[MRSA in bacterial infections].
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Biomedical subjects
Publications and source records attributed to K Hara.
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To elucidate the effects of endothelium-derived relaxing factor (EDRF) released from vascular endothelial cells (ECs) on handling of intracellular calcium ion (Ca2+i) in ECs themselves and vascular smooth muscle cells (VSMCs), we measured the Ca2+i by two-dimensional digital image analysis of fura-2-loaded ECs and VSMCs in tissue culture. In isoculture of one cell type, adenosine triphosphate (ATP, 1 microM) transiently increased the Ca2+i of both ECs and VSMCs. High-K+ depolarization or angiotensin II also elevated the Ca2+i of VSMCs, whereas neither stimulants changed the Ca2+i of ECs. In coculture of ECs with VSMCs, the same dose of ATP rapidly increased the Ca2+i of ECs and then transiently decreased the Ca2+i of VSMCs to below the resting level. The maximal Ca2+i-modulating effects of ATP on both cell types were reproducible after the second application of ATP. Three kinds of EDRF blockers (L-NG-monomethylarginine, methemoglobin, or methylene blue) potentiated the ATP-induced Ca2+i rise in ECs and attenuated the Ca2+i reduction in VSMCs, suggesting the autocrine and paracrine effects of EDRF on ECs and VSMCs, respectively. However, neither indomethacin, superoxide dismutase, nor neutralizing monoclonal antibody to endothelin-1 altered the second responses. Thus, two-dimensional Ca2+i image analysis of ECs and VSMCs in coculture enabled direct visualization of the EDRF actions in ECs and VSMCs and their modifications.
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We previously have shown that insulin treatment of cells greatly increases the activity of phosphatidylinositol (PI) 3-kinase in immunoprecipitates made with an antibody to phosphotyrosine. However, the association of PI 3-kinase activity with the activated insulin receptor is not significant under these conditions. In the present study, we have attempted to reconstitute the association of PI 3-kinase activity with the activated insulin receptor in vitro. PI 3-kinase activity does indeed associate with the autophosphorylated insulin receptor in our in vitro system. The autophosphorylation of the insulin receptor and/or its associated conformational change appear to be necessary for the association of PI 3-kinase activity with the receptor, since kinase negative receptor failed to bind PI 3-kinase activity. After binding, PI 3-kinase or its associated protein seems to be released from the activated receptor after the completion of its tyrosine phosphorylation by the receptor. Tyr960 in the juxtamembrane region of the insulin receptor beta-subunit seems to be involved in the association of PI 3-kinase activity with the receptor, but not C terminus region of the beta-subunit including two tyrosine autophosphorylation sites (Tyr1316 and Tyr1322). The in vitro assay system for the association of PI 3-kinase activity with the insulin receptor can be utilized to study the mechanism of interaction of these molecules and will be an useful method to detect other associated molecules with the insulin receptor.
Activity of prolyl endopeptidase (EC 3.4.21.26) which hydrolyses the Pro7-Phe8 bond in angiotensin II has been found to elevate in experimentally produced granulomatous inflammation in liver and skin. We purified the enzyme 1,536-fold by 6 steps from murine hepatic granulomas. The purified enzyme has a molecular weight of 79 kDa and physicochemical properties equivalent to those previously reported for prolyl endopeptidase purified from other sources. By HPLC analysis, the cleavage of Phe8-Leu10 and Phe8 from angiotensin I and II, respectively, was detected and quantified. Monospecific IgG was prepared from serum of rabbits injected with purified enzyme. Concentration of the enzyme was immunohistochemically detected in cells which form granulomatous organization, but not in inflammatory cells surrounding the foci. The antibody, however, cross reacted with the enzyme in adjacent liver cells and weakly stained their cytoplasm. The findings indicate that this enzyme, in addition to angiotensin converting enzyme, may serve as a useful biochemical marker for granulomatous tissue reactions.
An indirect method to measure beta-glucan, a major structural component of yeast cell walls, is available, but has the disadvantage of requiring the combined use of two assays. Recent reports describe the fungal index, which measures the difference between the conventional limulus test, in which factors C and G react with endotoxin and beta-glucan, and a new endotoxin-specific test, in which only factor C reacts with endotoxin. The G test was developed as a direct method to measure beta-glucan, and contains only factor G reacting with beta-glucan alone. In this study, the G test was examined in sera of rabbits with experimental systemic candidiasis, and compared with the fungal index and mannan assay. The G test showed positive in all rabbits with systemic candidiasis faster and with higher titers than with the fungal index. Three rabbits with fulminant systemic candidiasis showed higher levels of reactivity with the G test and the fungal index than two rabbits with mild reactions. Mannan was positive by at least one serum in four of five rabbits by the latex agglutination test, and there was a good correlation between these assays. The G test is a good serodiagnostic method for the detection of candidiasis.
