Impacts of polypropylene microplastics on the distribution of cadmium, enzyme activities, and bacterial community in black soil at the aggregate level.

Microplastics (MPs) can co-occur widely with heavy metals in soil. This study intended to investigate the influences of the co-exposure of polyethylene MPs (0.5 %, w/w) and cadmium (Cd) in black soil on the Cd distribution, enzyme activities, and bacterial communities in both bulk soil and different sized soil aggregates (> 1, 0.50-1, 0.25-0.50, and < 0.25 mm aggregates) after a 90-day incubation. Our results showed that the existence of MPs increased the distributions of Cd in >1 mm and < 0.25 mm soil aggregates and decreased its distributions in 0.50-1 mm and 0.25-0.50 mm soil aggregates. About 12.15 %-17.65 % and 9.03 %-11.13 % of Cd were distributed in the exchangeable and oxidizable forms in bulk soil and various sized soil aggregates after the addition of MPs which were higher than those in the only Cd-treated soil (11.17 %-14.72 % and 8.66 %-10.43 %, respectively), while opposite tendency was found for Cd in the reducible form. Urease and ß-glucosidase activities in the Cd-treated soils were 1.14-1.18 and 1.07-1.31 times higher than those in the Cd-MPs treated soils. MPs disturbed soil bacterial community at phylum level and increased the bacteria richness in bulk soil. The levels of predicted functional genes which are linked to the biodegradation and metabolism of exogenous substances and soil C and N cycles were altered by the co-exposure of Cd and MPs. The findings of this study could help deepen our knowledge about the responses of soil properties, especially microbial community, to the co-occurrence of MPs and heavy metals in soil.

Levels of heavy metal in soil and vegetable and associated health risk in peri-urban areas across China.

Peri-urban vegetable field plays an essential role in providing vegetables for local residents. Because of its particularity, it is affected by both industrial and agricultural activities which have led to the accumulations of heavy metal in soil. So far, information on heavy metal pollution status, spatial features, and human health risks in peri-urban vegetable areas across China is still scarce. To fill this gap, we systematically compiled soil and vegetable data collected from 123 articles published between 2010 and 2022 at a national level. The pollution status of heavy metals (i.e., cadmium (Cd), mercury (Hg), arsenic (As), lead (Pb), chromium (Cr), copper (Cu), nickel (Ni), and zinc (Zn)) in peri-urban vegetable soils and vegetables were investigated. To evaluate the levels of heavy metal pollution in soil and human health risks, the geoaccumulation index (Igeo) and target hazard quotient (HQ) were calculated. The results showed that mean concentrations of Cd, Hg, As, Pb, Cr, Cu, Ni, and Zn in peri-urban vegetable soils were 0.50, 0.53, 12.03, 41.97, 55.56, 37.69, 28.55, and 75.38 mg kg-1, respectively. The main pollutants in peri-urban vegetable soil were Cd and Hg, and 85.25% and 92.86% of the soil samples had Igeo > 1, respectively. The mean Igeo values of this regions followed the order of northwest > central > south > north > east > southwest > northeast for Cd and northeast > northwest > north > southwest > east > central > south for Hg. The mean Cd, Hg, As, Pb, Cr, Cu, Ni, and Zn concentrations in vegetables were 0.30, 0.26, 0.37, 0.54, 1.17, 6.17, 1.96, and 18.56 mg kg-1, respectively. Approximately 87.01% (Cd), 71.43% (Hg), 20% (As), 65.15% (Pb), 27.08% (Cr) of the vegetable samples exceeded the safety requirement values. The vegetables grown in central, northwest, and northern China accumulated much more heavy metals than those grown in other regions. As the HQ values for adults, 53.25% (Cd), 71.43% (Hg), 84.00% (As), and 58.33% (Cr) of the sampled vegetables were higher than 1. For children, the HQ values were higher than 1 for 66.23% (Cd), 73.81% (Hg), 86.00% (As), and 87.50% (Cr) of the sampled vegetables. The findings of this study demonstrate that the situation of heavy metal pollution in peri-urban vegetable areas across China are not optimistic and residents who consume the vegetables are at high risk of health issues. To ensure soil quality and human health, strategies should be taken to guide vegetable production and remedy soil pollution in peri-urban areas with the rapidly urbanizing China.

