Metastasis-associated alterations in phospholipids and fatty acids of human prostatic adenocarcinoma cell lines.

Metastatic variants of human prostatic adenocarcinoma cell lines (DU-145, LNCaP, and ND-1) were studied by using soft agar colony forming efficiency, nude mice tumorigenicity, in vitro invasion assay, and type IV collagenase assay. The DU-145 and ND-1 cell line showed higher metastatic potential than LNCaP. Lipids from DU-145, ND-1, and LNCaP cells were extracted and analyzed by thin-layer chromatography and gas-liquid chromatography. The major lipids were phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, fatty acids, and cholesterol. The sphingomyelin level was significantly higher in highly metastatic cells (DU-145 and ND-1) compared with the lower metastatic variant (LNCaP). The increase in the synthetic pathway and decrease in degradation pathway of sphingomyelin in microsomal fractions was sufficient to account for the measured increase in sphingomyelin in DU-145 cells compared with LNCaP cells. The major fatty acids of these lipids were palmitic (16:0), stearic (18:0), oelic (18:1), and arachidonic acid (20:4). The arachidonic acid level was significantly decreased in DU-145 and ND-1 compared with LNCaP cells. Electron microscopic studies showed no significant changes in the morphology of DU-145, ND-1, and LNCaP cells. The results of these investigations demonstrate for the first time that sphingomyelin and arachidonic acid contents are different in high and low metastatic variants of human prostatic adenocarcinoma cell lines.     

Dideoxy fingerprinting (ddE): a rapid and efficient screen for the presence of mutations.

We describe dideoxy fingerprinting (ddF), a hybrid between dideoxy sequencing and SSCP that can detect the presence of single base and other sequence changes in PCR-amplified segments. As implemented herein, ddF involves a Sanger sequencing reaction with one dideoxynucleotide followed by nondenaturing gel electrophoresis. When ddF was used to examine segments of the human factor IX gene, 84 of 84 different mutations were detected with a very low rate of false positive signals. The approximate locations of the sequence changes could be determined from the ddF pattern and samples with different sequence changes had different fingerprints. In addition, large segments could be amplified and rapidly screened by ddF in multiple smaller subsegments. The patterns observed with ddF are instructive in that they suggest an inherent limitation in the detection of certain mutations by SSCP.     

Macrophage oxidation of L-arginine to nitrite and nitrate: nitric oxide is an intermediate

Previous studies have shown that murine macrophages immunostimulated with interferon gamma and Escherichia coli lipopolysaccharide synthesize NO2-, NO3-, and citrulline from L-arginine by oxidation of one of the two chemically equivalent guanido nitrogens. The enzymatic activity for this very unusual reaction was found in the 100,000g supernatant isolated from activated RAW 264.7 cells and was totally absent in unstimulated cells. This activity requires NADPH and L-arginine and is enhanced by Mg2+. When the subcellular fraction containing the enzyme activity was incubated with L-arginine, NADPH, and Mg2+, the formation of nitric oxide was observed. Nitric oxide formation was dependent on the presence of L-arginine and NADPH and was inhibited by the NO2-/NO3- synthesis inhibitor NG-monomethyl-L-arginine. Furthermore, when incubated with L-[guanido-15N2]arginine, the nitric oxide was 15N-labeled. The results show that nitric oxide is an intermediate in the L-arginine to NO2-, NO3-, and citrulline pathway. L-Arginine is required for the activation of macrophages to the bactericidal/tumoricidal state and suggests that nitric oxide is serving as an intracellular signal for this activation process in a manner similar to that very recently observed in endothelial cells, where nitric oxide leads to vascular smooth muscle relaxation [Palmer, R. M. J., Ashton, D. S., & Moncada, S. (1988) Nature (London) 333, 664-666].     

