Insights into the binding interaction of substrate with catechol 2,3-dioxygenase from biophysics point of view.

This study aims to clarify the interaction mechanism of substrate with catechol 2,3-dioxygenase (C23O) through multi-technique combination. A novel C23O (named C23O-2G) was cloned, heterogeneously expressed, and identified as a new member in subfamily I.2 of extradiol dioxygenases. Based on the simulations of molecular docking and dynamics, the exact binding sites of catechol on C23O-2G were identified, and the catalytic mechanism mediated by key residues was proposed. The roles of the predicted residues during catalysis were confirmed by site-directed mutagenesis, and the mutation of Thr254 could significantly increase catalytic efficiency and substrate specificity of C23O-2G. The binding and thermodynamic parameters obtained from fluorescence spectra suggested that catechol could effectively quench the intrinsic fluorescence of C23O-2G via static and dynamic quenching mechanisms and spontaneously formed C23O-2G/catechol complex by the binding forces of hydrogen bond and van der Waals force. The results of UV-vis spectra, synchronous fluorescence, and CD spectra revealed obvious changes in the microenvironment and conformation of C23O-2G, especially for the secondary structure. The atomic force microscope images further demonstrated the changes from an appearance point of view. This study could improve our mechanistic understanding of representative dioxygenases involved in aromatic compound degradation.

An explanation of soil amendments to reduce cadmium phytoavailability and transfer to food chain.

Cadmium contamination in soil, water and food has become a global problem since last century's industrial and agricultural revolution. It is a highly toxic metal with serious consequences on human and animal health. Different natural and anthropogenic sources are responsible for Cd release in the soil which ultimately leads to the food chain. Cd persists in soil for long durations due to its minimal microbial or chemical loss. There are various physical, chemical or biological techniques which are helpful to minimize Cd risk in food chain. Among them, in-situ immobilization with organic, inorganic or clay amendments is a cost-effective and an environment friendly strategy to remediate Cd polluted sites. Lime, biochar, organic wastes, phosphorus fertilizers, sepiolite, zeolite, hydroxyapatite and bentonite are commonly used amendments for amelioration of Cd contaminated soils. These amendments reduce Cd uptake and enhance immobilization by adsorption, complexation, and precipitation processes. This review is aimed to provide a comprehensive note on Cd toxicity in humans and environment, its immobilization by different agents through variety of processes, and comparison of technologies for Cd removal from contaminated sites.

Comparative efficacy of organic and inorganic amendments for cadmium and lead immobilization in contaminated soil under rice-wheat cropping system.

Field experiments were conducted in two consecutive rice-wheat cropping seasons on a yellow clay soil to assess the efficacy of organic and inorganic amendments for cadmium (Cd) and lead (Pb) immobilization. Amendments were applied alone and in combinations to compare their efficacy for metals immobilization. Composite amendment of GSA-4 (Green Stabilizing Agent) and biochar resulted in higher biomass and grains yield for both rice (Oryza sativa L.) and wheat (Triticum aestivum L.). Liming, DEK1 (Di Kang No. 1) or GSA-4 amendment increased soil pH from 6.34 to 7.35, 7.20 and 7.15, respectively. Soil amendments significantly reduced DTPA extractable Cd and Pb in soil at wheat harvest. Cadmium and Pb fractionation showed a significant decrease in the extractable fractions by the amendment of biochar (34% and 25%) or GSA-4 (35% and 26%, respectively). GSA-4 and biochar amendment enhanced metals immobilization and reduced their uptake by plant and subsequent accumulation in the grains of rice and wheat, particularly with GSA-4. These results indicate that GSA-4 and biochar, especially their combination, have great potential for application to remediate Cd and Pb contaminated soils.

Effect of lead on plant availability of phosphorus and potassium in a vegetable-soil system.

Two typical red soils were sequentially cultivated with celery (Apium graveolens L.) and Chinese cabbage (Brassica chinensis L.) in a greenhouse to determine the effect of lead (Pb) on plant availability of phosphorus (P) and potassium (K) in the soils. The concentrations of available P as estimated by the 0.05 mol L-1 HCl-0.025 mol L-1 (1/2 H2SO4) extraction and available K estimated by the NH4OAc extraction method in the crop-free soils were not affected by Pb treatment. Plant P concentrations in the above-ground part of celery and Chinese cabbage exposed to Pb were either lower or showed no significant difference to the control.