We report herein a case of myasthenia gravis associated with thymoma and occult thyroid carcinoma with metastasis to a cervical lymph node, which was treated in two stages by a total thymectomy and radical dissection of the bilateral neck after a total thyroidectomy. A 48 year old woman was admitted with right blepharoptosis and hypodynamia of the proximal muscles. After various examinations, a diagnosis of myasthenia gravis was made. The association of thymoma was clarified upon CT scanning and a total thymectomy performed. However, after surgery, swelling of the cervical lymph nodes became apparent and a biopsy revealed metastasis of thyroid carcinoma in the lymph nodes. A diagnosis of occult thyroid carcinoma with metastasis to a cervical lymph node was subsequently made and a total thyroidectomy and radical dissection of the bilateral neck performed 37 days after the total thymectomy. The patient's postoperative course was uneventful and for the time being, no recurrence is expected. It is considered better to perform two-stage operations for easier and safer postoperative management when myasthenia gravis associated with occult thyroid carcinoma is treated, although it depends on the development of thyroid carcinoma being occult.
The toxic effects of bis (tributyltin) oxide (TBTO) on the rat liver were studied with an electron microscope and the accumulation sites of tin were determined with an X-ray microanalyzer. The activities of serum enzymes and the concentration of serum bilirubin were also analyzed. Male Wistar rats received an intramuscular injection of 0.5 ml/kg of TBTO. Marked swelling of the mitochondria appeared in the hepatocytes 4 h after injection of TBTO. Cytoplasmic vacuoles, which contained degenerated mitochondria, gradually increased in number in these hepatocytes. This in turn may have caused a decrease in the volume of hepatic cell cords and an enlargement of sinusoids in the entire hepatic lobule. However, fine structures of intrahepatic bile ducts were not altered. By X-ray microanalysis, tin peaks were preferentially obtained from swollen mitochondria of the hepatocytes. By polarographic analysis of the respiratory responses of mitochondria, it was demonstrated that rates of state 4 respiration and respiratory control ratio were significantly disturbed in TBTO-treated rats in comparison with those of controls. The activities of AST (aspartate aminotransferase) and ALT (alanine aminotransferase) were significantly increased after TBTO treatment, but those of ALP (alkaline phosphatase), LAP (leucine aminopeptidase) and total bilirubin were not changed. These results indicated that parenterally administered TBTO accumulated in the liver cell mitochondria and disturbed oxidative phosphorylation. Mitochondrial dysfunction might induce severe damage of the hepatocytes. Four days after injection of TBTO, hepatic structures and chemical indices were almost restored by the regeneration of hepatocytes.
Porphyromonas gingivalis has been implicated as a major pathogen in periodontitis. To determine the role of T cells in the regulation of this disease, a method was developed for the generation and characterization of rat T-cell clones with antigen specificity to P. gingivalis whole cells. The clones studied so far demonstrated a T-helper (Th) phenotype W3/13+, W3/25+, OX8- and OX22-. These T-cell clones proliferated in vitro in response to P. gingivalis, but not to other bacteria (Prevotella intermedia, Actinobacillus actinomycetemcomitans, Wolinella recta, Fusobacterium nucleatum, Streptococcus sanguis). Limiting dilution analysis showed W3/25+, OX8- T cells preferentially respond to P. gingivalis, rather than W3/25-, OX8+ T cells. P. gingivalis-reactive W3/25+ T cells belonged to the OX22- population, suggesting that the OX22- T cells may represent memory cells. All clones tested produced interferon gamma, but not interleukin 2. The cloned T-cell F1 significantly enhanced P. gingivalis-specific antibody production (p < 0.03). The availability of these cloned T cells should bring new insight into the mechanism by which T cells regulate oral health and periodontal disease.
The role of the submandibular glands in the excretion of parenterally administered bis (tributyltin) oxide (TBTO) was studied. Fine structural alterations of the submandibular glands were observed with an electron microscope. Accumulation sites of TBTO were determined with an X-ray microanalyzer and tin concentrations in saliva and blood were measured by atomic absorption spectrophotometry.
Invasive carcinomas of the uterine cervix of 38 patients were examined for the presence of human papillomavirus (HPV) genomes and for the state of the c-myc and Ha-ras oncogenes. A combination of Southern blot hybridization and polymerase chain reaction revealed the presence of the genome of HPV type 16 in 17 tumors (45%), that of HPV type 18 in 3 tumors (8%), and that of unknown types in 16 others (42%), while no viral DNA sequences were detected in 2 tumors. Of the 38 tumors, c-myc amplification was found in only 1 tumor, while there was no Ha-ras amplification. Overexpression of the c-myc gene was observed in 15 (44%) of the 34 tumors analyzed, while there was no overexpression of Ha-ras. Of the 23 squamous cell carcinomas analyzed, relapse-free rates at 24 months were 55% in tumors with c-myc overexpression and 100% in case of tumors with no c-myc overexpression, respectively. The results suggest the possibility that activation of the c-myc oncogene is involved in tumor progression.
