Shoji Tsunekawa

Inhibition of tumor necrosis factor-induced apoptosis in transgenic mouse liver expressing creatine kinase

Abstract: Background: The mitochondrion acts as a pivotal decision center in many types of apoptotic responses. To clarify the effects of the enhanced mitochondrial function on tumor necrosis factorα (TNFα)‐induced apoptosis, we studied hepatic injuries in transgenic mice whose livers express creatine kinase (CK).

A useful ELISA system for human liver-type arginase, and its utility in diagnosis of liver diseases.

OBJECTIVES To develop a new ELISA system for liver-type arginase using monoclonal antibodies against the enzyme, and to verify the utility of the arginase in diagnosis of hepatic disorders. DESIGN AND METHODS We have developed an enzyme-linked immunosorbent assay (ELISA), using two kinds of monoclonal antibodies (Mo6G3 and Mo9C5) for human liver-type arginase as the first and second antibodies respectively. We have also developed a new method to eliminate the influence of erythrocyte-derived arginase contamination in hemolytic samples. This ELISA was applied to specimens received from patients with acute and chronic hepatic disease and also patients who had undergone partial hepatectomy. RESULTS This assay is sensitive and reproducible for the measurement of liver-type arginase in the sera of patients with liver dysfunction, and enabled us to detect enzyme concentrations as low as 27 pmol/L without any processing of the samples. The assay showed within-run coefficients of variation (CV) ranging from 1.9 to 4.1% and between-day CV from 3.6 to 5.1% for arginase concentrations varying from 57.1 to 1200 pmol/L. The recovery was 113% (mean) with a range of 96 to 129%. These antibodies reacted strongly with both recombinant and native liver-type arginases, while, to some extent, with erythrocyte-derived arginase. Correction for erythrocyte-derived arginase contamination in hemolytic samples was, however, easily made by assaying peroxidase-like activity of hemoglobin. From the view of a limited localization of arginase in the liver, the marked increase of the enzyme in serum reflects initiation of liver injury, while the rapid decrease reflects termination of the damage. Such quick normalization in circulating liver-type arginase indicated another merit of the enzyme in diagnosis of liver diseases. CONCLUSIONS The changes in circulating liver-type arginase level could be helpful not only in the diagnosis of liver diseases but also subsequent treatment of the patients with liver damage.

Liver-type arginase in serum during and after liver transplantation: a novel index in monitoring conditions of the liver graft and its clinical significance.

We quantified liver-type arginase in sera of 47 patients undergoing partial liver transplantation with use of an ELISA method. The level of liver-type arginase fluctuated slightly beyond the normal range in successful liver recipients, while it changed more drastically or precipitously in unsuccessful ones, accompanying or unaccompanying elevation of AST and ALT levels. A higher elevation pattern of the arginase level (above 100 ng ml-1) was observed in each of the unsuccessful recipients with critical condition, except for one patient. Other hepatic markers (LDH, ALP, and T-BIL) remained relatively unchanged until the terminal stage of deceasing patients. The finding that the liver-type arginase emerged in large quantity in the blood stream immediately after reperfusion of the liver graft indicates that the enzyme leaks out of hepatocytes damaged, presumably, by storage in the absence of circulation. A half-life of the liver-type arginase in the human blood was estimated to be 1 h, that is clearly shorter than that of AST. The short half-life of the arginase appears to be ascribable, at least partly, to formation of an immune complex with circulating autoantibody which appears in many liver recipients. These results suggest that liver-type arginase behaves uniquely in the serum among many hepatic enzymes, and could serve as a distinct marker of hepatic lesions, particularly during and after liver transplantation.

Calpain proteolysis of free and bound forms of calponin, a troponin T-like protein in smooth muscle.