Colloid-facilitated mobilization of cadmium: Comparison of spring freeze-thaw event and autumn freeze-thaw event.

Freeze-thaw action has the potential to facilitate the mobilization of colloid-associated contaminants in soil. However, the differences in colloid-associated contaminants following autumn freeze-thaw (AFT) events and spring freeze-thaw (SFT) events remain unclear. In this study, the potential influence mechanisms of AFT and SFT on both the generation and migration of colloids and colloid-associated cadmium (Cd) in soil were explored. Higher aggregate stabilities were found in soils after AFT compared with after SFT. After SFT, lower Cd concentrations were found in soil aggregates of 0.25-0.50 mm and <0.106 mm and higher concentrations were found in 0.106-0.25 mm aggregates. Moreover, SFT generated higher amounts of colloidal Cd than AFT, while AFT increased the total Cd concentration in leachates. Additionally, compared with SFT, AFT led to higher Cd concentrations in dissolved and colloid-associated forms in leachates. These findings demonstrate that higher amounts of colloid and fewer loadings of Cd in colloids in Cd contaminated soil can be found after SFT events. Thus, to better understand the environmental risk of contaminants in areas subject to seasonal freeze-thaw cycles, the differences between freeze-thaw processes in spring and autumn should be considered.

Effects of soil aging conditions on distributions of cadmium distribution and phosphatase activity in different soil aggregates.

Aging behaviors of metals in the field differ from those in a controlled laboratory environment. Whether aging conditions influence the fates of metals in soil remains unclear. In this study, distributions of cadmium (Cd) and phosphatase activity were compared in soil aggregates (i.e., >2, 1-2, 0.25-1, and <0.25 mm) along a profile (0-5, 5-10, and 10-15 cm) at the end of 500-day aging experiments under both controlled laboratory and field conditions. Cd concentration in the 0-5 cm layer was lower and Cd concentration in the 5-10 cm layer was higher in field-aged soil compared to laboratory-aged soil. 25.26-35.62% of soil Cd was loaded in >2 mm aggregates of field-aged soils, and 58.41-66.95% was in laboratory-aged soils. Higher loadings of Cd in 0.25-1 and <0.25 mm aggregates were found in field-aged soil. A higher proportion of exchangeable Cd fraction (20.93% of total soil Cd) was found in the 0-5 cm layer of field-aged soil than in laboratory-aged soil (17.63%), while the opposite tendency was found in deeper soil layers. Soil phosphatase activities in field-aged soils were 1.13-1.26 times higher than in laboratory-aged soils. Phosphatase loadings in the >2 mm aggregates were lower and loadings in both the 1-2 and 0.25-1 mm aggregates were higher in field-aged soils than in laboratory-aged soils. Furthermore, correlation analysis and principal component analysis indicated that available Cd fractions accounted for most of the variations in phosphatase activities. In summary, the fates of the exogenous metal Cd differed between field and controlled laboratory conditions. To better understand the behaviors of heavy metals in soil, especially in a seasonal freeze-thaw area, further field studies are needed.

Do freeze-thaw cycles affect the cadmium accumulation, subcellular distribution, and chemical forms in spinach (Spinacia oleracea L.)?

To date, although there are many studies investigating the toxicity of heavy metal to plant, little research exists in the seasonal freeze-thaw (FT) regions where FT cycles often happen during the plant growing process. To reveal the adaptive mechanisms of plants to the combination stresses of cadmium (Cd) and FT, the Cd accumulation, subcellular distribution, chemical forms, and antioxidant enzyme activity (peroxidase (POD)) were investigated in spinach (Spinacia oleracea L.) growing under different soil Cd levels (i.e., 0.10 mg Cd kg-1 soil (low), 1.21 mg Cd kg-1 soil (medium), and 2.57 mg Cd kg-1 soil (high)). Compared to the non-freeze-thaw (NFT) treatments, higher Cd concentrations in the root and lower translocation factors from root to leaf were found for the plants experiencing FT cycles. FT significantly decreased the Cd concentrations in the leaves under the low- and medium-Cd treatments, while similar values were found for the high-Cd treatments. Generally, FT could decrease the concentrations and proportions of Cd stored in the cell wall and soluble fractions and increase them in the organelle fractions for the medium- and high-Cd treatments, while opposite tendency was found for the low-Cd treatments. Moreover, larger Cd amounts in the inorganic and water-soluble forms were found for the low- and medium-Cd treated plants under FT, while lower values were found for the high-Cd treatments. Additionally, POD, which presented higher activities at the low- and medium-Cd treatments and lower activities at the high-Cd treatments under FT, were also significantly influenced by the Cd × FT interaction. This study indicated that FT could significantly change the accumulations of Cd in plant, and it provided a new insight into the Cd accumulation by plants in the seasonal FT region.

