Nobuo Ohya

Effects of glycyrrhizin and glycyrrhetinic acid on growth and melanogenesis in cultured B16 melanoma cells.

The effects of glycyrrhizin (GL) and its aglycone, glycyrrhetinic acid (GA), on the growth and differentiation of mouse melanoma (B16) cells in culture were studied. GA inhibits the growth of B16 melanoma cells, causes morphological alterations and stimulates melanogenesis. GL also resulted in the same changes but only when the concentration was about 20 times more than that needed for GA. When GA was removed after 84 h of treatment, the growth rate recovered slightly, but the doubling time was about twice that of the control. Cytofluorometric analysis showed that the growth inhibition of GA is the result of inhibition of the transfer from G1 to S phase.

Ischemia-reperfusion lung injury in rabbits: mechanisms of injury and protection

To study the mechanisms responsible for ischemia-reperfusion lung injury, we developed an anesthetized rabbit model in which the effects of lung deflation, lung inflation, alveolar gas composition, hypothermia, and neutrophils on reperfusion pulmonary edema could be studied. Rabbits were anesthetized and ventilated, and the left pulmonary hilum was clamped for either 2 or 4 h. Next, the left lung was reperfused and ventilated with 100% oxygen. As indexes of lung injury, we measured arterial oxygenation, extravascular lung water, and the influx of a vascular protein (131I-labeled albumin) into the extravascular space of the lungs. The principal results were that 1) all rabbits with the deflation of the lung during ischemia for 4 h died of fulminant pulmonary edema within 1 h of reperfusion; 2) inflation of the ischemic lung with either 100% oxygen, air, or 100% nitrogen prevented the reperfusion lung injury; 3) hypothermia at 6-8°C also prevented the reperfusion lung injury; 4) although circulating neutrophils declined during reperfusion lung injury, there was no increase in interleukin-8 levels in the plasma or the pulmonary edema fluid, and, furthermore, neutrophil depletion did not prevent the reperfusion injury; and 5) ultrastructural studies demonstrated injury to both the lung endothelium and the alveolar epithelium after reperfusion in deflated lungs, whereas the inflated lungs had no detectable injury. In summary, ischemia-reperfusion injury to the rabbit lung can be prevented by either hypothermia or lung inflation with either air, oxygen, or nitrogen.To study the mechanisms responsible for ischemia-reperfusion lung injury, we developed an anesthetized rabbit model in which the effects of lung deflation, lung inflation, alveolar gas composition, hypothermia, and neutrophils on reperfusion pulmonary edema could be studied. Rabbits were anesthetized and ventilated, and the left pulmonary hilum was clamped for either 2 or 4 h. Next, the left lung was reperfused and ventilated with 100% oxygen. As indexes of lung injury, we measured arterial oxygenation, extravascular lung water, and the influx of a vascular protein (131I-labeled albumin) into the extravascular space of the lungs. The principal results were that 1) all rabbits with the deflation of the lung during ischemia for 4 h died of fulminant pulmonary edema within 1 h of reperfusion; 2) inflation of the ischemic lung with either 100% oxygen, air, or 100% nitrogen prevented the reperfusion lung injury; 3) hypothermia at 6-8 degreesC also prevented the reperfusion lung injury; 4) although circulating neutrophils declined during reperfusion lung injury, there was no increase in interleukin-8 levels in the plasma or the pulmonary edema fluid, and, furthermore, neutrophil depletion did not prevent the reperfusion injury; and 5) ultrastructural studies demonstrated injury to both the lung endothelium and the alveolar epithelium after reperfusion in deflated lungs, whereas the inflated lungs had no detectable injury. In summary, ischemia-reperfusion injury to the rabbit lung can be prevented by either hypothermia or lung inflation with either air, oxygen, or nitrogen.