Functional genomic analysis of phthalate acid ester (PAE) catabolism genes in the versatile PAE-mineralising bacterium Rhodococcus sp. 2G.

Microbial degradation is considered the most promising method for removing phthalate acid esters (PAEs) from polluted environments; however, a comprehensive genomic understanding of the entire PAE catabolic process is still lacking. In this study, the repertoire of PAE catabolism genes in the metabolically versatile bacterium Rhodococcus sp. 2G was examined using genomic, metabolic, and bioinformatic analyses. A total of 4930 coding genes were identified from the 5.6 Mb genome of the 2G strain, including 337 esterase/hydrolase genes and 48 transferase and decarboxylase genes that were involved in hydrolysing PAEs into phthalate acid (PA) and decarboxylating PA into benzoic acid (BA). One gene cluster (xyl) responsible for transforming BA into catechol and two catechol-catabolism gene clusters controlling the ortho (cat) and meta (xyl &mhp) cleavage pathways were also identified. The proposed PAE catabolism pathway and some key degradation genes were validated by intermediate-utilising tests and real-time quantitative polymerase chain reaction. Our results provide novel insight into the mechanisms of PAE biodegradation at the molecular level and useful information on gene resources for future studies.

Variations in cadmium and nitrate co-accumulation among water spinach genotypes and implications for screening safe genotypes for human consumption.

Vegetables are important constituents of the human diet. Heavy metals and nitrate are among the major contaminants of vegetables. Consumption of vegetables and fruits with accumulated heavy metals and nitrate has the potential to damage different body organs leading to unwanted effects. Breeding vegetables with low heavy metal and nitrate contaminants is a cost-effective approach. We investigated 38 water spinach genotypes for low Cd and nitrate co-accumulation. Four genotypes, i.e. JXDY, GZQL, XGDB, and B888, were found to have low co-accumulation of Cd (<0.71 mg/kg dry weight) and nitrate (<3100 mg/kg fresh weight) in the edible parts when grown in soils with moderate contamination of both Cd (1.10 mg/kg) and nitrate (235.2 mg/kg). These genotypes should be appropriate with minimized risk to humans who consume them. The Cd levels in the edible parts of water spinach were positively correlated with the concentration of Pb or Zn, but Cd, Pb, or Zn was negatively correlated with P concentration. These results indicate that these three heavy metals may be absorbed into the plant in similar proportions or in combination, minimizing the influx to aerial parts. Increasing P fertilizer application rates appears to prevent heavy metal and nitrate translocation to shoot tissues and the edible parts of water spinach on co-contaminated soils.

MACF1 knockdown in glioblastoma multiforme cells increases temozolomide-induced cytotoxicity / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the role of microtubule-actin crosslinking factor 1 (MACF1) in the response of glioma cells to temozolomide (TMZ).</p><p><b>METHODS</b>TMZ was applied to a human gliomablastoma cell line (U87) and changes in the protein expression and cellular localization were determined with Western blot, RT-PCR, and immunofluorescence. The responses of the cells with MACF1 expression knockdown by RNA interference to TMZ were assessed. TMZ-induced effects on MACF1 expression were also assessed by immunohistochemistry in a nude mouse model bearing human glioblastoma xenografts.</p><p><b>RESULTS</b>TMZ resulted in significantly increased MACF1 expression (by about 2 folds) and changes in its localization in the gliomablastoma cells both in vitro and in vivo (P<0.01). Knockdown of MACF1 reduced the proliferation (by 45%) of human glioma cell lines treated with TMZ (P<0.01). TMZ-induced changes in MACF1 expression was accompanied by cytoskeletal rearrangement.</p><p><b>CONCLUSION</b>MACF1 may be a potential therapeutic target for glioblastoma.</p>

