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Biomedical subjects

K Ochi

Publications and source records attributed to K Ochi.

At least 19 recordsLinked to original sources

Polyacrylamide gel electrophoresis analysis of ribosomal protein: a new approach for actinomycete taxonomy.

The ribosomal (r)-proteins from eleven Streptomyces strains representing various numerical taxonomic clusters were compared by two-dimensional polyacrylamide-gel electrophoresis (2D-PAGE). The protein patterns were specific for each species. An attempt was made to identify one strain of Streptomyces by both traditional taxonomic methods and 2D-PAGE analysis of the r-protein patterns. Both methods identified the strain as Streptomyces lavendulae, and protein pattern analysis also showed that S. griseolavendus was a variant of S. lavendulae. Actinomycete r-protein AT-L30 exhibited electrophoretic mobility that is specific for each genus. On the basis of this observation, we analyzed AT-L30 r-proteins from numerous strains of species belonging to the genera Actinomadura, Microtetraspora, Streptosporangium, Nocardia, Rhodococcus and mycolate-less wall chemotype-IV actinomycetes. The results strongly supported the conclusions of previous work and thus proved the efficacy of r-protein analysis as a novel approach for taxonomy of actinomycetes.

Actinomycetales

Enzyme immunoassay for specific analysis of pancreatic stone proteins in human pancreatic juice.

In order to study the concentration of pancreatic stone protein (PSP) in human pancreatic juice, we investigated the influence of the insoluble form of PSP-S1 converted from PSP-S2-5 on PSP determination and the assay method for PSP-S1 precipitate after solubilizing PSP-S1. When bovine trypsin was added to pancreatic juice, PSP-S1 was converted from PSP-S2-5 and precipitated about 45-85% after 1 h. The precipitated PSP-S1 was dissolved in 0.1 M sodium acetate buffer, pH 4.0, and the concentration was measured by the enzyme immunoassay, with similar reactivity to PSP-S1 and PSP-S2-5. The proposed method can offer accurate and specific analysis of the PSP level in pancreatic juice. The results of the fractionation of pancreatic juice and duodenal juice on Mono S cation-exchange chromatography suggested that the major component of PSP was PSP-S2-5 in pancreatic juice and PSP-S1 in duodenal juice.

Calcium-Binding Proteins

In vitro and in vivo effects of diethylene triamine penta-acetic acid on the distribution of indium-111 monoclonal antibody metabolism.

The effects of diethylene triamine penta-acetic acid (DTPA) on indium-111 monoclonal antibody (MoAb) metabolism were examined. Sequential analysis of 111In-MoAb incubated in serum at 37 degrees C by high performance liquid chromatography (HPLC) and electrophoresis revealed that the radioactivity gradually moved from the MoAb to a 70-90 kDa molecular weight fraction. DTPA inhibited the transchelation of 111In to this fraction. It also decreased 111In uptake by isolated rat hepatocytes but did not remove 111In incorporated in hepatocytes. The daily in vivo administration of DTPA (0.5-2.0 mg/mouse daily) to athymic mice after 111In-MoAb injection significantly reduced the 111In uptake in the liver and kidney. The tumour uptake was decreased somewhat but not significantly. The serum radioactivity in the 70-90 kDa fraction was also decreased. Scintigraphic examination demonstrated a decreased liver uptake in the DTPA-treated group of mice. Our results show that 111In released from the DTPA-MoAb conjugate in serum binds to molecules of 70-90 kDa and that DTPA decreases the 111In uptake in this fraction, which induces a decrease of 111In accumulation in normal tissues.

Animals

Immunohistochemical and ultrastructural studies of intraluminal crystalloids in human prostatic carcinomas.

Intraluminal crystalloids (ICr) observed in 19 cases of incidental or invasive human prostatic carcinoma (PCa) and in a case of benign prostatic hyperplasia were examined extensively by immunohistochemistry and electron microscopy. They were brilliantly eosinophilic with haematoxylin and eosin, manifesting needle-like, triangular, rectangular, hexagonal and irregular lump-like in shape. They were strongly positive, dark blue, with phosphotungstic acid -haematoxylin (PTAH) stain in all cases examined. Among the human antibodies tested, epithelial membrane antigen (EMA) gave specifically positive immunostainability with ICr in all cases. Annual ring-like lamellar or concentric structures were detected by electron microscopy. Positive staining of ICr with PTAH and anti-EMA antibody is very useful as a diagnostic marker for PCa in human prostatic tissues.

