The Wilson disease protein ATP7B resides in the late endosomes with Rab7 and the Niemann-Pick C1 protein.
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Wilson disease is a genetic disorder characterized by the accumulation of copper in the body due to a defect of biliary copper excretion. Although the Wilson disease gene has been cloned, the cellular localization of the gene product (ATP7B) has not been fully clarified. Therefore, the precise physiological action of ATP7B is still unknown. We examined the distribution of ATP7B using an anti-ATP7B antibody, green fluorescent protein (GFP)-ATP7B (GFP-ATP7B) and ATP7B-DsRed in various cultured cells. Intracellular organelles were visualized by fluorescence microscopy. The distribution of ATP7B was compared with that of Rab7 and Niemann-Pick C1 (NPC1), proteins that localize in the late endosomes. U18666A, which induces the NPC phenotype, was used to modulate the intracellular vesicle traffic. GFP-ATP7B colocalized with various late endosome markers including Rab7 and NPC1 but not with Golgi or lysosome markers. U18666A induced the formation of late endosome-lysosome hybrid organelles, with GFP-ATP7B localized with NPC1 in these structures. We have confirmed that ATP7B is a late endosome-associated membrane protein. ATP7B appears to translocate copper from the cytosol to the late endosomal lumen, thus participating in biliary copper excretion via lysosomes. Thus, defective copper ATPase activity of ATP7B in the late endosomes appears to be the main defect of Wilson disease. (+info)
Endo-1,4-beta-xylanase B from Aspergillus cf. niger BCC14405 isolated in Thailand: purification, characterization and gene isolation.
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During the screening of xylanolytic enzymes from locally isolated fungi, one strain BCC14405, exhibited high enzyme activity with thermostability. This fugal strain was identified as Aspergillus cf. niger based on its morphological characteristics and internal transcribed spacer (ITS) sequences. An enzyme with xylanolytic activity from BCC14405 was later purified and characterized. It was found to have a molecular mass of ca. 21 kDa, an optimal pH of 5.0, and an optimal temperature of 55 degrees C. When tested using xylan from birchwood, it showed K(m) and V(max) values of 8.9 mg/ml and 11,100 U/mg, respectively. The enzyme was inhibited by CuSO(4) EDTA, and by FeSO(4) The homology of the 20-residue N-terminal protein sequence showed that the enzyme was an endo-1,4-beta-xylanase. The full-length gene encoding endo-1,4-beta-xylanase from BCC14405 was obtained by PCR amplification of its cDNA. The gene contained an open reading frame of 678 bp, encoding a 225 amino acid protein, which was identical to the endo-1,4-a-xylanase B previously identified in A. niger. (+info)
Effects of algicide (copper sulfate) application on short-term fluctuations of phytoplankton in Lake Paranoa, central Brazil.
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Short-term fluctuations and structure of phytoplankton were examined for two months in the dry season (August-September/1997) in a eutrophic reservoir of central Brazil. Algicide treatment for the control of Microcystis aeruginosa bloom influenced the short-term variation pattern of the phytoplankton community. Algicide treatment was effective in controlling M. aeruginosa bloom, but it also influenced the Cylindrospermopsis raciborskii population. These species retained low densities, showing small colonies and trichomes, respectively. Drops in Cyanobacteria populations following algicide treatment were succeeded by progressive increase in Chlorophyta. The shifts in environmental conditions may have allowed the development of this group. In this study correspondence analysis of abundance data for phytoplankton assemblage in lake Paranoa revealed that available light, rather than chlorophyll-a, total suspended material, and water temperature, accounted for most of the short-term fluctuation in phytoplankton structure during algicide application. Canonical correspondence analysis (CCA) showed the primary importance of water transparency changes in abundance of taxa in the community. Algicide treatment for the control of nuisance blooms is discussed as a contribution to improved efficiency in reservoir management. (+info)
Chemical modification of proteins during peroxidation of phospholipids.
