Intracellular recordings were made to investigate the mechanism, site, and ionic basis of generation of the rapid depolarization induced by superfusion with ischemia-simulating medium in hippocampal CA1 pyramidal neurons of rat tissue slices. Superfusion with ischemia-simulating medium produced a rapid depolarization after approximately 6 min of exposure. When oxygen and glucose were reintroduced, the membrane potential did not repolarize but depolarized further, reaching 0 mV approximately 5 min after reintroduction. Simultaneous recordings of changes in cytoplasmic Ca2+ concentration ([Ca2+]i) and membrane potential recorded from 1-[6-amino-2-(5-carboxy-2-oxazolyl)-5-benzofuranyloxy]-2-(2- amino-5-methylphenoxy)-ethane-N,N,N',N'-tetraacetic acid pentaacetoxymethyl ester (Fura-2/AM) loaded slices revealed a rapid increase in [Ca2+]i in all CA1 layers corresponding to the rapid depolarization of the soma membrane. The result suggests that the rapid depolarization is generated not only in the soma but also in the apical and basal dendrites. Application of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), DL-2-amino-4-phosphonobutyric acid, and DL-2-amino-3-phosphonopropionic acid or bicuculline did not affect the amplitude and the maximal slope. Reduction in the concentration of extracellular Ca2+ or addition of CNQX or DL-2-amino-5-phosphonopentanoic acid delayed the onset of the rapid depolarization. The amplitude of the rapid depolarization recorded with Cs acetate electrodes in tetraethylammonium-containing medium had a linear relationship to the membrane potential between -50 and 20 mV. The reversal potential was shifted in the hyperpolarizing direction by a decrease in either [Na+]o or [Ca2+]o, whereas the reversal potential was shifted in the depolarizing direction by a decrease in [Cl-]o or using CsCl electrodes. An increase or decrease in [K+]o did not affect the reversal potential. These results indicate that the rapid depolarization is Na+, Ca2+, and Cl- dependent. The lack of effects of changes in [K+]o is probably due to the accumulation of interstitial K+ before generating the rapid depolarization. Prolonged application of ouabain (30 microM) caused an initial small hyperpolarization, a subsequent slow depolarization, and a rapid depolarization. In summary, the present study has demonstrated that the rapid depolarization is voltage-independent and is probably due to a nonselective increase in permeability to all participating ions, which may occur only in pathological conditions. The underlying conductance change is primarily the result of inhibition of Na,K-ATPase activity in the recorded neuron.

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Although hepatitis C virus (HCV)-related cirrhosis has been suggested as a risk factor for intrahepatic cholangiocarcinoma (ICC), few sizeable studies have tested this hypothesis. We investigated ICC risk factors, with special reference to HCV infection. We conducted a hospital-based case-control study including 50 ICC patients and 205 other surgical patients without primary liver cancer. HCV seropositivity was detected in 36% of ICC patients and 3% of controls. By univariate analysis, the odds ratio (OR) for association of anti-HCV antibodies with development was 16.87 (95% confidence interval (CI), 5.69 to 50.00). History of blood transfusion or diabetes mellitus, elevated serum total bilirubin, elevated aspartate aminotransferase and alanine aminotransferase, decreased serum albumin and decreased platelet count were identified as other possible ICC risk factors. By multivariate analysis, anti-HCV antibodies (adjusted OR, 6.02; 95% CI, 1.51 to 24.1), elevated alanine aminotransferase, decreased serum albumin, and decreased platelet count were found to be independent risk factors for ICC development. As liver status worsened, the adjusted OR for ICC tended to increase. HCV infection is a likely etiology of ICC in Japan. ntrahepatic cholangiocarcinoma (ICC), a malignant tumor arising from bile duct epithelium, is the second most common primary liver cancer, following hepatocellular carcinoma (HCC). Established risk factors for HCC include chronic active hepatitis, and hepatic fibrosis induced by hepatitis virus infection or heavy alcohol intake. On the other hand, risk factors for ICC remain uncertain. Hepatitis C virus (HCV)-related cirrhosis has been suggested as a risk factor for ICC, 1, 2) but confirmation is required. We therefore investigated risk factors for ICC, with special reference to HCV infection. Patients and MethodsWe conducted a hospital-based study using a matched case-control design, including 50 patients with pathologically diagnosed ICC and 205 other surgical patients without primary liver cancer including ICC.Patients. Fifty patients were treated for ICC between January 1991 and December 2002 in the two major medical centers of Osaka City (Department of Gastroenterological and HepatoBiliary-Pancreatic Surgery, Osaka City University Hospital; Department of Gastrointestinal Surgery, Osaka City General Hospital). ICC was diagnosed by pathologic examination. Two to five control patients at the two medical centers were matched with each ICC patient according to gender, 5-year age group, and operation date (within 1 year). Patients with HCC were excluded from the control group since hepatitis B virus (HBV) infection and HCV infection are established etiologic agents of HCC. In all, control patients numbered 205. This study was conducted in accordance with the Helsinki Declaration and the guidelines of the ethics committees at our institutions.Etiologic factors and data collection. Factors evaluated for association with ICC risk included habits such as alcohol consumption and smoking; family histor...

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine released from T-cells and macrophages. Although a detailed understanding of the biological functions of MIF has not yet been clarified, it is known that MIF catalyzes the tautomerization of a nonphysiological molecule, D-dopachrome. Using a structure-based computer-assisted search of two databases of commercially available compounds, we have found 14 novel tautomerase inhibitors of MIF whose K(i) values are in the range of 0.038-7.4 microM. We also have determined the crystal structure of MIF complexed with the hit compound 1. It showed that the hit compound is located in the active site of MIF containing the N-terminal proline which plays an important role in the tautomerase reaction and forms several hydrogen bonds and undergoes hydrophobic interactions. A crystallographic study also revealed that there is a hydrophobic surface which consists of Pro-33, Tyr-36, Trp-108, and Phe-113 at the rim of the active site of MIF, and molecular modeling studies indicated that several more potent hit compounds have the aromatic rings which can interact with this hydrophobic surface. To our knowledge, our compounds are the most potent tautomerase inhibitors of MIF. One of these small, drug-like molecules has been cocrystallized with MIF and binds to the active site for tautomerase activity. Molecular modeling also suggests that the other hit compounds can bind in a similar fashion.

The KCNE1-D85N polymorphism was significantly more frequent in our LQTS probands. The functional variant is a disease-causing gene variant of LQTS phenotype that functions by interacting with KCNH2 and KCNQ1. Since its allele frequency was approximately 1% among control individuals, KCNE1-D85N may be a clinically important genetic variant.