Biomedical subjects
Publications and source records attributed to K Iguchi.
Two new marine prostanoids-17,18-dehydroclavulone I (1) and clavulolactone I (2)-were isolated from the Okinawan soft coral, Clavularia viridis. Their structures, including absolute configurations, were determined based on the results of spectroscopic analysis and chemical conversions.
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Zinc exhibits inhibitory effects on apoptosis, and a deficiency in this metal generally causes this type of cell death to occur. In the present study, we found that exposure to zinc results in necrosis of prostate carcinoma cells. When zinc acetate was added to LNCaP or PC-3 cells in monolayer culture, they began to detach from the culture dishes, and viability was lost after 4-8 h. Most of the cell death was found to be due to necrosis as determined by double staining with fluorescein-isothiocyanate-labeled annexin V and ethidium bromide, and by detection of hypodiploid cells. Associated with the induction of necrosis was an increase in low molecular-mass proteins, identified by HPLC analysis to be thymosin beta10, parathymosin and GAGE in LNCaP cells, and thymosin beta4, parathymosin and metallothionein in PC-3. The time course of the increase of thymosin beta10 in LNCaP cells and thymosin beta4 in PC-3 cells was consistent with that of appearance of cell detachment and dead cells. These results indicate that zinc can induce necrosis and suggest that production of proteins including beta-thymosins is involved in induction of processes leading to cell detachment.
Monkey-specific C-reactive protein (CRP) assay methods (enzyme-linked immunosorbent assay (ELISA) and turbidimetric immunoassay (TIA)) were developed. The anti-monkey CRP serum was prepared by immunization of rabbits with the immune complex formed between the acute-phase serum from turpentine oil-inoculated monkeys and goat anti-human CRP serum. The specificity of the rabbit anti-monkey CRP serum was confirmed by immunoelectrophoresis and Western blotting. The purity of monkey CRP prepared by chromatography procedures was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The serum CRP levels in nine normal monkeys, as measured by sandwich ELISA were ranged from 0.26 to 1.42 microg/ml (mean 0.71+/-0.37). The CRP levels in five acute-phase sera of turpentine oil-inoculated monkeys were 248-451 microg/ml (mean 371.2+/-73.8). This monkey-specific CRP assay method was found more sensitive than the human-specific CRP assay method in detecting monkey CRP by TIA.
Pituitary adenylate cyclase-activating peptide (PACAP)-immunoreactive (IR) neurons in the myenteric and submucosal plexus of the rat small and large intestine were examined by immunostaining with purified polyclonal antiserum against PACAP (1-15), using both light and electron microscopy. Many PACAP-IR neuronal cell bodies and fibers were found in the myenteric and submucosal plexus. Many of the PACAP-IR fibers originated from the cell bodies of the myenteric and submucosal ganglia. The ganglia were also innervated by PACAP-IR fibers. PACAP-IR fibers penetrated both the circular and longitudinal muscle layers, confirming the previous observations indicating that PACAP neurons act as motor neurons. Ultrastructural study demonstrated that PACAP-IR nerve terminals formed synaptic contacts with PACAP-IR nerve cell bodies or dendritic processes. This observation suggests that PACAP-IR neurons innervate other PACAP-IR neurons, and that PACAP neurons work as interneurons in the enteric nervous system. PACAP-IR nerve cells received not only PACAP-positive nerve terminal input also PACAP-negative nerve terminal input. It also suggests that PACAP neurons are regulated not only by PACAP-IR enteric neurons, but also by neurons originating elsewhere. Our observations support the view that PACAP-IR neurons are involved in the control of gut motility.
BACKGROUND: The concentration of ECP in serum has been proposed as a marker of airway inflammation in asthma. However, its clinical significance is still to be determined. OBJECTIVES: This study was performed to determine whether concentration of ECP in serum reflects clinical status in asthma and can serve as a predictive parameter. METHODS: Cross-sectional analysis was performed in 28 children with asthma. A total of 91 blood samples was obtained to determine levels of ECP in serum and eosinophil counts. Forced expiratory volume in 1 s was also determined at the time of the sampling. Data were analysed on the basis of asthma symptoms in the 4 weeks before and the 4 weeks after sampling. RESULTS: Serum levels of ECP were significantly lower in patients who had been asymptomatic for 3 or 4 weeks before sampling than in patients who had been symptomatic or asymptomatic for only 1 or 2 weeks. In the former group, serum levels of ECP were higher when patients became symptomatic after sampling than when they remained stable, a finding that suggests that serum levels of ECP may have a predictive value in certain situations. Although the concentration of ECP in serum was not proved to be predictive in the latter symptomatic group, the concentration of ECP was significantly lower when measured again 4 weeks later when the patients' symptoms had resolved. In contrast, levels of ECP were unchanged when patients remained symptomatic, a finding that suggests serum levels of ECP may reflect the clinical response to therapy. CONCLUSIONS: Serum ECP may be a useful marker for monitoring and predicting the clinical course in asthma.