Laboratory versus field soil aging: Impacts on cadmium distribution, release, and bioavailability.

To date, most studies about the aging of metals in soil were based on the controlled laboratory experiments, and few works have attempted to investigate how aging process influences the distribution and bioavailability of metals in soil under the field condition. The purpose of this study was to compare the aging of cadmium (Cd) in soils under the controlled laboratory and the field by monitoring time-dependent soil Cd speciation changes, Cd release kinetics, and Cd bioavailability to plant through the 438-day aging experiments. During the aging process, the proportions of Cd associated with the most weakly bound fraction tended to decrease, with corresponding increases in the more stable binding fractions. After aging, a higher concentration of available Cd was found in the field aging soil (0.74 mg kg-1) than the laboratory aging soil (0.65 mg kg-1). The Elovich equation was the best model to describe the soil available Cd aging process. The constant b in the Elovich equation, which was defined as the transformation rate, was in the order of laboratory aging soil > field aging soil. Moreover, higher Cd release amounts were found for the field aging soil (2.74 mg kg-1) than the laboratory aging soil (2.57 mg kg-1) at the end of aging. Additionally, higher body Cd concentrations were found for the vegetables grown in the field aging soils (1.49 mg kg-1, fresh weight) than those grown in the laboratory aging soils (1.32 mg kg-1, fresh weight). Therefore, this study indicated that the metal distribution process and its bioavailability may be overestimated or underestimated if research data from the laboratory experiments are used to derive soil quality criteria or investigate soil metal bioavailability.

[Characteristics of seawater nutrients during the occurrence of brown tide in the coastal area of Qinhuangdao, China]. / ç§¦çš‡å²›æµ·åŸŸè¤æ½®ç”Ÿæ¶ˆè¿‡ç¨‹ä¸­è¥å »ç›ç‰¹å¾.

Brown tide that occurred in Qinhuangdao coastal waters from 2009 to 2015 caused huge losses of local marine aquaculture and coastal tourism, with devastating effects on marine ecosystems. Nutrients are important biogenic elements for algal growth. It is of great significance to examine the fluctuation characteristics of nutrients in the process of brown tide to understand the nutritional mechanism of brown tide. Based on the survey data of 30 stations located in Qinhuangdao coastal area from April to June 2014, we analyzed nutrient characteristics during the occurrence of brown tide and its relationship with the population dynamics of Aureococcus anophagefferens. The results showed that the concentration of dissolved nitrogen (DN) in April, May and June 2014 was 265.65, 355.36 and 323.71 µg·L-1 respectively, and the concentration of dissolved organic nitrogen (DON) was 196.98, 242.88 and 177.69 µg·L-1, accounting for 74.2%, 68.3% and 54.9% of DN, respectively. The concentration of dissolved phosphorus (DP) in April, May and June was 15.95, 11.39 and 11.14 µg·L-1 respectively. In April and May, PO43--P accounted for a large proportion of the DP, 74.8% and 80.9% respectively. In June, the proportion of PO43--P in DP fell to 33.8%, and the proportion of dissolved organic phosphorus (DOP) in DP rose to 66.2%. The concentration of SiO32--Si in April, May and June was 70.95, 181.13 and 120.68 µg·L-1, respectively. Except for dissolved inorganic nitrogen (DIN) in May and DON, the distribution of other nutrients had clear characteristics that it decreased gradually from inshore to the offshore, with the relatively high concentrations in river mouth. Through R-factor analysis and nutrient structure analysis, it was found that in April, brown tide was at the development stage, and DOP might be the main driving factor for the growth of A. anophagefferens. In May, brown tide was at the maintenance stage, and water temperature became the main controlling factor. When water temperature was higher than 12 ℃, brown tide could occur. In June, brown tide began to decay, and PO43--P had greater effects on the community structure of phytoplankton. DON was the important factor causing the outbreak of brown tide, with a concentration threshold of 150 µg·L-1 and the ratio DON/DIN being greater than 1.

