Copper Deficiency Induces Oxidative Stress in Liver of Mice by Blocking the Nrf2 Pathway.

Copper (Cu) is an essential metal required for many physiological processes and biological reactions. Liver is the main organ of metabolism of Cu and is also the site where synthesis of some metalloproteins. The purpose of this study is to explore the effects of Cu deficiency on the liver and to evaluate the changes in liver oxidative stress levels to reveal its possible impact mechanisms. Mice were feed to a nutritional Cu-deficiency diet from weaning and injected with copper sulfate (CuSO4) intraperitoneally to correct Cu deficiency. Cu deficiency resulted in reduced liver index, liver histological alteration, and oxidative stress; decreased the contents of Cu and ALB; elevated ALT and AST concentrations in serum together with decreased mRNA and protein expressions of Nrf2 pathway related molecules (Nrf2, HO-1, NQO1); and increased mRNA and protein expressions of Keap1. However, the supplement of copper sulfate (CuSO4) significantly ameliorated the changes mentioned above. Our results indicate that Cu deficiency can cause hepatic damage in mice is associated with the activation of oxidative stress and inhibition of Nrf2 pathway.

Vitamin D alleviates cognitive dysfunction and brain damage induced by copper sulfate intake in experimental rats: focus on its combination with donepezil.

This study aimed to demonstrate the potential benefits of donepezil (DPZ) and vitamin D (Vit D) in combination to counteract the neurodegenerative disorders induced by CuSO4 intake in experimental rats. Neurodegeneration (Alzheimer-like) was induced in twenty-four male Wistar albino rats by CuSO4 supplement to drinking water (10 mg/L) for 14 weeks. AD rats were divided into four groups: untreated AD group (Cu-AD) and three treated AD groups; orally treated for 4 weeks with either DPZ (10 mg/kg/day), Vit D (500 IU/kg/day), or DPZ + Vit D starting from the 10th week of CuSO4 intake. Another six rats were used as normal control (NC) group. The hippocampal tissue content of ß-amyloid precursor protein cleaving enzyme 1 (BACE1), phosphorylated Tau (p-tau), clusterin (CLU), tumor necrosis factor-α (TNF-α), caspase-9 (CAS-9), Bax, and Bcl-2 and the cortical content of acetylcholine (Ach), acetylcholinesterase (AChE), total antioxidant capacity (TAC), and malondialdehyde (MDA) were measured. Cognitive function tests (Y-maze) and histopathology studies (hematoxylin and eosin and Congo red stains) and immunohistochemistry for neurofilament. Vit D supplementation alleviated CuSO4-induced memory deficits including significant reduction hippocampal BACE1, p-tau, CLU, CAS-9, Bax, and TNF-α and cortical AChE and MDA. Vit D remarkably increased cortical Ach, TAC, and hippocampal Bcl-2. It also improved neurobehavioral and histological abnormalities. The effects attained by Vit D treatment were better than those attained by DPZ. Furthermore, Vit D boosted the therapeutic potential of DPZ in almost all AD associated behavioral and pathological changes. Vit D is suggested as a potential therapy to retard neurodegeneration.

Effects of Various Copper Sources and Concentrations on Performance, Skeletal Growth, and Mineral Content of Excreta in Broiler Chickens.

