Akira Tamura

WFS1 protein modulates the free Ca2+ concentration in the endoplasmic reticulum

The WFS1 gene, encoding an endoplasmic reticulum (ER) membrane glycoprotein, is mutated in Wolfram syndrome characterized by diabetes mellitus and optic atrophy. Herein, Ca2+ dynamics were examined in WFS1‐knockdown and ‐overexpressing HEK293 cells. Studies using ER‐targeted Ca2+‐sensitive photoprotein aequorin demonstrated WFS1 protein to positively modulate ER Ca2+ levels by increasing the rate of Ca2+ uptake. Furthermore, Ca2+ imaging with Fura‐2 showed the magnitude of the store‐operated Ca2+ entry to parallel WFS1 expression levels. These data indicate that WFS1 protein participates in the regulation of cellular Ca2+ homeostasis, at least partly, by modulating the filling state of the ER Ca2+ store.

Distribution of two types of lymphocytes (intraepithelial and lamina-propria-associated) in the murine small intestine

The intestine, which is exposed to nutrition and to food-derived antigens and microbes including viruses and bacteria, might be an important site for the immune response. Crucial structural and functional differences exist between the small and large intestine, regional differences even having been demonstrated within the small intestine. Accordingly, intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs) might be heterogeneous among the different intestinal regions. The aim of this study has been to describe, as accurately as possible, the numbers and T-cell receptor (TCR) phenotypes of IELs and LPLs present in distinct regions of the murine small intestine under physiological conditions. Using an immunohistological technique to differentiate IELs from LPLs, the differential enumeration of IELs and LPLs in distinct regions of the murine small intestine, based upon their definition originally determined by their location, has been performed for the first time and has demonstrated that (1) there are more IELs than LPLs in the duodenum and jejunum, but more LPLs than IELs in the ileum, (2) in the duodenum and jejunum, TCRγδ IELs account for 70%–75% of the total CD3+ IELs, a much greater percentage than previously reported, (3) the ratio of TCRγδ to TCRαβ IELs is inverted in the ileum, with more than 75% IELs being TCRαβ-positive, (4) the lamina propria forms one functional unit throughout the small intestine in terms of the TCR subset components (TCRαβ:TCRγδ=3:1), and (5) the ileum is entirely different from other regions of the small intestine. To deepen our understanding of the functional significance of the small intestine as an immunologically competent organ, the precise distributions of IELs and LPLs, the ratio of their various subsets, and the strict distinction of IELs and LPLs, as described in this study, is indispensable.

Glucose transport into human erythrocytes treated with phospholipase A2 or C

Phospholipase A2 induced crenation of human erythrocytes and decreased glucose transport activity (influx rate) by 40% when 51% of phosphatidylcholine (PC) in the membrane was hydrolyzed. On the other hand, phospholipase C induced invagination of the cells and negligibly affected the glucose transport in the case of 21% hydrolysis of the PC. By altering the pH of the medium for suspending cells treated with phospholipase A2 from 7.4 to 6.0, cell shape was changed from clear crenation to slight invagination, but glucose transport activity was not affected. Cells that were treated with phospholipase A2 and then washed with albumin to remove free fatty acids produced in the cell membrane showed an almost normal cell shape and slightly higher glucose transport activity than did untreated cells. The ratios of beta-D-glucose transport rate to alpha-D-glucose transport rate in untreated cells, cells treated with phospholipase A2 and cells treated with phospholipase C were 1.13, 1.04, and 1.20, respectively. These results demonstrate that the drastic morphological change (invagination or crenation) induced by the treatment with phospholipases bears no clear relationship to the activity of glucose transport and suggest that the increase in the volume of the outer half of the lipid bilayer might reduce the rate of glucose transport across the human erythrocyte membrane and change the anomeric preference of glucose transport.

Efficacy of laparoscopic surgery for recurrent hepatocellular carcinoma.

BACKGROUND/AIMS Although laparoscopic hepatectomy is increasingly performed for hepatocellular carcinoma, few studies have investigated the efficacy of laparoscopic surgery, including hepatectomy for the treatment of recurrent hepatocellular carcinoma. We report the results of our study on the efficacy of laparoscopic surgery. METHODOLOGY Forty-three of 123 hepatocellular carcinoma patients underwent laparoscopic surgery in 2002-2009; 16 were treated for recurrent hepatocellular carcinoma. The characteristics and postoperative outcomes of these 16 patients were retrospectively investigated. RESULTS Seven patients underwent laparoscopic hepatectomy, 1 underwent laparoscopic radiofrequency ablation, 7 underwent resection of an extrahepatic metastatic tumor and 1 patient received diagnostic assessment. Twelve of the 15 surgical patients had a repeat recurrence after surgery. Survival at 2 years was significantly higher in patients with intrahepatic vs. extrahepatic recurrence (100% vs. 42.9%). In addition, overall survival (51.2 vs. 23.0 months) was significantly longer in patients with intrahepatic recurrence, although disease-free survival (19.2 vs. 10.6 months) was not so. CONCLUSIONS Laparoscopic surgery for recurrent hepatocellular carcinoma enabled precise tumor localization, more accurate diagnosis, and more careful selection of therapy based on hepatic functional reserve and recurrence type. Laparoscopic surgery appears to be an effective, minimally invasive option for the diagnosis and treatment of recurrent hepatocellular carcinoma.