RNA interference of PC4 and SFRS1 interacting protein 1 inhibits invasion and migration of U87 glioma cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effect of small interfering RNA (siRNA)-mediated silencing of PC4 and SFRS1 interacting protein 1 (PSIP1) on invasion and migration of human glioma U87 cells.</p><p><b>METHODS</b>Chemically synthesized siRNA targeting PSIP1 gene was transfected into U87 cells via lipofectamine, and the gene silencing effect was determined using real-time PCR. The changes in the invasion and migration abilities of the transfected cells were assessed with Transwell assay and wound healing assay, respectively. Western blotting was used to analyze the expression of N-cadherin, β-catenin and the transcription factor Slug.</p><p><b>RESULTS</b>The mRNA and protein level of PSIP1 was significantly reduced in U87 cells after transfection with PSIP1 siRNA (P<0.0001). PSIP1 knockdown in U87 cells resulted in significant suppression of cell invasion and migration abilities (P<0.01) and also reduced N-cadherin, β-catenin and Slug expressions.</p><p><b>CONCLUSION</b>s Silencing of PSIP1 impairs the invasion and migration abilities of glioma cells and lowers the expressions of N-cadherin, β-catenin and Slug, suggesting that PSIP1 may regulate Slug by classical Wnt/β-catenin signaling pathway to modulate epithelial-mesenchymal transition and promote the invasion and migration of glioma cells.</p>

Sorption of ammonium and phosphate from aqueous solution by biochar derived from phytoremediation plants.

The study on biochar derived from plant biomass for environmental applications is attracting more and more attention. Twelve sets of biochar were obtained by treating four phytoremediation plants, Salix rosthornii Seemen, Thalia dealbata, Vetiveria zizanioides, and Phragmites sp., sequentially through pyrolysis at 500 °C in a N2 environment, and under different temperatures (500, 600, and 700 °C) in a CO2 environment. The cation exchange capacity and specific surface area of biochar varied with both plant species and pyrolysis temperature. The magnesium (Mg) content of biochar derived from T. dealbata (TC) was obviously higher than that of the other plant biochars. This biochar also had the highest sorption capacity for phosphate and ammonium. In terms of biomass yields, adsorption capacity, and energy cost, T. dealbata biochar produced at 600 °C (TC600) is the most promising sorbent for removing contaminants (N and P) from aqueous solution. Therefore, T. dealbata appears to be the best candidate for phytoremediation application as its biomass can make a good biochar for environmental cleaning.

Differential iron-bioavailability with relation to nutrient compositions in polished rice among selected Chinese genotypes using Caco-2 cell culture model.

Genotypic variation of iron bioavailability and the relationship between iron bioavailability and nutrient composition in polished rice among 11 rice genotypes were assessed using an in vitro digestion/Caco-2 cell model. The results indicated that significant differences in iron bioavailability were detected among tested rice genotypes, with a 3-fold range, suggesting a possibility of selecting high bioavailable iron by plant breeding. Although iron bioavailability was not significantly correlated with Fe concentration in polished rice among tested rice genotypes, the results also indicated that most of the iron dense genotypes showed relatively high ferritin formation in Caco-2 cell and transported iron. Additionally, iron bioavailability in polished rice was enhanced by addition of ascorbic acid, with a much wider range of Fe bioavailability variation in polished rice with addition of ascorbic acid than that without addition of ascorbic acid. The positive relationship between iron bioavailability in polished rice and cysteine concentration (R = 0.669) or sulfur (S) concentration (R = 0.744) among tested rice genotypes, suggests that cysteine and sulfur concentration in polished rice could be used as an indicator for high iron bioavailability.

Bioremediation of Cd-DDT co-contaminated soil using the Cd-hyperaccumulator Sedum alfredii and DDT-degrading microbes.

