Blue LED light exposure develops intracellular reactive oxygen species, lipid peroxidation, and subsequent cellular injuries in cultured bovine retinal pigment epithelial cells.

The effects of blue light emitter diode (LED) light exposure on retinal pigment epithelial cells (RPE cells) were examined to detect cellular damage or change and to clarify its mechanisms. The RPE cells were cultured and exposed by blue (470 nm) LED at 4.8 mW/cm(2). The cellular viability was determined by XTT assay and cellular injury was determined by the lactate dehydrogenase activity in medium. Intracellular reactive oxygen species (ROS) generation was determined by confocal laser microscope image analysis using dihydrorhodamine 123 and lipid peroxidation was determined by 4-hydroxy-2-nonenal protein-adducts immunofluorescent staining (HNE). At 24 h after 50 J/cm(2) exposures, cellular viability was significantly decreased to 74% and cellular injury was significantly increased to 365% of control. Immediately after the light exposure, ROS generation was significantly increased to 154%, 177%, and 395% of control and HNE intensity was increased to 211%, 359%, and 746% of control by 1, 10, and 50 J/cm(2), respectively. These results suggest, at least in part, that oxidative stress is an early step leading to cellular damage by blue LED exposure and cellular oxidative damage would be caused by the blue light exposure at even lower dose (1, 10 J/cm(2)).

Salt is an oxidative stressor for gastric epithelial cells.

Salt/NaCl has been reported to induce necrosis in gastric mucosal cells, however, the mechanisms for gastric injury by salt are not clarified. In this study, we elucidated whether salt is an oxidative stress inducer via mitochondrial injury on rat gastric epithelial cells (RGM-1) in 300, 450, 650 and 1000 mM of NaCl-contained medium. To clarify whether salt-induced reactive oxygen species (ROS) is derived from mitochondria, we also investigated a salt-induced ROS production in manganese superoxide dismutase overexpressing cells (RGM-MnSOD). MnSOD is a specific scavenger for superoxide anion produced from mitochondria. The results showed that cellular injuries in RGM-MnSOD were significantly less severe than that in normal RGM-1. The electron paramagnetic resonance (EPR) studies also provided an evidence that the salt-derived superoxide production in RGM-MnSOD was less than that in normal RGM-1. These results indicated that salt is not merely a necrotizing factor for gastric epithelial cells, but also an oxidative stress inducer.

Rebamipide attenuates nonsteroidal anti-inflammatory drugs (NSAID) induced lipid peroxidation by the manganese superoxide dismutase (MnSOD) overexpression in gastrointestinal epithelial cells.

Nonsteroidal anti-inflammatory drugs (NSAIDs) often cause gastrointestinal complications such as gastric ulcers and erosions. Recent studies on the pathogenesis have revealed that NSAIDs induce lipid peroxidation in gastric epithelial cells by generating superoxide anion in mitochondria, independently with cyclooxygenase-inhibition and the subsequent prostaglandin deficiency. Although not clearly elucidated, the impairment of mitochondrial oxidative phosphorylation, or uncoupling, by NSAIDs is associated with the generation of superoxide anion. Physiologically, superoxide is immediately transformed into hydrogen peroxide and diatomic oxygen with manganese superoxide dismutase (MnSOD). Rebamipide is an antiulcer agent that showed protective effects against NSAID-induced lipid peroxidation in gastrointestinal tracts. We hypothesized that rebamipide may attenuate lipid peroxidation by increasing the expression of MnSOD protein in mitochondria and decreasing the leakage of superoxide anion in NSAID-treated gastric and small intestinal epithelial cells. Firstly, to examine rebamipide increases the expression of MnSOD proteins in mitochondria of gastrointestinal epithelial cells, we underwent Western blotting analysis against anti-MnSOD antibody in gastric RGM1 cells and small intestinal IEC6 cells. Secondly, to examine whether the pretreatment of rebamipide decreases NSAID-induced mitochondrial impairment and lipid peroxidation, we treated these cells with NSAIDs with or without rebamipide pretreatment, and examined with specific fluorescent indicators. Finally, to examine whether pretreatment of rebamipide attenuates NSAID-induced superoxide anion leakage from mitochondria, we examined the mitochondria from indomethacin-treated RGM1 cells with electron spin resonance (ESR) spectroscopy using a specific spin-trapping reagent, CYPMPO. Rebamipide increased the expression of MnSOD protein, and attenuated NSAID-induced mitochondrial impairment and lipid peroxidation in RGM1 and IEC6 cells. The pretreatment of rebamipide significantly decreased the signal intensity of superoxide anion from the mitochondria. We conclude that rebamipide attenuates lipid peroxidation by increasing the expression of MnSOD protein and decreasing superoxide anion leakage from mitochondria in both gastric and small intestinal epithelial cells.

