Chylomicron-remnant-like particles inhibit the basal nitric oxide pathway in porcine coronary artery and aortic endothelial cells. (49/283)

The effects of chylomicron remnants on the activity of basally produced nitric oxide (NO) from porcine coronary artery rings and porcine aortic endothelial cells were studied by investigating the effects of chylomicron-remnant-like particles (CMR-LPs) containing porcine apolipoprotein E on the vessel tone of porcine coronary arteries and on cGMP release by aortic endothelial cells. CMR-LPs were oxidized by incubation with CuSO(4) (10 microM) for 18 h at 37 degrees C. N (omega)-nitro-L-arginine (L-NOARG) and oxidized CMR-LPs (oxCMR-LPs), but not native CMR-LPs, increased the vessel tone of static porcine coronary artery rings (increase in tone as a percentage of the tone induced by depolarizing Krebs-Henseleit solution: L-NOARG, 14.24 +/- 2.09; oxCMR-LPs, 4.98 +/- 0.88; and native CMR-LPs, 0.47 +/- 0.21). L-NOARG, endothelium removal and oxCMR-LPs also all significantly increased the maximum relaxation of the vessels to S -nitroso- N -acetyl-DL-penicillamine. In addition, oxCMR-LPs reduced the amounts of cGMP released by porcine aortic endothelial cells into the culture medium from 116 +/- 12.0 to 84.2 +/- 11.6 fmol/microg of cellular protein, mimicking the effects of L-NOARG. These results indicate that oxCMR-LPs, but not native CMR-LPs, inhibit the activity, production or release of NO from unstimulated porcine coronary and aortic endothelial cells. oxCMR-LPs mimicked the addition of L-NOARG and endothelium removal in these experimental systems, suggesting that the lipoproteins were interfering with the L-arginine/NO pathway. This study provides further evidence to support a role of chylomicron remnants in the development of atherosclerosis.  (+info)

PAA1, a P-type ATPase of Arabidopsis, functions in copper transport in chloroplasts. (50/283)

Copper (Cu) is an essential trace element with important roles as a cofactor in many plant functions, including photosynthesis. However, free Cu ions can cause toxicity, necessitating precise Cu delivery systems. Relatively little is known about Cu transport in plant cells, and no components of the Cu transport machinery in chloroplasts have been identified previously. Cu transport into chloroplasts provides the cofactor for the stromal enzyme copper/zinc superoxide dismutase (Cu/ZnSOD) and for the thylakoid lumen protein plastocyanin, which functions in photosynthetic electron transport from the cytochrome b(6)f complex to photosystem I. Here, we characterized six Arabidopsis mutants that are defective in the PAA1 gene, which encodes a member of the metal-transporting P-type ATPase family with a functional N-terminal chloroplast transit peptide. paa1 mutants exhibited a high-chlorophyll-fluorescence phenotype as a result of an impairment of photosynthetic electron transport that could be ascribed to decreased levels of holoplastocyanin. The paa1-1 mutant had a lower chloroplast Cu content, despite having wild-type levels in leaves. The electron transport defect of paa1 mutants was evident on medium containing <1 micro M Cu, but it was suppressed by the addition of 10 micro M Cu. Chloroplastic Cu/ZnSOD activity also was reduced in paa1 mutants, suggesting that PAA1 mediates Cu transfer across the plastid envelope. Thus, PAA1 is a critical component of a Cu transport system in chloroplasts responsible for cofactor delivery to plastocyanin and Cu/ZnSOD.  (+info)

Copper modulates the degradation of copper chaperone for Cu,Zn superoxide dismutase by the 26 S proteosome. (51/283)

Copper chaperones are copper-binding proteins that directly insert copper into specific targets, preventing the accumulation of free copper ions that can be toxic to the cell. Despite considerable advances in the understanding of copper transfer from copper chaperones to their target, to date, there is no information regarding how the activity of these proteins is regulated in higher eukaryotes. The insertion of copper into the antioxidant enzyme Cu,Zn superoxide dismutase (SOD1) depends on the copper chaperone for SOD1 (CCS). We have recently reported that CCS protein is increased in tissues of rats fed copper-deficient diets suggesting that copper may regulate CCS expression. Here we show that whereas copper deficiency increased CCS protein in rats, mRNA level was unaffected. Rodent and human cell lines cultured in the presence of the specific copper chelator 2,3,2-tetraamine displayed a dose-dependent increase in CCS protein that could be reversed with the addition of copper but not iron or zinc to the cells. Switching cells from copper-deficient to copper-rich medium promoted the rapid degradation of CCS, which could be blocked by the proteosome inhibitors MG132 and lactacystin but not a cysteine protease inhibitor or inhibitors of the lysosomal degradation pathway. In addition, CCS degradation was slower in copper-deficient cells than in cells cultured in copper-rich medium. Together, these data show that copper regulates CCS expression by modulating its degradation by the 26 S proteosome and suggest a novel role for CCS in prioritizing the utilization of copper when it is scarce.  (+info)

