Biomedical subjects
Publications and source records attributed to H Ohshima.
A theory of electroosmosis in an array of parallel cylindrical fibers of Kozak and Davis (J. Colloid Interface Sci., 112, 403 (1986)), in which the liquid flow is normal to the axes of the cylinders, is developed to derive a simple approximate expression for the electroosmotic velocity for low zeta potentials without involving numerical integration. The relative error is less than 6%. Copyright 1999 Academic Press.
Nitric oxide reacts rapidly with superoxide to form the strong nitrating agent peroxynitrite, which is responsible for much of the tissue damage associated with diverse pathophysiological conditions such as inflammation. The occurrence of free or protein-bound nitrotyrosine (NTYR) has been considered as evidence for in vivo formation of peroxynitrite. However, various agents can nitrate tyrosine, and their relative significance in vivo has not been determined due to lack of a sensitive method to analyze NTYR in tissue proteins and biological fluids. We have developed a new HPLC-electrochemical detection method to analyze NTYR in protein hydrolyzates or biological fluids. The sample is injected directly into a reversed-phase HPLC column and NTYR is subsequently reduced by a platinum column to 3-aminotyrosine, which is quantified with an electrochemical detector. The method is simple, selective, and sensitive (detection limit, 0.1 pmol per 20-microl injection). We have applied this method to compare in vitro the ability of various nitrating agents to form NTYR in bovine serum albumin and human plasma. Yields of NTYR formed in human plasma proteins incubated with 1 or 10 mM nitrating agent decreased in the following order: synthetic peroxynitrite > 3-morpholinosydonimine, a generator of both NO and superoxide > Angeli's salt, which forms nitroxyl anion (NO-) > spermine-NONOate, which releases NO > sodium nitrite plus hypochlorite, which forms the nitrating agent nitryl chloride (NO2Cl). A simple purification method using a C18 Sep-Pak cartridge is also described for analysis of free NTYR in human plasma.
Cannulation of the ascending aorta is the recognized method to achieve arterial return during cardiopulmonary bypass. Mediastinal infection after cardiac surgery can cause disruption at the point of insertion, and give rise to a pseudoaneurysm in the ascending aorta. Although rare, a pseudoaneurysm can occur without clear evidence of infection. Here we report two cases that received aortic valve replacement and that developed ascending aortic aneurysms after surgery. Based on our clinical findings, these aneurysms were related to the arterial cannulation into the aorta and not to any infection.
Peroxynitrite, the reaction product between nitric oxide (.NO) and superoxide, has been presumed to be a mediator of cellular and tissue injury in various pathological situations. It is formed at the convergence of two independent radical-generating metabolic pathways. Its biological effects are due to its reactivity towards a large range of molecules including amino acids such as cysteine, methionine, tyrosine and tryptophan, nucleic bases and antioxidants (e.g. phenolics, selenium- and metal-containing compounds, ascorbate and urate). Peroxynitrite reactions involve oxidation and nitration. The chemical properties depend on the presence of CO2 and metallic compounds as well as the concentrations of reagents and kinetic laws. This complex chemistry can be explained by the formation of several structural forms and active intermediates released from peroxynitrite.
The distribution and ultrastructure of class II major histocompatibility complex (MHC)-positive cells were investigated in human dental pulp, employing immunohistochemistry using an anti-human leukocyte antigen (HLA)-DR-monoclonal antibody. HLA-DR-immunopositive cells, appearing spindle-like or dendritic in profile, were densely distributed throughout the dental pulp. Under the electron microscope, these cells exhibited various sizes of vesicles containing clear or opaque contents, multivesicular bodies and characteristic fine tubulovesicular structures in their cytoplasm. Some reactive cells possessed coated pits and vesicles including electron-dense materials, indicating an active endocytosis. At the periphery of the pulp tissue, the HLA-DR-immunopositive cells were predominantly situated in the subodontoblastic layer, with some located in the odontoblast layer and/or predentin and extending their cytoplasmic processes into the dentinal tubules. Cell processes of these cells occasionally made contact with several odontoblast processes in the same way as the nerve fibers in the predentin. These cells never contained the typical phagosomes frequently observed in the HLA-DR-immunoreactive macrophages in the subodontoblastic layer and the pulp core. The results suggest that the HLA-DR-immunopositive cells in the odontoblast layer and/or predentin have some regulatory function on the odontoblasts under physiological conditions, in addition to their involvement in the initial defense reaction after tooth injury.
