GOLPH3 Regulates EGFR in T98G Glioblastoma Cells by Modulating Its Glycosylation and Ubiquitylation

Protein trafficking is altered when normal cells acquire a tumor phenotype. A key subcellular compartment in regulating protein trafficking is the Golgi apparatus, but its role in carcinogenesis is still not well defined. Golgi phosphoprotein 3 (GOLPH3), a peripheral membrane protein mostly localized at the trans-Golgi network, is overexpressed in several tumor types including glioblastoma multiforme (GBM), the most lethal primary brain tumor. Moreover, GOLPH3 is currently considered an oncoprotein, however its precise function in GBM is not fully understood. Here, we analyzed in T98G cells of GBM, which express high levels of epidermal growth factor receptor (EGFR), the effect of stable RNAi-mediated knockdown of GOLPH3. We found that silencing GOLPH3 caused a significant reduction in the proliferation of T98G cells and an unexpected increase in total EGFR levels, even at the cell surface, which was however less prone to ligand-induced autophosphorylation. Furthermore, silencing GOLPH3 decreased EGFR sialylation and fucosylation, which correlated with delayed ligand-induced EGFR downregulation and its accumulation at endo-lysosomal compartments. Finally, we found that EGF failed at promoting EGFR ubiquitylation when the levels of GOLPH3 were reduced. Altogether, our results show that GOLPH3 in T98G cells regulates the endocytic trafficking and activation of EGFR likely by affecting its extent of glycosylation and ubiquitylation.     

Chronic fluoride exposure does not cause detrimental, extraskeletal effects in nutritionally deficient rats

On the basis of observations that endemic fluorosis occurs more often in malnourished populations, a series of studies tested the hypothesis that deficient dietary intake of calcium, protein or energy affects fluoride metabolism so that the margin of safe fluoride exposure may be reduced. The objective of the investigation was to determine whether changes in fluoride metabolism in nutritionally deficient rats resulted in manifestation of any extraskeletal toxic fluoride effects not observed in healthy animals. This investigation included two studies, one that monitored the effect of calcium deficiency on the effects of chronic fluoride exposure, and a second study that observed fluoride effects in rats that were deficient either in protein or in energy and total nutrient intake. Control and experimental rats received drinking water containing 0, 0.26 (5), 0.79 (15) or 2.63 (50) mmol fluoride/L (mg/L) for 16 or 48 wk. Control rats were fed optimal diets and experimental rats were fed diets deficient in calcium (Study 1) or protein (Study 2). An additional group of experimental rats (Study 2) was provided with a restricted amount of diet; thus these rats were deficient in energy and total nutrient intake. The intake, excretion and retention of fluoride were monitored; after the rats were killed, tissue fluoride levels and biochemical markers of tissue function were analyzed. Bone marrow cells were harvested from some of the rats, after 48 wk of treatment, for determining the frequency of sister chromatid exchange, a marker of genetic damage. Although there were significant differences among fluoride treatment groups in fluoride excretion and retention that resulted in significantly greater fluoride levels in tissues of the experimental rats, we were unable to detect any harmful, extraskeletal biochemical, physiologic or genetic effects of fluoride in the nutritionally deficient rats.     

Analysis of Combined Systems of Two Endoreversible Engines

A single endoreversible engine can operate as a cooler, a true heat engine, a heat pump, or a refrigerator. We investigate how many different modes of operation a combined system of two endoreversible engines may display. Special attention is paid to the independent combined system which neither consumes nor supplies power.     

An alcohol expectancy-challenge prevention program for at risk college women.

Alcohol expectancy-challenge programs are effective in changing expectancies and reducing drinking in college men (J. Darker & M. S. Goldman. 1993. 1998); however, recent evidence suggests this protocol might not be effective for women (M. E. Dunn, C. Lau, & I. Y. Cruz, 2000). This study was designed to reevaluate the effectiveness of a social/sexual expectancy-challenge intervention in college women reporting moderate to heavy alcohol use. Forty-six undergraduate women were randomly assigned to the prevention or control condition. Participants reported alcohol expectancies at pre- and posttest and monitored their drinking patterns daily for 6 weeks. The program was effective in changing some expectancies but did not reduce drinking. This further confirms differences in the mechanisms by which expectancy-challenge programs function for men and women. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Tetradecylthioacetic acid incorporated into very low density lipoprotein: changes in the fatty acid composition and reduced plasma lipids in cholesterol-fed hamsters

