We used an oligonucleotide to introduce an A --T transversion at nudeotide position 1178 in polyoma virus DNA. The single effect of this mutation is to substitute phenylalanine for tyrosine at residue 315 of the middle-sized tumor (mT) protein (antigen). This site was previously identified as a major phosphate acceptor in the protein kinase reaction of immunocomplexes containing mT antigen. Reconstituted polyoma virus with the transversion, Py-1178-T, produces an altered mT protein that shows about 20% of the activity of wild-type mT antigen in the immunocomplex kinase assay. This residual activity appears to be directed primarily at another tyrosine at position 322 in the mT protein. The transforming ability of Py-1178-T is drastically reduced compared to wild-type virus. The efficiency of transformation by the mutant is <1% of that of wild type in focus assays and <0.1% in soft-agar growth assays. Cells identified in focus assays with Py-1178-T are generally less transformed in their phenotype than wild-type transformed cells.The demonstration of a protein kinase activity associated with the product of the transforming gene of Rous sarcoma virus (1, 2) has been followed by reports of a similar activity in a number of different tumor virus systems. The tyrosine specificity of these tumor virus-related protein kinases was first shown for the polyoma virus middle-sized tumor (mT) antigen (3), a protein that is essential, though not sufficient, for transformation (4-7). Tyrosine-specific kinase activities also have been shown for the Abelson virus transforming protein (8) and in other virus systems (9, 10). In most instances the activity is measured in vitro by incubating immunocomplexes containing the viral protein with [y-32P]ATP. The heavy chains of immunoglobulin often serve as the major phosphate acceptor in these reactions, although the viral gene product or products themselves also may be preferred substrates. In the case of Rous and Abelson viruses, the activity seems to be intrinsic to the viral proteins as shown by copurification and by the presence of tyrosine-specific protein kinase activity in the viral gene products cloned and expressed in Escherichia coli (11,12).In the case of polyoma virus, the source of the activity seen in immunocomplexes is unclear. The mT antigen appears to lack ATP-binding activity (13), and at least two laboratories have succeeded in expressing mT protein in E. coli without attendant kinase activity (unpublished data; K. Palme and W. Eckhart, personal communication). The possibility that mT antigen associates with cell-specified tyrosine kinase(s) has been supported recently by evidence showing the presence of c-src antigen in anti-polyoma-tumor (T) antigen immunocomplexes (14). In addition to serving as an acceptor for a tyrosine kinase activity measured in vitro, polyoma mT antigen is also a substrate for kinases in vivo with either or both serine and threonine being the major phosphoamino acids (15, 16).Two considerations point to the importance of these phosphory...

The hr-t gene of polyoma virus encodes both the small and middle T (tumor) antigens and exerts pleiotropic effects on cells. By mutating the 3' splice site for middle T mRNA, we have constructed a virus mutant, Py8O8A, which fails to express middle T but encodes normal small and large T proteins. The mutant failed to induce morphological transformation or growth in soft agar, but did stimulate postconfluent growth of normal cells. Cells infected by Py8O8A became fully agglutinable by lectins while retaining normal actin cable architecture and normal levels of extracellular fibronectin. These properties of Py8O8A demonstrated the separability of structural changes at the cell surface from those in the cytoskeleton and extracellular matrix, parameters which have heretofore been linked in the action of the hr-t and other viral oncogenes.

Andrographolide, the main active component extracted from Andrographis paniculata (Burm.f.) Wall. ex Nees, exerts anti-inflammatory effects; however, the principal molecular mechanisms remain unclear. The objective of this study was to investigate the molecular mechanisms of Andrographolide in modifying lipopolysaccharide- (LPS-) induced signaling pathway in RAW264.7 cells. An in vitro model of inflammation was induced by LPS in mouse RAW264.7 cells in the presence of Andrographolide. The concentration and expression levels of proinflammatory cytokines were determined by an enzyme-linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (qRT-PCR), respectively. The nuclear level of NF-κB was measured by an electrophoretic mobility shift assay (EMSA). The expression levels of NF-κB, p38, ERK, and JNK were determined by western blot. Andrographolide dose-dependently inhibited the release and mRNA expression of TNF-α, IL-6, and IL-1β in LPS-stimulated RAW264.7 cells. The nuclear level of p65 protein was decreased in Andrographolide treatment group. Western blot analysis showed that Andrographolide suppressed LPS-induced NF-κB activation and the phosphorylation of IkBa, ERK1/2, JNK, and p38. These results suggest that Andrographolide exerts an anti-inflammatory effect by inhibiting the activation of NF-κB/MAPK signaling pathway and the induction of proinflammatory cytokines.

Ependymal cells (ECs) are multiciliated neuroepithelial cells that line the ventricles of the brain and the central canal of the spinal cord (SC). How ependymal motile cilia are maintained remains largely unexplored. Here we show that zebrafish embryos deficient in Wnt signaling have defective motile cilia, yet harbor intact basal bodies. With respect to maintenance of ependymal motile cilia, plcδ3a is a target gene of Wnt signaling. Lack of Connexin43 (Cx43), especially its channel function, decreases motile cilia and intercellular Ca2+ wave (ICW) propagation. Genetic ablation of cx43 in zebrafish and mice diminished motile cilia. Finally, Cx43 is also expressed in ECs of the human SC. Taken together, our findings indicate that gap junction mediated ICWs play an important role in the maintenance of ependymal motile cilia, and suggest that the enhancement of functional gap junctions by pharmacological or genetic manipulations may be adopted to ameliorate motile ciliopathy.