The experiment was designed to study the effect of supplemental sources and concentrations of copper on the performance and development and mineralization of tibia bones in broiler chickens. A 42-day feeding experiment was conducted utilising three copper sources, including copper sulphate (CuS), copper chloride (CuCl), and copper propionate (CuP), each with four different concentrations, i.e. 8, 100, 150, and 200 mg/kg. The body weight gain with 200 mg Cu/kg food was noticeably higher during the first 4-6 weeks of age. Due to the interaction between Cu sources and levels, there was no significant change in the body weight gained. The feed intake during various growing phases did differ significantly neither the main effect nor the interaction between different copper sources and levels. A CuP-supplemented diet (200 mg/kg food) considerably (P ≤ 0.05) improved the feed conversion ratio between 4-6 and 0-6 weeks. At the end of the experiment, a total of 72 tibia bones, i.e. six for each treatment were collected. A metabolic trial was conducted to look into mineral retention in broiler chickens on the final 3 days of the trial (40-42 days). Increased tibia bone zinc (Zn) levels were seen with the addition of 8 mg Cu/kg of Cu chloride, 100 mg Cu/kg of Cu propionate, 8 mg Cu/kg of Cu sulphate, and 8 mg/kg of Cu propionate to the diet. At higher levels of Cu (150 and 200 mg/kg diet), there was a significantly (P ≤ 0.01) reduced tibia Zn content. Cu sulphate treatment group had higher (P ≤ 0.01) tibia Cu content (8 mg Cu/kg diet). Cu sulphate supplemented diet had a greater excreta Zn content (P ≤ 0.01) than Cu chloride supplemented diet, and Cu propionate supplemented diet had the lowest excreta Zn content. Excreta with a higher Fe concentration were found in diets supplemented with copper sulphate and copper chloride (P ≤ 0.05) than in diets supplied with copper propionate. Thus, it may be concluded that feeding dietary Cu concentrations up to 200 mg Cu/kg diet, regardless of the different sources, had no negative effects on bone morphometry and mineralization parameters with the exception of a decrease in the tibia's zinc content.

D-ribose-L-cysteine reduces oxidative stress and inflammatory cytokines to mitigate liver damage, and memory decline induced by copper sulfate in mice.

BACKGROUND: Current evidences have implicated copper in amyloid aggregation that trigger the downstream oxidative stress-mediated neuroinflammation that characterized memory deterioration in patients with Alzheimer's disease (AD). Thus, this study was designed to evaluate the effect of D-Ribose-L-Cysteine (DRLC), a potent antioxidant agent, on copper sulfate (CuSO4)-induced memory deterioration and the biochemical mechanisms underpinning its action in mice. METHODS: Male Swiss mice were randomly distributed into 5 groups (n = 10/group). Mice in group 1 were given distilled water (control), group 2 CuSO4 (100 mg/kg) while groups 3-5 were pretreated with CuSO4 (100 mg/kg) 30 min before administration of DRLC (10, 25 and 50 mg/kg). Treatments were given through oral gavage, daily for 28 days. Memory function was evaluated on day 28 using Y-maze test. The isolated liver and brain tissues were then processed for oxidative stress biomarkers, and proinflammatory cytokines [tumor necrosis factor- α (TNF-α) and interleukin-6)] assays. Brian acetylcholinesterase (AChE) and liver enzymes [aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities were also determined. RESULTS: DRLC reversed memory impairment and dysregulated levels of malondialdehyde, glutathione, nitrite and glutathione S-transferase in the liver and brain tissues of mice pretreated with CuSO4. The increased proinflammatory cytokines concentrations in the liver and brain tissues of mice pretreated with CuSO4 were reduced by DRLC. The elevated brain AChE and liver enzymes activities induced by CuSO4 were also reduced by DRLC. CONCLUSION: Taken together, these findings suggest that DRLC attenuates CuSO4-induced memory dysfunctions in mice through enhancement of antioxidative pathway, inhibition of pro-inflammatory cytokines and augmentation of liver function.

ß-Carotene extracted from Blakeslea trispora attenuates oxidative stress, inflammatory, hepatic injury and immune damage induced by copper sulfate in zebrafish (Danio rerio).

