Radical resection for pancreatic cancer

Ductal adenocarcinoma of the pancreas is still characterized by (1) poor prognosis after surgery and (2) extreme difficulty in early diagnosis, and we need a breakthrough. For the first problem, we have performed a wide range of lymphatic and connective tissue clearance (extended pancreatectomy) which has succeeded in improving the 5-year survival rate from 8% to 24% via decreasing the incidence of locoregional recurrence. When liver perfusion chemotherapy via the hepatic artery and the portal vein was added to the patients who had received extended pancreatectomy, the 5-year survival rate was further elevated to 40% via decreasing the incidence of hepatic metastasis. We conclude that pancreatic cancer should be treated by the better-balanced treatments between locoregional control and prevention of hepatic metastasis. For the second problem, we have more actively collected pancreatic juice to perform cytodiagnosis even though no obvious tumor was delineated by the conventional imaging diagnoses. When cancer cells were detected in the pancreatic juice, our method of intraoperative cytology was very useful in precisely locating the occult lesion indicating an appropriate range of pancreatectomy. The resected pancreas was proven to have included borderline malignancy and in situ or minimally-invasive carcinoma by the postoperative histology, and disease-free 5-year survival rate was 100%. In the future, we need to detect patients with a high risk of pancreatic cancer and develop a less-painful method to collect the pancreatic juice.     

Localization of mature neprilysin in lipid rafts

Alzheimer's disease (AD) is characterized by senile plaques caused by amyloid-β peptide (Aβ) accumulation. It has been reported that Aβ generation and accumulation occur in membrane microdomains, called lipid rafts, which are enriched in cholesterol and glycosphingolipids. Moreover, the ablation of cholesterol metabolism has been implicated in AD. Neprilysin (NEP), a neutral endopeptidase, is one of the major Aβ-degrading enzymes in the brain. Activation of NEP is a possible therapeutic target. However, it remains unknown whether the activity of NEP is regulated by its association with lipid rafts. Here we show that only the mature form of NEP, which has been glycosylated in the Golgi, exists in lipid rafts, where it is directly associated with phosphatidylserine. Moreover, the localization of NEP in lipid rafts is enhanced by its dimerization, as shown using the NEP E403C homodimerization mutant. However, the protease activities of the mature form of NEP, as assessed by in vitro peptide hydrolysis, did not differ between lipid rafts and nonlipid rafts. We conclude that cholesterol and other lipids regulate the localization of mature NEP to lipid rafts, where the substrate Aβ accumulates but does not modulate the protease activity of NEP.     

Mutation (Ser284Leu) of neuronal nicotinic acetylcholine receptor alpha 4 subunit associated with frontal lobe epilepsy causes faster desensitization of the rat receptor expressed in oocytes

To date five mutations in two major constituents of neuronal nicotinic acetylcholine receptor (nAChR) in the brain, i.e. alpha4 and beta2 subunits, have been identified to be associated with autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). Among them, only Ser284Leu, a point mutation in alpha4 subunit identified in ADNFLE as well as in a sporadic case with nocturnal frontal lobe epilepsy, remains to be characterized electrophysiologically. We examined the properties of rat nAChR harboring Ser284Leu reconstituted on Xenopus oocytes. Currents elicited in response to application of acetylcholine to oocytes expressing wild type or mutant nAChR were measured by a standard two-microelectrode voltage clamp method. Compared with wild-type nAChR, the mutant nAChR had a comparable EC(50) value for acetylcholine whereas it showed faster desensitization and lower Cs(+)/Na(+) permeability ratio. Ser284Phe, a putative mutation constructed for comparison, exhibited similar properties. These findings indicate that Ser(284) plays an important role in gating of nAChR along with Thr(276) and Ser(280), and suggest that mutation at Ser(284) could reduce nAChR activity similar to other mutations of alpha4 subunit found in ADNFLE.     

Usefulness of body surface mapping to differentiate patients with Brugada syndrome from patients with asymptomatic Brugada syndrome

We attempted to determine the usefulness of body surface mapping (BSM) for differentiating patients with Brugada syndrome (BS) from patients with asymptomatic Brugada syndrome (ABS). Electrocardiograms (ECG) and BSM were recorded in 7 patients with BS and 35 patients with ABS. Following the administration of Ic antiarrhythmic drugs, BSM was recorded in 5 patients with BS and 16 patients with ABS. The maximum amplitudes at J0, J20, J40 and J60 were compared between the 2 groups, as were 3-dimensional maps. The maximum amplitudes at J0, J20 and J60 under control conditions were larger in patients with BS than in patients with ABS (P < 0.05). A three-dimensional map of the ST segments under control conditions in patients with BS showed a higher peak of ST elevation in the median precordium compared to that for patients with ABS. Increases in ST elevation at J20, J40 and J60 following drug administration were greater in patients with BS than in patients with ABS (P < 0.05). Evaluation of the change in amplitude of the ST segment at E5 caused by Ic drug administration was also useful for differentiating between the 2 groups. In conclusion, BSM was useful for differentiating patients with BS from those with ABS.     

