[Distribution Characteristics and Pollution Evaluation of Heavy Metals in Greenbelt Soils of Nanjing City].

Concentrations and the spatial distribution of heavy metals (Cr, Cu, Zn, Pb, and Cd) in greenbelt soils in the main areas of Nanjing City, Jiangsu Province, were investigated and pollution levels were assessed using the single factor pollution index (SFPI), Nemerow integrated pollution index (NIPI), and potential ecological risk coefficient (PERC). The results showed that the average concentrations of heavy metals in greenbelt soils exceeded their background values, with the SFPI results ranked Cd > Pb > Cr > Cu > Zn, and the mean NIPI was 2.72 indicating that the greenbelt soils are moderately polluted. The PERC of each heavy metal (Cr, Cu, Zn, and Pb) was less than 10, indicating that the greenbelt soils present a slight ecological risk, while the PERC of Cd reached 97.32, indicating a strong ecological risk. The composite PERC of all heavy metals was less than 150, indicating a slight ecological risk overall. The five heavy metals showed a patchy spatial distribution, with high concentrations of Cr, Cu, Zn, and Pb in the greenbelt soils of northeast areas, and high concentrations of Cd in the southwest and northwest. Based on the results of study, Cd pollution in greenbelt soils in main areas of Nanjing City is relatively serious, which requires further attention.

The pro-survival function of DLEC1 and its protection of cancer cells against 5-FU-induced apoptosis through up-regulation of BCL-XL.

The tumor suppressor DLEC1 has been shown to promote cell proliferation when AP-2α2 is down-regulated in HCT116 stable clones, suggesting its pro-survival nature. However, the pro-survival function of DLEC1 has not been confirmed in other cells and its underlying mechanisms remain elusive. Therefore, we knocked down DLEC1 in a panel of cell lines and found that DLEC1 depletion caused various extents of cell death through intrinsic pathway. DLEC1 overexpression promoted cell survival and reduced cell death in cancer cells after 5-FU treatment, while DLEC1 down-regulation sensitized cancer cells to 5-FU. Further studies demonstrated that DLEC1 attenuated the increase in cleaved PARP, caspase-3 and caspase-7, the activity of caspase-9 and the diffusion of cytosolic cytochrome c from mitochondria. Our data also showed that BCL-XL was up-regulated by DLEC1 in stable clones after 5-FU treatment. Altogether, these results indicated that DLEC1 protects cells against cell death induced by 5-FU through the attenuation of active proteins in caspase cascade and the up-regulation of BCL-XL. Therefore, DLEC1 can be a pro-survival protein under certain circumstances and a potential therapeutic target for increasing sensitivity of cancer cells to 5-FU.

Effects of Silicon Application on Uptake of Arsenic and Phosphorus and Formation of Iron Plaque in Rice Seedlings Grown in an Arsenic-Contaminated Soil.

Silicon (Si) plays important roles in improving rice growth and mitigating rice arsenic (As) uptake. In the present study, a pot experiment was carried out to investigate effects of Si application on uptake of As and phosphorus (P) and formation of iron (Fe) plaque on root surface of two rice cultivars (Zhendao 10 and Nanjing 44) grown in a high As-contaminated soil. The results showed that dry weights of shoots and roots for both rice cultivars didn't significantly varied under low Si level, but significantly increased for Zhendao 10, while decreased for Nanjing 44 under high Si level (p < 0.05). As concentrations in shoots and roots of Nanjing 44 significantly decreased for low Si level, while significantly increased for high Si level (p < 0.05). Different from Nanjing 44, effect of Si application on As concentrations in the plants of Zhendao 10 wasn't significant (p > 0.05). Si significantly increased concentrations of P in shoots and roots of both rice cultivars (p < 0.05). However, Si didn't significantly affect formation of Fe plaque on root surface. These results suggest that the effects of Si application on rice growth and As uptake in As-contaminated soils may depend on type of rice cultivar and Si application level. Rice cultivar and Si application rate should be considered when Si application is used to mitigate As accumulation in rice.