Novel derivatives of 9,10-anthraquinone are selective algicides against the musty-odor cyanobacterium Oscillatoria perornata. (52/283)

Musty "off-flavor" in pond-cultured channel catfish (Ictalurus punctatus) costs the catfish production industry in the United States at least 30 million US dollars annually. The cyanobacterium Oscillatoria perornata (Skuja) is credited with being the major cause of musty off-flavor in farm-raised catfish in Mississippi. The herbicides diuron and copper sulfate, currently used by catfish producers as algicides to help mitigate musty off-flavor problems, have several drawbacks, including broad-spectrum toxicity towards the entire phytoplankton community that can lead to water quality deterioration and subsequent fish death. By use of microtiter plate bioassays, a novel group of compounds derived from the natural compound 9,10-anthraquinone have been found to be much more selectively toxic towards O. perornata than diuron and copper sulfate. In efficacy studies using limnocorrals placed in catfish production ponds, application rates of 0.3 micro M (125 micro g/liter) of the most promising anthraquinone derivative, 2-[methylamino-N-(1'-methylethyl)]-9,10-anthraquinone monophosphate (anthraquinone-59), dramatically reduced the abundance of O. perornata and levels of 2-methylisoborneol, the musty compound produced by O. perornata. The abundance of green algae and diatoms increased dramatically 2 days after application of a 0.3 micro M concentration of anthraquinone-59 to pond water within the limnocorrals. The half-life of anthraquinone-59 in pond water was determined to be 19 h, making it much less persistent than diuron. Anthraquinone-59 appears to be promising for use as a selective algicide in catfish aquaculture.  (+info)

Involvement of the macrophage low density lipoprotein receptor-binding domains in the uptake of oxidized low density lipoprotein. (53/283)

Macrophages, unlike most other cells, possess both low density lipoprotein (LDL) and scavenger receptors. The scavenger receptor has been shown to mediate the uptake of oxidized LDL (ox-LDL), which ultimately leads to cholesterol loading of the macrophages. The present study was undertaken to define epitopes on ox-LDL that are important for lipoprotein binding to macrophages and to ascertain whether ox-LDL can bind to the LDL receptor. Monoclonal antibodies (Mabs) directed against several epitopes along the apolipoprotein B-100 (apo B-100) molecule were used. LDL (300 micrograms/ml) was oxidized by incubation with 10 microM CuSO4 for 24 hours. Ox-LDL, as opposed to acetylated LDL (ac-LDL), reacted with Mabs directed against the LDL receptor-binding domains (Mabs B1B6 and B1B3). Similarly, uptake of ox-LDL but not ac-LDL by a murine J774 macrophage-like cell line was inhibited by as much as 40% after using Mab B1B6. The anti-LDL receptor antibody IgG-C7 also inhibited 125I-ox-LDL uptake by macrophages by 60%. Chromatography on heparin-Sepharose columns of LDL that was partially oxidized for only 3 hours resulted in two fractions: an unbound fraction with characteristics similar to those of ox-LDL and a bound fraction similar to native LDL. Macrophage degradation of the unbound fraction was inhibited by Mab IgG-C7 and Mab B1B6, which are directed toward the LDL receptor and the LDL receptor-binding domains on apo B-100, respectively. When incubated with three types of macrophages, J774 macrophage cells, mouse peritoneal macrophages, and human monocyte-derived macrophages, excess amounts of unlabeled ox-LDL, like native LDL but unlike ac-LDL, substantially suppressed the uptake and degradation of 125I-labeled LDL. Similar studies with fibroblasts, however, revealed that unlabeled LDL but not unlabeled ox-LDL or ac-LDL competed with 125I-LDL for cellular uptake and degradation. Mab directed against epitopes on the amino terminus domain of apo B-100 (C14) demonstrates a similar immunoreactivity with ox-LDL and native LDL but a much lower reactivity with ac-LDL. Mab C14 inhibited macrophage degradation of ox-LDL by 34% but had no inhibitory effect on the uptake of native LDL or ac-LDL. Thus, the ac-LDL and LDL receptor-binding domains as well as a unique epitope on the amino terminus of apo B-100 may be involved in macrophage binding of ox-LDL.(ABSTRACT TRUNCATED AT 400 WORDS)  (+info)