Measurement of serum levels of ECP has been widely used for monitoring airway inflammation in bronchial asthma and recently been applied to measure anti-inflammatory effect of theophylline. However, reduced levels of ECP in theophylline-administered patients may express not only in vivo effect of theophylline but also in vitro effect after sampling because serum ECP measures released ECP during coagulation and theophylline has been reported to inhibit eosinophil degranulation in vitro. In order to answer the question, we tested whether theophylline added to blood after sampling reduces measured levels of serum ECP. Various concentrations of theophylline were added to SST tube, to which venous blood from atopic patients was drawn. Serum was, then, obtained by centrifugation after 15 min to 6 hours of incubation at room temperature. Theophylline significantly reduced serum ECP in a concentration-dependent manner. Percent reduction of ECP levels at 1 hour of incubation were 11.9%, 18.7%, 22.8%, and 51.7% at theophylline levels of 5, 12.5, 22.5, and 120 micrograms/ml, respectively. Kinetics of serum ECP release was also inhibited in the presence of theophylline. These results suggest that in vitro effect of theophylline on serum ECP levels should be considered when data of serum ECP in patients who take theophylline are interpreted.
A reversed passive latex agglutination test (RPLA) using anti-canine haemoglobin (Hb) antibody was developed for detecting bleeding in the lower digestive organs in dogs, and its applicability as a simple test for faecal occult blood was assessed. In Ouchterlony's gel immunodiffusion test, the anti-canine Hb antibody used to sensitize the latex reacted with canine Hb but not with Hbs, plasmas or meat extracts from pigs, goats, sheep, cattle, horses or chickens, or with fish extracts. Using latex sensitized with 50 micrograms/mg of anti-canine Hb IgG antibody, the lowest limit of detection for canine Hb was 21 micrograms/ml, and the latex reacted negatively with all test specimens other than canine Hb. In an in vitro experiment with a mixture of canine faeces and erythrocytes, the antigenicity of the Hb was found to undergo only very slight changes even when the specimens were allowed to stand for 12 h at room temperature. Hb could not be detected by RPLA in any of four successive faecal samples from three experimental dogs after infusion of autologous blood (5, 3 or 1 ml) into the stomach. In 3 other experimental dogs given an infusion of autologous blood (5, 3 or 1 ml) into the ascending colon, the presence of Hb was confirmed by RPLA in all four successive faecal samples obtained from those which received 5 or 3 ml of blood and in all except that obtained following the first defecation from the animal which had received 1 ml of blood.
The ability of IL-5 to induce eosinophil degranulation was investigated. Peripheral blood eosinophils from patients with mildly allergic individuals were isolated with CD16- selection method. Eosinophils were then incubated with interleukin-5 (IL-5) (0.1-100 ng/ml) for 1-48 h and EPX in the supernatants were measured with RIA. We found that IL-5 induced significant amount of eosinophil protein X in a concentration-dependent manner at 24 h. Eosinophil viability was about 90% either in the presence or absence of IL-5 at 24 h. Eosinophils stimulated with IL-5 adhered to the plate. Anti-CD18 mAb blocked adhesion and degranulation induced by IL-5. Dexamethasone and TGFbeta significantly inhibited degranulation in a concentration-dependent manner. These results suggest that IL-5 may be a strong secretagogue for eosinophils, that adhesion via beta2 integrin is a requisite for degranulation, and that the anti-inflammatory effect of corticosteroids and TGFbeta may be exerted at least in part, through the inhibition of eosinophil degranulation.
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Serum and dialysate boron levels in 17 patients with long term hemodialysis (HD) were determined by inductively coupled plasma emission spectrometry (ICPES). Serum boron level was compared with the value of age matched 467 healthy controls and the relationship between serum and dialysate boron level was analyzed. The results showed that serum boron level was significantly higher at the beginning of HD, and lower at the completion of HD in comparison with controls. Although the dialysate was contaminated with trace boron, HD resulted in an excessive decrease of serum boron, rather than boron exposure from the dialysate. Boron hemodialyzability was almost proportional to the gradient of the boron level at the beginning of HD and it could be controlled by the adjustment of the gradient. In conclusion, the serum boron level was very much disturbed in long term HD patients. If boron excess in serum at the beginning of HD, or deficiency at the completion of HD may contribute to the complications of HD patients, fine adjustment and close surveillance of the gradient should be taken into account.