The distribution and ultrastructure of glycogen deposits were investigated in the murine tooth germ by histochemical periodic acid-Schiff (PAS) staining and transmission electron microscopy. Lower and upper first molars were examined in mouse embryos at embryonic days 11.5-17 (E11.5-E17) and in 2-day-old postnatal (P2) mice. The oral and dental epithelia and the mesenchymal cells were generally PAS-positive during tooth morphogenesis. PAS-negative cells were present at E13 in the distal tip of the tooth bud epithelium and in the contacting mesenchyme, and this complete lack of PAS reactivity continued in the dental papilla mesenchyme and inner enamel epithelium during the cap and bell stages. The lack of glycogen deposits in the interacting epithelium and mesenchyme during early morphogenesis may be associated with their demonstrated high signaling activities. Mesenchymal cells in the dental follicle consistently possessed small clusters or large pools of glycogen, which disappeared by P2. Since an intense PAS reaction was seen in mesenchymal cells at future bone sites, the glycogen in the dental follicle cells may be associated with their development into hard-tissue-forming cells. Ultrastructural observation of the enamel organ cells from the cap to early bell stages (E14-E15) revealed the occurrence of glycogen pools, which were associated with the Golgi apparatus and with vesicles having amorphous contents. Glycogen particles were also occasionally present inside vesicles or in the extracellular matrix. These may be associated with the exocytosis of glycosaminoglycan components into extracellular spaces and the formation of the stellate reticulum.
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The recent case-control studies in Thailand indicate that a high incidence of liver cancer in Thailand has not been associated with common risk factors such as hepatitis B infection, aflatoxin intake and alcohol consumption. While the infestation by the liver fluke Opisthorchis viverrini (OV) accounted for the high risk in north-east Thailand, there was no such exposure in the other regions of the country where the incidence of liver cancer is also high. Case-control studies suggest that exposure to exogenous and possibly endogenous nitrosamines in food or tobacco in betel nut and cigarettes may play a role in the development of hepatocellular carcinoma (HCC), while OV infestation and chemical interaction of nitrosamines may also be aetiological factors in the development of cholangiocarcinoma (CCA). Over 1800 samples of fresh and preserved food were systematically collected and tested between 1988 and 1996. All the food items identified by anthropological studies to be consumed frequently in four major regions of Thailand were analysed for volatile nitrosamines using gas chromatography combined with a thermal energy analyser. Relatively high levels of N-nitrosodimethylamine (NDMA), N-nitrosopiperidine (NPIP) and N-nitrosopyrrolidine (NPYR) were detected in fermented fish ("Plasalid"). NDMA was also detected at levels ranging from trace amounts to 66.5 microg/kg in several salted and dried fish ("Larb-pla" and "Pla-siu"). NDMA and NPYR were frequently detected in several vegetables, particularly fermented beans ("Tau-chiau") at levels ranging between 1 and 95.1 microg/kg and 0-146 microg/kg, respectively. The possible role of nitrosamines in Thai food in the aetiology of liver cancer (HCC, CCA) is discussed.
Nitroxyl anion (NO-), the one-electron reduction product of nitric oxide (NO*), has been reported to be formed under various physiological conditions and to be cytotoxic, although the mechanism responsible for the toxic effects has not been identified. We have studied the effects of NO- generated from Angeli's salt (sodium trioxodinitrate) or Piloty's acid (N-hydoxybenzenesulfonamide) on DNA strand breakage and DNA base oxidation in vitro. Induction of strand breakage was dose- and time-dependent upon incubation of plasmid pBR322 with Angeli's salt or Piloty's acid. Similarly, 8-oxo-2'-deoxyguanosine and malondialdehyde were formed when calf-thymus DNA or 2'-deoxyribose, respectively, were incubated with Angeli's salt. Electron acceptors (ferricyanide, 4-hydroxy-TEMPO), that convert NO to NO*, inhibited the reactions, indicating that NO , but not NO*, is responsible for the reactions. Furthermore, the reactions were also inhibited by the presence of hydroxyl radical (HO*) scavengers, antioxidants, metal chelators and superoxide dismutase and catalase, implying involvement of free HO*. These results suggest that NO- is a possible endogenous source of HO*, that may be formed either directly from the reaction product of NO- with NO* (N2O2*-) or indirectly through H2O2 formation. Thus NO may play an important role as a cause of diverse pathophysiological conditions such as inflammation and neurodegenerative diseases.