Stimulatory effect of phospholipase A2 treatment on glucose utilization in human erythrocytes

We examined whether modification of membrane phospholipids of human erythrocytes by hydrolysis with phospholipase A2 (PLA2 from bee venom) would affect glucose utilization, chosen as a typical model of intracellular metabolism, and, if so, intended to clarify the mechanism of the alteration of glycolysis. Treatment of erythrocytes with PLA2 induced a marked shape change (i.e., crenation) and significantly increased the rate of lactate production from glucose. Available evidence indicated that there is no relevance of this cell-shape change to the alteration of glycolysis. The lack of a detectable effect of papain treatment on glycolysis in PLA2-treated cells suggested that the increase in glycolysis by PLA2 treatment might not be caused by the conformational change of band-3 protein through modulation of membrane phospholipids. The result of the measurement of lactate production in the presence and absence of ouabain did not support the idea that hydrolysis of phospholipids by PLA2 treatment makes plasma membranes leaky to Na+ and consequently enhances glycolysis through activation of Na+/K(+)-ATPase. The action of PLA2 on glycolysis was abolished by extraction of free fatty acids in the cell membrane with bovine serum albumin. Loading erythrocytes with free fatty acid (oleic acid, linoleic acid, or arachidonic acid) caused a significant increase in glycolysis. Analysis of glycolytic intermediates suggested that the enhancement of glycolysis was induced by activation of 6-phosphofructokinase. The data, thus, indicate that treatment of human erythrocytes with PLA2 significantly accelerates glucose utilization and suggest that the stimulation of glycolysis is caused by activation of 6-phosphofructokinase through liberation of free fatty acids of membrane phospholipids by PLA2.

Effectiveness of laparoscopic subtotal cholecystectomy: perioperative and long-term postoperative results.

BACKGROUND/AIMS We analyzed perioperative and long-term postoperative data to determine the effectiveness of laparoscopic subtotal cholecystectomy (LSC). METHODOLOGY 89 LSCs done at our center were classified into 3 groups according to the operative procedure required to treat severe cholecystitis. Perioperative and long-term postoperative results were then compared with standard laparoscopic cholecystectomy (s-LC). RESULTS Operative time was longer and postoperative CRP level was higher for LSC. Significantly more bleeding and longer operative times were seen only in the LSC-II subgroup, and average postoperative hospital stay was longer only in the LSC-III subgroup. As for long-term (1.5 to 8 years) postoperative results, 3 of 26 LSC-III cases had a relapse of cholecystolithiasis in remnant gallbladder tissue 2 years or later after initial surgery. CONCLUSIONS There were no serious intraoperative complications such as the bile duct injury, and a smaller proportion of procedures were intraoperatively converted to open laparotomy. It is considered that LSC is a safe, useful surgical procedure to the patients in whom the neck of the gallbladder is anatomically unclear due to cholecystitis or fibrosis although patients undergoing LSC-III should be monitored for cholecystolithiasis in remnant gallbladder tissue. LSC is often the procedure of choice for patients.

Stimulatory effect of fatty acid treatment on glucose utilization in human erythrocytes

We previously reported that treatment of human erythrocytes with bee venom phospholipase A2 increased the rate of lactate production from glucose. This increase was suggested to be mediated through liberation of free fatty acids from membrane phospholipids. So, in the present study we examined the mechanism of stimulation of glycolysis by fatty acids. Treatment of intact erythrocytes with most of the 15 fatty acids tested resulted in stimulation of lactate production from glucose. Among the fatty acids tested, myristoleic acid showed the highest stimulatory activity. The ratio of moles of lactate produced to those of glucose utilized was about 1.9 in both myristoleic acid-treated and untreated cells. Treatment of erythrocytes with myristoleic acid did not affect the amount of 2,3-bisphosphoglycerate. Lactate production from D-glyceraldehyde, which is thought to be phosphorylated to D-glyceraldehyde 3-phosphate and then metabolized in the glycolytic pathway, was not at all affected by treatment of cells with myristoleic acid. The cross-over plot of glycolytic intermediates suggested that the enhancement of glycolysis was induced by activation of the 6-phosphofructokinase (PFK) step. Fatty acids incorporated into erythrocytes were found to be present predominantly in the cytoplasm rather than in the plasma membrane. The PFK activity, but not the hexokinase activity, in hemolysates was clearly increased by a set of fatty acids, and myristoleic acid was again the most potent. However, partially purified human erythrocyte PFK was not activated by the acid. We conclude that fatty acids stimulate glycolysis through activation of PFK in cooperation with some other component(s) in erythrocytes.

Laparoscopic Hepatectomy for the Patients with Hepatocellular Carcinoma.