Effects of Dissolved Organic Matter on Uptake and Translocation of Lead in Brassica chinensis and Potential Health Risk of Pb.

Dissolved organic matter (DOM) can affect the bioavailability of heavy metals in soil, especially in soils used for vegetable production, where intensive organic fertilization is applied. The present study examined the effects of DOM derived from commercial organic fertilizers (COF), cow manure (COM) and chicken manure (CHM), on uptake and translocation of lead (Pb) in Brassica chinensis in a pot experiment. The results indicate that DOM derived from CHM (DOMCHM) significantly increased Pb concentrations in roots of B. chinensis (p < 0.05). By contrast, there was no significant increase in shoot Pb concentration for all the DOM treatments except the high DOMCHM treatment in the soil with 800 mg·kg(-1) Pb. Consistent with the Pb concentrations in shoots, translocation factor of Pb from soil to shoot and specific lead uptake (SLU) by B. chinensis were significantly increased for the high DOMCHM treatment in the high Pb soil, but not for other DOM treatments. Based on the results of this study, the application of DOM to the soil with 800 mg·kg(-1) Pb could result in an increase in total Pb annually ingested by the inhabitants of Nanjing City in the range of 2018-9640 kg, with the highest estimates resulting from the high DOMCHM treatment. This study suggests the risk may rise under some conditions as indicated in the high DOMCHM treatment and high Pb pollution level.

Tumor Suppressor DLEC1 can Stimulate the Proliferation of Cancer Cells When AP-2ɑ2 is Down-Regulated in HCT116.

BACKGROUND: The molecular mechanisms of tumor suppressor gene DLEC1 are largely unknown. OBJECTIVES: In this study, we established DLEC1 over-expression stable clones to study the cellular function of DLEC1 in the colorectal cancer cell line, HCT116. MATERIALS AND METHODS: Stable clones with DLEC1 over-expression were first established by the transfection of DLEC1 expression construct pcDNA31DLEC1 in HCT116. On G418 selection, positive stable clones were screened for DLEC1 expression level by conventional reverse transcription-polymerase chain reaction (RT-PCR), and verified by real-time RT-PCR and Western blotting. Subsequently, these stable clones were subjected to colony formation and cell cycle analyses and identification of factors involved in G1 arrest. Lastly, three stable clones, DLEC1-7 (highest DLEC1 expression), DLEC1-3 (lowest expression) and pcDNA31 vector control, were employed to analyze cell proliferation and cell cycle after AP-2α2 knockdown by siRNAs. RESULTS: The DLEC1 over-expression was found to reduce the number of colonies in colony formation and to induce G1 arrest in seven clones, and apoptosis in one clone in the cell cycle analysis. Furthermore, regardless of the different cell cycle defects in all eight stable clones, the expression level of transcriptional factor AP-2α2 was found to be elevated. More interestingly, we found that when AP-2α2 was knocked down, DLEC1 over-expression neither suppressed cancer cell growth nor induced G1 arrest, yet, instead promoted cell growth and decreased cells in the G1 fraction. This promotion of cell proliferation and release of G1 cells also seemed to be proportional to DLEC1 expression levels in DLEC1 stable clones. CONCLUSIONS: DLEC1 suppresses tumor cell growth the presence of AP-2α2 and stimulates cell proliferation in the down-regulation of AP-2α2 in DLEC1 over-expression stable clones of HTC116.

Distinctive pharmacological differences between liver cancer cell lines HepG2 and Hep3B.