Integration of chemical and toxicological tools to assess the bioavailability of copper derived from different copper-based fungicides in soil.

Because the extensive use of Cu-based fungicides, the accumulation of Cu in agricultural soil has been widely reported. However, little information is known about the bioavailability of Cu deriving from different fungicides in soil. This paper investigated both the distribution behaviors of Cu from two commonly used fungicides (Bordeaux mixture and copper oxychloride) during the aging process and the toxicological effects of Cu on earthworms. Copper nitrate was selected as a comparison during the aging process. The distribution process of exogenous Cu into different soil fractions involved an initial rapid retention (the first 8 weeks) and a following slow continuous retention. Moreover, Cu mainly moved from exchangeable and carbonate fractions to Fe-Mn oxides-combined fraction during the aging process. The Elovich model fit well with the available Cu aging process, and the transformation rate was in the order of Cu(NO3)2 > Bordeaux mixture > copper oxychloride. On the other hand, the biological responses of earthworms showed that catalase activities and malondialdehyde contents of the copper oxychloride treated earthworms were significantly higher than those of Bordeaux mixture treated earthworms. Also, body Cu loads of earthworms from different Cu compounds spiked soils were in the following order: copper oxychloride > Bordeaux mixture. Thus, the bioavailability of Cu from copper oxychloride in soil was significantly higher than that of Bordeaux mixture, and different Cu compounds should be taken into consideration when studying the bioavailability of Cu-based fungicides in the soil.

Adsorption behaviors of fungicide-derived copper onto various size fractions of aggregates from orchard soil.

Although the gradual accumulations of Cu in orchard soils due to the application of Cu-based fungicides have been widely reported, limited information is available about the retention characteristics of fungicide-derived Cu in soil, especially in various size soil aggregates. This study described the adsorption characteristics of Cu from commonly used fungicide, Bordeaux mixture (CuSO4 + Ca(OH)2), onto various aggregate fractions (2000-1000, 1000-500, 500-250, 250-106, and <106 µm) of orchard soil. The Cu(NO3)2 was selected as a comparison. Two different types of adsorption experiments were conducted as follows: variable pH and variable Cu concentration experiments. The adsorption processes of Bordeaux mixture and Cu(NO3)2 onto the studied soil samples followed well with the Freundlich isotherm, and the adsorption isotherms were the S shaped. The adsorption amounts of Cu from different Cu compounds differed, and Bordeaux mixture can result in more Cu retention in soil than Cu(NO3)2. The adsorption ability of different size soil aggregates varied, and it was mainly governed by soil properties. The findings of this study suggested that both the chemical compositions of Cu compounds and the soil physical structure should be taken into account when performing soil Cu retention experiments with fungicide-derived Cu.

Enhanced-electrokinetic remediation of copper-pyrene co-contaminated soil with different oxidants and pH control.

Electrokinetic (EK) remediation has potential to simultaneously remove heavy metals and organic compounds from soil, but the removal percent of these pollutants is very low in general if no enhancing treatment is applied. This study developed a new enhanced-EK remediation technology to decontaminate a heavy metal-organic compound co-contaminated soil by applying different oxidants and pH control. A red soil was used as a model clayed soil, and was spiked with pyrene and Cu at about 500 mg kg(-1) for both to simulate real situation. Bench-scale EK experiments were performed using four oxidants (H(2)O(2), NaClO, KMnO(4), and Na(2)S(2)O(8)) and controlling electrolyte pH at 3.5 or 10. After the treatments with 1.0 V cm(-1) of voltage gradient for 335 h, soil pH, electrical conductivity, and the concentrations and chemical fractionations of soil pyrene and Cu were analyzed. The results showed that there was significant migration of pyrene and Cu from the soil, and the removal percent of soil pyrene and Cu varied in the range of 30-52% and 8-94%, respectively. Low pH favoured the migration of soil Cu, while KMnO(4) was the best one for the degradation of pyrene among the tested oxidants, although it unfortunately prevented the migration of soil Cu by forming Cu oxide. Application of Na(2)S(2)O(8) and to control the catholyte pH at 3.5 were found to be the best operation conditions for decontaminating the Cu-pyrene co-contaminated soil.