ß-Carotene, as a kind of potent antioxidant compounds, has gained extensive attention. Blakeslea trispora, a filiform aerobic fungus, has been proposed as a natural source of ß-carotene for commercial exploitation. However, it has not yet been investigated whether ß-carotene extracted from Blakeslea trispora can attenuate oxidative stress, inflammatory, liver injury and immune damage of zebrafish (Danio rerio) exposed to copper sulfate (CuSO4). In this study, we evaluated the effects of ß-carotene on migration of GFP-labeled neutrophils, histological changes of liver, markers of oxidative, inflammatory cytokines and transaminase analysis, as well as the expression and activities of apoptosis, immune-related certain genes in zebrafish treated with different concentrations of ß-carotene (0, 10, 20, 40 µg/mL) after exposure to CuSO4. The results indicated that ß-carotene reduced migration of neutrophils and released liver damage. What's more, ß-carotene was found to reduce the index levels of oxidative stress response (HMOX-1, reactive oxygen species (ROS), NADPH, MDA), inflammatory factors (interleukine-1ß (IL-1ß), interleukine-6 (IL-6), interleukine-8 (IL-8), tumor necrosis factor-α (TNF-α)), liver function protein (AST, ALT) which increased by CuSO4. ß-Carotene also promoted the activities of SOD, GSH-Px, ACP, AKP and LZM and increased the protein of immune-related factors, IgM and IFN-γ after exposure to CuSO4. Thus, our results demonstrate that ß-carotene has an antioxidant, anti-inflammatory and hepatoprotective activity and participation in immunoregulation.

Sources and levels of copper affect liver copper profile, intestinal morphology and cecal microbiota population of broiler chickens fed wheat-soybean meal diets.

Super dosing copper (Cu) has long been used as an alternative to antibiotic growth-promoters in broiler chickens' diet to improve gut health. This study was designed to compare nutritional and growth-promoting levels of Cu hydroxychloride (CH) with CuSO4 on gut health bio-markers and liver mineral profile of broiler chickens. Ross 308 chicks (n = 864) were randomly assigned to eight treatments, as basal diet containing no supplemental Cu; the basal diet with 15 or 200 mg/kg Cu as CuSO4; or 15, 50, 100, 150 or 200 mg/kg Cu from CH. The highest liver Cu content was observed in birds fed the diets with 200 mg/kg CuSO4 (P < 0.01). Serum FITC-d concentration as the leaky gut marker, and liver malondialdehyde concentration were not affected. Copper level or source had no effect on cecal short chain fatty acid and the mRNA expression of five jejunal genes involved in gut integrity. Negative linear responses of Cu were observed on Lactobacillus (P = 0.032), Bacteroides (P = 0.033), and Enterobacteriaceae (P = 0.028) counts. The jejunal villus height increased in birds fed CH at 200 and 100 mg/kg (P < 0.05). Increasing Cu levels, linearly and quadratically (P < 0.001), increased Cu excretion.

Evaluation of Zebrafish Toxicology and Biomedical Potential of Aeromonas hydrophila Mediated Copper Sulfide Nanoparticles.

The present study deals with extracellular synthesis and characterization of copper sulfide (CuS) nanoparticles using Aeromonas hydrophila, and the biological applications of the synthesized CuS like antibacterial, anti-inflammatory, and antioxidant activity were reported. Further, the toxicological effects of the CuS were evaluated using zebrafish as an animal model. The primary step of the synthesis was carried out by adding the precursor copper sulfates to the culture supernatant of Aeromonas hydrophila. The UV-visible spectrophotometer was used to characterize the synthesized nanoparticles, and the peak was obtained at 307 nm through the reduction process. Fourier transform infrared spectroscopy (FTIR) was involved to find out the functional groups (carboxylic acid, alcohols, alkanes, and nitro compounds) associated with copper sulfide nanoparticles (CuS-NPs). Atomic force microscopy (AFM) was used to characterize the CuS topographically, and a scanning electron microscope (SEM) revealed about 200 nm sized CuS nanoparticles with agglomerated structures. Overall, the characterized nanoparticles can be considered as a potential candidate with therapeutic proficiencies as antibacterial, antioxidant, and anti-inflammatory mediator/agents.

Characterizing effects of excess copper levels in a human astrocytic cell line with focus on oxidative stress markers.

