE-DNA probes for MAC were 97.2 and 100%, respectively, for the conventional method and were both 100% with the 125I-labeled DNA probes. Because the procedure is simple, reliable, rapid (it can be completed within an hour), and safe (it does not use radioisotopes), it can easily be performed in any clinical laboratory.

Acridines↗