A quantitative radiological assessment of outcomes of autogenous bone graft combined with platelet-rich plasma in the alveolar cleft.

This longitudinal study evaluated the outcomes of secondary autogenous bone graft combined with platelet-rich plasma (PRP) in the alveolar cleft. Thirty-five alveolar clefts in 30 patients with grafted autogenous bone and PRP (PRP group), and 36 clefts in 30 patients with grafted autogenous bone alone (non-PRP group) were enrolled. PRP was extracted from autogenous blood using a plasma centrifuge system (SmartPReP SMP-1000). The density and resorption of grafted bone were evaluated at 1 week, and 1, 3, 6 and 12 months postoperatively. Bone density was quantitatively assessed as an aluminum-equivalence (Al-Eq) value. Moreover, relationships between bone resorption rate and prognostic factors were discussed. Al-Eq values decreased significantly until 3 months, and then increased up to 12 months in both groups. The Al-Eq rate in the PRP group was significantly smaller than that in the non-PRP group at 3 months. No significant differences were observed in the bone resorption rate between the groups. Regarding prognostic factors, continuous mechanical stress affected bone resorption with or without PRP. The authors suggest that PRP may enhance bone remodeling in the early phase, however, PRP seems to be insufficient as a countermeasure against bone resorption following secondary bone graft in the long term.

Pleomorphic adenoma presenting low T2 signal-intensity on magnetic resonance imaging.

We report an unusual case of pleomorphic adenoma of the submandibular gland in a 48-year-old female. The present case appeared as a relatively homogeneous, low to intermediate signal-intensity on the T(2) weighted magnetic resonance (MR) images. To our knowledge, the MR feature of low T(2) signal-intensity of pleomorphic adenoma has not been reported in the literature.

Irreversible morphological changes in leg bone following chronic gravitational unloading of growing rats.

Effects of gravitational unloading or loading on the growth and development of hindlimb bones were studied in rats. Male Wistar rats were hindlimb-unloaded or loaded at 2-G from the postnatal day 4 to month 3. The morphology and mineral content of tibia and fibula, as well as the mobility of ankle joints, were measured at the end of 3-month suspension or loading, and 1, 2, and 3 months after ambulation recovery. Growth-related increases of bone weight and mineral density were inhibited by unloading. But they were gradually recovered toward the control levels, even though they were still less than those in the age-matched controls after 3 months. None of the parameters were influenced by 2-G loading. However, here we report that chronic unloading causes abnormal morphological development in hindlimb bone of growing rats. Irreversible external bend of the shaft and rotation of the distal end of tibia, which limit the dorsiflexion of ankle joints, were induced following chronic gravitational unloading during developing period. It is also suggested that such phenomena are caused by the abnormal mechanical forces imposed by muscle utilization with altered patterns. The activity of ankle dorsiflexor was increased and that of plantarflexor was inhibited during unloading.

Expression of permeability-glycoprotein (P-gp) and uptake of technetium-99m-hexakis-2-methoxy-isobutyl-isonitrile (99Tcm-MIBI) in malignant tumour of the head and neck.

OBJECTIVES: The purpose of this study is to estimate the role of permeability-glycoprotein (P-gp) in the technetium-99m-hexakis-2-methoxy-isobutyl-isonitrile (99Tc(m)-MIBI) scintigraphy. METHODS: 71 patients with squamous cell carcinoma (39 patients with well differentiated, 19 with moderately differentiated and 13 with poorly differentiated tumour) were examined. Eighteen of these patients underwent 99Tc(m)-MIBI scintigraphy (early and delayed scans). The tumour retention index, obtained from the ratio of the accumulation of the delayed scan to that of the early scan, was divided into three groups. The immunohistochemical evaluation of P-gp expression was performed in all 71 patients. Levels of the P-gp expression were classified into three grades (score 0, 1 and 2). Correlations among the tumour retention index, the P-gp expression and the tumour tissue differentiation were evaluated. RESULTS: 17 of 18 patients showed a decreasing of the tumour retention index ranging from 0.70 to 0.93 (mean+/-SD=0.850+/-0.071). The tumour retention index showed a statistical correlation with the P-gp expression and the tumour tissue differentiation (chi-squared=7.802>7.779, P=0.10 and 16.835>14.860, P=0.005, respectively). Moreover, there was a statistical correlation between the P-gp expression and the tumour tissue differentiation (chi-squared=14.863>14.860, P=0.005). CONCLUSION: There is a possibility that the P-gp expression is high in the high-grade malignant tumours and P-gp causes the decrease of tumour retention index.

