Cloning and characterization of a novel cry8Ab1 gene from Bacillus thuringiensis strain B-JJX with specific toxicity to scarabaeid (Coleoptera: Scarabaeidae) larvae

Isolation of Bacillus thuringiensis (Bt) strain or its cry gene encoding insecticidal crystal protein (ICP) with specific toxicity is of great importance to biological control of insect pests. In this study, by screening 66 strains of Bt isolated from soil samples collected in Shandong Province, China, a new cry8-type gene from Bt strain B-JJX was identified via PCR-RFLP method. This novel gene, cry8Ab1, was cloned from the Bt strain B-JJX and expressed in an acrystalliferous mutant strain HD-73^?. The open reading frame of the cry8Ab1 gene consists of 3543 bp with a G + C content of 37.99% and encodes a protein of 1180 amino acids with a putative MW of 133.3 kDa which was confirmed by SDS-PAGE analysis. The Cry8Ab1 protein was expressed and released as spherical parasporal crystals from Bt acrystalliferous mutant strain HD-73^? along with the presence of spores. In bioassays, this protein was toxic to 3-day-old larvae of the scarabaeid pests, Holotrichia oblita and H. parallela, with an LC₅₀ of 5.72 and 2.00 μg toxin g^?1 soil, respectively. The results are in accordance with the insecticidal activities of the original Bt strain B-JJX, which had an LC₅₀ of 1.72 and 0.96 μg toxin g^?1 soil against H. oblita and H. parallela, respectively.     

Bioprocess development of the production of the mutant P-219-L human d-amino acid oxidase for high soluble fraction expression in recombinant Escherichia coli

In order to examine the structure–activity relationship and the substrate specificity of human d-amino acid oxidase (h.DAO), a single amino acid mutation had been established as proline-219-luecine (P-219-L). The gene encoding mutant h.DAO has been cloned and expressed in Escherichia coli BL21 (DE3). It was observed that the host cell was negatively affected by the expressed mutant h.DAO, resulting in a remarkable decrease in the cell growth and consequently the amount of the produced enzyme. To overcome this problem, we investigated several factors that may affect the cell growth rate and the mutant h.DAO production such as optimization of the glucose concentration as a main carbon source and the yeast extract concentration as a main nitrogen source, optimization of dissolved oxygen (DO%) concentration and the addition of benzyl alcohol (BA, which can artificially induce a strong heat shock response at low temperature), to enhance the production of natively folded soluble fraction of the recombinant protein. These parameters were tested on both shake flask level and fed-batch bioreactor level. The Western blot analysis and the enzyme activity assay indicated the higher level of the mutant expression towards enhancement of the conditions by using our designed approach.The specific activity (which was used as an indicator for the level of the desired protein produced = U/mg protein) and the OD_600 nm of the host cells (which was used as an indicator for the cell growth), reached to be 0.061 U/mg protein and 3.44, respectively upon using fed-batch culture system containing the optimized medium composition (15 g/l glucose and 5 g/l yeast extract). While upon using the shake flask level, these values were 0.032 and 1.1, respectively. Enhancement of the cell growth and the enzyme production was noticed after DO% optimization upon using 500 rpm agitation speed and 1.8 v.v.m. (volume volume minute) aeration. The specific activity for the mutant enzyme and the OD_600 nm of the host cells reached to be 0.14 U/mg protein and 7.1, respectively. Finally upon using the optimized culture composition (15 g/l glucose and 5 g/l yeast extract), optimized DO% (using 500 rpm agitation speed and 1.8 v.v.m.) and 0.1 mM BA at the fed-batch bioreactor level, the specific activity and the OD_600 nm of the host cells increased significantly to be 0.21 U/mg protein and 11.3, respectively at 24 h culture. These results indicate the importance of our approaches to overproducing mutant h.DAO in soluble form in E. coli.     

