In vitro inactivation of the rabies virus by ascorbic acid.
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OBJECTIVE: The current recommended inactivating agent for the rabies virus, beta propiolactone (BPL) is very expensive and potentially carcinogenic. There is a need to evaluate alternative chemicals, which will inactivate the virus without affecting its antigenicity. In this study the effect of ascorbic acid on the infectivity of the rabies virus has been investigated. METHOD: Vero cell grown fixed rabies virus CVS strain was treated with 0.1 mg/ml, 0.5 mg/ml and 1mg/ml final concentrations of ascorbic acid and 5 microg/ml of copper sulfate and kept at 4 degrees C along with untreated virus material. Each aliquot was titrated after various intervals for viral infectivity using both mice inoculation and titration in vero cells. The antigenicity of the virus material was determined by antibody induction in mice and modified NIH tests in parallel with virus material inactivated with a 1:4000 concentration of BPL. RESULTS: An optimal concentration of 0.5 mg/ml of ascorbic acid and 5 microg/ml of copper sulfate completely inactivated the virus after 72 hours. The inactivated virus retained good antigenicity and potency value, which was comparable with using BPL. CONCLUSION: These findings suggest that ascorbic acid can be used as an inactivating agent for fixed rabies virus grown in cell culture particularly for the preparation of diagnostic reagents. Further studies are required to evaluate its effect on the cell associated virus, probable therapeutic potential and feasibility of replacing BPL in production of inactivated rabies vaccine. (+info)
Transition metal ions significantly decrease phospholipase C activity degrading phosphatidylinositol-4,5-bisphosphate in the brain cortex.
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Highly reactive transition metals, such as copper and iron play an obligatory role in generating of reactive oxygen species (ROS). Many neurodegenerative diseases including Alzheimer's disease (AD) and Parkinson's disease (PD) show increased accumulation of these metals. Phosphoinositide metabolism is altered in neurodegenerative diseases. In the present study, we examined the effect of CuSO(4) and FeCl(2) on phospholipase C (PLC) activity degrading phosphatidylinositol-4,5-bisphosphate (PIP(2)) and phosphatidylinositol (PI) in synaptic plasma membranes (SPM) from the rat brain cortex. We report that 25 microM CuSO(4) and FeCl(2) decreased PIP(2)-PLC activity by 60% and 75%, respectively. However, both compounds had no effect on PI-PLC activity. These data indicated that exclusively PIP(2)-PLC is sensitive to transition metal ions. We suggest that chelators of these metals may protect brain against alteration of phosphoinositide metabolism and might be beneficial in the treatment of neurodegenerative diseases. (+info)
Extracellular norepinephrine reduces neuronal uptake of norepinephrine by oxidative stress in PC12 cells.
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Cardiac norepinephrine (NE) uptake activity is reduced in congestive heart failure. Our studies in intact animals suggest that this effect on the cardiac sympathetic nerve endings is caused by oxidative stress and/or NE toxic metabolites derived from NE. In this study, we investigated the direct effects of NE on neuronal NE uptake activity and NE transporter (NET), using undifferentiated PC12 cells. Cells were incubated with NE (1-500 microM) either alone or in combination of Cu(2+) sulfate (1 microM), which promotes free radical formation by Fenton reaction for 24 h. NE uptake activity was measured using [(3)H]NE. Cell viability was determined with the use of Trypan blue exclusion and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay, and cellular oxidative stress by dichlorodihydrofluorescein fluorescence and the GSH/GSSG ratio. Cell viability was reduced by NE >100 microM. At lower doses, NE produced oxidative stress and a dose-dependent reduction of NE uptake activity without affecting cell viability significantly. Cu(2+), which has no direct effect on NE uptake activity, potentiated oxidative stress and reduction of NE uptake activity produced by NE. This decrease of NE uptake activity was associated with reductions of NE uptake binding sites and NET protein expression by using the radioligand assay and Western blot analysis, but no changes in NET gene expression. In addition, the free-radical scavenger mannitol, and antioxidant enzymes superoxide dismutase and catalase, reduced oxidative stress and attenuated the reductions of NE uptake activity and NET protein produced by NE/Cu. Thus our results support a functional role of oxidative stress in mediating the neuronal NE uptake reducing effect of NE and that this effect of NE on NET is a posttranscriptional event. (+info)
Population genetic variation in gene expression is associated with phenotypic variation in Saccharomyces cerevisiae.