Scintigraphy for interpretation of malignant tumours of the head and neck: comparison of technetium-99m-hexakis-2-methoxyisobutylisonitrile (Tc-MIBI) and thallium-201-chloride (Tl-201).

OBJECTIVES: The purpose of this study was to compare the usefulness of technetium-99m-hexakis-2-methoxyisobutylisonitrile (99Tc(m)-MIBI) and thallium-201-chloride (Tl-201) as scintigraphic agents. METHODS: Dynamic and static scintigraphic imaging with 99Tc(m)-MIBI and Tl-201 were performed on patients with a variety of malignant and benign tumours. Factors of the grade of the static scan, the blood flow index, the early and delayed retention indexes, and the tumour retention index were obtained from the scintigraphy. In addition to these factors, the grade of tissue differentiation and tumour size were evaluated to clarify the difference between 99Tc(m)-MIBI and Tl-201 for the diagnosis of malignant tumours of the head and neck. RESULTS: 99Tc(m)-MIBI accumulation depended upon the blood flow index in the early static scan, but this accumulation did not correlate with tumour size. The accumulation in most subjects decreased in the delayed static scan, and the tumour retention index had a tendency to decrease with the grade of tissue differentiation. Tl-201 accumulation depended upon the blood flow index in the early static scan similar to 99Tc(m)-MIBI, and the accumulation correlated with tumour size, unlike 99Tc(m)-MIBI. The tumour retention index had a tendency to increase with the grade of tissue differentiation. Thus, the tumour retention indexes showed opposite behaviours between 99Tc(m)-MIBI and Tl-201, but they both accurately determined tumour malignancy. CONCLUSIONS: There was no major difference between 99Tc(m)-MIBI and Tl-201scintigraphy with respect to accuracy of diagnosis of malignant tumours of the head and neck. However, 99Tc(m)-MIBI was superior to Tl-201 for small-size tumours and Tl-201 was useful for large-size tumours.

Thallium-201 chloride (Tl-201) accumulation and Na+/K+-ATPase expression in tumours of the head and neck.

OBJECTIVES: The purpose of this report was to evaluate the relationship between the tumour retention index of thallium-201 chloride (Tl-201) scintigraphy and the Na+/K+-ATPase expression in tumours of the head and neck. METHODS: Tl-201 scintigraphy was performed in 146 patients (129 with malignant tumours, ten with benign tumours and seven with inflammation). The tumour retention index was obtained from the early and delayed dynamic Tl-201 scans. The Na+/K+-ATPase expression was evaluated immunohistochemically in 61 of 129 patients with malignant tumour. Furthermore, another 22 patients with benign tumour were evaluated immunohistochemically as a benign control. Comparison of the correlations between the grade of histopathological differentiation of tumour, the tumour retention index of Tl-201 scintigraphy and the Na+/K+-ATPase expression was performed. RESULTS: The grade of histopathological differentiation of tumour, the tumour retention index of Tl-201 scintigraphy and the expression of Na+/K+-ATPase showed a good correlation indicating that Na+/K+-ATPase plays an important role in transportation for Tl-201 to go through the tumour cell membrane. CONCLUSIONS: Na+/K+-ATPase is one of the most important factors for Tl-201 accumulation in tumour.

A trial for histopathological subclassification of papillary cystadenoma lymphomatosum by 99Tcm-pertechnetate in a patient with multiple bilateral lesions of the parotid glands.

OBJECTIVES: The purpose of this report was to evaluate the possibility of subclassification of papillary cystadenoma lymphomatosum (PCL) with 99Tc(m)-pertechnetate. METHODS: A patient with multiple bilateral PCLs in the parotid glands was examined by using 99Tc(m)-pertechnetate. RESULTS: All PCLs of the present case, which were diagnosed as the subtype-II histopathologically, showed similar radioactive indexes in scintigraphy (the mean radioactive index = 3.62), although tumours were different in size. The mean radioactive index corresponded well with that from four cases of subtype-II of our previous report (the mean radioactive index = 3.84). CONCLUSIONS: The results of the present report suggest a possibility of histopathological subclassification of PCLs into subtypes by 99Tc(m)-pertechnetate scintigraphy.

Increased expression of humanin peptide in diffuse-type pigmented villonodular synovitis: implication of its mitochondrial abnormality.

