Concomitant Loss of p120-Catenin and β-Catenin Membrane Expression and Oral Carcinoma Progression with E-Cadherin Reduction

The binding of p120-catenin and β-catenin to the cytoplasmic domain of E-cadherin establishes epithelial cell-cell adhesion. Reduction and loss of catenin expression degrades E-cadherin-mediated carcinoma cell-cell adhesion and causes carcinomas to progress into aggressive states. Since both catenins are differentially regulated and play distinct roles when they dissociate from E-cadherin, evaluation of their expression, subcellular localization and the correlation with E-cadherin expression are important subjects. However, the same analyses are not readily performed on squamous cell carcinomas in which E-cadherin expression determines the disease progression. In the present study, we examined expression and subcellular localization of p120-catenin and β-catenin in oral carcinomas (n = 67) and its implications in the carcinoma progression and E-cadherin expression using immunohitochemistry. At the invasive front, catenin-membrane-positive carcinoma cells were decreased in the dedifferentiated (p120-catenin, P < 0.05; β-catenin, P < 0.05) and invasive carcinomas (p120-catenin, P < 0.01; β-catenin, P < 0.05) and with the E-cadherin staining (p120-catenin, P < 0.01; β-catenin, P < 0.01). Carcinoma cells with β-catenin cytoplasmic and/or nuclear staining were increased at the invasive front compared to the center of tumors (P < 0.01). Although the p120-catenin isoform shift from three to one associates with carcinoma progression, it was not observed after TGF-β, EGF or TNF-α treatments. The total amount of p120-catenin expression was decreased upon co-treatment of TGF-β with EGF or TNF-α. The above data indicate that catenin membrane staining is a primary determinant for E-cadherin-mediated cell-cell adhesion and progression of oral carcinomas. Furthermore, it suggests that loss of p120-catenin expression and cytoplasmic localization of β-catenin fine-tune the carcinoma progression.     

Prevention of LLC-PK(1) cell overgrowth in a bioartificial renal tubule device using a MEK inhibitor, U0126

A common approach to construct a bioartificial renal tubule system is to utilize renal tubular cells seeded in porous polymer membrane hollow fibers. We have reported that overgrowth of renal tubular cells was not beneficial for the transport and reabsorption functions of bioartificial tubules. Therefore, long-term maintenance of a confluent monolayer of cells in hollow fibers is essential and technically challenging. In this study, we examined whether MEK inhibitor, U0126, could maintain the monolayer of Lewis-lung cancer porcine kidney 1 (LLC-PK(1)) cells on polystyrene plates and in a dialysis module housing hollow fibers made of ethylene vinyl alcohol (EVAL). We also evaluated the leakage of urea nitrogen (UN) and creatinine (Cr) through the cell-lined hollow fibers, and reabsorption of glucose and sodium by the cells, comparing the U0126-treated cells with nontreated cells in the module. Treatment with 50micromol l(-1) U0126 prevented the overgrowth of cells cultured on polystyrene plates. Moreover, U0126-treatment reduced the leakage of UN, and increased the reabsorption of electrolytes in 65cm(2) modules. Scanning electron microscopy revealed that it also prevented the overconfluence of cells in modules. Therefore, application of U0126 is a potentially effective method to improve the performance of the device.     

WRN counteracts the NHEJ pathway upon camptothecin exposure

We investigated the function of the interaction between WRN (Werner syndrome gene product) and Ku70 and between WRN and DNA-PKcs, which are components of the DNA-PKcs/Ku70/Ku80 complex, by generating KU70(-/-)/WRN(-/-) and DNA-PKcs(-/-/-)/WRN(-/-) double-gene knockout chicken DT40 cells. When treated with camptothecin (CPT), an inhibitor of DNA topoisomerase I, WRN(-/-) cells showed higher sensitivity than wild-type cells, whereas KU70(-/-) and DNA-PKcs(-/-/-) cells showed hyper-resistance. Disruption of KU70 or DNA-PKcs suppressed the sensitivity of WRN(-/-) cells to CPT, rendering them as resistant to CPT treatment as KU70(-/-) and DNA-PKcs(-/-/-) cells. On the other hand, CPT sensitivity of BLM(-/-) cells, which are defective in a RecQ helicase similar to WRN, was enhanced by deletion of KU70. The implications for the function of WRN in the non-homologous end-joining pathway of DNA repair involving Ku70 and DNA-PKcs, which may be the cause of lethality in the presence of CPT, will be discussed.     

