Papillary dilation vs sphincterotomy in endoscopic removal of bile duct stones a randomized trial with manometric function

To circumvent the long-term effects of papillary ablation for extracting common bile duct stones (<12 mm in diameter) in endoscopic sphincterotomy (EST), endoscopic papillary dilation (EPD) was attempted in 20 patients. To evaluate papillary function before and after the procedures, manometry of the sphincter of Oddi was carried out in 13 with EPD and 10 of 20 patients with EST. Extraction of all stones was successful (100%) in both groups at an equal rate. Repeated numbers of procedures were common in both groups. However, the mean duration of the procedure was high in EPD compared to EST (63 min vs 42 min,P<NS). Adjunctive therapies like mechanical lithotripsy (ML), nasobiliary drainage, and choledochoscopy were included in EPD, while EST required a basket catheter and ML. There was no significant difference on manometry before and after the procedures (P=NS), although papillary function was found to have decreased after the EPD. In contrast, all patients in the EST group lost papillary function after the procedure. Thirty-day morbidity and mortality rate were absent in both groups. Immediate and 2.5-year follow up complications were uncommon in both groups. As a simple method, EPD may be an effective and safe alternative to EST in the management of patients with bile duct stones who require maintenance of papillary function.     

Identification of human hepatic cytochrome P450 sources of N-alkylprotoporphyrin IX after interaction with porphyrinogenic xenobiotics, implications for detection of xenobiotic-induced porphyria in humans.

Porphyrinogenicity of certain xenobiotics depends upon mechanism-based inactivation of specific cytochrome P450 (P450) enzymes, followed by formation of N-alkylprotoporphyrin IX (N-alkylPP). Examination of the porphyrinogenicity of xenobiotics in animals and extrapolation of the results to humans is associated with ambiguity due, in part, to differences between P450 enzymes. The goal of this study was to develop an in vitro test for the detection of N-alkylPPs, produced in human liver after administration of xenobiotics found to be porphyrinogenic in animals. This goal was achieved using fluorometry to detect N-alkylPP formation following mechanism-based inactivation by porphyrinogenic xenobiotics of single cDNA-expressed human P450 enzymes in microsomes prepared from baculovirus-infected insect cells (Supersomes) and in human liver microsomes. The following combinations of P450 enzymes were major sources of N-alkylPPs in Supersomes: CYP3A4 [3-[(arylthio)-ethyl]sydnone (TTMS)]; CYP1A2 and 2C9 [3,5-diethoxycarbonyl-1,4-dihydro-2,6-dimethyl-4-ethylpyridine (4-ethyl DDC)]; and CYP2C9, 2D6, and 3A4 [allylisopropylacetamide (AIA)]. Whereas similarities were found between results with human enzymes in Supersomes and their rat orthologs in rat liver microsomes, some differences were found. The results with TTMS and AIA, but not with 4-ethyl DDC, were the same in individual human enzymes expressed in Supersomes and human liver microsomes. We conclude that some differences exist between human liver P450 enzymes and their rat P450 orthologs in liver microsomes. It would therefore be prudent when dealing with xenobiotics in which porphyrinogenicity depends upon N-alkylPP formation to supplement animal data with studies using human P450 enzymes.     

Isolation, structure, and synthesis of novel 4-quinolinone alkaloids from Esenbeckia leiocarpa

Two new biocidal quinolinone alkaloids, 3-methoxy-1-methyl-2-propyl-4-quinolone [1] and 2(1'-ethylpropyl)-1-methyl-4-quinolone [2], were efficiently isolated using reversed-phase recycling hplc from the leaves of Esenbeckia leiocarpa. The structures were determined through spectroscopic data and confirmed by total synthesis. These alkaloids have antifeedant activities against the pink bollworm, Pectinophora gossypiella.     

Clinical reevaluation of continuous intravenous infusion of 5-fluorouracil--plasma concentrations and clinical dose by continuous intravenous and 60-min infusions

Daily and intermittent continuous intravenous infusions [by gravity drip, (IVG) or infusion pump, (IVP)] and intermittent short-time intravenous drip infusion of 5-FU were carried out on advanced cancer patients. The MTD and dose-limiting toxicity were investigated in relation to the plasma concentrations of 5-FU determined by HPLC. Responses in eleven patients receiving IVG administration daily at 8-21 mg/kg/day were NC, but those given 5-FU alone showed no adverse reactions. Plasma concentrations were too low to be determined. In 9 patients receiving IVG or IVP administration weekly at 60 mg/kg for 24 hr, 1 of the 5 evaluable patients showed reduced hepatic metastatic lesions. One of 4 patients receiving IVP administration weekly at 120 mg/kg for 48 hr showed a disappearance of metastatic lesions in the skeletal muscle, but bone marrow suppression was observed as dose-limiting toxicity. Pharmacokinetics were more stable in IVP than in IVG with less individual difference in the plasma concentrations. Among the outpatients receiving short-time iv, IVG administration once or twice a week, 2 patients given weekly administrations at 20 mg/kg for 60 min showed slight adverse reactions. In 6 patients given high-dose administrations, bone marrow suppression was observed. When pharmacokinetics in the patients given 5-FU for 60 min were compared between the IVG and IVP groups, there were individual differences in plasma concentrations, but the differences were not significant. It was concluded from above results that the following practical dose schedules would be recommendable: 60 mg/kg for 24hr/week by IVP for inpatients and 20 mg/kg for 60 min/week by IVG for outpatients.     

