Effects of sodium arsenite on metallothionein isoforms gene expression in cultured human lymphocytes / 中国地方病学杂志

Objective To explore the effects of sodium arsenie on Metallothionein(MT) isoforms genes expression,and to study the relevance between the MT expression and cell survival percentage. Methods Healthy persons blood was extracted aseptically and the lymphocytes were separated. The lymphocytes were treated by 0 (control) ,2,5,10,15,30,60 μmol/L sodium arsenites, respectively. The cell survival percentage was determined by methyl thiazolyl tetrazolium(MTT) reduction assay at 24,48,72 h intervals, while the expression of MT-1 and MT-2 were examined by RT-PCR in 72 h. Results The cell survival percentage in 2,5μmoL/L groups were (115.50± 11.80)% and (130.49±8.28)%,which were all higher than those in the control group [(100.00±0.00)%,all P < 0.05]at any intervals. In 10,15,30,60 μmol/L groups,the cell survival percentage[(78.12±9.33)%,(71.62± 10.82)%,(52.06±3.05)%,(40.98±5.41)%]increased along with the decrease of concentration,which showed a significant difference between these groups and the control group and 2,5μmol/L groups(all P < 0.05). There was no significant difference on MT-1 expression after exposed to different concentration sodium arsenites (0,2,5,10,15,30,60 μmol/L) for72 h(0.925±0.123,1.082±0.504,1.103±0.170,0.927±0.056,0.730±0.307,0.604± 0.173,0.540±0.075,all P > 0.05). The expression of MT-2 in 2,5μmol/L groups(1.503±0.212,1.557±0.377) was up regulated compared with that in control group(0.702±0.112) and MT-2 also expressed more than that in other groups(all P < 0.05). However,the expression of MT-2 declined when the concentraion was 10,15,30,60 μmol/L(0.814±0.139,0.679±0.201,0.607±0.229,0.533±0.102). There was no significant difference among the groups (all P > 0.05). The expression level oE MT-1,MT-2 was positively correlated with the cell survival pereentage(r = 0.955,0.909,all P < 0.05). Conclusions The sodium arsenite at concentration of 10 μmoL/L might inhibit the expression of MT of lymphocytes and low concentration sodium arsenite (2,5 μmol/L) might stimulate the lymphocytes to regulate the expression of MT-2 to higher levels,which can increase the cell survival percentage and exert the function of protecting cells.

Analysis on the arsenic speciation in urine of rats treated with sodium arsenite and sodium arsenate / 中国地方病学杂志

Objective To analyze the levels and speciation of arsenic metabolites in urine of rats treated with sodium arsenite and sodium arsenate in order to investigate the different aspects of metabolism between sodium arsenite and sodium arsenate,thus to understand further the basic data about relationship between it's metabolism and mechanism of toxicity. Methods Seventy Wistar rats,weighting 80-120 g,were divided into 7 groups of 10 each,such as normal control group,high,middle and low sodium arsenite group and high,middle and low sodium arsenate group. After the animals were fed for one month,the urine was collected by metabolic cage in 12 hours. Applying the high efficiency liquid chromatography and hydride genesis atomic fluorescence spectroscopy (HPLC-HGAFS),the levels and speciation of arsenic metabolites were determined in urine of rats. Meanwhile,the recovery rate of dimethyl arsinic acid(DMA) would be determined to estimate the degree of accuracy of results. Results The levels of iAs~(3+),iAs~(5+) and DMA in middle sodium arsenite group[(121.66±1.26),(10.26±2.68),(200.91±0.56) μg/L]were higher than the high sodium arsenite group[(113.20±0.75),(5.16±1.32),(147.70±μ0.77)μg/L,all P < 0.05]and low sodium arsenite group[(79.35±2.12),(5.13±2.25),(56.35±1.23)μg/L,all P < 0.05]. The levels of iAs~(3+) and DMA in middle sodium arsenate group[(315.81±1.69),(245.12±1.18)μg/L]were higher than the high sodium arsenate group[(85.03±0.56),(110.34±1.04)μg/L,all P< 0.05]and low sodium arsenate group[(22.97±2.67),(15.75±2.15)μg/L,all P < 0.05]. Compared with sodium arsenate group,the levels of iAs~(3+) and DMA in high and low sodium arsenite group were higher(all P < 0.05) ; and the levels of iAs~(3+) and DMA in middle sodium arsenite group were lower(all P < 0.05). Meanwhile,the average urinary recovery rate of DMA of rats in different sodium arsenite group were 94.80%-102.70%,and the average urinary recovery rate of DMA of rats in different sodium arsenate group were 95.33%-108.40%. Conclusion The speciation and levels of arsenic are influenced by the external exposure dose,and some distinction appeared in the metabolism and metabolic path between sodium arsenite and sodium arsenate in urine in vivo.