Site-directed mutagenesis of an Aspergillus niger xylanase B and its expression,purification and enzymatic characterization in Pichia pastoris

Xylanase is an important industrial enzyme. In this research, to improve the thermostability and biochemical properties of a xylanase from Aspergillus niger F19, five arginine substitutions and a disulfide bond were introduced by site-directed mutagenesis. The wild-type gene xylB and the mutant gene xylCX8 were expressed in Pichia pastoris. Compare to those of the wild-type enzyme, the optimal reaction temperature for the mutant enzyme increased from 45 °C to 50 °C, the half-life of the mutant enzyme extended from 10 min to 180 min, and the specific activity increased from 2127 U/mg to 3330 U/mg. However, the V_max and K_m of the mutant xylanase decreased. The enzyme activity in broth obtained from shake flask cultures could be induced to 1850 U/mL in 7 days, which is higher than results reported previously. Furthermore, the highest achievable enzyme activity was 7340 U/mL from 140 g/L of biomass in a 3 L fermentor used in our study.     

Hyperuricemia influences tryptophan metabolism via inhibition of multidrug resistance protein 4 (MRP4) and breast cancer resistance protein (BCRP)

Hyperuricemia is related to a variety of pathologies, including chronic kidney disease (CKD). However, the pathophysiological mechanisms underlying disease development are not yet fully elucidated. Here, we studied the effect of hyperuricemia on tryptophan metabolism and the potential role herein of two important uric acid efflux transporters, multidrug resistance protein 4 (MRP4) and breast cancer resistance protein (BCRP). Hyperuricemia was induced in mice by treatment with the uricase inhibitor oxonic acid, confirmed by the presence of urate crystals in the urine of treated animals. A transport assay, using membrane vesicles of cells overexpressing the transporters, revealed that uric acid inhibited substrate-specific transport by BCRP at clinically relevant concentrations (calculated IC₅₀ value: 365 ± 13 μM), as was previously reported for MRP4. Moreover, we identified kynurenic acid as a novel substrate for MRP4 and BCRP. This finding was corroborated by increased plasma levels of kynurenic acid observed in Mrp4^?/? (107 ± 19 nM; P = 0.145) and Bcrp^?/? mice (133 ± 10 nM; P = 0.0007) compared to wild type animals (71 ± 11 nM). Hyperuricemia was associated with > 1.5 fold increase in plasma kynurenine levels in all strains. Moreover, hyperuricemia led to elevated plasma kynurenic acid levels (128 ± 13 nM, P = 0.005) in wild type mice but did not further increase kynurenic acid levels in knockout mice. Based on our results, we postulate that elevated uric acid levels hamper MRP4 and BCRP functioning, thereby promoting the retention of other potentially toxic substrates, including kynurenic acid, which could contribute to the development of CKD.     

Complete nucleotide sequence and organization of the mitochondrial genome of eri-silkworm,Samia cynthia ricini (Lepidoptera: Saturniidae)

Samia cynthia ricini is a commercial silk-producing insect that is now reared year-round in Korea, with the expectation of being utilized for diverse purposes. In this report, we present the complete mitochondrial genome (mitogenome) of S. c. ricini. The 15,384-bp long S. cynthia ricini mitogenome was amplified into 26 short fragments using three long overlapping fragments using primers designed from reported lepidopteran mitogenome sequences. The genome comprises 37 genes (13 protein-coding genes, two rRNA genes, and 22 tRNA genes), and one large non-coding region termed the A + T-rich region. The A/T content of the third codon position was 91.7%, which was 18.8% and 21.6% higher than those of first and second codon positions, respectively. The high A/T content in the genome is reflected in codon usage, accounting for 39.5% of A/T-composed codons (TTA, ATT, TTT, and ATA). Unlike a previous report on the start codon for the COI gene, the S. c. ricini COI gene commences with a typical ATT codon. A total of 221 bp of non-coding sequences are dispersed in 17 regions, ranging in size from 1 to 54 bp, which comprise 1.4% of the total genome. One of the non-coding sequence located between tRNA^Gln and ND2 (54 bp) has 77% sequence homology with the 5′-sequence of the neighboring ND2 gene, suggesting partial duplication of the sequence during evolution. The 361-bp long A + T-rich region contains an 18 bp-long poly-T stretch, ATAGA motif, ATTTA element, microsatellite-like A/T sequence, poly-A stretch and one tRNA-like sequence, as typically found in Lepidoptera including Bombycoidea.     