1. Cathepsin H was purified about 5400-fold from hepatopancreas of carp (Cyprinus carpio) by the method involving ammonium sulfate fractionation, and chromatography on S-Sepharose, DEAE-Sephacel, Ultrogel AcA54, Concanavalin A-Sepharose 4B and GPC on Protein-Pak 125. 2. The purified cathepsin H gave a single protein band on analytical-PAGE, but migrated as two bands of 27,000 and 23,000 mol. wt on SDS-PAGE. 3. Cathepsin H had a pH and temperature optimum of 6.5 and 45 degrees C using Arg-MCA as a substrate, respectively, and was activated by sulfhydryl compounds and inhibited by cysteine protease inhibitors and metal compounds having high reactivities at cysteine residue. 4. The carp hepatopancreas cathepsin H immunoreacted with the monospecific antibody against rat liver cathepsin H, and did not react with the antibodies against carp hepatopancreas cathepsins B and L by the method of immunoelectrophoretic blotting.
Three Japanese families with dysplastic nevus syndrome (DNS) are reported. Each family had at least two members with multiple dysplastic nevi (DN). Family members with DN or multiple moles had paler skin than average Japanese, usually sunburned, and tanned less than average, whereas members with darker skin almost never had DN. Each of two families had one cutaneous malignant melanoma (CMM) patient associated with multiple DN. Both CMMs were on the leg. In Japanese DNS, skin color and sun sensitivity seem to be closely related to DN. The locations of CMMs associated with DNS were unique: No CMMs were found on acral areas where they most frequently occur in Japanese.
The case of a 63-year-old man with acrosyringeal adenomatosis (eccrine syringofibroadenoma of Mascaro) is reported. Asymptomatic papular lesions had appeared on the dorsum of both of his feet 30 years earlier, then extended gradually and symmetrically to his hands, arms, legs, trunk, and auriculae. Although his hair, teeth, and nails were normal, he had glaucoma, chronic sinusitis, chronic otitis media, chronic pancreatitis, and gastrointestinal polyposis. No similar symptoms were found in the family history. Histopathologically, proliferations of pale epithelial cells extended from acrosyringia into the dermis, forming interconnected epithelial cords, within some of which were ductal structures. A syringoma and syringoma-like structures were closely associated with these lesions. Enzyme-histochemical, immunohistochemical, and ultrastructural studies supported the concept that this case represents a benign tumor derived from the acrosyringium, which is different from eccrine poroma. Acrosyringeal adenomatosis seems to be a more appropriate name for such a lesion than does eccrine syringofibroadenoma (Mascaro).
We examined the production of interleukin-6 (IL-6) by human gingival fibroblasts (ATCC CRL 1292) stimulated with lipopolysaccharide (LPS) from Porphyromonas gingivalis and Escherichia coli, or supernatant of human peripheral blood adherent cell culture medium incubated in the presence of IL-1 and the same two LPS. Confluent monolayers of gingival fibroblasts were incubated with stimulants for 6 h at 37 degrees C in 5% CO2 and air. After removal of stimulants, the cell cultures were incubated for an additional 2 or 24 h in the same environment. At the end of the culture period, supernatants were collected and assayed for IL-6 activity by stimulatory IgG production with the human B-lymphoblastoid cell line CESS. The direct effect of LPS on IL-6 production by gingival fibroblasts was much weaker than the indirect one via IL-1 production by adherent cells. The stimulating effect of culture supernatants of adherent cells stimulated with LPS on IL-6 production by gingival fibroblasts was as effective as that of recombinant IL-1, when this latter was added at a concentration equivalent to that contained in the culture supernatant of adherent cells. These results suggest that, although gingival fibroblasts may be involved in the pathogenesis of chronic periodontal disease by the production of cytokines, such a role may not result from a direct stimulation by periodontopathic bacteria. The phenomenon is more likely to be mediated indirectly by IL-1 produced by infiltrating inflammatory cells.
This study was performed to investigate the frequency and distribution of CD5-positive (CD5+) B cells in inflamed gingival tissues using flow cytometric and immunohistochemical analyses. The ability of CD5+ B cells to produce anti-type I collagen antibody was also examined. CD5+ B cells expressed "low" fluorescence intensity in the peripheral blood of both healthy subjects and patients with adult periodontitis. However, in inflamed gingival tissues the intensity of this surface marker was high. The percentage of B cells bearing CD5 surface marker was statistically higher in gingiva than in peripheral blood obtained from both the patients and healthy subjects. These CD5+ B cells were observed in gingival subepithelial connective tissues from the bottom to the middle of the periodontal pocket. This area showed destruction of collagen fibers and dense cell infiltrations. Anti-collagen IgG antibody level in patients' gingival crevicular fluids (GCF) was higher than that in sera from healthy subjects, and slightly higher than in autologous sera. IgM anti-collagen antibody in GCF was lower than in autologous sera and in sera from healthy subjects. EBV-transformed CD5+ B cells produced considerably more IgM and IgG antibody to collagen than CD5- B cells. Therefore CD5+ B cells may contribute to the pathogenesis of inflamed gingival tissues.