Aged

Heat-induced aggregation of recombinant erythropoietin in the intact and deglycosylated states as monitored by gel permeation chromatography combined with a low-angle laser light scattering technique.

Aggregate formation of recombinant human erythropoietin (r-EPO) on heat-treatment was followed by gel permeation chromatography combined with a low-angle laser light scattering technique under various conditions with respect to pH and salt concentration in order to provide basic knowledge about the change strictly required to be monitored for medicinal proteins. When heated at 60 degrees C at neutral pH, an aggregate with a limited size consisting of about 20 r-EPO molecules was formed. On heating at 50 degrees C at acidic pH, aggregation was unlimited. The aggregation proceeded non-covalently in acidic pH, but in alkaline pH covalent bond formation was also involved. Increase in salt concentration enhanced the aggregation. Deglycosylation of the N-linked oligosaccharides made r-EPO remarkably susceptible to aggregation on heat-treatment, indicating that the carbohydrate chains are essential to the stability of r-EPO.

Animals

Electrophoretic heterogeneity of ribosomal protein AT-L30 among actinomycete genera.

The ribosomal proteins from 17 type strains of species belonging to various actinomycete genera were compared by two-dimensional polyacrylamide gel electrophoresis. I detected a striking variability among certain ribosomal proteins (designated AT-L30 proteins) with respect to electrophoretic mobility in the first dimension. In contrast, such variability was not observed among ribosomal L30 proteins from other bacteria, such as Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, and Staphylococcus aureus. Although actinomycete AT-L30 proteins from different taxa exhibited considerable heterogeneity in electrophoretic mobility, within each genus the proteins had a specific mobility characteristic. On the basis of this observation, the ribosomal AT-L30 proteins from 11 type strains of species belonging to the mycolic acid-containing genera Nocardia, Rhodococcus, Gordona, and Tsukamurella were analyzed. The relative electrophoretic mobilities of AT-L30 protein preparations from these strains, as determined by two-dimensional gel electrophoresis, revealed that the genera Nocardia, Rhodococcus, Gordona, and Tsukamurella can be sharply separated from each other. My results are consistent with the previously discussed view that each of these genera merits separate genus status.

Actinomycetales

Polyacrylamide gel electrophoresis analysis of ribosomal protein AT-L30 from an actinomycete genus, Streptosporangium.

We analyzed the ribosomal AT-L30 proteins from 11 type strains of species belonging to the genus Streptosporangium. The electrophoretic mobilities of the AT-L30 preparations from these strains, as determined by two-dimensional polyacrylamide gel electrophoresis, revealed that they could be divided into three groups. The first group contained Streptosporangium viridogriseum, S. viridogriseum subsp. kofuense, and S. albidum, while the second group contained S. roseum, S. album, S. vulgare, S. nondiastaticum, S. fragile, S. violaceochromogenes, and S. amethystogenes. S. corrugatum was a member of the third group. These groups were completely consistent with Nonomura's previous classification, which was based on morphological criteria. The results of partial amino acid sequencing of AT-L30 preparations from several representative strains strongly supported the hypothesis that each of the three groups of the genus Streptosporangium merits separate generic status.

Actinomycetales

The possible role of ADP-ribosylation in sporulation and streptomycin production by Streptomyces griseus.

Mutants resistant to 3-aminobenzamide, a known inhibitor of ADP-ribosyltransferase, were obtained from Streptomyces griseus IFO 13189, a streptomycin-producing strain. One (strain no. 4), which had significantly reduced ADP-ribosyltransferase activity, was analysed in detail. Mutant 4 displayed a conditional phenotype with respect to cultivation temperature. At 30 degrees C, it exhibited severely reduced ability to produce aerial mycelium (on solid medium) and submerged spores and streptomycin (in liquid culture), but this ability was fully restored at 25 degrees C. The mutant produced A-factor normally, regardless of cultivation temperature, and exhibited normal ability to accumulate ppGpp intracellularly. SDS-PAGE analyses of cellular proteins labelled by [32P]NAD revealed that an ADP-ribosylated protein with a molecular size of 44 kDa, which appeared in sporulating cultures of the parent strain, was missing from the mutant grown at the non-permissive temperature (30 degrees C). Genetic analysis showed that the aba mutation conferring resistance to 3-aminobenzamide was tightly linked to the altered phenotype. Failure to ADP-ribosylate certain cellular protein(s), presumably due to the aba mutation, may be responsible for impaired differentiation in this mutant.