Folding and oxidation of recombinant human granulocyte colony stimulating factor produced in Escherichia coli. Characterization of the disulfide-reduced intermediates and cysteine----serine analogs. (54/283)

The folding and oxidation of recombinant human granulocyte colony-stimulating factor solubilized from Escherichia coli inclusion bodies was investigated. During the folding process, two intermediates, I1 and I2, were detected by kinetic studies using high performance liquid chromatography. I1 exists transiently and disappears quickly with the concomitant formation of I2. In contrast, I2 requires a longer time to fold into the final oxidized form, N. CuSO4 catalysis increases the folding rate of I2 from I1, while CuSO4 and elevated temperature (37 degrees C) have a dramatic effect on the folding rate of N from I2. These observations suggest the following sequential oxidative folding pathway. [sequence: see text] Peptide map analysis of the iodoacetate-labeled intermediates revealed that I1 represents the fully reduced granulocyte colony-stimulating factor containing 5 free cysteines; I2 is the partially oxidized species containing a single Cys36-Cys42 disulfide bond; and N, the final folded form, has two disulfide bonds. The physicochemical properties and biological activities of I1, I2, N, and several Cys----Ser analogs made by site-directed mutagenesis were further investigated. In guanidine hydrochloride-induced denaturation studies, the disulfide-reduced intermediates and the analogs missing either of the disulfide bonds are conformationally less stable than those of the wild type molecule or the analog with the free Cys at position 17 changed to Ser. Recombinant human granulocyte colony stimulating factor lacking either disulfide bond or both has overall secondary and tertiary structures different from those of the wild type molecule and exhibits lower biological activity. These studies show that disulfide bond formation is crucial for maintaining the molecule in a properly folded and biologically active form.  (+info)

Copper-induced oxidative damage on astrocytes: protective effect exerted by human high density lipoproteins. (55/283)

In the present study, we confirmed that copper ions induce oxidative damage in human astrocytes in culture, as demonstrated by the significant increase in the levels of hydroperoxides and in the fluorescence intensity of the fluorescent probe dichloro-dihydrofluorescein diacetate (H(2)DCFDA). The compositional changes were associated with a significant decrease in cell viability in astrocytes treated with 10 microM Cu(++) with respect to control cells. Astrocytes incubated with copper ions in the presence of high density lipoproteins (HDL) isolated from plasma of normolipemic subjects showed lower levels of hydroperoxides and a higher cell viability with respect to cells oxidized alone. Moreover, a significant decrease in the levels of hydroperoxides was observed in oxidized astrocytes treated with HDL. These results demonstrate that HDL exert a protective role against lipid peroxidation. The protective effect could be related to the ability of HDL to bind metal ions at the lipoprotein surface and/or to a stimulation of the efflux of lipid hydroperoxides from cell membranes as demonstrated in other cell types. Oxidative damage of astrocytes was induced at a copper concentration similar to that observed in cerebrospinal fluid (CSF) of patients affected by neurodegenerative diseases such as Alzheimer's (AD) and Parkinson's diseases (PD). Lipoprotein particles similar for density and chemical composition to plasma HDL were recently isolated in human CSF, therefore, the protective role exerted by HDL against Cu(++)-induced oxidative damage of astrocytes could be of physiological relevance.  (+info)

A quantitative study of magnetization transfer in MAGIC gels. (56/283)

Magnetization transfer (MT) has been measured quantitatively as a function of radiation dose in MAGIC polymer gels. The MT rates between the free and immobile macromolecular proton pools (kmr and kfm), and the ratio of the sizes of these coupled proton pools (Pm/Pf), were measured by analysing the response to an inversion recovery sequence. While pm/pf increases linearly with dose, the fast MT rate kmf also increases with dose, unlike previous measurements in BANG gels. This dependence of kmf on dose suggests there are additional factors that modify spin exchange in MAGIC gels as irradiation occurs.  (+info)