The Annona muricata leaf ethanol extract affects mobility and reproduction in mutant strain NB327 Caenorhabditis elegans

The C. elegans NB327 mutant strain is characterized for the knockdown of the dic-1 gene. The dic-1 gene is homologous to the dice-1 gene in humans, encoding the protein DICE-1 as a tumor suppressor. Absence or under-regulation of the dice-1 gene can be reflected in lung and prostate cancer [17], [18]. This study evaluated the effect of EEAML on the C. elegans NB327 mutant strain. Phenotypic aspects such as morphology, body length, locomotion, and reproductive behaviour were analyzed. It is important to emphasize that the strain presents a phenotype characteristic with respect to egg laying and hatching. Reported studies showed that Annona muricata extract and its active components evidence anti-cancer and anti-tumor effects, through experimentation in vivo and in vitro models. However, neurotoxicity has been reported as a side effect. The results showed that the mutant strain NB327 was exposed to EEAML (5 mg/ml) concentration, it showed a significant decrease in average locomotion, resulting in 13 undulations in 30 s. This contrasts with the control strain's 17.5 undulations in 30 s. Similarly, the number of progenies was reduced from 188 progenies (control strain) to 114 and 92 progenies at the dose of (1 mg/ml and 5 mg/m) EEAML. The results of this study suggest that EEAML has a possible neurotoxic effect in concentrations equal to or greater than 5 mg/ml. Also, it does not have positive effects on the mutant strain of Caenorhabditis elegans NB327 phenotype.     

2-DE based proteomic analysis of Saccharomyces cerevisiae wild and K+ transport-affected mutant (trk1,2) strains at the growth exponential and stationary phases

By using a 2-DE based workflow, the proteome of wild and potassium transport mutant trk1,2 under optimal growth potassium concentration (50 mM) has been analyzed. At the exponential and stationary phases, both strains showed similar growth, morphology potassium content, and Vmax of rubidium transport, the only difference found being the Km values for this potassium analogue transport, higher for the mutant (20 mM) than for the wild (3–6 mM) cells.Proteins were buffer-extracted, precipitated, solubilized, quantified, and subjected to 2-DE analysis in the 5–8 pH range. More differences in protein content (37–64 mg g^? 1 cell dry weight) and number of resolved spots (178–307) were found between growth phases than between strains. In all, 164 spots showed no differences between samples and a total of 105 were considered to be differential after ANOVA test. 171 proteins, corresponding to 71 unique gene products have been identified, this set being dominated by cytosolic species and glycolitic enzymes. The ranking of the more abundant spots revealed no differences between samples and indicated fermentative metabolism, and active cell wall biosynthesis, redox homeostasis, biosynthesis of amino acids, coenzymes, nucleotides, and RNA, and protein turnover, apart from cell division and growth. PCA analysis allowed the separation of growth phases (PC1 and 2) and strains at the stationary phase (PC3 and 4), but not at the exponential one. These results are also supported by clustering analysis. As a general tendency, a number of spots newly appeared at the stationary phase in wild type, and to a lesser extent, in the mutant. These up-accumulated spots corresponded to glycolitic enzymes, indicating a more active glucose catabolism, accompanied by an accumulation of methylglyoxal detoxification, and redox-homeostasis enzymes. Also, more extensive proteolysis was observed at the stationary phase with this resulting in an accumulation of low Mr protein species.     

Double-strand break repair and genetic recombination in topoisomerase and primase mutants of bacteriophage T4

The effects of primase and topoisomerase II deficiency on the double-strand break (DSB) repair and genetic recombination in bacteriophage T4 were studied in vivo using focused recombination. Site-specific DSBs were induced by SegC endonuclease in the rIIB gene of one of the parents. The frequency/distance relationship was determined in crosses of the wild-type phage, topoisomerase II mutant amN116 (gene 39), and primase mutant E219 (gene 61). Ordinary two-factor (i × j) and three-factor (i k × j) crosses between point rII mutations were also performed. These data provide information about the frequency and distance distribution of the single-exchange (splice) and double-exchange (patch) events. In two-factor crosses ets1 × i, the topoisomerase and primase mutants had similar recombinant frequencies in crosses at ets1–i distances longer than 1000 bp, comprising about 80% of the corresponding wild-type values. They, however, differ remarkably in crosses at shorter distances. In the primase mutant, the recombinant frequencies are similar to those in the wild-type crosses at distances less than 100 bp, being a bit diminished at longer distances. In two-factor crosses ets1 × i of the topoisomerase mutant, the recombinant frequencies were reduced ten-fold at the shortest distances. In three-factor crosses a6 ets1 × i, where we measure patch-related recombination, the primase mutant was quite proficient across the entire range of distances. The topoisomerase mutant crosses demonstrated virtually complete absence of rII⁺ recombinants at distances up to 33 bp, with the frequencies increasing steadily at longer distances. The data were interpreted as follows. The primase mutant is fully recombination-proficient. An obvious difference from the wild-type state is some shortage of EndoVII function leading to prolonged existence of HJs and thus stretched out ds-branch migration. This is also true for the topoisomerase mutant. However, the latter is deficient in the ss-branch migration step of the DSB repair pathway and partially deficient in HJ initiation. In apparent contradiction to their effects on the DSB-induced site-specific recombination, the topoisomerase and primase mutants demonstrated about 3–8-fold increase in the recombinant frequencies in the ordinary crosses, with the recombination running exclusively via patches. This implies that most of the spontaneous recombination events are not initiated by dsDNA ends in these mutants.     