As cellular models for in vitro liver cancer and toxicity studies, HepG2 and Hep3B are the two most frequently used liver cancer cell lines. Because of their similarities they are often treated as the same in experimental studies. However, there are many differences that have been largely over-sighted or ignored between them. In this review, we summarize the differences between HepG2 and Hep3B cell lines that can be found in the literature based on PubMed search. We particularly focus on the differential gene expression, differential drug responses (chemosensitivity, cell cycle and growth inhibition, and gene induction), signaling pathways associated with these differences, as well as the factors in governing these differences between HepG2 and Hep3B cell lines. Based on our analyses of the available data, we suggest that neither HBx nor p53 may be the crucial factor to determine the differences between HepG2 and Hep3B cell lines although HBx regulates the expression of the majority of genes that are differentially expressed between HepG2 and Hep3B. Instead, the different maturation stages in cancer development of the original specimen between HepG2 and Hep3B may be responsible for the differences between them. This review provides insight into the molecular mechanisms underlying the differences between HepG2 and Hep3B and help investigators especially the beginners in the areas of liver cancer research and drug metabolism to fully understand, and thus better use and interpret the data from these two cell lines in their studies.

Computed tomography scan use variation: patient, hospital, and geographic factors.

OBJECTIVES: To examine patient, hospital, and geographic characteristics influencing variation in computed tomography (CT) scan use in inpatients in New York State. STUDY DESIGN: Retrospective cohort study. METHODS: We used the 2007 healthcare cost and utilization project's state inpatient database from the agency for healthcare research and quality and applied descriptive univariate statistics and logistic regression models to quantify the influence of each factor on CT scan use. RESULTS: The primary contributors to variation in CT scan use were the inpatients' diagnosis, age, and hospital county, whereas inpatients' sex and method of payment and hospitals' teaching status and size had very little effect. Inpatients diagnosed with trauma had the highest CT scan use; CT scan use increased with age for inpatients over 30 years; and CT scan use varied widely between counties. CONCLUSIONS: After controlling for patient and hospital characteristics, significant geographic variation remained at the level of the county, which indicates that additional research investigating the use of CT scans is necessary to understand the reasons behind small-area variation. Understanding the distribution and practice patterns of specific physician specialties may be helpful in curtailing underuse and overuse.

Elite model for the generation of induced pluripotent cancer cells (iPCs).

The inefficiency of generating induced pluripotent somatic cells (iPS) engendered two contending models, namely the Stochastic model and Elite model. Although the former is more favorable to explain the inherent inefficiencies, it may be fallible to extrapolate the same working model to reprogramming of cancer cells. Indeed, tumor cells are known to be inherently heterogeneous with respect to distinctive characteristics thus providing a suitable platform to test whether the reprogramming process of cancer cells is biased. Here, we report our observations that all randomly picked induced pluripotent cancer cells (iPCs) established previously do not possess mutations known in the parental population. This unanticipated observation is most parsimoniously explained by the Elite model, whereby putative early tumor progenies were selected during induction to pluripotency.

Recognition and suppression of transfected plasmids by protein ZNF511-PRAP1, a potential molecular barrier to transgene expression.

Nonviral vectors present considerable advantages over viral counterparts in gene transfer. However, the poor expression efficiency of the transfected genes poses a challenge for their use in gene therapy, primarily due to the inability of these vectors to overcome various barriers, including the biological barriers. Here, we report that ZNF511-PRAP1 may be involved in the recognition and inactivation of transfected plasmids. ZNF511-PRAP1 is induced by transfection of plasmid DNA and suppresses the transcription of transfected plasmids. It binds directly to the p21 promoter in transfected plasmids but not the endogenous counterpart. Similarly, ZNF511-PRAP1 suppresses the expression of the green fluorescent protein reporter gene on transiently transfected plasmids but not an integrated red fluorescence reporter gene with the same cytomegalovirus (CMV) promoter. Therefore, ZNF511-PRAP1 is able to differentiate between exogenous/nonintegrated and endogenous/integrated DNA. The suppression by ZNF511-PRAP1 is independent of DNA methylation and can be abolished by trichostatin A (TSA) treatment and knockdown of HDAC2 and/or ZNF511-PRAP1. Furthermore, ZNF511-PRAP1 interacts directly with HDAC2. Our results revealed that transfected plasmids are recognized by ZNF511-PRAP1 and suppressed by a repressor complex comprising ZNF511-PRAP1 and HDAC2 and suggest that ZNF511-PRAP1 could play a role as a potential molecular barrier in nonviral transgene expression.