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Human liver cancers have been associated mainly with chronic inflammations such as viral hepatitis B or C. This suggests that prolonged cell damage by chronic inflammation is critical in cancer development. Overproduction of nitric oxide (NO.) and its derivative (NOx, peroxynitrite) has been implicated as a cause of tissue damage by inflammation, thus contributing to tumor promotion. We have demonstrated the expression of the inducible isoform of nitric oxide synthase (iNOS) and 3-nitrotyrosine, a marker of peroxynitrite formation, by immunohistochemistry in preneoplastic and neoplastic rat liver tissues induced by continuous infusion of N-nitrosodiethylamine with mini-pumps. The preneoplastic lesions were characterized by proliferation of phenotypically altered hepatic foci (PAHF), dysplastic hepatocytes and oval cells. Histologically, the tumors were hepatocellular carcinomas (HCCs) of trabecular, (pseudo)glandular and solid types with or without cholangiocellular involvement. iNOS was located mainly in oval cells, capillary endothelial and muscular cells, epithelia of cholangiomas and glandular HCCs. 3-Nitrotyrosine was observed in the cytoplasms of PAHF and dysplastic hepatocytes in preneoplasias and in the cytoplasms of some living or apoptotic HCC cells, connective tissues, proteinaceous fluids, sinusoidal endothelia of tumorous hepatocytes and cholangiomas in tumors. From these observations, we suggest that: (i) chronic tissue damage by chemical carcinogens may act to induce iNOS and peroxynitrite formation; (ii) oval cells play a key role in development and/or growth of tumor tissues by producing NO. via iNOS, which may also cause tissue damage by peroxynitrite; (iii) iNOS can be considered as a phenotypic marker in cells of oval cell lineage and neovascularized capillaries in tumor tissues.
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Recently it was shown that motility of Vibrio alginolyticus facilitated cell attachment to glass surfaces. In the present study the same relationship between motility and cell attachment was confirmed for Alcaligenes and Alteromonas spp. These findings clearly answer a long-standing question: does motility facilitate attachment? However, they are contradictory to a general view on cell attachment that the energy barrier due to electrostatic repulsion between negatively charged bacterial cells and a glass surface is much greater than both the thermal kinetic energy of the bacterial cell and the bacterial swimming energy. It is shown that the energy barrier becomes far less than that usually estimated when bacterial cells are rich in polymers at their surfaces. This finding reasonably explains the dependence of bacterial attachment rate on cell motility and demands reconsideration of the mechanism of bacterial attachment.
Twenty-eight cases of bacterial meningitis during the recent ten years were analyzed retrospectively, and the following results were obtained. 1. Pathogens were as follows; H. influenzae 13 (46.4%), S. pneumoniae 8 (28.6%), S. agalactiae 4 (14.3%), E. coli 2 (7.1%), and L. monocytogenes 1 case (3.6%). 2. Twelve out of the thirteen H. influenzae cases were caused by serotype b (Hib), and 2 strains were beta-lactamase producer. Fifty percent of the S. pneumoniae cases were caused by penicillin-resistant strains. And all these resistant strains belonged to serotype 19 or 23. 3. Underlying diseases related to the onset of meningitis were found in 46% of the cases, and these consisted of CNS shunt operated 5, asplenia or polysplenia 2, Mondini's anomaly 1, sacral dermal sinus 1, and neonate 4 cases. 4. Prognosis of these cases were three deaths, four with neurologic sequelae, and twenty-one complete recoveries. 5. On admission, 85% (17/20) of the cases were diagnosed correctly by the rapid antigen detection. Sensitivity and specificity of the rapid antigen detection by using latex particle agglutination is 90% and 100% in the Hib cases, and 83% and 100% in the S. pneumoniae cases respectively. Moreover, the bacteriologically unknown 2 cases caused by parenteral partial treatment were also diagnosed by the detection of antigen in concentrated urine.
The expression of heat shock protein 25 (Hsp 25) was investigated in the rat temporomandibular joint by immunocytochemistry combined with confocal and electron microscopy. Immunostaining with an antibody to Hsp25 was able to demonstrate various cellular elements in the synovial membrane of the joint. Intense immunoreaction for Hsp25 was recognized in certain cells comprising the synovial lining layer. Confocal microscopic observation revealed two characteristic profiles of the Hsp25-positive cells with cytoplasmic processes: one extended thick and long processes towards the articular cavity, and the other prejected horizontally slender processes which covered the synovial membrane. Under the electron microscope, the immunoreactive synovial lining cells were characterized by a well-developed rough endoplasmic reticulum and secretory granules, suggesting that they can be categorized as fibroblastic type B cells. The covering by the cytoplasmic extensions was confirmed by immuno-electron microscopic observations. This cytoplasmic covering presumably performs a barrier function and expedites the effective secretion/resorption of synovial fluids. Since it has been proposed that Hsp 25 is associated with an estrogen receptor, the immunopositive synovial lining cells were considered estrogen-target cells. Immunoreactivity for Hsp25 was also observed in the chondrocytes of the maturative and hypertrophic cell layers as well as in the cells of the articular disk. A suggestion was made that Hsp25 might be involved in the inhibition of apoptosis of those cells.