BACKGROUND: Being an essential trace element, copper is involved in diverse physiological processes. However, excess levels might lead to adverse effects. Disrupted copper homeostasis, particularly in the brain, has been associated with human diseases including the neurodegenerative disorders Wilson and Alzheimer's disease. In this context, astrocytes play an important role in the regulation of the copper homeostasis in the brain and likely in the prevention against neuronal toxicity, consequently pointing them out as a potential target for the neurotoxicity of copper. Major toxic mechanisms are discussed to be directed against mitochondria probably via oxidative stress. However, the toxic potential and mode of action of copper in astrocytes is poorly understood, so far. METHODS: In this study, excess copper levels affecting human astrocytic cell model and their involvement in the neurotoxic mode of action of copper, as well as, effects on the homeostasis of other trace elements (Mn, Fe, Ca and Mg) were investigated. RESULTS: Copper induced substantial cytotoxic effects in the human astrocytic cell line following 48 h incubation (EC30: 250 µM) and affected mitochondrial function, as observed via reduction of mitochondrial membrane potential and increased ROS production, likely originating from mitochondria. Moreover, cellular GSH metabolism was altered as well. Interestingly, not only cellular copper levels were affected, but also the homeostasis of other elements (Ca, Fe and Mn) were disrupted. CONCLUSION: One potential toxic mode of action of copper seems to be effects on the mitochondria along with induction of oxidative stress in the human astrocytic cell model. Moreover, excess copper levels seem to interact with the homeostasis of other essential elements such as Ca, Fe and Mn. Disrupted element homeostasis might also contribute to the induction of oxidative stress, likely involved in the onset and progression of neurodegenerative disorders. These insights in the toxic mechanisms will help to develop ideas and approaches for therapeutic strategies against copper-mediated diseases.

Gene expression profiling of copper-resistant Caco-2 clones.

The Caco-2 cell line is composed of a heterogeneous mix of cells; isolation of individual clonal populations from this mix allows for specific mechanisms and phenotypes to be further explored. Previously we exposed Caco-2 cells to inorganic copper sulphate or organic copper proteinate to generate resistant variant populations. Here we describe the isolation and characterisation of clonal subpopulations from these resistant variants to organic (clone Or1, Or2, Or3) or inorganic (clone In1 and In2) copper. The clones show considerable homogeneity in response to Cu-induced toxicity and heterogeneity in morphology with variations in level of cross-resistance to other metals and doxorubicin. Population growth was reduced for Cu-resistant clones In2 and Or3 in selective pressure relative to parental Caco-2 cells. Gene expression analysis identified 4026 total (2102 unique and 1924 shared) differentially expressed genes including those involved in the MAP Kinase and Rap1 signalling pathways, and in the focal adhesion and ECM-receptor contact pathways. Gene expression changes common to all clones included upregulation of ANXA13 and GPx2. Our analysis additionally identified differential expression of multiple genes specific to copper proteinate exposure (including overexpressed UPK1B) in isolated clones Or1, Or2 and Or3 and CuSO4 exposure (including decreased AIFM2 expression) in isolated clones In1 and In2. The adaptive transcriptional responses established in this study indicate a cohort of genes, which may be involved in copper resistance regulation and chronic copper exposure.

Effects of Organic Copper on Growth Performance and Oxidative Stress in Mice.

Copper (Cu) has been used as a feed additive for many years. However, high Cu amounts can cause oxidative stress and adversely affect animal performance. Such negative effects may depend on the amounts and forms of Cu. In the present study, the effects of inorganic Cu (CuSO4) and organic Cu (chelate-Cu) present in mice feed on daily growth rate and Cu deposition in the liver, kidneys, spleen, brain, and serum were assessed in addition to the oxidative stress levels in the liver and brain. Organic Cu at a concentration of 15 mg/kg significantly enhanced daily growth rate in mice, whereas Cu deposition in the livers was significantly lower than that in the inorganic Cu group. Glutathione peroxidase activity in the liver of the mice fed with organic Cu significantly improved, whereas malondialdehyde levels in the brain and liver were significantly lower than that in the inorganic Cu group. The different effects of organic Cu and inorganic Cu provide key evidence supporting the use of organic Cu in animal feeds.