Lung deflation impairs alveolar epithelial fluid transport in ischemic rabbit and rat lungs

BACKGROUND Because the fluid transport capacity of the alveolar epithelium after lung ischemia with and without lung deflation has not been well studied, we carried out experimental studies to determine the effect of lung deflation on alveolar fluid clearance. METHODS After 1 or 2 hr of ischemia, we measured alveolar fluid clearance using 125I-albumin and Evans blue-labeled albumin concentrations in in vivo rabbit lungs in the presence of pulmonary blood flow and in ex vivo rat lungs in the absence of any pulmonary perfusion, respectively. RESULTS The principal results were: (1) lung deflation decreased alveolar fluid clearance while inflation of the lungs during ischemia preserved alveolar fluid clearance in both in vivo and ex vivo studies; (2) alveolar fluid clearance was normal in the rat lungs inflated with nitrogen (thus, alveolar gas composition did not affect alveolar fluid clearance); (3) amiloride-dependent alveolar fluid clearance was preserved when the lungs were inflated during ischemia; (4) terbutaline-simulated alveolar fluid clearance was preserved in the hypoxic rat lungs inflated with nitrogen; (5) lecithinized superoxide dismutase, a scavenger of superoxide anion, and N(omega)-nitro-L-arginine methyl ester, an inhibitor of nitric oxide, preserved normal alveolar fluid clearance in the deflated rat lungs. CONCLUSION Lung deflation decreases alveolar fluid clearance by superoxide anion- and nitric oxide-dependent mechanisms.

Pharyngeal Cross-Sectional Area and Pharyngeal Compliance in Normal Males and Females

Obstructive sleep apnea syndrome is ascribed to pharyngeal dysfunction, but there are only a few reports about the normal morphological values in this anatomical region. We measured the pharyngeal cross-sectional area and the compliance (collapsibility), using the acoustic reflection technique with air breathing, in 181 healthy subjects (age 21–69 years). We assessed their sex-related differences, and the effects of age, body size and body postures on these parameters. The pharyngeal cross-sectional area, defined as the region from the fauces to the glottis, posturally changed with successive decreases in the sitting, left lateral decubitus and supine positions. The area was significantly greater in male than in female subjects in the sitting position (p < 0.01), but no difference was present in the recumbent positions. The pharyngeal cross-sectional area did not correlate with either age or body size. The specific pharyngeal compliance was greater in the males than in the females (p < 0.01) and increased with age only in the male subjects.

Expression and Localization of a Novel Rab Small G Protein (Rab38) in the Rat Lung

The Rab small G protein family participates in intracellular vesicle transport, including exocytosis and endocytosis. The cDNA encoding a novel Rab-related small G protein (Rab38) has been cloned from rat lung cDNA library and recorded in GenBank (accession no. M94043). However, the expression and localization of the protein in the lung remains primarily unknown. We produced polyhistidine-tagged recombinant Rab38 and a polyclonal antibody with a synthetic peptide. Immunohistochemistry demonstrated that the protein is specifically localized in alveolar type II cells and in bronchial epithelial cells. In situ hybridization using a digoxygenin-labeled RNA riboprobe clearly showed that the mRNA of the protein is localized in alveolar type II cells and bronchial epithelial cells, especially terminal airway epithelial cells. Western blot and reverse transcriptase-polymerase chain reaction showed distinct expression of the protein and mRNA in isolated alveolar type II cells, but not in alveolar macrophages. The native protein was predominantly hydrophobic and was enriched in a high-density vesicle fraction but was barely detectable in nuclear and lamellar body fractions in alveolar type II cells. Immunofluorescence cytochemistry performed on cultured alveolar type II cells showed that Rab38 distributed extensively in the cytoplasm with a distribution pattern similar to endoplasmic reticulum rather than other subcellular organelles. These results suggest that this novel rab small G protein (Rab38) mediates vesicular transport in terminal airway epithelium.