The development of an integrated strategy for the remediation of soil co-contaminated by heavy metals and persistent organic pollutants is a major research priority for the decontamination of soil slated for use in agricultural production. The objective of this study was to develop a bioremediation strategy for fields co-contaminated with cadmium (Cd), dichlorodiphenyltrichloroethane (DDT), and its metabolites 1, 1-dichloro-2, 2-bis (4-chlorophenyl) ethylene (DDE) and 1, 1-dichloro-2, 2-bis (4-chlorophenyl) ethane (DDD) (DDT, DDE, and DDD are collectively called DDs) using an identified Cd-hyperaccumulator plant Sedum alfredii (SA) and DDT-degrading microbes (DDT-1). Initially, inoculation with DDT-1 was shown to increase SA root biomass in a pot experiment. When SA was applied together with DDT-1, the levels of Cd and DDs in the co-contaminated soil decreased by 32.1-40.3% and 33.9-37.6%, respectively, in a pot experiment over 18 months compared to 3.25% and 3.76% decreases in soil Cd and DDs, respectively, in unplanted, untreated controls. A subsequent field study (18-month duration) in which the levels of Cd and DDs decreased by 31.1% and 53.6%, respectively, confirmed the beneficial results of this approach. This study demonstrates that the integrated bioremediation strategy is effective for the remediation of Cd-DDs co-contaminated soils.

Natural variation of folate content and composition in spinach (Spinacia oleracea) germplasm.

Breeding to increase folate levels in edible parts of plants, termed folate biofortification, is an economical approach to fight against folate deficiency in humans, especially in the developing world. Germplasm with elevated folates are a useful genetic source for both breeding and direct use. Spinach is one of the well-know vegetables that contains a relatively high amount of folate. Currently, little is known about how much folate, and their composition varies in different spinach accessions. The aim of this study was to investigate natural variation in the folate content and composition of spinach genotypes grown under controlled environmental conditions. The folate content and composition in 67 spinach accessions were collected from the United States Department of Agriculture (USDA) and Asian Vegetable Research and Development Center (AVRDC) germplasm collections according to their origin, grown under control conditions to screen for natural diversity. Folates were extracted by a monoenzyme treatment and analyzed by a validated liquid chromatography (LC) method. The total folate content ranged from 54.1 to 173.2 µg/100 g of fresh weight, with 3.2-fold variation, and was accession-dependent. Four spinach accessions (PI 499372, NSL 6095, PI 261787, and TOT7337-B) have been identified as enriched folate content over 150 µg/100 g of fresh weight. The folate forms found were H(4)-folate, 5-CH(3)-H(4)-folate, and 5-HCO-H(4)-folate, and 10-CHO-folic acid also varied among different accessions and was responsible for variation in the total folate content. The major folate vitamer was represented by 5-CH(3)-H(4)-folate, which on average accounted for up to 52% of the total folate pool. The large variation in the total folate content and composition in diverse spinach accessions demonstrates the great genetic potential of diverse genotypes to be exploited by plant breeders.

The impact of EDTA on lead distribution and speciation in the accumulator Sedum alfredii by synchrotron X-ray investigation.

The in vivo localization and speciation of lead (Pb) in tissues of the accumulator Sedum alfredii grown in EDTA-Pb and Pb(NO(3))(2) was studied by synchrotron X-ray investigation. The presence of EDTA-Pb in solution resulted in a significant reduction of Pb accumulation in S. alfredii. Lead was preferentially localized in the vascular bundles regardless of treatments but the intensities of Pb were lower in the plants treated with EDTA. Lead was predominantly presented as a Pb-cell wall complex in the plants regardless of its supply form. However, a relatively high proportion of Pb was observed as Pb-EDTA complex when the plant was treated with EDTA-Pb, but as a mixture of Pb(3)(PO(4))(2), Pb-malic, and Pb-GSH when cultured with ionic Pb. These results suggest that EDTA does not increase the internal mobility of Pb, although the soluble Pb-EDTA complex could be transported and accumulated within the plants of S. alfredii.

Uptake, translocation, and remobilization of zinc absorbed at different growth stages by rice genotypes of different Zn densities.