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BACKGROUND: The relationship between genetic variation in gene expression and phenotypic variation observable in nature is not well understood. Identifying how many phenotypes are associated with differences in gene expression and how many gene-expression differences are associated with a phenotype is important to understanding the molecular basis and evolution of complex traits. RESULTS: We compared levels of gene expression among nine natural isolates of Saccharomyces cerevisiae grown either in the presence or absence of copper sulfate. Of the nine strains, two show a reduced growth rate and two others are rust colored in the presence of copper sulfate. We identified 633 genes that show significant differences in expression among strains. Of these genes, 20 were correlated with resistance to copper sulfate and 24 were correlated with rust coloration. The function of these genes in combination with their expression pattern suggests the presence of both correlative and causative expression differences. But the majority of differentially expressed genes were not correlated with either phenotype and showed the same expression pattern both in the presence and absence of copper sulfate. To determine whether these expression differences may contribute to phenotypic variation under other environmental conditions, we examined one phenotype, freeze tolerance, predicted by the differential expression of the aquaporin gene AQY2. We found freeze tolerance is associated with the expression of AQY2. CONCLUSIONS: Gene expression differences provide substantial insight into the molecular basis of naturally occurring traits and can be used to predict environment dependent phenotypic variation. (+info)
Copper proteinate in weanling pig diets for enhancing growth performance and reducing fecal copper excretion compared with copper sulfate.
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Two 28-d experiments were conducted to evaluate the efficacy of low dietary concentrations of Cu as Cu-proteinate compared with 250 ppm Cu as CuSO4 with growth performance, plasma Cu concentrations, and Cu balance of weanling swine as the criteria. In the production study (Exp. 1), 240 crossbred pigs that averaged 19.8 d of age and 6.31 kg BW initially were group-fed (two or three pigs per pen) the basal diets (Phase 1: d 0 to 14 and Phase 2: d 14 to 28) supplemented with 0 (control), 25, 50, 100, or 200 ppm Cu as Cu-proteinate, or 250 ppm Cu as CuSO4 (as-fed basis). The basal diets contained 16.5 ppm Cu supplied as CuSO4 before supplementation with Cu-proteinate or 250 ppm Cu as CuSO4. There were quadratic responses (P < or = 0.05) in ADFI and ADG for wk 1, Phases 1 and 2, and overall because ADFI was higher for pigs fed 25 or 50 ppm Cu as Cu-proteinate, and ADG increased with increasing Cu-proteinate up to 50 ppm Cu. The Cu-proteinate treatment groups combined had a higher (P < or = 0.05) Phase 2 and overall ADFI and ADG than the CuSO4 group. In the mineral balance study (Exp. 2), 20 crossbred barrows that averaged 35 d of age and 11.2 kg/BW initially were placed in individual metabolism pens with total urine and fecal grab sample collections on d 22 to 26. Treatments were the basal Phase 2 diet supplemented with 0, 50, or 100 ppm Cu as Cu-proteinate, or 250 ppm Cu as CuSO4 (as-fed basis). Treatments did not differ in growth performance criteria. There were linear increases (P < 0.001) in Cu absorption, retention, and excretion (milligrams per day) with increasing Cu-proteinate. Pigs fed 100 ppm Cu as Cu-proteinate absorbed and retained more Cu and excreted less Cu (mg/d, P < or = 0.003) than pigs fed 250 ppm Cu as CuSO4. Plasma Cu concentrations increased linearly (P = 0.06) with increasing Cu-proteinate. In conclusion, weanling pig growth performance was increased by 50 or 100 ppm Cu as Cu-proteinate in our production Exp. 1, but not in our balance Exp. 2, compared with 250 ppm Cu as CuSO4. However, 50 or 100 ppm Cu as Cu-proteinate increased Cu absorption and retention, and decreased Cu excretion 77 and 61%, respectively, compared with 250 ppm Cu as CuSO4. (+info)
Effect of dietary copper source (cupric citrate and cupric sulfate) and concentration on growth performance and fecal copper excretion in weanling pigs.