l-cysteine reversibly inhibits glucose-induced biphasic insulin secretion and ATP production by inactivating PKM2

Increase in the concentration of plasma l-cysteine is closely associated with defective insulin secretion from pancreatic β-cells, which results in type 2 diabetes (T2D). In this study, we investigated the effects of prolonged l-cysteine treatment on glucose-stimulated insulin secretion (GSIS) from mouse insulinoma 6 (MIN6) cells and from mouse pancreatic islets, and found that the treatment reversibly inhibited glucose-induced ATP production and resulting GSIS without affecting proinsulin and insulin synthesis. Comprehensive metabolic analyses using capillary electrophoresis time-of-flight mass spectrometry showed that prolonged l-cysteine treatment decreased the levels of pyruvate and its downstream metabolites. In addition, methyl pyruvate, a membrane-permeable form of pyruvate, rescued l-cysteine–induced inhibition of GSIS. Based on these results, we found that both in vitro and in MIN6 cells, l-cysteine specifically inhibited the activity of pyruvate kinase muscle isoform 2 (PKM2), an isoform of pyruvate kinases that catalyze the conversion of phosphoenolpyruvate to pyruvate. l-cysteine also induced PKM2 subunit dissociation (tetramers to dimers/monomers) in cells, which resulted in impaired glucose-induced ATP production for GSIS. DASA-10 (NCGC00181061, a substituted N,N′-diarylsulfonamide), a specific activator for PKM2, restored the tetramer formation and the activity of PKM2, glucose-induced ATP production, and biphasic insulin secretion in l-cysteine–treated cells. Collectively, our results demonstrate that impaired insulin secretion due to exposure to l-cysteine resulted from its direct binding and inactivation of PKM2 and suggest that PKM2 is a potential therapeutic target for T2D.A metabolite, l-cysteine, is found in blood plasma, and its concentration is closely associated with an increase in fat mass and the body-mass index. These values are used as an index of obesity (1, 2), which is a major risk factor for type 2 diabetes (T2D) (3). The relationship between l-cysteine and diabetes has attracted attention because there is increasing evidence for a positive correlation between increases in plasma l-cysteine concentrations and the development and progression of diabetes. For example, increased plasma l-cysteine concentrations were associated with T2D in African American women (4), renal insufficiency [reduced glomerular filtration rate (GFR)] in T2D patients (5), obstructive sleep apnea [a risk factor for diabetes (6, 7)], and insulin resistance among Europeans (8).Reduced insulin secretion from pancreatic β-cells is the major cause of T2D (9, 10). Many investigators have studied the molecular mechanisms of glucose-stimulated insulin secretion (GSIS), which have been elucidated in detail. Elevated extracellular glucose concentration results in the enhancement of ATP production, an increased ATP/ADP ratio, the closure of ATP-sensitive K channels (K_ATP channels), and depolarization (11). The resulting activation of voltage-dependent Ca²⁺ channels (VDCCs) induces an influx of calcium ions and elevated intracellular Ca²⁺ concentrations, which triggers insulin secretion (11). Perifusion experiments have shown that insulin secretion could be categorized into two phases. The first phase involves a sharp increase in insulin secretion within ∼5 min, followed by a second phase, during which moderate insulin secretion lasts for hours (9, 12). A loss of the GSIS first phase is closely associated with the future development of T2D (9, 13, 14).Many recent studies have reported that l-cysteine is involved in GSIS. In mouse pancreatic islets and mouse insulinoma 6 (MIN6) cells, l-cysteine treatment decreased both intracellular ATP levels and insulin secretion (15). Ammon et al. also reported that the total amount and GSIS second phase were specifically inhibited by l-cysteine for rat pancreatic islets (16). Several groups showed that an increase in the H₂S moiety, which is generated from l-cysteine in cells (17), was one possible cause for l-cysteine–induced impairment of GSIS by inhibiting K_ATP channels and VDCCs (18–20). However, opposite results were reported in that l-cysteine increased the amount of total and first-phase insulin secretion by rat pancreatic islets (16, 21). Thus, the effects of l-cysteine on GSIS remain controversial. It should be noted that most of these studies on the effects of l-cysteine on GSIS were performed using experimental conditions in which insulin-secreting cells were only transiently exposed (∼1 h) to a high glucose solution that contained l-cysteine. However, in obese or T2D patients, insulin-secreting cells can be exposed to plasma that contains l-cysteine for prolonged periods of time; thus, continuous exposure to an l-cysteine–containing solution is necessary to investigate the precise effects of l-cysteine on GSIS in insulin-secreting cells.In this study, we found that l-cysteine treatment of statically incubated or perifused MIN6 cells and mouse pancreatic islets resulted in reversibly inhibiting GSIS. A comprehensive analysis of charged metabolites in l-cysteine–treated MIN6 cells by capillary electrophoresis time-of-flight mass spectrometry (CE-TOF-MS) showed significant accumulations of l-cysteine and, concomitantly, decreased levels of pyruvate and its downstream metabolites in the tricarboxylic acid (TCA) cycle in these cells. Biochemical experiments for pyruvate kinase activity in vitro and in MIN6 cells showed that l-cysteine specifically inhibited the activity of pyruvate kinase muscle isoform 2 (PKM2), an isoform of major pyruvate kinases in pancreatic islets (22) that catalyzes the glycolytic conversion of phosphoenolpyruvate (PEP) to pyruvate. PKM2 inactivation resulted from l-cysteine–induced subunit dissociation (tetramers to dimers/monomers) and inhibited glucose-induced ATP production for GSIS in MIN6 cells. Impaired GSIS due to l-cysteine was restored by treatment with methyl pyruvate, a membrane-permeable form of pyruvate, or DASA-10 (NCGC00181061, a substituted N,N′-diarylsulfonamide), a specific activator of PKM2 (23). Thus, we concluded that reduced PKM2 activity due to l-cysteine inhibited ATP production and subsequently inhibited GSIS by insulin-secreting cells.     