Comparative toxicity of nanoparticulate and ionic copper following dietary exposure to common carp (Cyprinus carpio).

Copper is an essential element for the normal growth and survival of all organisms including fish. However, its excessive presence in the environment can cause bioaccumulation and aquatic toxicology. The aim of the present study was to compare the dietary toxicity effects of two different Cu compounds, copper oxide nanoparticles (CuO-NPs) and ionic copper (CuSO4) in juvenile common carp, Cyprinus carpio. To prepare experimental diets, two nominal concentrations of 100 and 1000 mg Cu kg-1 diet were added to a basal diet. Carp (n = 450, average initial weight of 35.94 ± 5.35 g) were fed on the Cu-supplemented diets and basal diets for two 21-day courses as dietary exposure and recovery periods, respectively. The growth performance, survival rate and blood biochemical indices as well as copper accumulation in target organs of fish were investigated at the end of each exposure period. The results showed that the weight gain (WG) of carp significantly decreased coincident with increasing concentration of the both dietary Cu forms (P = 0.00). Both Cu sources at concentrations of 100 mg kg-1 diet decreased the survival rate of fish (P = 0.003), likely due to more feed intake and thus increased copper toxicity. The both forms of dietary Cu at two different concentrations significantly decreased the plasma glutamate oxaloacetate transaminase (GOT) level compared to the control group (P = 0.008). Fish exposed to diets containing Cu sources except 100 mg Cu kg-1 of CuO-NPs showed the lower glutamate pyruvate transaminase (GPT) activity in comparison to the control (P = 0.00). The plasma sodium level in1000 mg CuO-NPs kg-1 diet was significantly lower than the control (P = 0.001). The plasma potassium level increased in the all Cu-supplemented groups except 100 mg kg-1 of CuO-NPs after the dietary exposure period (P = 0.035). The copper accumulation was dose-dependent in all target organs. In 100 mg Cu kg-1 dietary groups, the liver showed the highest Cu accumulation (P = 0.00), while in 1000 mg Cu kg-1 dietary groups, the highest Cu content was observed in the intestine (P = 0.00). The results demonstrated the enhanced toxicological responses in fish after 21 days of dietary exposure, but the levels of most of biochemical indices and tissues Cu content decreased or returned to the control values after the recovery period.

Effects of soybean oil and dietary copper levels on nutrient digestion, ruminal fermentation, enzyme activity, microflora and microbial protein synthesis in dairy bulls.

The study evaluated the effects of soybean oil (SO) and dietary copper levels on nutrient digestion, ruminal fermentation, enzyme activity, microflora and microbial protein synthesis in dairy bulls. Eight Holstein rumen-cannulated bulls (14 ± 0.2 months of age and 326 ± 8.9 kg of body weight) were allocated into a replicated 4 × 4 Latin square design in a 2 × 2 factorial arrangement with factors being 0 or 40 g/kg dietary dry matter (DM) of SO and 0 or 7.68 mg/kg DM of Cu from copper sulphate (CS). The basal diet contained per kg DM 500 g of corn silage, 500 g of concentrate, 28 g of ether extract (EE) and 7.5 mg of Cu. The SO × CS interaction was significant (p < 0.05) for ruminal propionate proportion and acetate to propionate ratio. Dietary SO addition increased (p < 0.05) intake and total tract digestibility of EE but did not affect average daily gain (ADG) of bulls. Dietary CS addition did not affect nutrient intake but increased (p < 0.05) ADG and total tract digestibility of DM, organic matter, crude protein and neutral detergent fibre. Ruminal pH was not affected by treatments. Dietary SO addition did not affect ruminal total volatile fatty acids (VFA) concentration, decreased (p < 0.05) acetate proportion and ammonia N and increased (p < 0.05) propionate proportion. Dietary CS addition did not affect ammonia N, increased (p < 0.05) total VFA concentration and acetate proportion and decreased (p < 0.05) propionate proportion. Acetate to propionate ratio decreased (p < 0.05) with SO addition and increased (p < 0.05) with CS addition. Dietary SO addition decreased (p < 0.05) activity of carboxymethyl cellulase, cellobiase and xylanase as well as population of fungi, protozoa, methanogens, Ruminococcus albus and R. flavefaciens but increased (p < 0.05) α-amylase activity and population of Prevotella ruminicola and Ruminobacter amylophilus. Dietary CS addition increased (p < 0.05) activity of cellulolytic enzyme and protease as well as population of total bacteria, fungi, protozoa, methanogens, primary cellulolytic and proteolytic bacteria. Microbial protein synthesis was unchanged with SO addition but increased (p < 0.05) with CS addition. The results indicated that the addition of CS promoted nutrient digestion and ruminal fermentation by stimulating microbial growth and enzyme activity but did not relieve the negative effects of SO addition on ruminal fermentation in dairy bulls.