Calponin, a novel homologue of troponin T, purified from chicken gizzard was found to be one of the most susceptible proteins among smooth muscle contraction‐associated proteins to hydrolysis by calpain I purified from human red blood cells. The high susceptibility of calponin was comparable to that reported for troponin T. The rate of degradation of calponin, unlike caldesmon and myosin light chain kinase, was accelerated when bound to calmodulin. When calponin existed as a bound form in both reconstituted actin filament and native thin filament, the rate of proteolysis was markedly retarded, indicating close association of calponin with actin filament. These observations are compatible with the view that calponin is an integral part of the actin‐linked contractile machinery in smooth muscle.

Retroflexed endoscopic multiple band ligation of symptomatic internal hemorrhoids

Abstract Background Elastic band ligation is a well-established nonoperative method for treatment of internal hemorrhoids that give rise to symptoms. This study assessed the efficacy and safety of retroflexed endoscopic multiple band ligation, a procedure that involves extensive ligation of internal hemorrhoids, and the immediately proximal normal rectal mucosa, by means of a retroflexed endoscope. Methods Eighty-two patients with symptoms caused by internal hemorrhoids (15, stage I; 19, stage II; 47, stage III; 1, stage IV) were treated by retroflexed endoscopic multiple band ligation. Symptoms (prolapse, bleeding, pain with defecation) were graded from 0 to 3. Range and form of the internal hemorrhoids were evaluated endoscopically. Retroflexed endoscopic multiple band ligation was performed by using a flexible endoscope with an attached band ligation device in the retroflexed position. Results A mean of 8 bands (range 4-14) were placed per treatment session. Seventy-six patients were treated in a single session, 5 in two sessions, and one in 3 sessions. Symptom and endoscopic scores improved at 4 weeks after the retroflexed endoscopic multiple band ligation: bleeding, from 1.26 to 0.53 ( p p p =0.67); Goligher classification, from 2.41 to 1.09 ( p p p Conclusions Retroflexed endoscopic multiple band ligation is a safe and effective method for treatment for patients with symptoms caused by internal hemorrhoids.

Molecular damage to rat liver mitochondrial H+-ATpase during cold preservation with UW solution

Deterioration of rat liver mitochondrial function during cold preservation with UW solution was studied. Mitochondria were isolated from control liver (0-hr preservation), 24-hr preserved liver, and 48-hr preserved liver with UW solution at 4 degrees C. Respiration assay revealed that phosphorylation was damaged more rapidly than oxidation. Inside-out submitochondrial particles prepared from each sample by sonication in the presence of EDTA were subjected to ATPase assay. ATP hydrolyzing activity of H(+)-ATPase (ATP synthase), which plays a key role in phosphorylation in mitochondria, decreased markedly to 58% after 24-hr preservation. After 48-hr preservation, reduction to 40% of control was noted. When an intrinsic H(+)-ATPase inhibitor protein was removed from ESMP by Sephadex gel filtration, decrease of the ATPase activity was enhanced down to 49% and 29% of the control for 24-hr and 48-hr preserved liver, respectively. Molecular damage of the enzyme was confirmed by SDS-PAGE. Alpha subunit of the enzyme decreased time-dependently, and H(+)-ATPase molecules that lost alpha subunit seemed to lose their catalytic activity. Although the cause of the molecular damage of H(+)-ATPase is not clear yet, some mitochondrial protease(s) may be involved.

Postoperative monitoring of the oxygenation state of the graft liver in cases with hepatopulmonary syndrome.

Postoperative changes in the oxygen saturation of hemoglobin in the graft liver (graft SO2) were monitored by near-infrared spectroscopy in four cases complicated by hepatopulmonary syndrome. A plastic cylinder was placed in the abdominal wall, and optical measurements of the graft liver were obtained through this window. Our findings were as follows; (1) graft SO2 decreased after abdominal wall closure, and decreased further 1 day after surgery. (2) Graft SO2 was maintained despite severe hypoxemia, with partial pressure of oxygen in arterial blood as low as 50 mmHg. High hematocrit was beneficial for oxygenating the graft. (3) Graft livers could tolerate hypoxia with a graft SO2 as low as 20%. (4) It may be useful to monitor graft SO2 during the critical period after transplantation for the assessment of graft function.