[Analysis on heavy metal concentrations in agricultural soils of Baoshan, Shanghai].

Based on the acquisition of heavy metal data from 216 topsoil samples of the agricultural land in Baoshan District, a typical region of Shanghai City, the content, distribution characteristics and sources of the heavy metals in agricultural soil of Shanghai Baoshan District were analyzed with the methods of combining multivariate statistics and geo-statistical. The results show that the average concentrations of the Cd, Hg, As, Cr, Pb, Cu and Zn in agricultural soil of Baoshan District are 0.195, 0.148, 7.44, 82.5, 29.1, 33.2 and 124.5 mg x kg(-1), they are lower than the secondary standards of the national soil environment; but the average concentrations of the Cd, Hg, Cr, Pb, Cu and Zn are more than background values of soil in Shanghai. Especially the Cd, Hg, Zn, they are 1.50, 1.48, 1.45 times higher than the background values, showing a net cumulative trend. The results of correlation analysis and factor analysis show that the sources of these elements can be divided into three categories; the Zn, Cd, Hg and Pb as one class; the Cr and Cu as another class; the As in a separate category. The concentrations of the former two classes are much higher than the background value, implying mainly from various human activities; the content of the As is almost the same as the background value of soil, it has the lowest degree of variation. Spatial structure analysis shows that the distribution of the As is influenced by the soil structural variations, such as soil parent material, topography, and other elements are mainly affected by random factors of human activities. Through the probability distribution of contour lines of the critical value, it can be found that the Cd, Zn, Cr, Cu, Hg come mainly from the point sources of pollution, but the source of Pb is relatively dispersed.

The identification of lymphocyte-like cells and lymphoid-related genes in amphioxus indicates the twilight for the emergence of adaptive immune system.

To seek evidence of a primitive adaptive immune system (AIS) before vertebrate, we examined whether lymphocytes or lymphocyte-like cells and the related molecules participating in the lymphocyte function existed in amphioxus. Anatomical analysis by electron microscopy revealed the presence of lymphocyte-like cells in gills, and these cells underwent morphological changes in response to microbial pathogens that are reminiscent of those of mammalian lymphocytes executing immune response to microbial challenge. In addition, a systematic comparative analysis of our cDNA database of amphioxus identified a large number of genes whose vertebrate counterparts are involved in lymphocyte function. Among these genes, several genes were found to be expressed in the vicinity of the lymphocyte-like cells by in situ hybridization and up-regulated after exposure to microbial pathogens. Our findings in the amphioxus indicate the twilight for the emergence of AIS before the invertebrate-vertebrate transition during evolution.

New insights on macrophage migration inhibitory factor: based on molecular and functional analysis of its homologue of Chinese amphioxus.

Macrophage migration inhibitory factor (MIF) is an intricate cytokine. Many questions about it are not fully resolved. In order to identify the role of MIF in Chinese amphioxus, its genomic organization, transcription pattern and enzymatic activity were studied. It's found that MIF has multi-copy gene number in the Chinese amphioxus genome and special transcription pattern in reproductive organs. Interestingly, the recombinant Bbt-MIF has tantomerase and redox activity, but fails to utilize GSH to reduce insulin instead of DTT, strikingly different from MIF in mammalian. All these results indicate that MIF gene must have undergone important changes in structure and function during the transition of invertebrate/vertebrate and might exert important role in this primitive species, which may be quite different from those found in vertebrate.

Molecular cloning and characterization of CD9 cDNA from cartilaginous fish, red stingray, Dasyatis akajei.