Evaluation of laccase production by Ganoderma lucidum in submerged and solid-state fermentation using different inducers.

Laccases are multicopper oxidases with high potential for industrial applications. Several basidiomycete fungi are natural producers of this enzyme; however, the optimization of production and selection of inducers for increased productivity coupled with low costs is necessary. Lignocellulosic residues are important lignin sources and potential inducers for laccase production. Pinus taeda, a dominant source of wood-based products, has not been investigated for this purpose yet. The aim of this study was to evaluate the production of laccase by the basidiomycete fungus Ganoderma lucidum in the presence of different inducers in submerged and solid-state fermentation. The results of submerged fermentation in presence of 5 µM CuSO 4 , 2 mM ferulic acid, 0.1 g/L P. taeda sawdust, or 0.05 g/L Kraft lignin indicated that although all the tested inducers promoted increase in laccase activity in specific periods of time, the presence of 2 mM ferulic acid resulted in the highest value of laccase activity (49 U/L). Considering the submerged fermentation, experimental design following the Plackett-Burman method showed that the concentrations of ferulic acid and P. taeda sawdust had a significant influence on the laccase activity. The highest value of 785 U/L of laccase activity on submerged fermentation was obtained on the seventh day of cultivation. Finally, solid-state fermentation cultures in P. taeda using ferulic acid or CuSO 4 as inducers resulted in enzymatic activities of 144.62 and 149.89 U/g, respectively, confirming the potential of this approach for laccase production by G. lucidum.

Proton pump inhibitors versus Cryptococcus species: effects on in vitro susceptibility and melanin production.

Aim: This study aimed to evaluate the effects of proton pump inhibitors (PPIs) on growth and melanin production by Cryptococcus spp. Materials & methods: Minimum inhibitory concentrations (MICs) of omeprazole, esomeprazole, rabeprazole, pantoprazole and lansoprazole against Cryptococcus spp. were determined and the effect of PPIs on melanin production was evaluated, in the presence or absence of copper sulfate or glutathione. Results: PPIs showed MICs ranging from 125-1000 µg/ml and decreased melanization by Cryptococcus cells. Addition of copper sulfate or gluthatione restored melanogenesis of cells grown in the presence of PPIs. The presence of PPIs and glyphosate decreased copper sulfate toxicity (1 mM). Conclusion: PPIs inhibited melanogenesis of Cryptococcus spp., possibly by chelating copper or inhibiting copper ATPase transport.

Upscale and characterization of lignin-modifying enzymes from Marasmiellus palmivorus VE111 in a bioreactor under parameter optimization and the effect of inducers.

Testing different pHs, dissolved oxygen concentrations and temperatures, plus the addition of inducers, to optimize ligninolytic enzyme activity, resulted in increased production of laccases, total peroxidases and manganese peroxidases on the order of 2.1-fold, 4.6-fold and 10-fold, respectively; laccases reached 6588 U/mL, total peroxidases reached 3533 U/mL and manganese peroxidase achieved 60 U/mL. Furthermore, an increase in laccase volumetric productivity and in its specific activity was verified. The addition of inducers, such as copper sulphate and manganese sulphate, improved enzymatic activity. In addition, a new previously unidentified laccase isoform was documented by zymography. The present work successfully increased the production of ligninolytic enzymes.