The mechanism behind the hypolipidemic effect of tetradecylthioacetic acid (CMTTD, a non-beta-oxidizable 3-thia fatty acid) was studied in hamsters fed a high cholesterol diet (2%), which resulted in hyperlipidemia. Treating hyperlipidemic hamsters with CMTTD resulted in a progressive hypocholesterolemic and hypotriacylglycerolemic effect. Decreased plasma cholesterol was followed by a 39% and 30% reduction in VLDL-cholesterol and LDL-cholesterol, respectively. In contrast, the HDL-cholesterol content was not affected, thus decreasing the VLDL-cholesterol/HDL-cholesterol and LDL-cholesterol/HDL-cholesterol ratios. 3-Hydroxy-3-methylglutaryl- (HMG) CoA reductase activity and its mRNA level were unchanged after CMTTD administration. Also, the LDL receptor and LDL receptor-related protein (LRP-4) mRNAs were unchanged. The decrease in plasma triacylglycerol was accompanied by a 45% and 56% reduction in VLDL-triacylglycerol and LDL-triacylglycerol, respectively. The hypolipidemic effect of CMTTD was followed by a 1.4-fold increase in mitochondrial fatty acid oxidation and a 2.3-fold increase in peroxisomal fatty acid oxidation. CMTTD treatment led to an accumulation of dihomo-gamma-linolenic acid (20:3n-6) in liver, plasma, very low density lipoprotein, and heart. Noteworthy, CMTTD accumulated more in the heart, plasma, and VLDL particles compared to the liver, and in the VLDL particle alpha-linolenic acid (18:3n-3) decreased whereas eicosatetraenoic acid (20:4n-3) increased. In addition, linoleic acid (18:2n-6) and the total amount of polyunsaturated fatty acids decreased, the latter mainly due to a decrease in n-6 fatty acids. The present data show that CMTTD was detected in plasma and incorporated into VLDL, liver, and heart. The relative incorporation (mol%) of CMTTD was heart > VLDL > liver. In conclusion, CMTTD causes both a hypocholesterolemic and hypotriacylglycerolemic effect in hyperlipidemic hamsters.     

IL-1 in gingival crevicular fluid following closed root planing and papillary flap debridement

Interleukin (IL)-1 alpha and beta are cytokines which can mediate inflammatory, bone resorbing, and reparative effects in the periodontium, but few longitudinal data exist exploring their role following periodontal therapy. This study examined gingival crevicular fluid (GCF) concentrations of IL-1 alpha and IL-1 beta at sites with shallow sulci (SS) or inflamed moderate/advanced pockets (M/AP) before and 6 months after treatment with closed scaling/root planing (SC/RP) or papillary flap debridement (PFD), all in the same subject (n = 14 patients). No significant differences were noted in IL-1 alpha or beta concentrations (determined with two-site enzyme-linked immunosorbent assays) between SS and M/AP sites at baseline. While both therapies improved clinical parameters of periodontal disease, IL-1 alpha concentration increased significantly (p < 0.05) in M/AP-PFD sites 6 months after treatment, but were unchanged in other groups. IL-1 beta concentrations were numerically lower after therapy, except for a significant increase (p < 0.05) in M/AP-PFD sites. These data suggest that surgical wound healing in an inflamed, plaque-infected site (M/AP-PFD) results in prolonged production of IL-1, which may be a reflection of the extent of tissue trauma and delayed wound healing. In spite of increased IL-1 levels, these sites demonstrated significant short-term improvement in clinical attachment level (+ 1.8 mm, p < or = 0.001) postoperatively.     

Insulin-like growth factor I activates the invasion suppressor function of E-cadherin in MCF-7 human mammary carcinoma cells in vitro

The calcium-dependent cell-cell adhesion molecule E-cadherin has been shown to counteract invasion of epithelial neoplastic cells. Using three monoclonal antibodies, we have demonstrated the presence of E-cadherin at the surface of human MCF-7/6 mammary carcinoma cells by indirect immunofluorescence coupled to flow cytometry and by immunocytochemistry. Nevertheless, MCF-7/6 cells failed to aggregate in a medium containing 1.25 mM CaCl2, and they were invasive after confrontation with embryonic chick heart fragments in organ culture. Treatment of MCF-7/6 cells with 0.5 microgram ml-1 insulin-like growth factor I (IGF-I) led to homotypic aggregation within 5 to 10 min and inhibited invasion in vitro during at least 8 days. The effect of IGF-I on cellular aggregation was insensitive to cycloheximide. However, monoclonal antibodies that interfered with the function of either the IGF-I receptor (alpha IR3) or E-cadherin (HECD-1, MB2) blocked the effect of IGF-I on aggregation. The effects of IGF-I on aggregation and on invasion could be mimicked by 1 microgram ml-1 insulin, but not by 0.5 microgram ml-1 IGF-II. The insulin effects were presumably not mediated by the IGF-I receptor, since they could not be blocked by an antibody against this receptor (alpha IR3). Our results indicate that IGF-I activates the invasion suppressor role of E-cadherin in MCF-7/6 cells.