OBJECTIVES: To define the pathogenesis of pigmented villonodular synovitis (PVNS), by searching for highly expressed genes in primary synovial cells from patients with PVNS. METHODS: A combination of subtraction cloning and Southern colony hybridisation was used to detect highly expressed genes in PVNS in comparison with rheumatoid synovial cells. Northern hybridisation was performed to confirm the differential expression of the humanin gene in PVNS. Expression of the humanin peptide was analysed by western blotting and immunohistochemistry. Electron microscopic immunohistochemistry was performed to investigate the distribution of this peptide within the cell. RESULTS: 68 highly expressed genes were identified in PVNS. Humanin genes were strongly expressed in diffuse-type PVNS, but were barely detected in nodular-type PVNS, rheumatoid arthritis, or osteoarthritis. Humanin peptide was identified in synovium from diffuse-type PVNS, and most of the positive cells were distributed in the deep layer of the synovial tissue. Double staining with anti-humanin and anti-heat shock protein 60 showed that humanin was expressed mainly in mitochondria. Electron microscopy disclosed immunolocalisation of this peptide, predominantly around dense iron deposits within the siderosome. CONCLUSIONS: Increased expression of the humanin peptide in mitochondria and siderosomes is characteristic of synovial cells from diffuse-type PVNS. Humanin is an anti-apoptotic peptide which is encoded in the mitochondrial genome. Present findings suggest that mitochondrial dysfunction may be the principal factor in pathogenesis of diffuse-type PVNS and that humanin peptide may play a part in the neoplastic process in this form of PVNS.

Overexpression of mitochondrial manganese superoxide dismutase protects against radiation-induced cell death in the human hepatocellular carcinoma cell line HLE.

We investigated the potential role of mitochondrial manganese superoxide dismutase (Mn-SOD) in protective activity against irradiation by analyzing cell viability by a colony formation assay and by detecting apoptosis in stably human Mn-SOD gene-transfected HLE, a hepatocellular carcinoma cell line. We found that overexpression of Mn-SOD reduced the levels of reactive oxygen species in the mitochondria and intracellular phospholipid peroxidation product (4-hydroxy-2-nonenal) and prevented cell death. The production of intracellular nitric oxide after irradiation was not changed by Mn-SOD overexpression. The results suggested that Mn-SOD might play an important role in protecting cells against radiation-induced cell death by controlling the generation of mitochondrial reactive oxygen species and intracellular lipid peroxidation.

Prevention of mitochondrial injury by manganese superoxide dismutase reveals a primary mechanism for alkaline-induced cell death.

Alkalosis is a clinical complication resulting from various pathological and physiological conditions. Although it is well established that reducing the cellular proton concentration is lethal, the mechanism leading to cell death is unknown. Mitochondrial respiration generates a proton gradient and superoxide radicals, suggesting a possible link between oxidative stress, mitochondrial integrity, and alkaline-induced cell death. Manganese superoxide dismutase removes superoxide radicals in mitochondria, and thus protects mitochondria from oxidative injury. Cells cultured under alkaline conditions were found to exhibit elevated levels of mitochondrial membrane potential, reactive oxygen species, and calcium which was accompanied by mitochondrial damage, DNA fragmentation, and cell death. Overexpression of manganese superoxide dismutase reduced the levels of intracellular reactive oxygen species and calcium, restored mitochondrial transmembrane potential, and prevented cell death. The results suggest that mitochondria are the primary target for alkaline-induced cell death and that free radical generation is an important and early event conveying cell death signals under alkaline conditions.

Accumulation of mutant p53 and hsp72 by heat treatment, and their association in a human glioblastoma cell line.

The cellular content of mutant p53 and hsp72 proteins following gamma-ray irradiation, UV irradiation, and heat treatment was studied in A-7 cells, a human glioblastoma cell line. A-7 was found to contain a mutant p53 gene in which the arginine codon at position 175 was substituted by a histidine codon. Although the p53 gene was mutant, the phenotype of the p53 protein appeared wild-type since the cellular content of the p53 protein was limited under normal culturing conditions. The quantity of mutant p53 and hsp72 proteins in A-7 was increased by heat treatment as well as gamma-ray and UV irradiation. Furthermore, the mutant p53 protein was coimmunoprecipitated with anti-hsp72/hsc73 antibody. Additionally, hsp72 and hsc73 were coimmunoprecipitated with anti-p53 antibody. These results suggest that in A-7, p53 protein accumulation may be caused as a result of response to stressors, such as gamma-ray, UV and heat and that mutant p53 protein and hsp72/hsc73 may manage biological functions cooperatively after gamma-ray, UV and also heat treatments.