CD9 is a glycoprotein of the transmembrane 4 superfamily (TM4SF) and is involved in various cellular processes. In this study, we describe the isolation of the full-length cDNA encoding for CD9 molecule (daCD9) of red stingray, Dasyatis akajei. This 1252 bp cDNA was isolated from leukocyte cDNA library and contains 681 bp open reading frame encoding 226 amino acid residues. Amino acid sequences analysis and structure prediction display approximately 50% identity to higher vertebrates with the presence of conserved structures, including the four transmembrane domains and certain characteristic residues. Southern blot analysis shows that daCD9 exists as a single copy gene. Northern blot analysis reveals that daCD9 is highly expressed in gill and spleen although its expression can be found in other tissues suggesting daCD9 might play an important role in immune defense in this fish.

The phylogenetic analysis of tetraspanins projects the evolution of cell-cell interactions from unicellular to multicellular organisms.

In animals, the tetraspanins are a large superfamily of membrane proteins that play important roles in organizing various cell-cell and matrix-cell interactions and signal pathways based on such interactions. However, their origin and evolution largely remain elusive and most of the family's members are functionally unknown or less known due to difficulties of study, such as functional redundancy. In this study, we rebuilt the family's phylogeny with sequences retrieved from online databases and our cDNA library of amphioxus. We reveal that, in addition to in metazoans, various tetraspanins are extensively expressed in protozoan amoebae, fungi, and plants. We also discuss the structural evolution of tetraspanin's major extracellular domain and the relation between tetraspanin's duplication and functional redundancy. Finally, we elucidate the coevolution of tetraspanins and eukaryotes and suggest that tetraspanins play important roles in the unicell-to-multicell transition. In short, the study of tetraspanin in a phylogenetic context helps us understand the evolution of intercellular interactions.

Genes "waiting" for recruitment by the adaptive immune system: the insights from amphioxus.

In seeking evidence of the existence of adaptive immune system (AIS) in ancient chordate, cDNA clones of six libraries from a protochordate, the Chinese amphioxus, were sequenced. Although the key molecules such as TCR, MHC, Ig, and RAG in AIS have not been identified from our database, we demonstrated in this study the extensive molecular evidence for the presence of genes homologous to many genes that are involved in AIS directly or indirectly, including some of which may represent the putative precursors of vertebrate AIS-related genes. The comparative analyses of these genes in different model organisms revealed the different fates of these genes during evolution. Their gene expression pattern suggested that the primitive digestive system is the pivotal place of the origin and evolution of the AIS. Our studies support the general statement that AIS appears after the jawless/jawed vertebrate split. However our study further reveals the fact that AIS is in its twilight in amphioxus and the evolution of the molecules in amphioxus are waiting for recruitment by the emergence of AIS.

Macrophage migration inhibitory factor (MIF) in chinese amphioxus as a molecular marker of immune evolution during the transition of invertebrate/vertebrate.

Macrophage migration inhibitory factor (MIF) is an important cytokine related to host defenses and autoimmune diseases. Here, we reported two full-length cDNA clones isolated from Chinese amphioxus (Branchiostoma belcheri tsingtaunese). Amino acid sequences analysis and structure prediction of these two molecules, called Bbt-MIF-I and Bbt-MIF-II, respectively, indicated that several conservative domains existed in the two amphioxus MIFs and their sequences were highly homologous to their counterparts of other species. Intriguingly, the Bbt-MIFs gene is present in multi-copy per haploid genome, which is very unusual compared with vertebrate's MIF gene given the known genome duplication theory. The genomic copy number, expression pattern of MIF gene and phylogenetic analysis of MIF proteins all suggested that a leap forward happened for MIF gene during the evolution from invertebrate to vertebrate. Considering the crucial role of MIF in innate immunity, MIF might serve as one of key molecular markers of evolution of immune system.

Functional expression and characterization of an acidic actinoporin from sea anemone Sagartia rosea.

