A panel of human lung carcinoma lines: establishment, properties and common characteristics.

A panel of human lung carcinoma lines representing the four main histological types (squamous, small-cell, large-cell and adenocarcinoma), and derived from both primary and metastatic sites, has been established in xenograft and in tissue culture. The highest take rates were achieved when biopsy specimens were obtained from large tumour masses and cultured lines were most readily established after preliminary passages as xenografts. The established lines exhibited an overlapping spectrum of biochemical and morphological characteristics, and showed a tendency to change from one cell type to another, in keeping with the concept of a common endodermal cell of origin. Radiation resistance appeared to be related to the large-cell phenotype

Glutathione determination by the Tietze enzymatic recycling assay and its relationship to cellular radiation response.

Large fluctuations in glutathione content were observed on a daily basis using the Tietze enzyme recycling assay in a panel of six human cell lines of varying radiosensitivity. Glutathione content tended to increase to a maximum during exponential cell proliferation, and then decreased at different rates as the cells approached plateau phase. By reference to high-performance liquid chromatography and flow cytometry of the fluorescent bimane derivative we were able to verify that these changes were real. However, the Tietze assay was occasionally unable to detect glutathione in two of our cell lines (MGH-U1 and AT5BIVA), although the other methods indicated its presence. The existence of an inhibitory activity responsible for these anomalies was confirmed through spiking our samples with known amounts of glutathione. We were unable to detect a direct relationship between cellular glutathione concentration and aerobic radiosensitivity in our panel of cell lines

Hypoxia-inducible factor-1 (HIF-1) pathway activation by quercetin in human lens epithelial cells

Quercetin is a dietary bioflavonoid which has been shown to inhibit lens opacification in a number of models of cataract. The objectives of this study were to determine gene expression changes in human lens epithelial cells in response to quercetin and to investigate in detail the mechanisms underlying the responses. FHL-124 cells were treated with quercetin (10 µM) and changes in gene expression were measured by microarray. It was found that 65% of the genes with increased expression were regulated by the hypoxia-inducible factor-1 (HIF-1) pathway. Quercetin (10 and 30 µM) induced a time-dependent increase in HIF-1a protein levels. Quercetin (30 µM) was also responsible for a rapid and long-lasting translocation of HIF-1a from the cytoplasm to the nucleus. Activation of HIF-1 signaling by quercetin was confirmed by qRT–PCR which showed upregulation of the HIF-1 regulated genes EPO, VEGF, PGK1 and BNIP3. Analysis of medium taken from FHL-124 cells showed a sustained dose-dependent increase in VEGF secretion following quercetin treatment. The quercetin-induced increase and nuclear translocation of HIF-1a was reversed by addition of excess iron (100 µM). These results demonstrate that quercetin activates the HIF-1 signaling pathway in human lens epithelial cells

Structures of substrate- and product-bound forms of a multi-domain copper nitrite reductase shed light on the role of domain tethering in protein complexes

Copper-containing nitrite reductases (CuNiRs) are found in all three kingdoms of life and play a major role in the denitrification branch of the global nitrogen cycle where nitrate is used in place of dioxygen as an electron acceptor in respiratory energy metabolism. Several C- and N-terminal redox domain tethered CuNiRs have been identified and structurally characterized during the last decade. Our understanding of the role of tethered domains in these new classes of three-domain CuNiRs, where an extra cytochrome or cupredoxin domain is tethered to the catalytic two-domain CuNiRs, has remained limited. This is further compounded by a complete lack of substrate-bound structures for these tethered CuNiRs. There is still no substrate-bound structure for any of the as-isolated wild-type tethered enzymes. Here, structures of nitrite and product-bound states from a nitrite-soaked crystal of the N-terminal cupredoxin-tethered enzyme from the Hyphomicrobium denitrificans strain 1NES1 (Hd 1NES1NiR) are provided. These, together with the as-isolated structure of the same species, provide clear evidence for the role of the N-terminal peptide bearing the conserved His27 in water-mediated anchoring of the substrate at the catalytic T2Cu site. Our data indicate a more complex role of tethering than the intuitive advantage for a partner-protein electron-transfer complex by narrowing the conformational search in such a combined system.</jats:p