[Prokaryotic expression and immunogenicity analysis of the chimeric HBcAg containing APP beta cleavage site peptide and Aß(1-15);].

AIM: To construct the recombinant prokaryotic expression plasmid pET/c-ABCSP-Aß(15-c);, and evaluate the immunogenicity of the fusion protein expressed in E.coli. METHODS: The gene fragment HBc88-144 was amplified by PCR and subcloned to pUC19. The APP beta cleavage site peptide(ABCSP) and Aß(1-15); gene(ABCSP-Aß(15);) was amplified by PCR and inserted downstream of HBc1-71 in pGEMEX/c1-71. After restriction enzyme digestion, c1-17-ABCSP-Aß(15); were connected with HBc88-144, yielding the recombinant gene c-ABCSP-Aß(15-c);. c-ABCSP-Aß(15-c); gene was subcloned into pET-28a(+).The fusion protein expressed in transformed E.coli BL21 was induced with IPTG and analyzed by SDS-PAGE. The virus-like particles (VLP) formed by fusion protein was observed with Transmission Electron Microscope (TEM). 4 Kunming (KM) mice received intraperitoneal injection (i.p) of fusion protein VLP. The antibody was detected by indirect ELISA. RESULTS: The recombinant gene was confirmed by restriction enzyme digestion and DNA sequencing. After IPTG induction, fusion protein was expressed and mainly existed in the sediment of the bacterial lysate. The expression level was 40% of all the proteins in the sediment. The fusion protein could form VLP. After 5 times of immunization, the titer of anti-ABCSP and anti-Aßantibody in sera of KM mice reached up to 1:5 000 and 1:10 000 respectively, while the anti-HBc antibody was undetectable. CONCLUSION: Recombinant c-ABCSP-Aß(15-c); gene can be expressed in E.coli. The expressed protein could form VLP and has a strong immunogenicity. This study lays the foundation for the study of AD genetic engineering vaccine.

[Construction of a general AAV vector regulated by minimal and artificial hypoxic-responsive element].

AIM: To synthesize the minimal and artificial HRE, and to insert it into the anterior extremity of CMV promoter of a AAV plasmid, and then to construct the AAV regulated by hypoxic-responsive element which was introduced into 293 cell by method of Ca3(PO4)2 using three plasmids. Thus obtaining the adenoassociated virus vector regulated by hypoxic-responsive element was possibly used for gene therapy in ischemia angiocardiopathy and cerebrovascular disease. METHODS: Artificially synthesize the 36 bp nucleotide sequences of four connection in series HIF-binding sites A/GCGTG(4×HBS)and a 35 bp nucleotide sequences spacing inserted into anterior extremity of CMV promoter TATA Box, then amplified by PCR. The cDNA fragment was confirmed to be right by DNA sequencing. Molecular biology routine method was used to construct a AAV vector regulated by minimal hypoxic-responsive element after the normal CMV promoter in AAV vector was replaced by the CMV promoter included minimal hypoxic-responsive element. Then, NT4-6His-PR39 fusogenic peptide was inserted into MCS of the plasmid, the recombinant AAV vector was obtained by three plasmid co-transfection in 293 cells, in which we can also investigate the expression of 6×His using immunochemistry in hypoxia environment. RESULTS: Artificial HRE was inserted into anterior extremity of CMV promoter and there was a correct spacing between the HRE and the TATA-box. The DNA sequencing and restriction enzyme digestion results indicated that the AAV regulated by hypoxic-responsive element was successfully constructed. Compared to the control group, the expressions of 6×His was significantly increased in the experimental groups in hypoxia environment, which confirmed that the AAV effectually regulated by the minimal HRE was inserted into anterior extremity of CMV promoter. CONCLUSION: The HRE is inserted into anterior extremity of CMV promoter to lack incision enzyme recognition site by PCR. And eukaryotic expression vector regulated by hypoxic-responsive is constructed. The AAV effectually regulated by the minimal HRE inserted into anterior extremity of CMV promoter. The vector is successfully constructed and it has important theoretical and practical value in the synteresis and therapy of ischemia angiocardiopathy and cerebrovascular disease.