原発性肝癌に対し腹腔鏡下肝切除を施行し, その手術手技や適応について検討した. 手術手技: 肝実質切離にはマイクロ波凝固装置と高周波凝固機能内臓超音波吸引装置の併用やハーモニックスカルペルを使用する. 視野展開はCO2塞栓の危険性から気腹を避け腹壁つり上げ法にする. 脈管切離にはクリッピングを, グリソンや肝静脈などの太い脈管には腹腔鏡用自動縫合器を使用する. 結果: 肝部分切除術7例, 肝外側区域切除術2例に試み, 1例は止血のため開腹術に移行したが, 8例は安全に施行できた. 手術時間, 出血量は通常の開腹術と差がなかった. 術後疼痛は軽微で, 回復も早く, 経過も良好であった. 適応: 高度肝硬変は原則的に除外, 系統的肝切除は外側区域切除を除き適応外とし, 腫瘍径は5cm以下, 部位は肝下区域の表面や辺縁, 外側区域に局在するものが適応となる. 原発性肝細胞癌に対しても, 腹腔鏡下肝切除は根治性と低侵襲を兼ね備えた外科治療の1手段になるものと考える.

7105 Laparoscopic hepatectomy for liver tumor as a new surgical alternative.

The development of laparoscopic hepatectomy has been impeded by unsolved technical difficulties. However, a variety of laparoscopic instruments and devices have been developed, leading us to establish laparoscopic hepatectomy. Results of a clinical series of 23 patients are reported. Surgical procedure: A microwave tissue coagulator was used in combination with an ultrasonic surgical aspirator or a laparoscopic coagulating shears to divide hepatic parenchyma by using abdominal wall lift without pneumoperitoneum. Branched vessels and ducts were clipped and transected. The endoscopic linear stapler was used to transect the Glissons pedicle and left hepatic vein. Patients: The indications were hepatocellular carcinoma in 12 cases, isolated metastatic tumor in 4 cases, hemangioma in 4 cases, others in 3 cases. The underlying liver diseases was liver cirrhosis in 9 cases and chronic hepatitis in 5 cases. Result: Six patients underwent left lateral segmentectomy and seventeen patients underwent partial hepatectomy. The 22 procedures were performed safely. There were notable difference in blood loss and operating time compared with open hepatectomy. No postoperative complication including no bile leakage and abscess, except ascitis and pneumonia in one case was observed. The postoperative recovery such as oral intake and ambulatory was faster and pain was minimal. Indication: The preoperative assessment of liver function for laparoscopic and open hepatectomy is the same. Tumors smaller than 5 cm, and nodular types, especially extrahepatic growth types, are the best candidates for laparoscopic resection. Concerning location, tumors in the lower segment and left lateral segment were good indication. Conclusion: The laparoscopic hepatectomy, especially partial or left lateral segmentectomy, appears to be a viable surgical alternative in selected patients as minimal invasive surgery.

7107 Clinical significance of hepatocyte growth factor and portal blood flow as a parameter of hepatic function following cO2pneumoperitoneum in cirrhotic patients.

Background: Laparoscopic surgery under carbon dioxide (CO2) pneumoperitoneum has many benefits. However, little is known concerning the risks of CO2pneumoperitoneum for the patients with liver cirrhosis. Influence of CO2pneumoperitoneum for the cirrhotic patients was investigated by assessing hemodynamics of portal vein and change of cytokine. Material & Methods: Participants were cholelithiasis patients divided into two groups: Group I (with liver cirrhosis: LC, n=12), and Group II (without liver cirrhosis: Control, n=12). The cross section area of the portal vein trunk (S) and the velocity of the portal blood (V) were measured using a laparoscopic pulsed Doppler ultrasonic probe (Aloka Ind., Ltd., Tokyo, Japan) inserted through the epigastric trocar when the abdomen was insufflated to an intra-abdominal pressure (IAP) of 4mmHg to 15 mm Hg preoperatively. The mean portal blood flow Φ at each IAP was calculated from the measured variables of S and V. In addition, plasma level of hepatocyte growth factor (HGF), interleukin (IL)-6, IL-8, tumor necrosis factor (TNF) were measured during CO2pneumopeitoneum. Total bilirubin (Tbil), AST, ALT were measured at postoperative day 1(1POD). Results: Laparoscopic cholecystectomy was performed successfully without any complications in all cases. With increases in IAP, S and V value were significantly decreased only in the LC group. F value was also significantly decreased with elevation of IAP in the LC group. No significant differences in changes of IL-6, IL-8 and TNF value during the preoperative CO2pneumopeitoneum were found in both group, but only HGF showed significant differences in changes of IAP in LC group. HGF and F value in IAP of 15mmHg showed significant correlations with AST and ALT at 1POD. Conclusions: On the basis of these results, HGF and F value were considered to be a good indicator for evaluation of liver function and CO2pneumopeitoneum during laparoscopic surgery would be a deteriorating factor for the patients with liver cirrhosis.