Zinc (Zn) is an essential micronutrient for humans, and increasing Zn density in rice ( Oryza sativa L.) grains is important for improving human nutrition. The characteristics of Zn translocation and remobilization were investigated in high Zn density genotype IR68144, in comparison with the low Zn density genotype IR64. Stable isotope tracer (68)Zn was supplied at various growth stages, either to the roots in nutrient solution or to the flag leaves to investigate the contribution of (68)Zn absorbed at different growth stages to grain accumulation and the remobilization ability of (68)Zn within plants. Significant differences in (68)Zn allocation were observed between the two rice genotypes. Much higher (68)Zn concentrations were found in grains, stems, and leaves of IR68144 than in IR64, but higher (68)Zn was found in roots of IR64. More than half of the Zn accumulated in the grains was remobilized before anthesis, accounting for 63 and 52% of the total Zn uptake for IR68144 and IR64, respectively. Without supply of external Zn, at vegetative or reproductive stages, more (68)Zn was retranslocated from "old tissues" to "new tissues" in IR68144 than in IR64. Retranslocation of (68)Zn from flag leaves to grains was twice as high in the former when (68)Zn was applied to the flag leaves during booting or anthesis. These results indicate that Zn density in rice grains is closely associated with the ability to translocate Zn from old tissues to new tissues at both early and late growth stages and with phloem remobilization of Zn from leaves and stems to grains.

[Effects of Hg contamination on paddy soil microbial and enzymatic activities].

With pot experiment, the microbial and enzymatic activities in two paddy soils were investigated at different Hg2+ loadings. The results showed that after harvest, all measured microbiological and enzymological indices including microbial biomass carbon, respiration rate, and activities of urease, acid phosphatase, and dehydrogenase in test soils were increased under low Hg treatments ( <2 mg Hg x kg(-1)) while decreased under high Hg treatments ( > or =2 mg Hg x kg(-1)), except the basal respiration and metabolic quotient were partly increased with increasing Hg level. Among the test indices, soil microbial quotient was a microbiological index more sensitive to Hg contamination. The enzymatic activity in test yellowish red soil was higher than that in test silty loam soil. ED50 analysis indicated that Hg had a stronger ecological toxicity on soil urease in test silty loam soil and on acid phosphatase in test yellowish red soil.

Cadmium uptake and xylem loading are active processes in the hyperaccumulator Sedum alfredii.

Sedum alfredii is a well known cadmium (Cd) hyperaccumulator native to China; however, the mechanism behind its hyperaccumulation of Cd is not fully understood. Through several hydroponic experiments, characteristics of Cd uptake and translocation were investigated in the hyperaccumulating ecotype (HE) of S. alfredii in comparison with its non-hyperaccumulating ecotype (NHE). The results showed that at Cd level of 10 microM measured Cd uptake in HE was 3-4 times higher than the implied Cd uptake calculated from transpiration rate. Furthermore, inhibition of transpiration rate in the HE has no essential effect on Cd accumulation in shoots of the plants. Low temperature treatment (4 degrees C) significantly inhibited Cd uptake and reduced upward translocation of Cd to shoots for 9 times in HE plants, whereas no such effect was observed in NHE. Cadmium concentration was 3-4-fold higher in xylem sap of HE, as compared with that in external uptake solution, whereas opposite results were obtained for NHE. Cadmium concentration in xylem sap of HE was significantly reduced by the addition of metabolic inhibitors, carbonyl cyanide m-chlorophenylhydrazone (CCCP) and 2,4-dinitrophenol (DNP), in the uptake solutions, whereas no such effect was noted in NHE. These results suggest that Cd uptake and translocation is an active process in plants of HE S. alfredii, symplastic pathway rather than apoplastic bypass contributes greatly to root uptake, xylem loading and translocation of Cd to the shoots of HE, in comparison with the NHE plants.

Enhanced root-to-shoot translocation of cadmium in the hyperaccumulating ecotype of Sedum alfredii.