[Construction of a phage antibody library and screening of anti-epidermal growth factor receptor variant III single chain antibody].

OBJECTIVE: To obtain specific anti-epidermal growth factor receptor variant III (EGFRvIII) single chain antibody (ScFv) by phage antibody library display system. METHODS: The total RNA was extracted from the spleen B cells of BALB/c mice immunized with pep-3-OVA protein, and the first-strand cDNA was synthesized by reverse transcription. Antibody VH and VL gene fragments were amplified and joined to a ScFv gene with the linker. The ScFv gene was ligated into the phagemid vector pCANTAB5E, which was transformed into competent E. coli TG1. The transformed cells were then infected with M13KO7 helper phage to yield the recombinant phage to construct the phage ScFv library. Pep-3-BSA protein was used to screen the phage antibody library and ELISA carried out to characterize the activity of the antibody. RESULTS: The VH and VL gene fragments of the antibody were about 350 bp and 320 bp in length as analyzed by agarose gel electrophoresis. The ScFv gene was 780 bp, consistent with the expected length. The recombinant phagemid with ScFv gene insert was rescued, and an immune phage ScFv library with the content of 5.0x10(6) was constructed. The recombinant ScFv phage had a titer of 3.0x10(4) cfu/ml, and the fourth phage harvest yielded 56 times as much as that of the first one. SDS-PAGE demonstrated a molecular mass of the soluble ScFv of about 28 kD. ELISA results indicated good specificity of the ScFv to bind EGFRvIII. CONCLUSION: An immune phage ScFv library is successfully constructed, and the ScFv antibody fragment is capable of specific binding to EGFRvIII.

Secretory expression of Par-4 SAC-HA2TAT following adeno-associated virus-mediated gene transfer induces apoptosis in HepG2 cells.

Prostate apoptosis response-4 (Par-4) is a tumor-suppressor protein that induces apoptosis in cancer cells, but not in normal cells. The cancer-specific pro-apoptotic action of Par-4 is encoded in its centrally located SAC domain. In this study, to further enhance the anti-cancer effect of Par-4 in order to overcome the limitations of peptide therapy, a recombinant adeno-associated virus was constructed using the following strategies: the secretory expression of therapeutic peptide, a HA2TAT-mediated cytosolic delivery technique, and an adeno-associated virus gene transfer system. To test the hypothesis that Par-4 has an additive bystander effect as an anti-cancer therapy, we designed a secretory protein by adding a secretory signal peptide NT4(Si) to the Par-4 SAC-HA2TAT peptide gene sequence [NT4(Si)-Par-4 SAC-HA2TAT]. The results indicated that, compared to the normal NIH3T3 cell line, AAV-NT4(Si)-Par-4 SAC-HA2TAT significantly suppressed cell growth and induced rapid cell death in HepG2 cells in a time-dependent manner through successful gene transfer and secretory expression of therapeutic peptide at 48 h post-transfection. In addition, the secretory properties of Par-4 may greatly increase its effectiveness in cancer therapy when delivered in vivo.

Roles of abiotic losses, microbes, plant roots, and root exudates on phytoremediation of PAHs in a barren soil.

Phytoremediation is an emerging technology for the remediation of polycyclic aromatic hydrocarbons (PAHs). In this study, pot experiments were conducted to evaluate the efficacy of phytoremediation of phenanthrene and pyrene in a typical low organic matter soil (3.75 g kg(-1)), and the contribution proportions of abiotic losses, microbes, plant roots, and root exudates were ascertained during the PAHs dissipation. The results indicated that contribution of abiotic losses from this soil was high both for phenanthrene (83.4%) and pyrene (57.2%). The contributions of root-exudates-enhanced biodegradation of phenanthrene (15.5%) and pyrene (21.3%) were higher than those of indigenous microbial degradation. The role of root exudates on dissipation of phenanthrene and pyrene was evident in this experiment. By the way, with the increasing of ring numbers in PAHs structures, the root-exudates-enhanced degradation became more and more important. BIOLOG-ECO plate analysis indicated that microbial community structure of the soil receiving root exudates had changed. The removal efficiency and substrate utilization rate in the treatment with plant roots were lower than the treatment only with root exudates, which suggested that possible competition between roots and microbes for nutrients had occurred in a low organic matter soil.