Purification and characterization of a halotolerant intracellular protease from Bacillus subtilis strain FP-133

A halotolerant strain FP-133, able to grow at concentrations of 0-12.5% (w/v) NaCl, was isolated from a fish paste and identified as Bacillus subtilis . B. subtilis strain FP-133 produced an intracellular protease which showed catalytic activity under saline conditions. The enzyme was purified to homogeneity 143-fold with a yield of 0.9%. The purified enzyme showed an optimum activity at a concentration of 5% (w/v) NaCl. After storage in 7.5% (w/v) NaCl at 4 degrees C for 24 h, the enzyme kept 100% of its activity. The molecular mass of the protease was determined to be 59 kDa by gel filtration; the protein consisted of four subunits each with a molecular mass of 14 kDa. The enzyme showed aminopeptidase activity. It acted on L-leucyl-p-nitroanilide, L-leucyl-beta-naphthylamide, and oligopeptides containing glycine, L-histidine, or L-leucine. The K(m ) and V (max) values for L-leucyl-p-nitroanilide were 18 microm and 2.2 mm/h mg, respectively. The enzyme was activated by Fe(2+), Fe(3+), and Ni(2+) in synergism with Mg(2+).     

Oncocytic non-functioning endocrine tumor of the pancreas

Herein is presented the case of a malignant non-functioning endocrine tumor of the pancreas with oncocytic features, and a discussion on the high incidence of malignancy in oncocytic endocrine pancreatic tumors. The patient was a 65-year-old woman who showed no paraneoplastic symptoms produced by functioning pancreatic endocrine tumors. The primary tumor was located in the body and tail of the pancreas, and had metastasized to the liver. Tumor cells were arranged in a ribbon-like or trabecular pattern and had an abundant eosinophilic cytoplasm containing numerous mitochondria and neurosecretory granules. The cytoplasm of the tumor cells was intensely stained with an antimitochondrial antigen antibody. Most tumor cells stained positively with Grimelius stain and for chromogranin A. Some tumor cells also stained for synaptophysin. However, the tumor cells negatively stained for hormones such as insulin, glucagon, somatostatin, gastrin, vasoactive intestinal peptide and pancreatic polypeptide, for serotonin, and for pancreatic enzymes such as amylase and trypsin. Analysis of 18 oncocytic pancreatic endocrine tumors, consisting of those reported previously and that in the present case, suggests that the high incidence of malignancy in oncocytic endocrine tumors is associated with the high incidence of non-functioning endocrine tumors among them, most of which are malignant.     

Increased sinusoidal resistance is responsible for the basal state and endothelin-induced venoconstriction in perfused cirrhotic rat liver

The localization of increased intrahepatic vascular resistance and the segmental vascular responsiveness to endothelin-1 are not well known in liver cirrhosis. We determined the segmental vascular resistances and their response to endothelin-1 of isolated portally perfused bile duct ligation (BDL)-induced cirrhotic rat livers. The portal occlusion pressure (Ppo) and the hepatic venous occlusion pressure (Phvo) were obtained by analyzing the profiles of the portal (Ppv) and hepatic venous (Phv) pressures during the double occlusion maneuver of simultaneous occlusions of the inflow and outflow perfusion lines. From the pressure gradients among Ppv, Ppo, Phvo, and Phv, the portal-hepatic venous resistance was assigned to three segments of the portal [Rpv = (Ppv − Ppo)/blood flow (Q)], sinusoidal [Rsinus = (Ppo − Phvo)/Q] and hepatic venous [Rhv = (Phvo − Phv)/Q] resistances. Rsinus, but not Rpv or Rhv, was significantly greater in BDL livers than in sham livers. Endothelin-1 (0.1–1 nM) increased Rpv and Rsinus to a similar magnitude, but not Rhv, in both sham and BDL. At 3 nM, the responsiveness of Rpv was smaller in BDL than in sham, but that of Rsinus were similar between in BDL and sham. In conclusion, increased sinusoidal resistance accounts for increased intrahepatic resistance of BDL-induced liver cirrhosis. Endothelin-1 contracts portal veins and sinusoids, but not hepatic veins, in both sham and cirrhotic livers. Sinusoidal contractility to endothelin-1 is not impaired in cirrhotic livers.     

Histopathological predictor for regional lymph node metastasis in gastric cancer

Regional lymph node metastasis in gastric cancer is a definitive indicator of the patient’s prognosis. The goal of this study was to identify the predictors for lymph node metastasis among all the possible histopathological parameters, especially by conducting an objective discrimination of the lymphatic and blood vessels. A total of 210 resected primary gastric cancers with or without lymph node metastasis were evaluated based on the conventional histopathological parameters together with immunohistochemistry using antisera-recognizing lymphatic endothelial hyaluronan receptor-1 (LYVE-1), von Willebrand factor, and lymphangiogenesis promoter vascular endothelial growth factor-C (VEGF-C) antibodies. A multivariate regression analyses of the results indicated that only lymphatic invasion was a significant independent predictor of lymph node metastasis at any stage of cancer invasion. VEGF-C expression was partially related to lymph node metastasis in early gastric cancer. The identification of lymphatic invasion by LYVE-1 antibody is therefore useful to predict regional lymph node metastasis in gastric cancer.     

Induction of specific and flavivirus—Cross-reactive CTLs by immunization with a single dengue virus-derived CTL epitope peptide

Specificities of cytotoxic T lymphocyte (CTL) effector cells induced in BALB/c mouse by immunization with the single modified CTL epitope peptide derived from NS3 of dengue virus types 1 and 3, or that of dengue virus types 2 and 4 were examined. The effector cells included CTLs specific for the epitope peptide used for immunization and those cross-reactive to epitope peptides of other flaviviruses. A CTL clone, 2F7, was established from the effector cells. The clone 2F7 was specific for the epitope peptide used for immunization. Recognition by the effector cells was remarkably impaired by amino acid substitutions at positions 3, 5, and 6 of the epitope peptides. These results indicate that immunization with a single CTL epitope peptide of dengue viruses induces serotype-specific CTLs as well as CTLs cross-reactive to the other flaviviruses, and that the a.a. residues at positions 3, 5, and 6 are critical for cross-reaction.