Rapid linkage of indole carboxylic acid to the plant cell wall identified as a component of basal defence in Arabidopsis against hrp mutant bacteria

Changes occurring to plant cell walls were examined following inoculation of Arabidopsis leaves with pathogenic and non-pathogenic (hrpA mutant) strains of Pseudomonas syringae pv. tomato. We have targeted low molecular weight, cross-linked phenolic and indolic compounds that were released from wall preparations by alkaline hydrolysis at 70 °C and in a microwave bomb. Significantly higher concentrations of syringaldehyde, p hydroxybenzaldehyde and indole carboxylic acid were recovered from cell walls isolated from leaves 24 h after challenge with the hrpA mutant compared with wild-type DC3000. Time course experiments showed that the accumulation of indole carboxylic acid and the other group of differentiating metabolites had occurred within 12 h of inoculation. The callose synthase deficient mutant pmr4-1 was more resistant than wild-type Columbia plants to P. syringae pv. tomato. Restricted bacterial multiplication was associated with increased accumulation of indole carboxylic acid on the plant cell wall. In the absence of callose deposition in the pmr 4-1 mutant, indolic derivatives may serve as a structural scaffold for wall modifications following bacterial challenge.     

Evaluating controllability of pharmaceuticals and metabolites in biologically engineered processes,using corresponding octanol–water distribution coefficient

The efficiency of removing 9 different pharmaceuticals, 5 carbamazepine metabolites, and 1 personal care product through wastewater treatment plants and constructed wetlands was investigated. The compound concentrations were measured using solid phase extraction followed by liquid chromatography quadrupole tandem mass spectrometry. For extraction confirmation and better accuracy, isotopic dilution and standards addition methods were employed. The reporting limits for the investigated compounds were less than 10 ng/L except TCEP (24 ng/L). The removal efficiencies were found to be inversely proportional to the octanol–water partition coefficients (log K_ow) after modification with ionizable functional groups (log D_ow); compounds with a higher hydrophilicity were more efficiently removed in the engineered processes through biological treatment mechanisms. Carbamazepine metabolites that were formed in the early stages of certain metabolic reactions exhibited enhanced removal efficiencies due to a decreased log D_ow values. However, the removal efficiency of those formed in later stages did not increase, but rather fluctuated with large standard deviations. The removal behaviors of metabolites in biologically operating engineered systems need to be more extensively examined.     

Tolerance,accumulation and distribution of zinc and cadmium in hyperaccumulator Potentilla griffithii