BETA1-ADRENOCEPTOR STIMULATION BY HIGH-DOSE TERBUTALINE DOWNREGULATES TERBUTALINE-STIMULATED ALVEOLAR FLUID CLEARANCE IN EX VIVO RAT LUNG

Because high-dose terbutaline and isoproterenol (10-3

ONO-5046 is a potent inhibitor of neutrophil elastase in human pleural effusion after lobectomy

The imbalance of neutrophil elastase and alpha1-antitrypsin in pleural effusion after lobectomy and the effects of the neutrophil elastase inhibitors, sodium N-[2-[4-(2,2-Dimethylpropionyloxy)phenyl-sulfonylamino]benzo yl]aminoacetic acid (ONO-5046) and purified alpha1-antitrypsin, on neutrophil elastase activity were determined. The amount of neutrophil elastase complexed to alpha1-antitrypsin, measured by an enzyme-linked immunosorbent assay, was 170 times higher in pleural effusion than in blood 3 h after lobectomy. The alpha1-antitrypsin levels measured by laser nephelometry did not increase in either blood or pleural effusion. Although neutrophil elastase activity, measured by the hydrolysis of succinyl-(Ala)3-p-nitroanilide, was not detected in blood, it was increased in pleural effusion 3 h and 24 h after lobectomy. ONO-5046, but not alpha1-antitrypsin, reduced the neutrophil elastase activity in pleural effusion. There is an imbalance of neutrophil elastase and alpha1-antitrypsin in pleural effusion after lobectomy. ONO-5046 is a potent inhibitor of neutrophil elastase activity in human pleural effusion.

Inducible nitric oxide synthase expression and nuclear factor-κB activation in alveolar type II cells in lung injury

Alveolar type II cells (type II cells)play a crucial role in the progression and repair of lung inflammation and injury. We investigated whether inducible nitric oxide synthase (iNOS) was expressed and nuclear factor- κ B (NF- κ B)was activated in type IIcells in lung injury. After injecting lipopolysaccharide (LPS)or saline in the rat, the lungs were excised and type II cells were isolated. iNOS and its mRNAwere expressed both in lung tissue and isolated type II cells in response to LPS. The lungs from saline-treated rats showed only minimal expression ofiNOS. Electrophoretic mobility shift assay revealed that expression of NF- κ B in the nuclear extracts was augmented by LPS, and p50/NF- κ B was expressed in type II cells in LPS-treated rats. Intraperitoneal dexamethasone almost completely inhibited the iNOS expression and attenuated the activation ofNF- Bin the LPS-treated lung. These findings suggest that type II cells can be a source of NO production in lung injury, and that the effects ofcorticosteroids may be in part through inhibition of both iNOS expression and NF- κ B activation.

Differentiation of human pulmonary alveolar epithelial cells revealed by peroxisome changes in pulmonary proteinosis

Regenerating areas of human lungs in pulmonary fibrosis were observed electron microscopically, and peroxidatic activity of catalase in lung peroxisomes were demonstrated cytochemically. Proliferation of Type II cells was prominent there, and some of the cells extended their cytoplasms to cover the denuded basement membrane. Unusual intermediate cells between Type II and Type I cells were observed. The extension of cytoplasmic processes with new generation of pinocytotic vesicles strongly suggested a Type I cell profile. However, catalase-positive peroxisomes were found in these cells simultaneously. From these results it was concluded that Type I cells may originate from Type II cells in human lungs as they do in experimental animals.

Serum KL-6, a novel mucin-like glycoprotein, as an indicator of interstitial pneumonitis following lobectomy.

Serum KL-6 has been shown to be a useful marker of active interstitial pneumonitis in patients who have not undergone lobectomy. Considering that KL-6 is produced mainly in the distal airway epithelium, the present study was conducted to determine whether resected lung volume influenced the postoperative KL-6 levels, and also to evaluate whether it is a useful parameter in patients who have undergone lobectomy. The serum KL-6 levels decreased by 36% 1 week after lobectomy, but returned to the preoperative levels by 2 months postoperatively. Although the KL-6 levels increased by 100% 3 to 4 months after lobectomy, the levels were significantly lower than those in interstitial pneumonitis (P<0.05). The decrease in the KL-6 levels correlated with the number of resected lung segments, but not with the changes in white blood cell count, lactate dehydrogenase level, or C-reactive protein level. In comparison with the lobectomy patients, the serum KL-6 levels decreased by half in patients who had undergone partial resection (P<0.05). The results of this study suggest that the serum KL-6 level may be a useful indicator of interstitial pneumonitis after lobectomy. Serum KL-6 levels are influenced by the volume of the resected lung, and probably also by the upregulation of KL-6 production.