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In recent years, BK virus (BKV) nephritis after renal transplantation has become a severe problem. The exact mechanisms of BKV cell entry and subsequent intracellular trafficking remain unknown. Since human renal proximal tubular epithelial cells (HRPTEC) represent a main natural target of BKV nephritis, analysis of BKV infection of HRPTEC is necessary to obtain additional insights into BKV biology and to develop novel strategies for the treatment of BKV nephritis. We coincubated HRPTEC with BKV and the cholesteroldepleting agents methyl beta cyclodextrin (MBCD) and nystatin (Nys), drugs inhibiting caveolar endocytosis. The percentage of infected cells (detected by immunofluorescence) and the cellular levels of BKV large T antigen expression (detected by Western blot analysis) were significantly decreased in both MBCD-and Nys-treated HPRTEC compared to the level in HRPTEC incubated with BKV alone. HRPTEC infection by BKV was also tested after small interfering RNA (siRNA)-dependent depletion of either the caveolar structural protein caveolin-1 (Cav-1) or clathrin, the major structural protein of clathrin-coated pits. BKV infection was inhibited in HRPTEC transfected with Cav-1 siRNA but not in HRPTEC transfected with clathrin siRNA. The colocalization of labeled BKV particles with either Cav-1 or clathrin was investigated by using fluorescent microscopy and image cross-correlation spectroscopy. The rate of colocalization of BKV with Cav-1 peaked at 4 h after incubation. Colocalization with clathrin was insignificant at all time points. These results suggest that BKV entered into HRPTEC via caveolae, not clathrin-coated pits, and that BKV is maximally associated with caveolae at 4 h after infection, prior to relocation to a different intracellular compartment.

Abstract-Glycogen synthase kinase (GSK)-3, a negative regulator of cardiac hypertrophy, is inactivated in failing hearts. To examine the histopathological and functional consequence of the persistent inhibition of GSK-3␤ in the heart in vivo, we generated transgenic mice with cardiac-specific overexpression of dominant negative GSK-3␤ (Tg-GSK-3␤-DN) and tetracycline-regulatable wild-type GSK-3␤. GSK-3␤-DN significantly reduced the kinase activity of endogenous GSK-3␤, inhibited phosphorylation of eukaryotic translation initiation factor 2B, and induced accumulation of ␤-catenin and myeloid cell leukemia-1, confirming that GSK-3␤-DN acts as a dominant negative in vivo. Tg-GSK-3␤-DN exhibited concentric hypertrophy at baseline, accompanied by upregulation of the ␣-myosin heavy chain gene and increases in cardiac function, as evidenced by a significantly greater E max after dobutamine infusion and percentage of contraction in isolated cardiac myocytes, indicating that inhibition of GSK-3␤ induces well-compensated hypertrophy. Although transverse aortic constriction induced a similar increase in hypertrophy in both Tg-GSK-3␤-DN and nontransgenic mice, Tg-GSK-3␤-DN exhibited better left ventricular function and less fibrosis and apoptosis than nontransgenic mice. Induction of the GSK-3␤ transgene in tetracycline-regulatable wild-type GSK-3␤ mice induced left ventricular dysfunction and premature death, accompanied by increases in apoptosis and fibrosis. Overexpression of GSK-3␤-DN in cardiac myocytes inhibited tumor necrosis factor-␣-induced apoptosis, and the antiapoptotic effect of GSK-3␤-DN was abrogated in the absence of myeloid cell leukemia-1. These results suggest that persistent inhibition of GSK-3␤ induces compensatory hypertrophy, inhibits apoptosis and fibrosis, and increases cardiac contractility and that the antiapoptotic effect of GSK-3␤ inhibition is mediated by myeloid cell leukemia-1. Thus, downregulation of GSK-3␤ during heart failure could be compensatory. Key Words: GSK-3␤ Ⅲ heart failure Ⅲ cardiac hypertrophy Ⅲ apoptosis G SK-3␤ is a ubiquitously expressed serine/threonine kinase that has versatile biological functions in cells, including regulation of metabolism, cell growth/death, and protein translation and transcription. 1,2 Unlike most protein kinases, GSK-3␤ remains active in the resting state and is inactivated when cells are stimulated by mitogens, by other protein kinases, such as Akt, or by the Wnt pathway. In cardiac myocytes, GSK-3␤ phosphorylates ␤-catenin, 3 eukaryotic translation initiation factor (eIF)2B, 4 NFAT, 5 GATA4, 6 myocardin, 7 and other proteins, thereby negatively regulating protein synthesis and gene expression. GSK-3␤ downregulates SERCA2a 8 and enhances mitochondrial permeability transition, 9 thereby leading to an inability to normalize cytosolic Ca 2ϩ in diastole and reduced cell survival, respectively.GSK-3␤ is an important negative regulator of cardiac hypertrophy. 10 GSK-3␤ negatively regulates ␤-adrenergic and endothelin-induced cardiac hypertrophy in cultured ...

This paper studies the application of the discrete Fourier transform (DFT) to predict angular orientation distributions from images of fibers and cells. Angular distributions of fibers in composites define their material properties. In biological tissues, cell and fiber orientation distributions are important since they define their mechanical properties and function.We developed a filtering scheme for the DFT to predict angular distributions accurately. The errors involved in this DFT technique and their sources were quantified through Monte Carlo simulation of computer-generated images. The knowledge of these errors allows one to verify the suitability of the method for a particular application. We found that the DFT method is most accurate for slender fibers, and propose a means to minimize errors by optimizing parameters. This method was applied to predict orientation distribution of cells and actin fibers in bio-artificial tissue constructs.