In this study, zinc (Zn) and cadmium (Cd) tolerance, accumulation and distribution was conducted in Potentilla griffithii H., which has been identified as a new Zn hyperaccumulator found in China. Plants were grown hydroponically with different levels of Zn²⁺ (20, 40, 80 and 160 mg L^?1) and Cd²⁺ (5, 10, 20 and 40 mg L^?1) for 60 days. All plants grew healthy and attained more biomass than the control, except 40 mg L^?1 Cd treatment. Zn or Cd concentration in plants increased steadily with the increasing addition of Zn or Cd in solution. The maximum metal concentrations in roots, petioles and leaves were 14,060, 19,600 and 11,400 mg kg^?1 Zn dry weight (DW) at 160 mg L^?1 Zn treatment, and 9098, 3077 and 852 mg kg^?1 Cd DW at 40 mg L^?1 Cd treatment, respectively. These results suggest that P. griffithii has a high ability to tolerate and accumulate Cd and Zn, and it can be considered not only as Zn but also as a potential cadmium hyperaccumulator. Light microscope (LM) with histochemical method, scanning electron microscope combined with energy dispersive spectrometry (SEM-EDS) and transmission electron microscope (TEM) were used to determine the distribution of Zn and Cd in P. griffithii at tissue and cellular levels. In roots, SEM-EDS confirmed that the highest Zn concentration was found in xylem parenchyma cells and epidermal cells, while for Cd, a gradient was observed with the highest Cd concentration in rhizodermal and cortex cells, followed by central cylinder. LM results showed that Zn and Cd distributed mainly along the walls of epidermis, cortex, endodermis and some xylem parenchyma. In leaves, Zn and Cd shared the similar distribution pattern, and both were mostly accumulated in epidermis and bundle sheath. However, in leaves of 40 mg L^?1 Cd treatment, which caused the phytotoxicity, Cd was also found in the mesophyll cells. The major storage site for Zn and Cd in leaves of P. griffithii was vacuoles, to a lesser extent cell wall or cytosol. The present study demonstrates that the predominant sequestration of Zn and Cd in cell walls of roots and in vacuoles of epidermis and bundle sheath of leaves may play a major role in strong tolerance and hyperaccumulation of Zn and Cd in P. griffithii.     

Characterization and PCR applications of dUTPase from the hyperthermophilic euryarchaeon Thermococcus pacificus

We cloned and sequenced the gene encoding Thermococcus pacificus dUTPase (Tpa dUTPase). The Tpa dUTPase gene consists of 471 bp and encodes a 156-amino acid protein. The deduced amino acid sequence of Tpa dUTPase has high sequence similarity with other archaeal dUTPases. The Tpa dUTPase had an 18-kDa major protein band consistent with the 17,801 Da molecular mass calculated based on the amino acid sequence. The specific activity of Tpa dUTPase on dUTP at 85 °C was 90,909 U/mg. For Tpa dUTPase activity, we determined an optimum pH of 8.5 and temperature of 85 °C. Magnesium ions strongly induced activity, with an optimum concentration of 0.75 mM. The half-life of the enzyme at 94 °C was about 7 h. The specific activity of the Tpa dUTPase on dUTP was about 10–20-fold higher than that of Tpa dUTPase on dCTP. Tpa dUTPase enhanced the PCR amplification efficiency of long targets when Pfu and Vent DNA polymerases were used.     

Association analysis of single nucleotide polymorphisms of proinflammatory cytokine and their receptors genes with rheumatoid arthritis in northwest Chinese Han population

ObjectiveTo analyze the relationship of genetic polymorphisms in IL1β, IL6, TNF-α genes and their receptors genes with rheumatoid arthritis (RA) for northwest Han Chinese. This study also explores whether there are gene–gene interactions among these genetic polymorphisms.MethodsA total of 452 patients with RA and 373 matched healthy controls were enrolled to carry out a case-control study for 16 SNPs of IL1B-511 C > T, IL1B-31 T > C, IL1B+3954 C > T, IL1RN T > C, IL6-597 G > A, IL6-572 G > C, IL6-174 G > C, IL6R-183 G > A, IL6R exon2 T > A, IL6R exon1 A > C, TNFA-863 C > A, TNFA-857 C > T, TNFA-308 G > A, TNFA-238 G > A, TNFR1-383 A > C and TNFR2 T676G T > G from seven genes. Genotyping for the SNPs was conducted on the RotorGene 6000 PCR platform using in-house high resolution melting (HRM) approaches. Detection correctness was validated through direct sequencing. Generalized multifactor dimensionality reduction (GMDR) analysis was applied to discover likely gene–gene interaction model among the SNPs.ResultsThe results showed that the genotype distributions of TNFA-308, TNFA-857 and TNFA-863 are significantly different between case and control groups (P = 0.016, P = 0.048 and P = 0.016, respectively). Carriers of TNFA-857 mutant allele conferred risk to RA (OR = 1.525, 95% CI = 1.157–2.009) while those of TNFA-308 and TNFA-863 mutant alleles conferred protection to RA (OR = 0.459, 95% CI = 0.286–0.739; OR = 0.490, 95% CI = 0.329–0.732). GMDR analysis for the SNPs indicated that gene–gene interaction existed among IL1B-31, IL1RN, IL6-572, IL6R-183, IL6R-exon1 and TNFA-857. Thirteen of all genotypes of the six SNPs combination were discovered to have significant distribution difference between RA group and the control.ConclusionsThis study demonstrated that PCR-HRM assay is a highly efficient SNP genotyping method especially for the detection of large-scale samples. The SNPs of TNFA-308 and TNFA-863 are closely associated with RA susceptibility and that gene–gene interactions may occur among the six SNPs of IL1B-31, IL1RN, IL6-572, IL6R-183, IL6R-exon1 and TNFA-857 in RA patients from northwest Chinese Han population, especially these SNPs’ combination genotypes CT/TT/CC/GG/AC/CC, CT/TT/GC/AA/AC/CT and CT/CT/CC/GA/AC/CC to show high risk of RA susceptibility in our study.     