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Chemical modification of proteins by advanced glycation and lipoxidation end products is implicated in the pathogenesis of macrovascular disease in aging and diabetes. To identify biomarkers of the lipoxidative modification of protein, we studied the oxidation of phospholipids in the presence of the model protein RNase A and compared protein-bound products formed in these reactions with those formed during oxidation of plasma proteins. Metal-catalyzed oxidation of 1-palmitoyl-2-arachidonoyl-phosphatidylcholine or 1-palmitoyl-2-linoleoyl-phosphatidylcholine in the presence of RNase led to the loss of amino groups in RNase and the incorporation of phosphate, hexanoate, pentanedioate, nonanedioate, and palmitate into protein. Protein-bound palmitate and phosphate correlated strongly with one another, and protein-bound pentanedioate and nonanedioate, derived from arachidonate and linoleate, respectively, accounted for approximately 20% of the cross-linking of lipid phosphorus to protein. Similar results were obtained on oxidation of total plasma or isolated LDL. We conclude that alkanedioic acids are quantitatively important linkers of oxidized phospholipids to proteins and that measurement of protein-bound phosphate and long-chain fatty acids may be useful for assessing long-term lipid peroxidative damage to proteins in vivo. Analyses of plasma proteins from control and diabetic patients indicated significant increases in lipoxidative modification of protein in diabetic compared with control subjects. (+info)
Sub-lethal effects of a copper sulfate fungicide on development and reproduction in three coccinellid species.
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Copper-based fungicides reliably control various foliar diseases in citrus production, although they are suspected to exacerbate mite problems through various mechanisms. Studies have shown negative effects of various copper formulations on entomopathogenic fungi, nematodes, and parasitoids, but few have sought to measure its impact on the biology of predatory insects. We exposed the larvae of three species of ladybeetle (Coleoptera: Coccinellidae) to field rates of copper sulfate in combination with petroleum oil, a formulation commonly applied in Florida citrus. First instar larvae of Curinus coeruleus Mulsant, Harmonia axyridis Pallas, and Olla v-nigrum Mulsant received a 24 h exposure to residues on Petri dishes, and another 24 h exposure in the third instar. Treated larvae of all three species survived to adulthood at the same rate as control larvae, but larvae of O. v-nigrum experienced a significant increase in developmental time. Female adults of C. coeruleus and H. axyridis receiving copper sulfate exposures as larvae did not differ from control adults in pre-reproductive period, fecundity or fertility over ten days of reproduction. Treated O. v-nigrum females had significantly longer pre-reproductive periods than control females and laid significantly fewer eggs, although egg fertility was equivalent. We conclude that copper-sulfate fungicides are unlikely to disrupt biological control processes in citrus groves that are mediated by these coccinellid beetles. (+info)
Influence of dietary zinc oxide and copper sulfate on the gastrointestinal ecosystem in newly weaned piglets.
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Dietary doses of 2,500 ppm ZnO-Zn reduced bacterial activity (ATP accumulation) in digesta from the gastrointestinal tracts of newly weaned piglets compared to that in animals receiving 100 ppm ZnO-Zn. The amounts of lactic acid bacteria (MRS counts) and lactobacilli (Rogosa counts) were reduced, whereas coliforms (MacConkey counts) and enterococci (Slanetz counts, red colonies) were more numerous in animals receiving the high ZnO dose. Based on 16S rRNA gene sequencing, the colonies on MRS were dominated by three phylotypes, tentatively identified as Lactobacillus amylovorus (OTU171), Lactobacillus reuteri (OTU173), and Streptococcus alactolyticus (OTU180). The colonies on Rogosa plates were dominated by the two Lactobacillus phylotypes only. Terminal restriction fragment length polymorphism analysis supported the observations of three phylotypes of lactic acid bacteria dominating in piglets receiving the low ZnO dose and of coliforms and enterococci dominating in piglets receiving the high ZnO dose. Dietary doses of 175 ppm CuSO(4)-Cu also reduced MRS and Rogosa counts of stomach contents, but for these animals, the numbers of coliforms were reduced in the cecum and the colon. The influence of ZnO on the gastrointestinal microbiota resembles the working mechanism suggested for some growth-promoting antibiotics, namely, the suppression of gram-positive commensals rather than potentially pathogenic gram-negative organisms. Reduced fermentation of digestible nutrients in the proximal part of the gastrointestinal tract may render more energy available for the host animal and contribute to the growth-promoting effect of high dietary ZnO doses. Dietary CuSO(4) inhibited the coliforms and thus potential pathogens as well, but overall the observed effect of CuSO(4) was limited compared to that of ZnO. (+info)
Therapeutic evaluation of zinc and copper supplementation in acute diarrhea in children: double blind randomized trial.