Adenosine Diphosphate

Cytokine-related immunopotentiating activities of paramylon, a beta-(1-->3)-D-glucan from Euglena gracilis.

Paramylon, a beta-(1-->3)-D-glucan, isolated from Euglena gracilis, was tested for its adjuvant activity on the antibody response to sheep red blood cell (SRBC) in mice. Paramylon markedly enhanced anti-SRBC plaque-forming cell production at a dose of 10 mg/kg. It was also found that in vitro addition of lipopolysaccharide in culture to macrophages from paramylon-treated mice produced a large amount of interleukin 1 (IL-1) and there was a significant level of interleukin 6 (IL-6) induced transiently in the blood of these mice. As IL-1 and IL-6 play crucial roles in the immune response to T cell-dependent antigens like SRBC, the immunopotentiating effect of paramylon might be expressed through the action of these cytokines.

Adjuvants, Immunologic

Asthma classification by a score calculated from clinical findings and examinations in subjects sensitive to inhalant allergens.

Twenty-one patients with atopic asthma were classified into three types according to their symptoms (clinical diagnosis): Ia, simple bronchoconstriction; Ib, bronchoconstriction + hypersecretion; and II, bronchiolar obstruction, and this classification was compared with a classification made according to clinical findings and examinations (score diagnosis). Type Ib asthma was characterized by the increased incidence of eosinophils in bronchoalveolar lavage fluid (BALF), while type II was characterized by ventilatory dysfunction in small airways and the increased incidence of neutrophils in BALF. Four patients, whose expectoration was between 50 and 99ml/day, of the 12 with type Ia assessed by clinical diagnosis were evaluated as type Ib by score diagnosis. One patient with type II by clinical diagnosis was assessed as questionable type II by score diagnosis. In the other 16 patients, the clinical and score diagnoses were the same.

Administration, Inhalation

Spa therapy improves ventilatory function in the small airways of patients with steroid-dependent intractable asthma (SDIA).

The improvement of ventilatory function by spa therapy was examined in 37 patients with steroid-dependent intractable asthma (SDIA) in relation to clinical asthma types. All subjects had been on long-term corticosteroid therapy before spa therapy. Spa therapy was found to improve the values of ventilatory parameters. The percent improvement in all subjects was +4.1% in %FVC, +8.9% in FEV1.0%, +9.8% in %PEFR, +22.0% in %MMF, +19.7% in %V50 and +28.2% in %V25, respectively. Relating to clinical asthma types, moderate improvement in %MMF and %V25 from 16.7% to 16.9% was observed in type Ia cases, and marked increase in %MMF, %V50 and %V25 was observed from 42.2% to 43.2% in type Ib cases. However, no significant increase was found in these parameters of types Ia or Ib after spa therapy. In patients with type II, a significant increase was shown in %V50 (p less than 0.05) and %V25 (p less than 0.01) after spa therapy. The results show that spa therapy improves the condition of small airways disorder in patients with SDIA.

Adrenal Cortex Hormones

Cellular composition of fluid in the airways of patients with house dust sensitive asthma, classified by clinical symptoms.

The cellular composition of bronchoalveolar lavage (BAL) fluid was observed in 28 patients with house dust mite-sensitive asthma. The proportion of neutrophils in the BAL fluid was significantly higher in the patients between the ages of 50 and 59 and those over 60 than in the patients younger than age 39. There was a significant difference in the number of neutrophils in patients aged 30-39 years and in those over 40 at the onset of disease. The cellular composition varied among the three types of asthma; the mean percentage of cells in the BAL fluid was 1.1% neutrophils and 21.3% eosinophils in type Ia (simple bronchoconstriction), 9.1% neutrophils and 23.0% eosinophils in type Ib (bronchoconstriction + hypersecretion), and 34.4% neutrophils and 12.0% eosinophils in type II (bronchiolar obstruction). The results suggested that an increased frequency of both neutrophils and eosinophils correlates with the mechanism of hypersecretion, and that a marked increase in the proportion of neutrophils is involved in bronchiolar obstruction.

