Insulin receptor substrate 1 translocation to the nucleus by the human JC virus T-antigen

Insulin receptor substrate 1 (IRS-1) is the major signaling molecule for the insulin and insulin-like growth factor I receptors, which transduces both metabolic and growth-promoting signals, and has transforming properties when overexpressed in the cells. Here we show that IRS-1 is translocated to the nucleus in the presence of the early viral protein-T-antigen of the human polyomavirus JC. Nuclear IRS-1 was detected in T-antigen-positive cell lines and in T-antigen-positive biopsies from patients diagnosed with medulloblastoma. The IRS-1 domain responsible for a direct JC virus T-antigen binding was localized within the N-terminal portion of IRS-1 molecule, and the binding was independent from IRS-1 tyrosine phosphorylation and was strongly inhibited by IRS-1 serine phosphorylation. In addition, competition for the IRS-1-T-antigen binding by a dominant negative mutant of IRS-1 inhibited growth and survival of JC virus T-antigen-transformed cells in anchorage-independent culture conditions. Based on these findings, we propose a novel role for the IRS-1-T-antigen complex in controlling cellular equilibrium during viral infection. It may involve uncoupling of IRS-1 from its surface receptor and translocation of its function to the nucleus.     

Small changes in the activity of chloroplastic NADP(+)-dependent ferredoxin oxidoreductase lead to impaired plant growth and restrict photosynthetic activity of transgenic tobacco plants

A ferredoxin-NADP+ oxidoreductase (FNR) cDNA from tobacco (Nicotiana tabacum cv. Samsun) was cloned and sequenced. Comparison of the deduced amino acid sequence revealed high identity to FNR proteins from Capsicum annuum, Pisum sativum, Spinacia oleracea and Vicia faba. Transgenic tobacco plants were generated that constitutively express the FNR cDNA in reverse orientation between the CaMV 35S promoter and the polyadenylation signal of the octopine synthase gene. Plants expressing the FNR antisense gene showed lower levels of FNR mRNA and protein accumulation, which was paralleled by a decrease in FNR activity. As a consequence, NADPH levels declined whereas NADP+ levels increased, leading to an unaltered NADP(H) pool. Growth rates, chlorophyll content and net CO2 uptake rates at high and low irradiances were strongly reduced in FNR antisense tobacco plants. These changes were accompanied by an over-reduced state of P700 as estimated by absorption changes at 820 nm. FNR control coefficients determined for the photosynthetic rate at saturating (C(R) = 0.94) and limiting (C(R) = 0.70) light conditions revealed a prominent role of this reductase in the regulation of photosynthesis.     

In vivo functional dissection of human inner kinetochore protein CENP-C

CENP-C is a fundamental component of the inner kinetochore plate and contributes to the formation of functional centromeres in eukaryotic organisms. Recruitment of CENP-C to kinetochore requires other centromere proteins, particularly CENP-A, CENP-H, and CENP-I. However, how CENP-C is correctly localized at the kinetochore is not clearly determined, mainly due to the functional variety of its domains, which hints at a complex recruitment mechanism. Here, by both immunofluorescent labeling and chromatin/immunoprecipitation we could show that human CENP-C contains two distinct domains, one in the central region, between amino acids 426 and 537, and the second one in the carboxyl terminal region, between amino acids 638 and 943, which are both capable of localizing at centromeres and binding alpha-satellite DNA. The presence of two domains that iterate the same function despite being significantly different in their amino acid sequence and structure suggests that CENP-C may target the centromere by establishing multiple contacts with both the DNA and protein constituents of the kinetochore.     