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Eight new marine steroids were isolated from the Okinawan soft coral Clavularia viridis Quoy and Gaimard. Their structures were determined based on spectroscopic analysis, chemical conversion, and X-ray crystallographic analysis. Steroids 5 and 6 showed growth inhibition activity toward HeLa S3 cells and human diploid cells in vitro.
BACKGROUND: The efficacy of immunotherapy is still controversial. To elucidate the mechanisms of immunotherapy, we studied mite-specific induction of IL-2 receptor (IL-2R) expression on T lymphocytes from children with mite-sensitive asthma. METHODS: Peripheral blood mononuclear cells were obtained from 28 children with mite-sensitive asthma: 13 had never received house dust immunotherapy (nonimmunotherapy group), 15 had been receiving house dust immunotherapy at the time of the study (immunotherapy group). After a 6-day culture with or without Dermatophagoides farinae (Df) antigen, the expression of IL-2Rp55 (CD25) and p75 on CD4+ or CD8+ T lymphocytes was measured by flow cytometry. RESULTS: The nonimmunotherapy group showed significant Df-specific CD25 induction on CD4+ T lymphocytes (delta CD4+ CD25+) but little induction on CD8+ T lymphocytes (delta CD8+ CD25+). delta CD4+ CD25+ was correlated with the severity of the disease. In the immunotherapy group delta CD8+ CD25+ was significantly higher than in the nonimmunotherapy group or in normal subjects and correlated with Df-specific IgG4 and cumulative doses of house dust extract, whereas delta CD4+ CD25+ was similar in the nonimmunotherapy and the immunotherapy groups. IL-2Rp75 was not induced either on CD4+ or CD8+ T lymphocytes. CONCLUSIONS: Our data suggest that house dust immunotherapy may have induced Df-specific CD8+ T lymphocytes in patients with mite-sensitive asthma and that the efficacy of immunotherapy may be attributed to the generation of Df-specific CD8+ T lymphocytes.
In a culture filtrate of Trichosporan penicillatum B2, which is a gamma-ray irradiation mutant induced from T. penicillatum SNO3, we found three kinds of pectin-releasing enzymes, protopectinases SE1, SE2, and SE3, that have endo-polygalacturonase activity. These enzymes were purified to homogeneity with cation-exchange and size exclusion chromatographies. The major PPase in the culture filtrate was PPase SE1, which accounted for 75% of total activities in the culture filtrate, and the two others were 0.15% (PPase SE2) and 0.007% (PPase SE3). Their molecular masses were approximately 41, 41, and 42 kDa on SDS-PAGE, respectively. They had similar enzymatic properties but different PPase activity and pH- and thermo-stability. Antibody against PPase S, which is produced by strain SNO3, inhibited the activities of PPase SE1, SE2, and SE3. However PPase SE1 was completely inhibited by treatment with the anti-PPase S antibody, but the activities of PPases SE2 and SE3 remained at 20 and 50% of the original activity, respectively.
We studied the correlation of eosinophil viability enhancing activity (EVEA) in sputum from asthmatic children and clinical symptoms. Sputum from asthmatic children and equal volume of saline were mixed with a Vortex mixer and centrifuged at 40000 G. Clear supernatants were obtained and filtered with 0.22 micron membrane filter. Periphral blood eosinophils purified by Percoll density gradient centrifugation and CD16 negative selection/ immunomagnetic beads technique were incubated with sputum extract for 4 days. Eosinophil viability was examined by staining the cells with fluorescen diacetate and propidium iodide and expressed as eosinophil viability enhancing activity (EVEA). Eosinophil cationic protein (ECP) concentrations in sputum were also measured by a radioimmunoassay. Correlation between EVEA in sputum and pulmonary functions, attack score, treatment score, or sputum ECP was determined by Spearman correlation test. Sputum EVEA was correlated 1) inversely with pulmonary functions on sampling, 2) with attack score of 2 weeks period before sampling, 3) with treatment score of 1 month to 1 week before and 1 to 2 weeks after sampling, and 4) with sputum ECP in individual cases. Sputum EVEA may serve as a suitable parameter for monitoring airway inflammation in asthma.
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