The interplay of processivity, substrate inhibition and a secondary substrate binding site of an insect exo-beta-1,3-glucanase

Abracris flavolineata midgut contains a processive exo-beta-glucanase (ALAM) with lytic activity against Saccharomyces cerevisiae, which was purified (yield, 18%; enrichment, 37 fold; specific activity, 1.89 U/mg). ALAM hydrolyses fungal cells or callose from the diet. ALAM (45 kDa; pI 5.5; pH optimum 6) major products with 0.6 mM laminarin as substrate are beta-glucose (61%) and laminaribiose (39%). Kinetic data obtained with laminaridextrins and methylumbelliferyl glucoside suggest that ALAM has an active site with at least six subsites. The best fitting of kinetic data to theoretical curves is obtained using a model where one laminarin molecule binds first to a high-affinity accessory site, causing active site exposure, followed by the transference of the substrate to the active site. The two-binding-site model is supported by results from chemical modifications of amino acid residues and by ALAM action in MUbetaGlu plus laminarin. Low laminarin concentrations increase the modification of His, Tyr and Asp or Glu residues and MUbetaGlu hydrolysis, whereas high concentrations abolish modification and inhibit MUbetaGlu hydrolysis. Our data indicate that processivity results from consecutive transferences of substrate between accessory and active site and that substrate inhibition arises when both sites are occupied by substrate molecules abolishing processivity.     

濒危帽贝铁锈笠螺的活动节律和采食行为的初步观察

铁锈笠螺是地中海最濒危的海洋无脊椎动物,对其生物学知之甚少,缺少对其活动节律和采食行为的了解。使用环氧树脂Eporai1127(原位标记了20个不同外壳长度的个体,并在每个外壳上标有不同的数字,作者在白昼或黑夜的高潮和低潮期收集了有关数据。可能由于云斑厚纹蟹(Pachygrapsus marmoratus)的捕食影响,铁锈笠螺白天的活动和运动多于夜间,但铁锈笠螺采食行为似乎仅限于高潮期。此外,汹涌的海面条件诱导了铁锈笠螺的活动和运动。     

Lack of Correlation between the Kinase Activity of LRRK2 Harboring Kinase-Modifying Mutations and Its Phosphorylation at Ser910, 935, and Ser955

Leucine-rich repeat kinase 2 (LRRK2) is extensively phosphorylated in cells within a region amino-terminal to the leucine-rich repeat domain. Since phosphorylation in this region of LRRK2, including Ser910, Ser935, Ser955, and Ser973, is significantly downregulated upon treatment with inhibitors of LRRK2, it has been hypothesized that signaling pathways downstream of the kinase activity of LRRK2 are involved in regulating the phosphorylation of LRRK2, although the precise mechanism has remained unknown. Here we examined the effects of LRRK2 inhibitors on the phosphorylation state at Ser910, Ser935, and Ser955 in a series of kinase-inactive mutants of LRRK2. We found that the responses of LRRK2 to the inhibitors varied among mutants, in a manner not consistent with the above-mentioned hypothesis. Notably, one of the kinase-inactive mutants, T2035A LRRK2, underwent phosphorylation, as well as the inhibitor-induced dephosphorylation, at Ser910, Ser935, and Ser955, to a similar extent to those observed with wild-type LRRK2. These results suggest that the kinase activity of LRRK2 is not involved in the common mechanism of inhibitor-induced dephosphorylation of LRRK2.     

Identification of Novel Regulatory Cholesterol Metabolite, 5-Cholesten, 3β,25-Diol,Disulfate

Oxysterol sulfation plays an important role in regulation of lipid metabolism and inflammatory responses. In the present study, we report the discovery of a novel regulatory sulfated oxysterol in nuclei of primary rat hepatocytes after overexpression of the gene encoding mitochondrial cholesterol delivery protein (StarD1). Forty-eight hours after infection of the hepatocytes with recombinant StarD1 adenovirus, a water-soluble oxysterol product was isolated and purified by chemical extraction and reverse-phase HPLC. Tandem mass spectrometry analysis identified the oxysterol as 5-cholesten-3β, 25-diol, disulfate (25HCDS), and confirmed the structure by comparing with a chemically synthesized compound. Administration of 25HCDS to human THP-1-derived macrophages or HepG2 cells significantly inhibited cholesterol synthesis and markedly decreased lipid levels in vivo in NAFLD mouse models. RT-PCR showed that 25HCDS significantly decreased SREBP-1/2 activities by suppressing expression of their responding genes, including ACC, FAS, and HMG-CoA reductase. Analysis of lipid profiles in the liver tissues showed that administration of 25HCDS significantly decreased cholesterol, free fatty acids, and triglycerides by 30, 25, and 20%, respectively. The results suggest that 25HCDS inhibits lipid biosynthesis via blocking SREBP signaling. We conclude that 25HCDS is a potent regulator of lipid metabolism and propose its biosynthetic pathway.