Binding of an antitumor drug to DNA, Netropsin and C-G-C-G-A-A-T-T-BrC-G-C-G

The antitumor antibiotic netropsin has been co-crystallized with a double-helical B-DNA dodecanucleotide of sequence: C-G-C-G-A-A-T-T-BrC-G-C-G, and the structure of the complex has been solved by X-ray diffraction at a resolution of 2.2 A. The structure has been refined independently by Jack-Levitt and Hendrickson-Konnert least-squares methods, leading to a final residual error of 0.257 by the Jack-Levitt approach (0.211 for two-sigma data) or 0.248 by the Hendrickson-Konnert approach, with no significant difference between refined structures. The netropsin molecule displaces the spine of hydration and fits snugly within the minor groove in the A-A-T-T center. It widens the groove slightly and bends the helix axis back by 8 degrees, but neither unwinds nor elongates the double helix. The drug molecule is held in place by amide NH hydrogen bonds that bridge adenine N-3 and thymine O-2 atoms, exactly as with the spine of hydration. The requirement of A X T base-pairs in the binding site arises because the N-2 amino group of guanine would demand impermissibly close contacts with netropsin. It is proposed that substitution of imidazole for pyrrole in netropsin should create a family of "lexitropsins" capable of reading G X C-containing base sequences.     

Helix geometry and hydration in an A-DNA tetramer: IC-C-G-G

The DNA oligomer of sequence IC-C-G-G has been synthesized, and its X-ray crystal structure solved at a resolution of 2.0 A, using anomalous scattering from iodines in phase analysis: 48 cycles of Jack-Levitt restrained least-squares refinement resulted in a residual error of 19.9% over all data, or 16.5% for two-sigma data. Two double-helical tetramers stack in the crystal to form a continuous octamer, except for the two missing phosphate connections across the center. The octamer has a mean helix rotation of 33.7 degrees (10.7 base-pairs per turn), rise of 2.87 A, mean inclination angle of base-pairs of 14 degrees, and mean base-pair propeller twist of +16.3 degrees. Local variations in both helix rotation and base plane roll angles, including those across the center of the octamer, are as predicted from base sequence by sum functions sigma 1 and sigma 2. The three known DNA octamers: IC-C-G-G/IC-C-G-G, G-G-T-A-T-A-C-C and G-G-C-C-G-G-C-C, make up a graded series in this order, with monotonically changing structural parameters. An exhaustive comparison of torsion angle correlations among the known A helices confirms some structural expectations and reveals some new features. 86 water molecules have been located per double-helical IC-C-G-G tetramer (the asymmetric unit), of which 451/2 per tetramer lie within a first hydrogen-bonded shell of hydration. No ordered water structure is observed comparable to the minor groove spine of hydration in B-DNA.     

Reduced synthesis of basement membrane heparan sulfate proteoglycan in streptozotocin-induced diabetic mice

In diabetes, certain basement membranes become thicker yet more porous than normal. To identify possible changes in the basement membrane, we have grown the Engelbreth-Holm-Swarm tumor, a tissue that produces quantities of basement membrane in normal mice and in streptozotocin-treated, insulin-deficient, diabetic mice. The level of laminin, a basement membrane-specific glycoprotein, and the level of total protein were slightly elevated in the diabetic tissue. In contrast, the level of the basement membrane specific heparan sulfate proteoglycan was only 20% of control. The synthesis of this proteoglycan was also reduced in the diabetic animals, while the synthesis of other proteoglycans by tissues such as cartilage was normal. The synthesis of the heparan sulfate proteoglycan in diabetic animals was inversely related to plasma glucose levels showing an abrupt decrease above the normal range of plasma glucose. Insulin restored synthesis to normal but this required doses of insulin that maintained plasma glucose at normal levels for several hours. Since the heparan sulfate proteoglycan in the basement membrane restricts passage of proteins, its absence could account for the increased porosity of basement membrane in diabetes. A compensatory synthesis of other components could lead to their increased deposition and the accumulation of basement membrane in diabetes.     

Morphology and life history of Halopteris filicina (Sphacelariales, Phaeophyceae) from Korea

The vegetative morphology and life history of Halopteris filicina (Grateloup) Kutzing, collected from Korea, were examined in laboratory culture. Field plants attaining 3–5 cm in height were epilithic, tufted, yellowish-brown, and produced numerous erect axes with alternately distichous branches from compact basal discs. They were cultured under a 12:12 h LD photoperiod at 10°-C, 15°C and 20°C to observe the influence of temperature on reproduction. At 10°C plants grew only vegetatively, whereas at 15°C and 20°C they produced unilocular sporangia. Unispores released from sporangia developed into monoecious, anisogamous gametophytes that formed plurilocular female and male gametangia on the same lateral branches. The zygotes, by fusion of female macrogametes and male microgametes, developed into sporophytes bearing unilocular sporangia, whereas the unfused female gametes germinated parthenogenetically. This species was confirmed to have an isomorphic life history, basically similar to the other species of Sphacelariales.