Gap Junctions Mediate Glucose Transport Between GLUT1-Positive and -Negative Cells in the Spiral Limbus of the Rat Cochlea

To elucidate the role of the spiral limbus in glucose transport in the cochlea, we analyzed the expression and localization of GLUT1, connexin26, connexin30, and occludin in the spiral limbus of the rat cochlea. GLUT1 and occludin were detected in blood vessels. GLUT1, connexin26, connexin30, and occludin were also expressed in fibrocytes just basal to the supralimbal lining cells. Connexin26 and connexin30 were present among not only these GLUT1-positive fibrocytes but also GLUT1-negative fibrocytes. In vivo glucose imaging using 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-6-deoxyglucose (6-NBDG, MW 342) together with Evans Blue Albumin (EBA, MW 68,000) showed that 6-NBDG was rapidly distributed throughout the spiral limbus, whereas EBA was localized only in the vessels. Moreover, the gap junctional uncoupler heptanol inhibited the distribution of 6-NBDG. These findings suggest that gap junctions play an important role in glucose transport in the spiral limbus, i.e., that gap junctions mediate glucose transport from GLUT1-positive fibrocytes to GLUT1-negative fibrocytes in the spiral limbus.     

MiR-376c Down-Regulation Accelerates EGF-Dependent Migration by Targeting GRB2 in the HuCCT1 Human Intrahepatic Cholangiocarcinoma Cell Line

MicroRNA miR-376c was expressed in normal intrahepatic biliary epithelial cells (HIBEpiC), but was significantly suppressed in the HuCCT1 intrahepatic cholangiocarcinoma (ICC) cell line. The biological significance of the down-regulation of miR-376c in HuCCT1 cells is unknown. We hypothesized that miR-376c could function as a tumor suppressor in these cells. To test this hypothesis, we sought the targets of miR-376c, and characterized the effect of its down-regulation on HuCCT1 cells. We performed proteomic analysis of miR-376c-overexpressing HuCCT1 cells to identify candidate targets of miR-376c, and validated these targets by 3′-UTR reporter assay. Transwell migration assays were performed to study the migratory response of HuCCT1 cells to miR-376c overexpression. Furthermore, microarrays were used to identify the signaling that were potentially involved in the miR-376c-modulated migration of HuCCT1. Finally, we assessed epigenetic changes within the potential promoter region of the miR-376c gene in these cells. Proteomic analysis and subsequent validation assays showed that growth factor receptor-bound protein 2 (GRB2) was a direct target of miR-376c. The transwell migration assay revealed that miR-376c significantly reduced epidermal growth factor (EGF)-dependent cell migration in HuCCT1 cells. DNA microarray and subsequent pathway analysis showed that interleukin 1 beta and matrix metallopeptidase 9 were possible participants in EGF-dependent migration of HuCCT1 cells. Bisulfite sequencing showed higher methylation levels of CpG sites upstream of the miR-376c gene in HuCCT1 relative to HIBEpiC cells. Combined treatment with the DNA-demethylating agent 5-aza-2′-deoxycytidine and the histone deacetylase inhibitor trichostatin A significantly upregulated the expression of miR-376c in HuCCT1 cells. We revealed that epigenetic repression of miR-376c accelerated EGF-dependent cell migration through its target GRB2 in HuCCT1 cells. These findings suggest that miR-376c functions as a tumor suppressor. Since metastasis is the major cause of death in ICC, microRNA manipulation could lead to the development of novel anti-cancer therapy strategies for ICC.     

On computational efficiency of the hybrid Monte Carlo method applied to the multicanonical ensemble

Abstract

In the present paper, computational efficiency of the hybrid Monte Carlo (HMC) method applied to the multicanonical ensemble is studied; the HMC is an equation of motion guided Monte Carlo method. As in the standard HMC for the canonical ensemble, the multicanonical HMC calculations with high acceptance ratio show better efficiency; about 60% acceptance yields the best performance for the system examined.     

Effects of temperature on development and growth in the tick,Haemaphysalis longicornis

As a part of ecological studies onHaemaphysalis longicornis, the effects of controlled temperatures (12, 15, 20, 25 and 30°C; 100% RH) on development and growth of the tick were investigated and the critical low temperature for each stage in the life cycle was estimated. As the temperature became low, the periods of preoviposition, oviposition, egg hatching (incubation) and moulting were prolonged. At 12°C, however, oviposition, egg hatching and moulting of the larva and nymph did not occur. The critical low temperatures for oviposition, egg hatching (developmental zero) and larval and nymphal moulting which were calculated theoretically from the regression equations, were 11.1, 12.2, 10.2 and 11.8°C, respectively. The temperature also affected the egg productivity and hatch-ratio. The number of deposited eggs per mg of body weight decreased markedly at 15°C, and the hatch-ratio was lowered with dropped temperatures.     