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In each of two experiments, 924 pigs (4.99 kg BW; 16 to 18 d of age) were assigned to 1 of 42 pens based on BW and gender. Pens were allotted randomly to dietary copper (Cu) treatments that consisted of control (10 ppm Cu as cupric sulfate, CuSO4 x 5H2O) and supplemental dietary Cu concentrations of 15, 31, 62, or 125 ppm as cupric citrate (CuCit), or 62 (Exp. 2 only), 125 (Exp. 1 only), or 250 ppm as CuSO4. Live animal performance was determined at the end of the 45-d nursery phase in each experiment. On d 40 of Exp. 2, blood and fecal samples were collected from two randomly selected pigs per pen for evaluation of plasma and fecal Cu concentrations and fecal odor characteristics. In Exp. 1, ADG, ADFI, and G:F were increased (P < 0.05), relative to controls, when pigs were fed diets containing 250 ppm Cu as CuSO4. Pigs fed diets containing 125 ppm Cu as CuCit had increased (P < 0.05) ADG compared with pigs fed diets supplemented with 15 or 62 ppm Cu as CuCit. The ADG, ADFI, and G:F did not differ among pigs fed diets containing 125 and 250 ppm Cu as CuSO4 or 125 ppm Cu as CuCit. In Exp. 2, pigs fed diets containing 250 ppm Cu as CuSO4 had improved (P < 0.05) ADG, ADFI, and G:F compared with controls. In addition, ADG, ADFI, and G:F were similar when pigs were fed diets containing either 250 ppm Cu as CuSO4 or 125 ppm Cu as CuCit. Pigs fed diets containing 62 ppm Cu as CuSO4 or CuCit had similar ADG, ADFI, and G:F. Plasma Cu concentrations were not affected by dietary Cu source or concentration, but fecal Cu concentrations were increased (P < 0.05) as the dietary concentration of Cu increased. Pigs consuming diets supplemented with 125 ppm Cu as CuCit had fecal Cu concentrations that were lower (P < 0.05) than pigs consuming diets supplemented with 250 ppm Cu as CuSO4. Fecal Cu did not differ in pigs receiving diets supplemented with 62 ppm Cu as CuSO4 or CuCit. Odor characteristics of feces were not affected by Cu supplementation or source. These data indicate that 125 and 250 ppm Cu gave similar responses in growth, and that CuCit and CuSO4 were equally effective at stimulating growth and improving G:F in weanling pigs. Fecal Cu excretion was decreased when 125 ppm Cu as CuCit was fed compared with 250 ppm Cu as CuSO4. Therefore, 125 ppm of dietary Cu, regardless of source, may provide an effective environmental alternative to 250 ppm Cu as CuSO4 in weanling pigs. (+info)
Protection of chylomicron remnants from oxidation by incorporation of probucol into the particles enhances their uptake by human macrophages and increases lipid accumulation in the cells.
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The effects of protection of chylomicron remnants from oxidation on their uptake and induction of lipid accumulation in macrophages were investigated using chylomicron remnant-like particles (CRLPs) containing the lipophilic antioxidant drug, probucol, and macrophages derived from the human monocyte cell line, THP-1. The total lipid content of THP-1 macrophages was markedly higher (x2.2) after 48 h of incubation of THP-1 macrophages with CRLPs containing probucol (pCRLPs) when compared to CRLPs without probucol, and this was because of increases in triacylglycerol (x2.3) and cholesterol (x1.8) levels, while cholesteryl ester concentrations were not significantly changed. Determination of the uptake of CRLPs and pCRLPs by the cells using particles labelled with the fluorescent probe 1,1'-dioctadecyl-3,3,3'3'-tetramethylindo-carbocyanine perchlorate showed that pCRLPs are taken up at a faster rate than CRLPs. The synthesis of triacylglycerol, as measured by the incorporation of [(3)H]oleate and [(3)H]glycerol, was also increased in macrophages incubated with pCRLPs as compared to CRLPs without probucol, but phospholipid and cholesteryl ester formation from [(3)H]oleate was unaffected. In addition, no differences between the effects of CRLPs and pCRLPs on the expression of mRNA for a range of genes believed to be involved in lipoprotein uptake, intracellular lipid metabolism and the efflux of cholesterol from macrophages was detected. These results suggest that antioxidants carried in chylomicron remnants enhance lipid accumulation in macrophages by increasing the rate of uptake of the particles and raising the intracellular synthesis of triacylglycerol, but not cholesteryl ester, and that these effects are brought about by changes at the post-transcriptional level. Antioxidants carried in chylomicron remnants therefore may promote the development of atherosclerosis, and this is likely to be particularly important in conditions where clearance of remnants from the circulation is delayed. (+info)
Performance and carcass characteristics of swine when fed diets containing canola oil and added copper to alter the unsaturated:saturated ratio of pork fat.
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A 3 x 2 trial was conducted to determine the effects of adding canola oil (0, 5, or 10%) and copper sulfate (0 or 250 ppm Cu) to diets of growing-finishing swine on performance, carcass characteristics, and carcass fat fatty acid composition. The trial used 180 pigs (27 kg). Grower diets (.80% lysine for 0% canola oil diet) were given from 27 to 57 kg of BW and finisher diets (.64% lysine) from 57 to 102 kg. Diets were formulated to constant ME:lysine ratio within the grower and finisher phases. Over the entire growing-finishing period, the addition of canola oil to the diets resulted in linear improvements in rate of gain (P less than .05) and feed efficiency (P less than .01). Dietary additions of canola oil had no effect (P greater than .10) on resulting backfat thickness or longissimus muscle area but resulted in reductions (P less than .01) in loin marbling and color and carcass fat firmness, mostly noted in pigs fed the diets with 10% canola oil. Canola oil additions at 5 and 10% levels, respectively, resulted in a 23 and 37% reduction (P less than .01) in saturated fatty acids, 3 and 8% increase (P less than .01) in monounsaturated fatty acids, and 37 and 77% increase (P less than .01) in polyunsaturated fatty acids in the carcass fat compared with the diets without canola oil. The addition of canola oil to diets of growing-finishing swine had a favorable influence on animal performance and on increasing the unsaturated:saturated ratio of the carcass fat.(ABSTRACT TRUNCATED AT 250 WORDS) (+info)