Adult

[Biopsy of head and neck lesions with a Biopty biopsy instrument].

Recently, "Biopty-Gun" technique has been introduced as a new biopsy technique specially in urological field. We applied it to obtain histopathological specimens of head and neck lesions. 25 specimens were taken with 18-gauge needle, using ultrasonic guidance in cases where the targets were very close to arteries or very small or cystic lesions. All these 25 specimens could be obtained without any complication such as crushing of tissue and they were all sufficiently clear and high-quality specimens. In 23 of these 25 specimens, exact histopathological diagnosis could be made. "Biopty-Gun" minimizing possible movement of tissue which may often occur with the conventional biopsy technique, permitted obtaining tissue specimens without crushing or damaging cells of tissue to be sampled, and it was concluded to be a highly safe and useful biopsy procedure.

Adolescent

[Potential application of DTPA for improving the image quality in radioimmunoscintigraphy with 111In labeled monoclonal antibodies].

Studies on application of diethylenetriamine-pentaacetic acid (DTPA) to decrease the non-specific localization of 111In-labeled antibodies (111In-MoAb) to the liver were performed. The transchelation of 111In to the molecule of M.W. 70,000-90,000 by the incubation of 111In-MoAb in human serum was inhibited by adding DTPA. These in vitro findings were also demonstrated by serum analysis after 111In-MoAb injection to the normal mice and postinjection of DTPA. By addition of DTPA, the uptake of 111In-MoAb by isolated rat hepatocytes was inhibited, but the 111In-release was unaffected. The addition of DTPA had no effect on uptake of 111In-MoAb and 111In-release in tumor cells. These results suggest that DTPA administration following 111In-MoAb injection may inhibit transchelation of 111In to transferrin or other serum proteins, so decrease non-specific liver localization of 111In and give rapid blood clearance.

Animals

Clinical trial with a protease inhibitor gabexate mesilate in acute pancreatitis.

To evaluate whether early administration of protease inhibitors could improve mortality and morbidity in acute pancreatitis (AP), we made a retrospective analysis of 23 patients with severe AP and 88 with mild to moderate AP who were treated in our institute and four affiliated medical centers during the 10-y period from 1980 to 1990. Intravenous infusion of a protease inhibitor, Gabexate Mesilate (FOY), was started within 24 h from onset of AP (early administration) in 17 patients with severe AP and 51 with mild to moderate AP. The remaining patients were put on FOY later than 24 h from onset of AP (late administration). Comparison of the mortality and morbidity between the two groups, early vs late administration of FOY, led to the following conclusions: (1) Early administration of FOY significantly improved mortality (29.4 vs 83.3%) in severe AP, although the improvement in mortality was not directly proportional to the shortening of the time lag between the onset of AP and the start of FOY, and (2) earlier administration of FOY brought about significantly earlier recovery of abdominal pain, hyperamylasemia, and leucocytosis in mild to moderate AP.

Acute Disease

Pleiotropic effects of a relC mutation in Streptomyces antibioticus.

Ochi (Agric. Biol. Chem. 51:829-835, 1987) has isolated a relaxed mutant of Streptomyces antibioticus, designated relC49, relC49 accumulates significantly lower levels of ppGpp than the parent stain, IMRU3720. At its maximum, the ppGpp level in relC49 was only one-fourth that observed in strain IMRU3720. Interestingly, a burst of ppGpp synthesis between 18 and 22 h of growth in IMRU3720 coincided with the onset of actinomycin production in that strain. As shown previously, the activity in protein synthesis of ribosomes from strain IMRU3720 decreases with the age of the culture. The decrease in activity was less pronounced in cultures of relC49. relC49 mycelium contains reduced levels of phenoxazinone synthase, a key enzyme involved in actinomycin biosynthesis. The rel mutation prevents the normal increase in the activity of one of the other enzymes required for production of the antibiotic, 3-hydroxyanthanilate-4-methyltransferase, and a third enzyme, actinomycin synthetase I, appears to be completely absent from relC49 mycelium. Levels of phenoxazinone synthease mRNA were examined by RNA dot blotting with the cloned phenoxazinone synthase gene as a probe. mRNA levels for phenoxazinone synthase were dramatically reduced in relC49 compared with strain IMRU3720. These results are discussed in terms of the possible regulation of the onset of actinomycin production by ppGpp.

Dactinomycin