The Growth Rate, Immune Status, Duodenal Development, and Cecal Microbial Diversity of 24-Day-Old Offspring of SD Rats Received Bacillus subtilis-Cu or CuSO4 During Pregnancy and Lactation Periods.

A total of 108 pregnant and lactation SD rats were divided into six groups, daily and orally dosed with Bacillus subtilis-copper (CuBs) and CuSO4 (CuS) both at three doses equivalent to 1.5 mg (Cu deficiency), 3 mg, and 6 mg Cu per kg diet, and the effects of the Cu source and dosage on the growth rate, immune status, duodenal development, and cecal microbial diversity were examined on 24-day-old offspring rats. The six offspring rats from each group were randomly selected for measuring the body weight gain and taking blood samples, and three rats were sacrificed for taking duodenum and cecum content samples. We found CuBs increased the body weight gain, development of duodenal villi, and survival rate of the offspring; increased the IgM content and lysozyme activity in serum; reduced the intestinal permeability; and increased the abundances of Lachnospiraceae, Ruminococcaceae, and Intestinibacter in the cecal content, when compared with CuS. We also found that Cu deficiency showed detrimental effects on the body weight gain and length, the survival rate of the offspring, and the immune indices in serum, as well as the increased intestinal permeability. We concluded that CuBs is better Cu source than CuSO4 for reproductive rats.

Copper and zinc sources and levels of zinc inclusion influence growth performance, tissue trace mineral content, and carcass yield of broiler chickens.

A 35-d experiment was conducted in broilers to study the effect of supplementation of sulfate or hydroxychloride forms of Zn and Cu at 2 supplemental Zn levels on growth performance, meat yield, and tissue levels of Zn. On day 0, 900 male Ross 308 broiler chicks (45 ± 1.10 g) were allocated to 4 treatments in a randomized complete block design and 2 × 2 factorial arrangement of treatments. The factors were 2 sources (sulfate or hydroxychloride) of Zn and Cu and 2 levels (low or high) of Zn. The Zn sources were zinc sulfate monohydrate (ZSM) or hydroxychloride Zn. Copper sources were copper (II) sulfate pentahydrate or hydroxychloride Cu. Each of the 4 treatments had 15 replicates and 15 birds per replicate. Birds were weighed on days 0, 21, and 35 for growth performance. On day 35, left tibia bone, liver, and blood were collected from 4 randomly selected birds per pen. In addition, 7 birds per pen were used for carcass evaluation. There was no significant source × level interaction on any of the growth performance response. Broiler chickens receiving hydroxychloride Zn and Cu had greater (P < 0.05) gain: feed, whereas broiler chickens receiving lower Zn level had greater (P < 0.01) weight gain. There was no source × level interaction on meat yield. Broiler chickens receiving hydroxychloride Zn and Cu had greater (P < 0.05) % breast yield than those receiving sulfate Zn and Cu. Higher level of Zn, irrespective of source, produced greater (P < 0.01) tibia and plasma Zn levels, whereas liver Cu was greater (P < 0.05) in broiler chickens receiving hydroxychloride Zn and Cu. It was concluded that hydroxychloride Zn and Cu were more efficacious than sulfate Zn and Cu in promoting growth performance and enhancing meat yield in the current study.

Copper requirements of broiler breeder hens.