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OBJECTIVE: To test the hypothesis that daily supplementation of zinc and copper mixed with the oral rehydration solution (ORS) reduces the duration and the severity of acute diarrhea in children. METHODS: In a randomized, double blind, placebo controlled trial children aged 6 months to 59 months in an urban hospital with acute diarrhea, were assigned to receive the intervention of once daily 40 mg of zinc sulfate and 5 mg of copper sulfate dissolved in a liter of standard ORS (n = 102) or placebo (50 mg of standard ORS powder) dissolved in a liter of ORS (n = 98). RESULT: The baseline characteristics in the two groups were similar. The mean survival time (days) (SE) with diarrhea was not significantly different in the treatment (4.34 (0.2)) as compared to the placebo group (4.48 (0.2)), nor was there any difference in the median time to cure. Cure was less likely with longer duration of diarrhea prior to enrollment (P < 0.001), if the time taken for rehydration was more (P = 0.001) and if intravenous fluids were used (P = 0.03) regardless of the micronutrient supplementation. The proportion of children with diarrhea > 4 days was 46% in the placebo group with an adjusted odds ratio (OR) (95% CI) of 1.19 (1.58, 0.9; P = 0.2) as compared to 39% in the supplemented group. The most important risk factor for diarrhea > 4 days was diarrheal duration prior to enrollment with OR = 6.25 (3.7, 11.1). The supplemented group however had less severity of diarrhea with a lower proportion of children requiring unscheduled intravenous fluids (OR = 0.4; 95% CI 0.05, 2.2), with weight loss (OR = 0.7; 95% CI; 0.4, 1.3), with complications (OR = 0.15; 0.01, 1.3) and had no deaths as compared to two in the placebo group. CONCLUSIONS: This study showed that the most important predictor for duration of diarrhea in children was the severity of the disease at enrollment, and, not the supplementation. There were clinical beneficial effects of supplementation on rate of any complications and mortality. A larger trial is warranted before supplementation of micronutrients mixed with ORS are recommended for management of acute diarrhea. (+info)
Chronic depletion of glutathione (GSH) and minimal modification of LDL in vivo: its prevention by glutathione mono ester (GME) therapy.
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A decline in reduced glutathione (GSH) level is associated with aging and free radical mediated diseases. The objective of this study was to determine whether the chronic depletion of extra cellular GSH causes oxidative damage to the circulating macromolecules such as lipoproteins. Decreased concentrations of plasma glutathione, vitamin E and ascorbic acid were recorded in the rats treated with buthionine sulfoximine (BSO), a selective GSH inhibitor. In LDL isolated from BSO-treated animals, the concentration of malondialdehyde (MDA) and conjugated dienes were significantly increased (P<0.01), whereas the levels of vitamin E were decreased (P<0.01). The analysis of total and LDL cholesterol revealed significant changes between the control and experimental groups. Of interest, altered concentrations of lyso-phosphatidyl choline (Lyso-PC) and phosphatidyl choline (PC) were recorded from the BSO mediated minimally modified LDL. A negative correlation between LDL-BDC/MDA and its antioxidant capacity was noted. Upon in vitro oxidation with CuSO(4), the electrophoretic behavior of purified LDL-apoprotein-B on agarose gel showed an increased mobility in BSO-treated rats, indicative of in vivo modification of LDL to become susceptible for in vitro oxidation. The increased mobility of LDL (after in vitro oxidation) isolated from the BSO-treated animals correlates with a decrease in its amino groups, as determined by the trinitrobenzene sulfonic acid (TNBS) reactants. However, the mobility of LDL molecule was not altered due to BSO treatment in vivo. Interestingly, the minimal modification on LDL does not lead to any vascular damage in the dorsal aorta of the rats injected with BSO. The administration of glutathione monoester (GME), at a dose of 5 mmol/kg body weight, twice a day, for 30 days, to animals treated with l-buthionine-SR-sulfoximine (BSO, 4 mmol/kg body weight, twice a day, for 30 days) normalized the antioxidant status and prevented the minimal modifications on LDL. Thus, increasing the cellular GSH levels may trigger beneficial effects against oxidative stress. (+info)