Polyethylene terephthalate membrane as a support for covalent immobilization of uricase and its application in serum urate determination

A method is described for covalent immobilization of uricase onto polyethylene terephthalate (PET) membrane with a conjugation yield of 4.44 μg/cm² and 66.6% retention of initial activity of free enzyme. The enzyme exhibited an increase in optimum pH from pH 7.0 to 8.5 and K_m for uric acid from 0.075 mM to 0.13 mM but slight decrease in temp. for maximum activity from 37 °C to 35 °C after immobilization. A colorimetric method for determination of serum uric acid was developed using immobilized uricase, which is based on measurement of H₂O₂ by a color reaction consisting of 3,5-dichlorobenzene sulphonic acid (DHBS), 4-aminoantipyrine and peroxidase as chromogenic system. Minimum detection limit of the method was 0.05 mM. Analytical recovery of added uric acid (5 mg/dl and 10 mg/dl) was 94.3% and 89.8%, respectively. Within and between batch coefficient of variation (CV) were <3.2% and <4.3%, respectively. A good correlation (r = 0.98) was found between uric acid values by standard enzymic colorimetric method and the present method. The immobilized uricase was reused 100 times during the span of 60 days without any considerable loss of activity, when stored in reaction buffer at 4 °C. The support chosen for the present study was biocompatible, antimicrobial, inert, impact resistant, light weight and had good shelf life.     

The association of IL-8-251T/A polymorphism with complicated malaria in Karbi Anglong district of Assam

Amongst host genetic factors, cytokine gene polymorphism can be anticipated to be an important factor as qualitative, quantitative and time of secretion play an important role in disease outcome. We have investigated association of cytokine promoter SNPs with risk of Plasmodium falciparum malaria and disease severity in a case control study in malaria endemic Karbi Anglong district of Assam, India.Frequency of IL-8-251T/A (p = 0.03 and p = 0.01) and TGF-β1-509C/T (p = 0.02 and p = 0.03) was higher in malaria in comparison to control participants and non-malarial fever controls. Interestingly, a higher frequency of mutant allele of IL-10-819T/C was observed in non-malarial fever controls compared to malaria thus suggesting its role as a distinguishing marker of the two disease groups. Higher IL-8 expression and increased frequency of IL-8-251T/A in complicated malaria (p = 0.002) was reported indicating its role in susceptibility to complicated malaria.In conclusion, our study suggests the role of mutant genotype of IL-8-251T/A as a marker of complicated malaria in our population. Surprisingly, decreased expression of TGF-β1 in uncomplicated malaria even in presence of high expressing mutant genotype was observed and needs to be investigated in context of the pool of activated cells producing the cytokine.     

Putative P1B-type ATPase from the bacterium Achromobacter xylosoxidans A8 alters Pb2 +/Zn2 +/Cd2 +-resistance and accumulation in Saccharomyces cerevisiae