One-hundred-twenty Cobb 500 hens, 20 wk of age, were randomly allocated into individual cages with the objective of estimating Cu requirements. After being fed a Cu deficient diet for 4 wk, hens were fed diets with graded increments of supplemental Cu (0.0; 3.5; 7.0; 10.5; 14; and 17.5 ppm) from Cu sulfate (CuSO4 5H2O), totaling 2.67; 5.82; 9.38; 12.92; 16.83; and 20.19 ppm analyzed Cu in feeds for 20 weeks. Estimations of Cu requirements were done using exponential asymptotic (EA), broken line quadratic (BLQ), and quadratic polynomial (QP) models. Obtained Cu requirements for hen d egg production and total settable eggs per hen were 6.2, 7.3, and 12.9 ppm and 8.1, 9.0, and 13.4 ppm, respectively, using EA, BLQ, and QP models. The QP model was the only one having a fit for total eggs per hen with 13.1 ppm Cu as a requirement. Hemoglobin, hematocrit, and serum Cu from hens had requirements estimated as 13.9, 11.3, and 18.5, ppm; 14.6, 13.0, and 19.0 ppm; and 16.2, 14.6, and 14.2 ppm, respectively, for EA, BLQ, and QP models. Hatching chick hemoglobin was not affected by dietary Cu, whereas requirements estimated for hatching chick hematocrit and body weight and length were 10.2, 12.3, and 13.3 ppm using EA, BLQ, and QP models; and 6.8 and 7.1 ppm, and 12.9 and 13.9 ppm Cu using EA and BLQ models, respectively. Maximum responses for egg weight, yolk Cu content, and eggshell membrane thickness were 14.9, 12.7, and 15.1 ppm; 15.0, 16.3, and 15.7 ppm; and 7.3, 7.8, and 14.0 ppm Cu, respectively, for EA, BLQ, and QP models. Yolk and albumen percentage were adjusted only with the QP model and had requirements estimated at 11.0 ppm and 11.3 ppm, respectively, whereas eggshell mammillary layer was maximized with 10.6, 10.1, and 14.4 ppm Cu using EA, BLQ, and QP models, respectively. The average of all Cu requirement estimates obtained in the present study was 12.5 ppm Cu.

Laccase production in bioreactor scale under saline condition by the marine-derived basidiomycete Peniophora sp. CBMAI 1063.

Laccase production in saline conditions is still poorly studied. The aim of the present study was to investigate the production of laccase in two different types of bioreactors by the marine-derived basidiomycete Peniophora sp. CBMAI 1063. The highest laccase activity and productivity were obtained in the Stirred Tank (ST) bioreactor, while the highest biomass concentration in Air-lift (AL) bioreactor. The main laccase produced was purified by ion exchange and size exclusion chromatography and appeared to be monomeric with molecular weight of approximately 55 kDa. The optimum oxidation activity was obtained at pH 5.0. The thermal stability of the enzyme ranged from 30 to 50 °C (120 min). The Far-UV Circular Dichroism revealed the presence of high ß-sheet and low α-helical conformation in the protein structure. Additional experiments carried out in flask scale showed that the marine-derived fungus was able to produce laccase only in the presence of artificial seawater and copper sulfate. Results from the present study confirmed the fungal adaptation to marine conditions and its potential for being used in saline environments and/or processes.

Bimodality of gene expression from yeast promoter can be instigated by DNA context, inducing conditions and strain background.

Bimodality in gene expression is thought to provide a high phenotypic heterogeneity that can be favourable for adaptation or unfavourable notably in industrial processes that require stable and homogeneous properties. Whether this property is produced or suppressed in different conditions has been understudied. Here we identified tens of Saccharomyces cerevisiae genomic fragments conferring bimodal yEGFP expression on centromeric plasmid and studied some of these promoters in different DNA contexts, inducing conditions or strain backgrounds. First, we observed that the bimodal behaviour identified on plasmid is generally suppressed at the genomic level. Second, an inducible promoter such as the copper-regulated CUP1 promoter can produce bimodal expression in a time- and dose-dependent fashion. For a given copper sulphate concentration, a constant proportion of the subpopulation is induced and only the induction level of this subpopulation changed with induction duration, while for a same induction time, higher copper sulphate concentrations induced more cells at higher levels. Third, we showed that bimodality conferred by the CUP1 promoter in expression profile is strain background dependent, revealing epistasis in the generation of bimodality. The influence of these parameters on bimodality has to be taken into account when considering transgene expression for industrial microbial productions.