Cell-cell communication is essential for tissue homeostasis, but its contribution to disease prevention remains to be understood. We demonstrate the involvement of connexin 43 (Cx43, also known as GJA1) and related gap junction in epithelial homeostasis, illustrated by polarity-mediated cell cycle entry and mitotic spindle orientation (MSO). Cx43 localization is restricted to the apicolateral membrane of phenotypically normal breast luminal epithelial cells in 3D culture and in vivo. Chemically induced blockade of gap junction intercellular communication (GJIC), as well as the absence of Cx43, disrupt the apicolateral distribution of polarity determinant tight junction marker ZO-1 (also known as TJP1) and lead to random MSO and cell multilayering. Induced expression of Cx43 in cells that normally lack this protein reestablishes polarity and proper MSO in 3D culture. Cx43-directed MSO implicates PI3K-aPKC signaling, and Cx43 coprecipitates with signaling node proteins β-catenin (CTNNB1) and ZO-2 (also known as TJP2) in the polarized epithelium. The distribution of Cx43 is altered by pro-inflammatory breast cancer risk factors such as leptin and high-fat diet, as shown in cell culture and on tissue biopsy sections. The control of polarity-mediated quiescence and MSO may contribute to the tumor-suppressive role of Cx43.

Japanese encephalitis was recently reported from individuals in the mountain districts of Nepal without travel history to Japanese encephalitis virus (JEV) endemic areas. We performed a cross-sectional study to estimate the seroprevalence of JEV in pigs and subsequently conducted a survey of farmers to identify risk factors associated with seropositivity. In July and August, 2010, 454 pig serum samples were collected and tested by competitive ELISA. Data from a 35-question survey of 109 pig owners were analysed using multivariate logistic regression. Seventy-six (16.7, 95% CI 13.6-20.4) pigs tested positive for anti-JEV antibodies, none of which had been vaccinated against JEV or sourced from JEV endemic areas. Risk factors associated with JEV seropositivity were 'summer abortion', 'wells as a water source', 'urban location', 'reported presence of mosquitoes' and 'lower elevation'. Our results suggest that JEV is likely circulating in the mountain districts of Nepal, and that locally acquired JEV should be considered a risk for residents and travellers in these areas.

A fluorescent labeling method was developed to study plasminogen (PG) concentration and location in simulated bovine milk. Activity and stability of PG labeled with Alexa Fluor 594 (PG-594) were comparable to those of native PG. The fluorescent signal of PG-594 exhibited pH, temperature, and storage stability, and remained stable throughout typical sample treatments (stirring, heating, and ultracentrifugation). These characteristics indicate broad applicability of the fluorescent labeling technique for milk protease characterization. In an example application, PG-594 was added to simulated milk samples to study effects of heat and beta-lactoglobulin (beta-LG) on the distribution of PG. Before heating, about one-third of the PG-594 remained soluble in the whey fraction (supernatant) whereas the rest became associated with the casein micelle. Addition of beta-LG to the system slightly shifted PG-594 distribution toward the whey fraction. Heat-induced PG-594 binding to micelles in whey-protein-free systems was evidenced by a decrease of PG-594 from 31 to 15% in the whey fraction accompanied by an increase of PG-594 from 69 to 85% in casein micelle fractions. When beta-LG was present during heating, more than 95% of PG-594 became associated with the micelle. A comparison with the distribution pattern of PG-derived activities revealed that heat-induced PG binding to micelles accompanies heat-induced PG inactivation in the micelle fraction. Incubation of the casein micelles with the reducing agent beta-mercaptoethanol revealed that disulfide bonds formed between PG and casein or between PG and casein-bound beta-LG are the mechanisms for heat-induced PG binding to casein micelles. Western blotting and zymography results correlated well with fluorescent labeling studies and activity studies, respectively. Theoretically important findings are: 1) when heated, serum PG is capable of covalently binding to micellar casein or complexing with beta-LG in whey and then coadhering to micelles, and 2) PG that associated with micellar casein through lysine binding sites before heating is capable of developing heat-induced disulfide bonds with casein. The overall results are PG covalently binding to micelles and inactivation thereafter. Our results suggest that, instead of thermal denaturation through irreversible unfolding, covalent bond formation between PG and other milk proteins is the mechanism of PG inhibition during thermal processing.

Results of the inhibition of alpha-lytic proteinase by two standard mechanism serine proteinase inhibitors, turkey ovomucoid third domain (OMTKY3) and eglin C, and many of their variants are presented. Despite similarities, including an identical P1 residue (Leu) in their primary contact regions, OMTKY3 and eglin C have vastly different association equilibrium constants toward alpha-lytic proteinase, with Ka values of 1.8 x 10(3) and 1.2 x 10(9) M(-1), respectively. Although 12 of the 13 serine proteinases tested in our laboratory for inhibition by OMTKY3 and eglin C are more strongly inhibited by the latter, the million-fold difference observed here with alpha-lytic proteinase is the largest we have seen. The million-fold stronger inhibition by eglin C is retained when the Ka values of the P1 Gly, Ala, Ser, and Ile variants of OMTKY3 and eglin C are compared. Despite the small size of the S1 pocket in alpha-lytic proteinase, interscaffolding additivity for OMTKY3 and eglin C holds well for the four P1 residues tested here. To better understand this difference, we measured Ka values for other OMTKY3 variants, including some that had residues elsewhere in their contact region that corresponded to those of eglin C. Assuming intrascaffolding additivity and using the Ka values obtained for OMTKY3 variants, we designed an OMTKY3-based inhibitor of alpha-lytic proteinase that was predicted to inhibit 10,000-fold more strongly than wild-type OMTKY3. This variant (K13A/P14E/L18A/R21T/N36D OMTKY3) was prepared, and its Ka value was measured against alpha-lytic proteinase. The measured Ka value was in excellent agreement with the predicted one (1.1 x 10(7) and 2.0 x 10(7) M(-1), respectively). Computational protein docking results are consistent with the view that the backbone conformation of eglin C is not significantly altered in the complex with alpha-lytic proteinase. They also show that the strong binding for eglin C correlates well with more favorable atomic contact energy and desolvation energy contributions as compared to OMTKY3.