Isopycnic sedimentation of DNA in metrizamide: the effect of low concentrations of ions on buoyant density and hydration

Metrizamide, an inert, non-ionic organic compound, dissolves in water to give a dense solution in which DNA bands isopycnically at a density corresponding to that of fully hydrated DNA. Density-gradient centrifugation in solutions of metrizamide has been used to determine the effects of very dilute solutions of salts on the buoyant density of native and denatured DNA. It has been shown that the buoyant density of DNA is dependent on both the counter-cation and the anion present. Interpretation of the data in terms of the degree of hydration of the macromolecule indicates that (i), NaDNA is more highly hydrated than CsDNA; and (ii), the hydration of NaDNA varies with anion in the order sulphate< fluoride< chloride< bromide< iodide. It is suggested that isopycnic centrifugation in metrizamide is a simple method for determining the effects of salts (and other small molecules) on the hydration of nucleic acids under conditions of high ratios of salt to DNA (> 5 × 10(3) moles/mole) while high (0.999) water activity is maintained

Comparative genomic hybridization analysis of primary colorectal carcinomas and their synchronous metastases

We have analyzed 26 tumors from 12 patients with metastatic colorectal adenocarcinoma by comparative genomic hybridization (CGH). Primary tumors and their lymph node metastases from five Dukes' C patients as well as primary tumors and their liver metastases from seven Dukes' D patients were used to assess the extent of genetic differences between primary and secondary colorectal carcinomas from the same patients, to calculate the degree of clonal divergence and genetic heterogeneity in metastatic colorectal cancer, and to determine the differences in genetic imbalances between Dukes' C and D stage tumors. We show that the same genetic aberrations were frequently found in the primary tumors and their metastases. However, metastases often contained genetic aberrations not found in the corresponding primary tumors. The comparison of Dukes' stages C and D revealed genetic aberrations common to both. However, reduced copy number of chromosome arm 17p (5/5 vs. 0/7; &lt;I &gt;P&lt;/I &gt; = 0.001) was significantly associated with Dukes' stage C and lymph node metastases, while increased copy number of chromosome arms 6p (6/7 vs. 0/5; &lt;I &gt;P&lt;/I &gt; = 0.007) and 17q (5/7 vs. 0/5; &lt;I &gt;P&lt;/I &gt; = 0.027) was associated more with Dukes' stage D and liver metastases. Our results established a repertoire of chromosomal alterations associated with metastatic colorectal cancer and suggest that Dukes' C (lymph node metastasis) tumors are not always simply an earlier stage of Dukes' D (liver metastasis) tumors and, thus, in some instances at least, they are distinct forms of the disease

Expression of the components and regulatory proteins of the alternative complement pathway and the membrane attack complex in normal and diseased synovium

We have studied synthesis of the complement components and regulatory proteins of the alternative pathway and the membrane attack complex in synovial membrane. RNA was extracted from synovial tissue of patients with rheumatoid arthritis (RA) or osteoarthritis (OA) as well as from normal synovial membrane. Dot blot analysis showed the presence of mRNAs for all the complement components and regulatory proteins (C3, factor B, factor D, C5, C6, C7, C9, factor H, factor I, S-protein, SP-40, 40, DAF, MCP, CR1, CD59), except for properdin, C8α, C8β and C8γ in all three types of synovial membrane studied. In an attempt to determine which components were synthesised by each cell type, monocytes (mononuclear phagocytes), human umbilical vein endothelial cells (HUVEC), synovial membrane fibroblasts (from normal, OA and RA synovial membrane) and peripheral blood lymphocytes were cultured in vitro and secretion rates of individual components were measured and total cellular RNA analysed by northern blotting. Monocytes secreted properdin, C3, and factor H but not factor B, factor I, C5, C6, C7, C8 or C9. Fibroblasts and endothelial cells secreted factor B, factor H and factor I, but not properdin, C5, C6, C7, C8 or C9. Lymphocytes did not secrete any of these components. mRNAs encoding C3, factor B, factor H, S-protein, SP-40, 40, MCP and DAF were detected in all three other cell types (monocytes, fibroblasts and HUVEC), but factor I and CD59 mRNAs were not detected in monocytes. C5, C6, C7, C8α, C8β, CD8γ and C9 mRNAs were not detected in any of the cell types studied. Cell-specific differences were observed in the expression of the different mRNA species for DAF, MCP and CD59. The results of the present study demonstrate that synthesis of many complement components occurs in normal, RA and OA synovial membrane, and that this may be explained in part by synthesis in mononuclear phagocytes, endothelial cells and fibroblasts. The cellular sources of C5, C6, C7 and C9 mRNAs in synovial membrane have not been determined. The data also show that there are important cell-specific differences in the expression of the genes encoding both the alternative complement pathway components and the membrane regulatory components. These differences require further investigation