Bilirubin diglucuronide transport by rat liver canalicular membrane vesicles: stimulation by bicarbonate ion

The purpose of this study was to provide further insight into the mechanism of bilirubin diglucuronide excretion through the hepatocyte canalicular membrane by investigating the uptake of (3H)bilirubin diglucuronide by purified canalicular membrane vesicles of rat liver. The uptake was analyzed by a rapid filtration technique. The difference between vesicle-associated (3H)bilirubin diglucuronide at 37 degrees C and at 0 degree C during the initial 1 min was regarded as uptake. Twenty second uptake was saturated by increasing the (3H)bilirubin diglucuronide concentration at a vesicle-inside-directed 100 mmol/L KCl gradient (Km = 75 mumol/L, Vmax = 320 pmol/mg protein.20 sec at 37 degrees C). No sodium dependency was observed. When canalicular membrane vesicles were preincubated with nonlabeled bilirubin diglucuronide, the uptake increased 1.3-fold (transstimulation). Vesicle-inside-positive potential induced by valinomycin and potassium caused a 1.4-fold increase in the uptake. When Cl- was replaced by equivalent ion concentrations of SO4(2-), HCO3-, NO3- and SCN-, the uptake was 78%, 244%, 68% and 50%, respectively, and specific stimulation by HCO3- was observed (Km = 75 mumol/L, Vmax = 700 pmol/mg protein.20 sec at a vesicle-inside-directed 100 mmol/L KHCO3 gradient at 37 degrees C). The uptake was inhibited in a dose-dependent manner by the addition of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. The uptake was ATP independent. From these results, it was concluded that bilirubin diglucuronide transport through the canalicular membrane is carrier mediated, electrogenic and stimulated by HCO3-.     

Treatment of paroxysmal supraventricular tachycardia with intravenous injection of adenosine triphosphate

Intravenous adenosine triphosphate rapidly terminated all 11 episodes of paroxysmal supraventricular tachycardia in 10 patients. Eight patients reported side effects but these resolved within 20 seconds and did not require treatment. Adenosine triphosphate is a suitable agent for the rapid termination of paroxysmal supraventricular tachycardia.     

Assessment of Prognostic Factors in Follicular Lymphoma Patients

Prognostic factors, including clinical, biological, and histological parameters, were assessed for 94 patients with follicular lymphomas at our institute. Follicular lymphomas constituted 7.7% (94/1208) of malignant lymphomas in this study. Eighteen patients were diagnosed with stage I follicular lymphoma, 20 with stage II, 23 with stage III, and 33 with stage IV. The cases of follicular lymphoma were subclassified as: follicular small cleaved cell lymphoma (FSC) in 20 cases, follicular mixed cell lymphoma (FMX) in 59 cases, and follicular large cell lymphoma (FLC) in 15 cases. The patients comprised 49 men and 45 women with a median age of 54 years (range, 25-84 years). The complete response rate was 76.5%, and the median survival time was 13 years. The expected 10-year overall survival and event-free survival rates were 61.9% and 38.2%, respectively. Univariate analysis identified the factors associated with poor survival as elevated serum lactate dehydrogenase (LDH) level (P < .0001), age of >60 (P < .0001), Ann Arbor stage III/IV (P < .01), and Eastern Cooperative Oncology Group performance status (PS) of 2 to 4 (P = .048). Multivariate analysis showed that LDH, age, and PS were independent predictors. After application of the International Prognostic Index (IPI), the 10-year survival rates for the low-risk, low-intermediate risk, high-intermediate risk and high-risk groups were 80.4%, 48.7%, 21.9%, and 0.0%, respectively. The differences among these groups were significant at P < .01. The IPI for aggressive non-Hodgkin's lymphoma was found to be applicable to survival prediction for Japanese follicular lymphoma patients.