Digital photography provides a fast,reliable, and noninvasive method to estimate anthocyanin pigment concentration in reproductive and vegetative plant tissues

Anthocyanin pigments have become a model trait for evolutionary ecology as they often provide adaptive benefits for plants. Anthocyanins have been traditionally quantified biochemically or more recently using spectral reflectance. However, both methods require destructive sampling and can be labor intensive and challenging with small samples. Recent advances in digital photography and image processing make it the method of choice for measuring color in the wild. Here, we use digital images as a quick, noninvasive method to estimate relative anthocyanin concentrations in species exhibiting color variation. Using a consumer‐level digital camera and a free image processing toolbox, we extracted RGB values from digital images to generate color indices. We tested petals, stems, pedicels, and calyces of six species, which contain different types of anthocyanin pigments and exhibit different pigmentation patterns. Color indices were assessed by their correlation to biochemically determined anthocyanin concentrations. For comparison, we also calculated color indices from spectral reflectance and tested the correlation with anthocyanin concentration. Indices perform differently depending on the nature of the color variation. For both digital images and spectral reflectance, the most accurate estimates of anthocyanin concentration emerge from anthocyanin content‐chroma ratio, anthocyanin content‐chroma basic, and strength of green indices. Color indices derived from both digital images and spectral reflectance strongly correlate with biochemically determined anthocyanin concentration; however, the estimates from digital images performed better than spectral reflectance in terms of r² and normalized root‐mean‐square error. This was particularly noticeable in a species with striped petals, but in the case of striped calyces, both methods showed a comparable relationship with anthocyanin concentration. Using digital images brings new opportunities to accurately quantify the anthocyanin concentrations in both floral and vegetative tissues. This method is efficient, completely noninvasive, applicable to both uniform and patterned color, and works with samples of any size.     

The inhibition of sphingomyelin synthase 1 activity induces collecting duct cells to lose their epithelial phenotype

Epithelial tissue requires that cells attach to each other and to the extracellular matrix by the assembly of adherens junctions (AJ) and focal adhesions (FA) respectively. We have previously shown that, in renal papillary collecting duct (CD) cells, both AJ and FA are located in sphingomyelin (SM)-enriched plasma membrane microdomains. In the present work, we investigated the involvement of SM metabolism in the preservation of the epithelial cell phenotype and tissue organization. To this end, primary cultures of renal papillary CD cells were performed. Cultured cells preserved the fully differentiated epithelial phenotype as reflected by the presence of primary cilia. Cells were then incubated for 24 h with increasing concentrations of D609, a SM synthase (SMS) inhibitor. Knock-down experiments silencing SMS 1 and 2 were also performed. By combining biochemical and immunofluorescence studies, we found experimental evidences suggesting that, in CD cells, SMS 1 activity is essential for the preservation of cell-cell adhesion structures and therefore for the maintenance of CD tissue/tubular organization. The inhibition of SMS 1 activity induced CD cells to lose their epithelial phenotype and to undergo an epithelial-mesenchymal transition (EMT) process.     

Identification of a human clustered G + C-rich DNA family of repeats (Sau3A family)

Sau3A digestion of human G + C-rich DNA molecules yields discrete bands of approximately 70 and 140 base-pairs, under-represented in A + T-rich DNA molecules and in total DNA. We have cloned the 70 base-pair band in a plasmid vector and isolated a representative recombinant clone that identifies a new human family of repeats, the Sau3A family. The new family has been characterized for a number of parameters: genomic organization; reiteration frequency; sequence analysis; and distribution in a human genomic library. The Sau3A sequence (68 base-pairs in length, 53% G + C) is present in approximately 4 X 10(4) copies/haploid genome; the family is characterized by a cluster organization and is confined to a limited fraction (0.5%) of phages of a human genomic library. Southern blot hybridizations of the cloned sequence to restriction digests of total human DNA and of isolated genomic clones does not show the involvement of Sau3A blocks in long-range periodicities for any of the enzymes tested. The data suggest either a high sequence variability in the family or a complex organization of Sau3A sequence domains.     

External K+ affects the internal acidification caused by the addition of glucose to yeast cells

In glucose-grown cells of Saccharomyces cerevisiae, collected during the stationary phase of growth, the addition of K+ to the external medium reversed glucose-induced internal acidification in 2 min. However, in ethanol-grown cells external K+ did not reverse the effect of glucose even after 20 min. The presence or absence of external K+ did not alter the modification of trehalase and fructose-1,6-bisphosphatase induced by glucose. It is concluded that transient acidification may be sufficient to cause the associated transient increase in cAMP.