Immune response to Toxoplasma gondii--analysis of suppressor T cells in a patient with symptomatic acute toxoplasmosis

Unresponsiveness of antigen-dependent (Toxoplasma-specific and purified protein derivative of tuberculin [PPD]-specific) T-cell proliferative responses of peripheral blood leukocytes (PBL) was observed in a patient with symptomatic acute toxoplasmosis. The immunosuppression of T-cell responses was mediated by Leu 1+, Leu 2a+, and Leu 3a- suppressor T cells that were induced by Toxoplasma gondii antigen and suppressed both Toxoplasma-specific and PPD-specific PBL T-cell responses from a patient with chronic toxoplasmosis when PBL of these patients were mixed and cocultured in vitro. Participation of class II molecules of HLA in Toxoplasma-specific proliferative T-cell responses and activation of suppressor T cells was examined by using monoclonal antibodies specific for HLA-DR and HLA-DQ molecules. Anti-HLA-DQ monoclonal antibody released the suppressive activity, while anti-HLA-DR monoclonal antibody inhibited Toxoplasma-specific T-cell responses. Thus, the suppressive effect of PBL from a patient with acute toxoplasmosis on antigen-dependent PBL T-cell responses from a patient with chronic toxoplasmosis was mediated by HLA-DQ molecules. By contrast, Toxoplasma-specific T-cell responses were activated by HLA-DR molecules (presumably present on antigen-presenting cells).     

Purification of Protein Body-I of Rice Seed and its Polypeptide Composition

Protein body type one (PB-I) was isolated and purified fromdeveloping rice grain by a combination of sucrose density gradientcentrifugation and treatment with pepsin. SDS-PAGE analysisshowed that isolated PB-I contains several polypeptide groups,the largest having an apparent molecular size of 13 kDa andtwo smaller ones of 10 kDa and 16 kDa. The 13-kDa group wasfound to be composed of two polypeptides of slightly differentmolecular sizes, 13a (larger component) and 13b (smaller component).Most of the 13a and 13b polypeptides were shown to be largelyprolamins, although there were also some salt- and alcohol-insolublepolypeptides with an apparent molecular size of 13 kDa. It wasconcluded that PB-I is the accumulation site of rice prolamin.It was further estimated that the protein amount in PB-I accountedfor about 20% of the total protein of rice endosperm. (Received March 20, 1987; Accepted September 8, 1987)     

Mutations Affecting Acetylcholine Levels in the Nematode Caenorhabditis elegans

Gene cha-1.unc-17 of the nematode Caenorhabditis elegans is a complex gene, consisting of at least two complementation groups. One part (cha-1 region) of the gene encodes the enzyme choline acetyltransferase (ChAT), but the function of the other part (unc-17 region) is still unclear. We measured the ChAT activity and ACh levels of the cha-1 and unc-17 complex gene mutants. We show here that alterations in ACh levels, rather than the ChAT activity, reflect abnormal phenotypes accompanying cha-1.unc-17 mutations, that is, the decreased ACh levels in cha-1 mutations and abnormal accumulation in unc-17 mutations. Our results suggest that the unc-17 region may encode functions necessary for storage and/or release of ACh at the presynaptic level.     

Correction of congenital microtia using the tissue expander

We attempted auricular reconstruction using Radovan-type inflatable silicone expanders in six children and one adult, with the complete hypoplastic, the conchal remnant, and constricted type of microtia. Ear frameworks, including the helix, anthelix, concha, and tragus, were prepared using autologous rib cartilage. Based on the surface area of the normal adult auricle, the silicone expander was tentatively shaped and sized into a rotated semiellipse and expanded with 70 cc saline. Auricular reconstruction on the framework was completed at the time of insertion in four of the seven patients, requiring no elevation of the ear. The reconstructed auricle was satisfactory in both color and texture and had nearly normal sensation. Mild complications were noted in three of the seven patients. However, no resorption of the inserted rib cartilage has been observed 14 months to 2 years and 5 months after the operation. Slight shrinkage of the expanded skin was noted in each patient.     

Exon-intron organization, expression, and chromosomal localization of the human motilin gene

The human motilin gene has been isolated and characterized. The gene spans about 9 kilobase pairs (kb) and the 0.7 kb motilin mRNA is encoded by five exons. The 22-amino-acid motilin sequence is encoded by exons 2 and 3. The human motilin gene was mapped to the p21.2----p21.3 region of chromosome 6 by hybridization of the cloned cDNA to DNAs from a panel of reduced human-mouse somatic cell hybrids and by in situ hybridization to human prometaphase chromosomes. RNA blotting using RNA prepared from various regions of the human gastrointestinal tract revealed high levels of motilin mRNA in duodenum and lower levels in the antrum of the stomach; motilin mRNA could not be detected by this procedure in the esophagus, cardia of the stomach, descending colon or gallbladder.