NT4(Si)-p53(N15)-antennapedia induces cell death in a human hepatocellular carcinoma cell line.

AIM: To construct the recombinant lentivirus expression plasmid, pLenti6/V5-NT4 p53(N15)-antennapedia (Ant), and study its effect on HepG2 cells. METHODS: Plasmid pLenti6/V5-NT4 p53(N15)-Ant was constructed incorporating the following functional regions, including signal peptide sequence and pro-region of neurotrophin 4, N-terminal residues 12-26 of p53 and 17 amino acid drosophila carrier protein, Ant. Hepatocellular carcinoma (HepG2) cells were used for transfection. 3-[4,5-dimethyl-thiazol-2yl]-2,5 diphenyl tetrazolium bromide (MTT) assay, lactate dehydrogenase (LDH) release assay, transmission electron microscopy (TEM) and flow cytometric analysis (FCM) were employed to investigate the effects of LV-NT4(Si)-p53(N15)-Ant in vitro on HepG2 cells. In vivo experiment was also performed to investigate the inhibitory effect of LV-NT4(Si)-p53(N15)-Ant on tumor growth in nude mice. RESULTS: LV-NT4(Si)-p53(N15)-Ant significantly suppressed the growth of HepG2 cells. MTT assay showed that the growth of HepG2 cells was mucj more significantly inhibited by LV-NT4(Si)-p53(N15)-Ant than by LV-EGFP. The inhibition rate for HepG2 cell growth in the two groups was 46.9% and 94.5%, respectively, 48 h after infection with LV-NT4(Si)-p53(N15)-Ant, and was 33.9% and 95.8%, respectively, 72 h after infection with LV-NT4(Si)-p53(N15)-Ant (P < 0.01). Light microscopy and TEM showed morphological changes in HepG2 cells infected with LV-NT4(Si)-p53(N15)-Ant, but no significant changes in HepG2 cells infected with LV-EGFP. Changes were observed in ultra-structure of HepG2 cells infected with LV-NT4(Si)-p53(N15)-Ant, with degraded membranes, resulting in necrosis. LDH release from HepG2 cells was analyzed at 24, 48, 72 and 96 h after infection with LV-NT4(Si)-p53(N15)-Ant and LV-EGFP, which showed that LDH release was significantly higher in LV-NT4(Si)-p53(N15)-Ant treatment group (682 IU/L) than in control group (45 IU/L, P < 0.01). The longer the time was after infection, the bigger the difference was in LDH release. FCM analysis showed that LV-NT4(Si)-p53(N15)-Ant could induce two different kinds of cell death: necrosis and apoptosis, with apoptosis being the minor type and necrosis being the main type, suggesting that LV-NT4(Si)-p53(N15)-Ant exerts its anticancer effect on HepG2 cells by inducing necrosis. The in vivo study showed that LV-NT4(Si)-p53(N15)-Ant significantly inhibited tumor growth with an inhibition rate of 66.14% in terms of tumor size and weight. CONCLUSION: LV-NT4(Si)-p53(N15)-Ant is a novel recombinant lentivirus expression plasmid and can be used in gene therapy for cancer.

[Secretory expression of PR39 following adeno-associated viral-encoding fusion gene transfer induces angiogenesis in hypoxia chick embryo].