Src I is the first reported acidic actinoporin from sea anemone Sagartia rosea with a pI value of 4.8 and comprises 13.9% alpha-helix, 65.1% beta-sheet, and 18.2% random coil. For structure-function studies, Src I was expressed in Escherichia coli as a cleavable fusion protein. Recombinant Src I exhibited obviously hemolytic activity, but the fusion protein Trx-Src I almost lost its hemolytic activity, suggesting the importance of the N-terminal amphiphilic alpha-helix for its functional activity. The cytotoxic effects of Src I depending on the toxin concentration and incubation time were also observed on cultured cells. Among five cell lines: NIH/3T3, U251, NSCLC, BEL-7402, and BGC-823, NSCLC was the most sensitive cells with ID(50) 2.8 microg/ml and BGC-823 was the least sensitive cells with ID(50) 7.4 microg/ml. After incubated with lipid SUVs, such as SM-SUVs and SM/PC-SUVs, the hemolytic activity of Src I was inhibited to some extent. When incubated with calcein-entrapped lipid LUVs, such as SM-LUVs, SM/PC-LUVs, and SM/PG-LUVs, Src I induced release of entrapped calcein. According to the interaction with lipid vesicles, we proposed that it was the membrane matrix made up of phospholipids, not a particular phospholipid that facilitates Src I to react properly.

cDNA cloning, identification and characterization of a novel cystatin from the tentacle of Cyanea capillata.

Cystatin is of interest from biochemical and evolutionary prospective, and also has been applied in biotechnology. In this paper, a novel cystatin was found by EST sequence analysis of the cDNA library of Cyanea capillata tentacle. The sequence of a full-length cDNA clone contained an open reading frame encoding a putative 18-residue signal peptide and a mature protein of 113 amino acids, which showed only 26% identities to Family 2 cystatins and had its own characteristic enzyme-binding motifs, Ser(97)-Trp(98), which had not been found in any other known cystatins. Thus, the novel cystatin cloned from jellyfish was designated as cystatin J, which may belong to a new family of cystatin, called Family 4. The mature cystatin J was produced in Escherichia coli as a thioredoxin (Trx) fusion protein using the pET expression system and purified by affinity and cation exchange chromatography. The recombinant cystatin J of approximately M(r) = 12,800 displayed an obvious inhibition of papain (K(i) value below 0.5 nM), in competition with substrate. Thus, the recombinant cystatin J was a functional cystatin in spite of relatively lower sequence similarity with other cystatins. Activity of the novel cystatin was stable at pH 4-11 at 4 degrees C, but unstable at neutral pH at >50 degrees C.

EST analysis of gene expression in the tentacle of Cyanea capillata.

Jellyfish, Cyanea capillata, has an important position in head patterning and ion channel evolution, in addition to containing a rich source of toxins. In the present study, 2153 expressed sequence tags (ESTs) from the tentacle cDNA library of C. capillata were analyzed. The initial ESTs consisted of 198 clusters and 818 singletons, which revealed approximately 1016 unique genes in the data set. Among these sequences, we identified several genes related to head and foot patterning, voltage-dependent anion channel gene and genes related to biological activities of venom. Five kinds of proteinase inhibitor genes were found in jellyfish for the first time, and some of them were highly expressed with unknown functions.

EST analysis of mRNAs expressed in neurula of Chinese amphioxus.

Amphioxus, a cephalochordate, is the closest living relative to the vertebrates. In order to investigate the molecular mechanisms of the early embryogenesis of amphioxus, we constructed a neurula embryo cDNA library of Chinese amphioxus (Branchiostoma belcheri tsingtauense) and generated 5235 expressed sequenced tags in the present study. The initial ESTs consisted of 638 clusters and 1855 singletons, which revealed approximately 2493 unique genes in the data set. Of these sequences, 35.52% ESTs matched to known genes, 12.76% matched to other ESTs, and 51.71% had no match to any known sequences in GenBank. Interestingly we found homologous genes related to neural development and human disease. Bioinformatic analysis showed the direct evidence that the gene homologue found only in vertebrates in previous studies also exists in the amphioxus genome. This study provides a preliminary view of the gene information involved in the development of neurula embryos of Chinese amphioxus and helps our understanding of vertebrate evolution at gene level.