PbtA, a putative P_1B-type ATPase from the Gram-negative soil bacterium Achromobacter xylosoxidans A8 responsible for Pb^2 +/Zn^2 +/Cd^2 +-resistance in Escherichia coli, was heterologously expressed in Saccharomyces cerevisiae. When present in Zn^2 +- and Pb^2 +/Cd^2 +-hypersensitive S. cerevisiae strains CM137 and DTY168, respectively, PbtA was able to restore Zn^2 +- and Pb^2 +-resistant phenotype. At the same time, the increase of Pb, Zn, and Cd accumulation in yeast was observed. However, Cd^2 +-tolerance of the pbtA-bearing yeasts dramatically decreased. The PbtA-eGFP fusion protein was localized primarily in the tonoplast and also in the plasma membrane and the perinuclear region corresponding to the endoplasmic reticulum at later growth stages. This indicates that PbtA protein is successfully incorporated into membranes in yeasts. Since PbtA caused a substantial increase of Pb^2 +/Zn^2 +-resistance and accumulation in baker's yeast, we propose its further use for the genetic modification of suitable plant species in order to obtain an effective tool for the phytoremediation of sites polluted by toxic transition metals.     

Effects of ambient temperature and methionine supplementation of a low protein diet on the performance of laying hens

The effects of ambient temperature and the supplementation of methionine to a low protein diet on egg production, egg quality, blood constituents and nitrogen excretion of laying hens were studied. The objective was to derive an environmental friendly feed formulation for warm climate. Seventy-two 29-week-old commercial White Leghorn hens of Babcock ISA white strain were used in this trial. The design is a completely randomized design with a 2×3 factors arrangement of treatments. Two constant ambient temperatures were 24±1°C and 34±1°C with 85% relative humidity. The three dietary treatments were 170 g kg⁻¹ crude protein, 140 g kg⁻¹ crude protein and 140 g kg⁻¹ crude protein supplemented with methionine 1.4 g kg⁻¹. Hens were allotted into six groups according to egg production and body weight. Birds were raised in individual wire cages for the experimental feeding period of five weeks. At the end of the feeding trial, one replicate of laying hens (four birds) from each treatment were selected for a four-day metabolic study for the daily collection of the excreta. The blood samples were withdrawn from the wing vein for analysis of hematocrit, blood glucose, sodium, potassium, magnesium, calcium, phosphorus and uric acid. Experimental results indicate that increases in ambient temperature significantly depress feed intake, egg production, egg weight and live weight of laying hens. High ambient temperature also caused inferior egg quality, including shell weight, shell thickness, shell breaking strength and specific gravity. Ambient temperature also changes the egg components with heavier egg albumin and yolk in the low-temperature group. Increasing ambient temperature also caused an increase in pH value in the plasma. This increase revealed a trend of depressed glucose (P<0.05) in the plasma of the laying hens. The dietary treatments, however, did not significantly influence feed intakes. Except egg weight, laying hens that were fed with the low protein (140 g kg⁻¹) with methionine supplemented diet produced similar numbers of egg and feed conversion as the layers fed with the high protein diet. The low protein with methionine supplemented diet produced significantly lighter eggs than the high dietary protein diet under the high ambient temperature, but produced heavier egg under the low ambient temperature. The concentration of uric acid in the plasma and nitrogen in the excreta of the high protein group was significantly higher than the other two low protein dietary groups (P<0.05).     

A multiple free-radical scavenging (MULTIS) study on the antioxidant capacity of a neuroprotective drug,edaravone as compared with uric acid,glutathione, and trolox

Edaravone (3-methyl-1-phenyl-2-pyrazoline-5-one) is a neuroprotective drug that has been used for brain ischemia injury treatment. Because its activity is speculated to be due to free radical scavenging activity, we carried out a quantitative determination of edaravone’s free radical scavenging activity against multiple free radical species. Electron spin resonance (ESR) spin trapping-based multiple free-radical scavenging (MULTIS) method was employed, where target free radicals were hydroxyl radical, superoxide anion, alkoxyl radical, alkylperoxyl radical, methyl radical, and singlet oxygen. Edaravone showed relatively high scavenging abilities against hydroxyl radical (scavenging rate constant k = 2.98 × 10¹¹ M⁻¹ s⁻¹), singlet oxygen (k = 2.75 × 10⁷ M⁻¹ s⁻¹), and methyl radical (k = 3.00 × 10⁷ M⁻¹ s⁻¹). Overall, edaravone’s scavenging activity against multiple free radical species is as robust as other known potent antioxidant such as uric acid, glutathione, and trolox. A radar chart illustration of the MULTIS activity relative to uric acid, glutathione, and trolox indicates that edaravone has a high and balanced antioxidant activity with low specificity.