Sedum alfredii (Crasulaceae) is the only known Cd-hyperaccumulating species that are not in the Brassica family; the mechanism of Cd hyperaccumulation in this plant is, however, little understood. Here, a combination of radioactive techniques, metabolic inhibitors, and fluorescence imaging was used to contrast Cd uptake and translocation between a hyperaccumulating ecotype (HE) and a non-hyperaccumulating ecotype (NHE) of S. alfredii. The K(m) of (109)Cd influx into roots was similar in both ecotypes, while the V(max) was 2-fold higher in the HE. Significant inhibition of Cd uptake by low temperature or metabolic inhibitors was observed in the HE, whereas the effect was less pronounced in the NHE. (109)Cd influx into roots was also significantly decreased by high Ca in both ecotypes. The rate of root-to-shoot translocation of (109)Cd in the HE was >10 times higher when compared with the NHE, and shoots of the HE accumulated dramatically higher (109)Cd concentrations those of the NHE. The addition of the metabolic inhibitor carbonyl cyanide m-chlorophenylhydrazone (CCCP) resulted in a significant reduction in Cd contents in the shoots of the HE, and in the roots of the NHE. Cd was distributed preferentially to the root cylinder of the HE but not the NHE, and there was a 3-5 times higher Cd concentration in xylem sap of the HE in contrast to the NHE. These results illustrate that a greatly enhanced rate of root-to-shoot translocation, possibly as a result of enhanced xylem loading, rather than differences in the rate of root uptake, was the pivotal process expressed in the Cd hyperaccumulator HE S. alfredii.

Phytoremediation of heavy metal polluted soils and water: progresses and perspectives.

Environmental pollution affects the quality of pedosphere, hydrosphere, atmosphere, lithosphere and biosphere. Great efforts have been made in the last two decades to reduce pollution sources and remedy the polluted soil and water resources. Phytoremediation, being more cost-effective and fewer side effects than physical and chemical approaches, has gained increasing popularity in both academic and practical circles. More than 400 plant species have been identified to have potential for soil and water remediation. Among them, Thlaspi, Brassica, Sedum alfredii H., and Arabidopsis species have been mostly studied. It is also expected that recent advances in biotechnology will play a promising role in the development of new hyperaccumulators by transferring metal hyperaccumulating genes from low biomass wild species to the higher biomass producing cultivated species in the times to come. This paper attempted to provide a brief review on recent progresses in research and practical applications of phytoremediation for soil and water resources.

Mechanisms and assessment of water eutrophication.

Water eutrophication has become a worldwide environmental problem in recent years, and understanding the mechanisms of water eutrophication will help for prevention and remediation of water eutrophication. In this paper, recent advances in current status and major mechanisms of water eutrophication, assessment and evaluation criteria, and the influencing factors were reviewed. Water eutrophication in lakes, reservoirs, estuaries and rivers is widespread all over the world and the severity is increasing, especially in the developing countries like China. The assessment of water eutrophication has been advanced from simple individual parameters like total phosphorus, total nitrogen, etc., to comprehensive indexes like total nutrient status index. The major influencing factors on water eutrophication include nutrient enrichment, hydrodynamics, environmental factors such as temperature, salinity, carbon dioxide, element balance, etc., and microbial and biodiversity. The occurrence of water eutrophication is actually a complex function of all the possible influencing factors. The mechanisms of algal blooming are not fully understood and need to be further investigated.

Microbial activity and community diversity in a variable charge soil as affected by cadmium exposure levels and time.

Effects of cadmium (Cd) on microbial biomass, activity and community diversity were assessed in a representative variable charge soil (Typic Aquult) using an incubation study. Cadmium was added as Cd(NO3)(2) to reach a concentration range of 0-16 mg Cd/kg soil. Soil extractable Cd generally increased with Cd loading rate, but decreased with incubation time. Soil microbial biomass was enhanced at low Cd levels (0.5-1 mg/kg), but was inhibited consistently with increasing Cd rate. The ratio of microbial biomass C/N varied with Cd treatment levels, decreasing at low Cd rate (<0.7 mg/kg available Cd), but increasing progressively with Cd loading. Soil respiration was restrained at low Cd loading (<1 mg/kg), and enhanced at higher Cd levels. Soil microbial metabolic quotient (MMQ) was generally greater at high Cd loading (1-16 mg/kg). However, the MMQ is also affected by other factors. Cd contamination reduces species diversity of soil microbial communities and their ability to metabolize different C substrates. Soils with higher levels of Cd contamination showed decreases in indicator phospholipids fatty acids (PLFAs) for Gram-negative bacteria and actinomycetes, while the indicator PLFAs for Gram-positive bacteria and fungi increased with increasing levels of Cd contamination.