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.     

Differentiation of Langerhans Cells from Interdigitating Cells Using CD1a and S-100 Protein Antibodies

The present study shows that Langerhans cells can be differentiated from Interdigitating cells at the light microscopic level. Superficial lymph nodes and skin taken from necropsies and the lymph nodes of dermatopathic lymphadenopathy (DPL) were used for this experiment. Sections of lymph node and skin were embedded using the acetone, methyl benzoate and xylene (AMeX) method and dendritic cells were immunostained with anti S-100 protein antibody (S-100, and OKT-6 (CD1a) using the restaining method. Langerhans cells in the skin were positive for both CD1a and S-100. Dendritic cells positive for both CD1a and S-100, and dendritic cells positive for S-100, but not for CD1a were observed in superficial lymph nodes. In normal superficial lymph nodes, there were more interdigitating cells than Langerhans cells. The majority of the dendritic cells in the DPL were Langerhans cells. We conclude that the S-100 and CD1a positive cells are Langerhans cells, and the S-100 positive-CD1a negative cells are interdigitating cells.     

Humans but Not Chimpanzees Vary Face-Scanning Patterns Depending on Contexts during Action Observation

Human and nonhuman primates comprehend the actions of other individuals by detecting social cues, including others’ goal-directed motor actions and faces. However, little is known about how this information is integrated with action understanding. Here, we present the ontogenetic and evolutionary foundations of this capacity by comparing face-scanning patterns of chimpanzees and humans as they viewed goal-directed human actions within contexts that differ in whether or not the predicted goal is achieved. Human adults and children attend to the actor’s face during action sequences, and this tendency is particularly pronounced in adults when observing that the predicted goal is not achieved. Chimpanzees rarely attend to the actor’s face during the goal-directed action, regardless of whether the predicted action goal is achieved or not. These results suggest that in humans, but not chimpanzees, attention to actor’s faces conveying referential information toward the target object indicates the process of observers making inferences about the intentionality of an action. Furthermore, this remarkable predisposition to observe others’ actions by integrating the prediction of action goals and the actor’s intention is developmentally acquired.     

Monodeiodination of thyroxine to 3, 5, 3'-triiodothyronine and to 3, 3', 5'-triiodothyronine in isolated dog renal cortical tubuli

Monodeiodination of T4 to T3 and rT3 in the intact cells of dog renal tubuli and glomeruli was investigated. The tubuli and glomeruli were obtained by a sieve method. T4 (2 micrograms/ml) was incubated in Tris-HCl buffer, pH 7.5, with renal cells (180 micrograms protein/ml) and 5 mM DTT for 1 h at 37 degrees C and the T3 and rT3 generated during incubation were measured by specific radioimmunoassays. In order of decreasing activity, dog renal cortical tubuli, cortical homogenate, glomeruli and medullary tubuli were capable of converting T4 to T3. Net rT3 production from T4 in cortical tubuli was also greater than that in cortical homogenate. The conversion of T4 to T3 and also to rT3 in cortical tubuli was enzymatic in nature, since the reactions showed dependence on time and protein concentration; instability to heating; temperature and pH optimum. The production of T3 and rT3 from T4 was maximum at pH 6.5 and at pH 9.5, respectively, indicating that two different enzymic systems, a 5- and a 5'-monodeiodinase, might be involved in the deiodination of the tyrosyl and the phenolic ring of T4 in dog kidney.     

Freeze-fracture localization of filipin-sterol complexes in plasma- and cyto-membranes of Pneumocystis carinii

The polyene antibiotic, filipin, was used as the probe for demonstrating sterols in the freeze-fractured plasma- and cytomembranes of Pneumocystis carinii. The distribution of filipin-sterol complexes was homogeneous on the plasma membrane throughout all developmental stages from trophozoite to cyst; however, the density of the complexes gradually decreased with the progress of development. In the trophozoite, the density of the complexes was 485 +/- 42/micron2 on the P face and 341 +/- 27/micron2 on the E face. It was 249 +/- 50 on the P face and 132 +/- 48 on the E face in the precyst and 138 +/- 24 and 59 +/- 20, respectively, in the cyst. The membranes of nucleus, mitochondria, and small round bodies showed more or fewer complexes while no complexes were found in the membranes of one endoplasmic reticulum. In nuclear and mitochondrial membranes, some small scattered clusters of complexes were observed. Two types of vacuoles were distinguished: one having many complexes in its membrane and the other having none at all.