OBJECTIVES: To investigate the impact of AAV-encoding NT4-TAT-His-PR39 fusion gene expression on HIF-1alpha level in ECV304 cultured under hypoxic condition (1%O(2)) and on angiogenesis in hypoxic chick embryo. METHODS: PR39 cDNA was connected with NT4, TAT, 6 x His cDNA by molecular biology methods. The recombinant AAV vector was obtained by three plasmid co-transfection in 293 cells. Then ECV304 were respectively infected with AAV-NT4-TAT-His-PR39, 6 x His expression and HIF-1alpha level in ECV304 were detected by immunocytochemistry. The chicken embryos were randomized into the AAV-PR39, EV and PBS groups (n = 10 each) subject to hypoxia (5%O(2), n = 15) or normoxia environments (n = 15), the vessel density of the chicken chorioallantoic membrane (CAM) were measured by Image Pro Plus (IPP) software. RESULTS: The expression of 6 x His protein was detected in AAV-PR39 infected ECV304 cells. HIF-1alpha protein activity was significantly increased in AAV-PR39 infected ECV304 underwent hypoxia compared to PBS and non-infected ECV304 groups (P < 0.05). The vessel density of chicken CAM in hypoxia environment but not in normoxia environment was also significantly higher in AAV-PR39 group than in EV group and PBS group (all P < 0.05). CONCLUSION: AAV-encoding NT4-TAT-His-PR39 fusion gene expression significantly increased HIF-1alpha level in ECV304 exposed to hypoxia and promoted angiogenesis in hypoxic chicken embryo.

Effects of electrokinetic treatment of a heavy metal contaminated soil on soil enzyme activities.

There is a growing concern on the potential application of a direct current (DC) electric field to soil for removing contaminants, but little is known about its impact on soil enzyme activities. This study investigated the change of enzyme activities of a heavy metal contaminated soil before and after electrokinetic (EK) treatments at lab-scale and the mechanisms of EK treatment to affect soil enzyme activities were explored. After treatments with 1-3 V cm(-1) of voltage gradient for 420 h, soil pH, electrical conductivity (EC), soil organic carbon, dissolved organic carbon (DOC), soil heavy metal concentration and enzyme activities were analyzed. The results showed that the average removal efficiencies of soil copper were about 65% and 83% without and with pH control of catholyte, respectively, and all the removal efficiencies of cadmium were above 90%. The soil invertase and catalase activities increased and the highest invertase activity was as 170 times as the initial one. The activities of soil urease and acidic phosphatase were lower than the initial ones. Bivariate correlation analyses indicated that the soil invertase and acidic phosphatase activities were significantly correlated with soil pH, EC, and DOC at P<0.05, but the soil urease activities had no correlation with the soil properties. On the other hand, the effects of DC electric current on solution invertase and catalase enzyme protein activities indicated that it had negative effect on solution catalase activity and little effect on solution invertase activity. From the change of invertase and catalase activities in soil and solution, the conclusion can be drawn that the dominant effect mechanism is the change of soil properties by EK treatments.

[Construction of a prokaryotic expression vector for apoptin and preparation of polyclonal antibody of apoptin].

OBJECTIVE: To construct a prokaryotic expression vector for apoptin and prepare polyclonal antibody of apoptin. METHODS: Apoptin gene amplified from pGEM-T/Apoptin plasmid by PCR was cloned into pET-28a (+). E.coli BL21 (DE3) was transformed by the recombinant plasmid, and apoptin protein expression induced by IPTG was analyzed by SDS-PAGE. BALB/c mice were immunized with the protein and the titer of the antibody was determined using indirect enzyme-linked immunosorbent assay (ELISA). RESULTS: Apoptin gene was successfully cloned into pET-28a (+), and the expression of a protein with relative molecular mass of about 17 000 was identified by SDA-PAGE. After 5 immunizations of the mice with the protein, the blood antibody titer reached 1:5x10(5). CONCLUSION: The prokaryotic expression vector for apoptin is successfully constructed and the polyclonal antibody of apoptin is obtained, which allows further functional study of apoptin.

Solid/solution Cu fractionations/speciation of a Cu contaminated soil after pilot-scale electrokinetic remediation and their relationships with soil microbial and enzyme activities.

The aim of this study was to investigate the detailed metal speciation/fractionations of a Cu contaminated soil before and after electrokinetic remediation as well as their relationships with the soil microbial and enzyme activities. Significant changes in the exchangeable and adsorbed-Cu fractionations occurred after electrokinetic treatment, while labile soil Cu in the solution had a tendency to decrease from the anode to the cathode, and the soil free Cu(2+) ions were mainly accumulated in the sections close to the cathode. The results of regression analyses revealed that both the soil Cu speciation in solution phase and the Cu fractionations in solid phase could play important roles in the changes of the soil microbial and enzyme activities. Our findings suggest that the bioavailability of soil heavy metals and their ecotoxicological effects on the soil biota before and after electroremediation can be better understood in terms of their chemical speciation and fractionations.