The postnatal expression of immunoreactivity for calretinin, one of the calcium binding proteins, and for protein gene product 9.5 (PGP 9.5), a general neuronal marker, was investigated in mechanoreceptive Ruffini endings in the periodontal ligament of the rat incisor. Age-related changes in the expression of these two proteins in periodontal nerves were further quantified with a computerized image analysis. At 1 day after birth, a few PGP 9.5-immunoreactive nerve fibers and a still smaller number of calretinin-positive fibers were found in the periodontal ligament: they were thin and beaded in appearance and no specialized nerve terminals were recognized. Tree-like terminals, reminiscent of immature Ruffini endings, were recognizable in 4-day-old rats by PGP 9.5-immunohistochemistry, while calretinin-immunostaining failed to reveal these specialized endings. At postnatal 7-11 days when PGP 9.5-immunostaining could demonstrate typical Ruffini endings, calretinin-immunopositive nerve fibers merely tapered off without forming the Ruffini type endings. A small number of Ruffini endings showing calretinin-immunoreactivity began to occur in the periodontal ligament at 24-26 days after birth when the occlusion of the first molars had been established. At the functional occlusion stage (60-80 days after birth), the Ruffini endings showing calretinin-immunoreactivity drastically increased in number and density, but less so than those positive for PGP 9.5-immunoreaction. The delayed expression of calretinin suggests that the function of the periodontal Ruffini endings is established after the completion of terminal formation because Ca2+, which binds to calcium binding proteins including calretinin with high affinity, plays an important role in mechano-electric transduction.
It has been pointed out in general that the resistance against infection is decreased in the diabetic patients. This problem is very important in patients undergoing major surgery. In cardiovascular surgery, the median wound infection may result in life threatening sequelae. We have used the internal thoracic artery as the appropriate graft for its excellent long-term patency. In this series of the diabetic patients who underwent coronary artery bypass grafting with bilateral internal thoracic artery grafts, the incidence of postoperative mediastinitis was significantly higher than other cases. Therefore the use of bilateral internal thoracic artery grafts should be avoided, if possible in the diabetic patients.
Peroxynitrite is a cytotoxic oxidant produced during shock, ischemia reperfusion, and inflammation. The cellular events mediating the cytotoxic effect of peroxynitrite include activation of poly(ADP-ribose) synthetase, inhibition of mitochondrial respiration, and activation of caspase-3. The aim of the present study was to investigate the role of intracellular calcium mobilization in the necrotic and apoptotic cell death induced by peroxynitrite. Peroxynitrite, in a low, pathophysiologically relevant concentration (20 microM), induces rapid (1 to 3 min) Ca(2+) mobilization in thymocytes. Inhibition of this early calcium signaling by cell-permeable Ca(2+) chelators [EGTA-acetoxymethyl ester (AM), 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM (BAPTA-AM), 8-amino-2-[(2-amino-5-methylphenoxy)methyl]-6-methoxyquinoline-N,N , N',N'-tetraacetic acid-tetra-AM] abolished cytotoxicity as measured by propidium iodide uptake. Intracellular Ca(2+) chelators also inhibited DNA single-strand breakage and activation of poly(ADP-ribose) synthase (PARS), which is a major mediator of cell necrosis in the current model. Intracellular Ca(2+) chelators also protected PARS-deficient thymocytes from peroxynitrite cytotoxicity, providing evidence for a PARS-independent, Ca(2+)-dependent cytotoxic pathway. Chelation of intracellular Ca(2+) blocked the peroxynitrite-induced decrease of mitochondrial membrane potential, secondary superoxide production, and mitochondrial membrane damage. Peroxynitrite-induced internucleosomal DNA cleavage was increased on BAPTA-AM pretreatment in the wild-type cells but decreased in the PARS-deficient cells. Two other apoptotic parameters (phosphatidylserine exposure and caspase 3 activation) were inhibited by BAPTA-AM in both the wild-type and the PARS-deficient thymocytes. Our findings provide evidence for the pivotal role of an early Ca(2+) signaling in peroxynitrite cytotoxicity.