Screening and cloning of hepatitis C virus non-structural protein 4B interacting protein gene in hepatocytes / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate biological functions of non-structural protein 4B (NS4B) of hepatitis C virus (HCV), yeast-two hybrid technique was performed to seek proteins in hepatocytes interacting with HCV NS4B.</p><p><b>METHODS</b>HCV NS4B bait plasmid was constructed by ligating the NS4B gene with carrier plasmid pGBKT7 and transformed into yeast cells AH109 (type alpha). The transformed yeast cells were amplified and mated with yeast cells Y187 (alpha type) containing liver cDNA library plasmid pACT2 in 2 x YPDA medium. Diploid yeast cells were plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) and synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing x-alpha-gal for selecting two times. After extracting plasmid from blue colonies, plasmid DNA was transformed into competent Escherichia coli and analysed by DNA sequencing and bioinformatics.</p><p><b>RESULTS</b>Five genes in eight positive colonies were obtained. There were one NADH dehydrogenase subunit 3, one cytochrome c oxidase subunit III, one retinol binding protein 4, one reticulon 3-A (RTN3) and one fibrinogen gamma polypeptide (FGG).</p><p><b>CONCLUSION</b>Genes of HCV NS4B interacting proteins in hepatocytes were successfully cloned and the results paved the way for studying the biological functions of NS4B and associated proteins.</p>

Screening and cloning target genes transactivated by hepatitis C virus F protein using suppression subtractive hybridization technique / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To identify and clone human genes transactivated by HCV F protein by constructing a cDNA subtractive library using the suppression subtractive hybridization technique.</p><p><b>METHODS</b>Suppression subtractive hybridization (SSH) and bioinformatics techniques were used for screening and cloning of the target genes transactivated by HCV F protein. The mRNA was isolated from HepG2 cells transfected with pcDNA3.1 (-)-F or with pcDNA3.1(-) empty vector as a control, and SSH method was employed to analyze the differentially expressed DNA sequence between the two groups. After restriction enzyme Rsa I digestion, small sized cDNAs were obtained. Then tester cDNA was divided into two groups and ligated to the specific adaptor 1 or adaptor 2. After tester cDNA was hybridized with driver cDNA twice and underwent two times of nested PCR, it was then subcloned into T/A plasmid vectors to set up the subtractive library. Amplification of the library was carried out with E. coli strain DH5 alpha. The cDNA was sequenced and analyzed in GenBank with blast search after PCR.</p><p><b>RESULTS</b>The subtractive library of genes transactivated by HCV F protein was constructed successfully. The amplified library contains 71 positive clones. Colony PCR shows that 56 clones contain 200-1000 bp inserts. Sequence analysis was performed on 28 clones randomly, and the full length sequences were obtained with using the bioinformatics method. Altogether 19 coding sequences were obtained, consisting of 17 known and 2 unknown.</p><p><b>CONCLUSIONS</b>The obtained sequences may be target genes transactivated by HCV F protein, and some gene coding proteins are those involved in cell cycle regulation, metabolism, and cell apoptosis.</p>

Screening and cloning of hepatitis C virus non-structural protein 4A interacting protein gene in hepatocytes / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate biological functions of hepatitis C virus (HCV) non-structural protein 4A (NS4A).</p><p><b>METHODS</b>Yeast-two hybrid technique was performed to seek proteins in hepatocytes interacting with HCV NS4A. HCV NS4A bait plasmid was constructed by ligating the NS4A gene with carrier plasmid pGBKT7, then it was transformed into yeast AH109 (alpha type). The transformed yeast cells were amplified and mated with yeast cells Y187 (alpha type) containing liver cDNA library plasmid pACT2 in 2 x YPDA medium. Diploid yeast cells were plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) and synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing X-alpha-gal for selection two times. After extracting plasmid from blue colonies, plasmid DNA was transformed into competent E.coli and analyzed by DNA sequencing and bioinformatics methods.</p><p><b>RESULTS</b>Among twenty-two positive colonies there were eleven positive for metallothionein 2A, three for eukaryotic translation elongation factor 1 alpha 1, two for albumin, two for RNA binding motif protein 21, two for myomesin, one for cytochrome C oxidase II, and one for ATPase.</p><p><b>CONCLUSIONS</b>Genes of HCV NS4A interacting proteins in hepatocytes were successfully cloned and the results pave the way for studying the biological functions of NS4A and associated proteins.</p>