Expression and regulation of 42Sp50 in spotted scat (Scatophagus argus).

42Sp50 is an isoform of the eukaryotic translation elongation factor 1 A (eEF1A) and is vital for fish ovarian development. Spotted scat (Scatophagus argus) is a popular marine cultured fish species in Southern Asia and China, and its artificial reproduction is complicated, with a relatively low success ratio in practice. In this study, the 42Sp50 gene was cloned from spotted scat. Tissue distribution analysis showed that 42Sp50 was mainly expressed in the ovary. qRT-PCR showed that 42Sp50 expression levels gradually decreased insignificantly in the ovaries from phase II to IV. Western blot analysis showed that 42Sp50 was highly expressed in the ovary, while it was almost undetectable in the testis. Immunohistochemistry analysis stained 42Sp50 mainly in the cytoplasm of the previtellogenic oocytes in ovaries of normal XX-female and sex-reversed XY-female. Aside from fish and amphibians, 42Sp50 was also identified in some reptile species using genomic database searching. Analyses of the transcriptome data from four different fish species (Hainan medaka (Oryzias curvinotus), silver sillago (Sillago sihama), Nile tilapia (Oreochromis niloticus), and Hong Kong catfish (Clarias fuscus)) revealed ovaries biased expression of 42Sp50 in all, similar to spotted scat. While the neighbor genes of 42Sp50 did not show ovary biased expression in the fish species analyzed. Bisulfite Sequencing PCR (BSP) results showed that the DNA methylation level of 42Sp50 promoter was low in ovaries, testes, and muscles. The luciferase reporter assay demonstrated that Dmrt4 activated 42Sp50 expression in the presence of Sf1 or Foxh1. These results suggest that 42Sp50 may be involved in regulating the early phase oocytes development of spotted scat.

Molecular cloning and expression of glutathione S-transferases involved in propargite resistance of the carmine spider mite, Tetranychus cinnabarinus (Boisduval).

The carmine spider mite (CSM) Tetranychus cinnabarinus has become a serious pest in China and has developed resistance to acaricide propargite as it is used to control mites worldwide including T. cinnabarinus. In this study, a resistant colony of T. cinnabarinus, PRR34 (37.78-fold resistant ratio), was established after 34 generations of propargite selection, and cross-resistance patterns of 7 other acaricides were determined in comparison with a susceptible strain (SS). The contribution of detoxification enzymes to propargite tolerance were investigated using biological, biochemical and molecular approaches. Enzyme inhibitor synergist tests suggested glutathione S-transferases (GST) involvement in propargite-resistance of PRR34, and GST activity against 1-chloro-2,4-dinitrobenzene (CDNB) was correlated with the development of resistance. Eight novel GST genes (TcGSTd1, TcGSTd2, TcGSTm1, TcGSTm2, TcGSTm3, TcGSTm4 and TcGSTm5) were cloned, and phylogenetic analysis showed that the eight GST genes were most closely related to GST family delta and mu from Tetranychusurticae. Quantitative RT-PCR revealed that the expression level of GSTs in PPR34 strain increased in larvae, nymphs and adults, while decreased in eggs compared with that of SS. Collectively, these results support a role of GSTs in mediating resistance to propargite in the PRR34 strain. TcGSTd1,TcGSTd2 and TcGSTm2 genes might play significant roles in propargite resistance of CSM, especially at adult stage. This is the first attempt to define specific genes involved in GST mediated propargite resistance of T. cinnabarinus at the transcriptional level.

[Research on treatment of high salt wastewater by the graphite and activated carbon fiber composite electrodes].

High salinity wastewater is one of the difficulties in the field of wastewater treatment. As a new desalination technology, electrosorption technology has many advantages. This paper studied a new type of carbon-based electrodes, the graphite and activated carbon fiber composite electrodes. And the influencing factors of electrosorption and its desalination effect were investigated. The electrosorption device had optimal desalination effect when the voltage was 1. 6 V, the retention time was 60 min and the plate spacing was 1 cm. The graphite and activated carbon fiber composite electrodes were used to treat the black liquor of refined cotton and sodium copper chlorophyll wastewater to investigate its desalination effect. When the electrodes were used to treat the black liquor of refined cotton after acid treatment, the removal rate of conductivity and COD reached 58. 8% and 75. 6% respectively when 8 pairs of electrodes were used. And when the electrode was used to treat the sodium copper chlorophyll wastewater, the removal rate of conductivity and COD reached higher than 50. 0% and 13. 5% respectively when 6-8 pairs of electrodes were used.

Association of MDR1 gene polymorphisms with susceptibility to hepatocellular carcinoma in the Chinese population.

OBJECTIVE: The objective of this study was to evaluate the association of MDR1 gene polymorphisms with susceptibility to hepatocellular carcinoma (HCC). METHODS: A total of 689 HCC patients and 680 cancer-free subjects were enrolled. Human MDR1 gene polymorphisms were investigated by created restriction site- polymerase chain reaction (CRS-PCR) and DNA sequencing methods. Multiple logistic regression models were applied to estimate the association between MDR1 gene polymorphisms and susceptibility to HCC. RESULTS: We detected a novel c.4125A>C polymorphism and our findings suggested that this variant was significantly associated with susceptibility to HCC. A significantly increased susceptibility to HCC was noted in the homozygote comparison (CC versus AA: OR=1.621, 95% CI 1.143-2.300, χ2=7.4095, P=0.0065), recessive model (CC versus AC+AA: OR=1.625, 95% CI 1.167-2.264, χ2=8.3544, P=0.0039) and allele contrast (C versus A: OR=1.185, 95% CI 1.011-1.389, χ2=4.4046, P=0.0358). However, no significant increase was observed in the heterozygote comparison (AC versus AA: OR=0.995, 95% CI 0.794-1.248, χ2=0.0017, P=0.9672) and dominant model (CC+AC versus AA: OR=1.106, 95% CI 0.894-1.369, χ2=0.8560, P=0.3549). CONCLUSIONS: These findings suggest that the c.4125A>C polymorphism of the MDR1 gene might contribute to susceptibility to HCC in the Chinese population. Further work will be necessary to clarify the relationship between the c.4125A>C polymorphism and susceptibility to HCC on larger populations of diverse ethnicity.

[Anti-hepatocarcinoma effect of recombinant adenovirus carrying melittin gene].

OBJECTIVE: To find a new method of treating hepatocellular carcinoma with melittin by way of using the melittin gene. METHODS: The recombinant adenoviruses carrying the melittin gene and alpha-fetoprotein (AFP) promoter (Ad-rAFP-Mel) were constructed through a bacterial homologous recombinant system. The efficiency of the adenovirus mediated gene transfer and the inhibition effect of Ad-rAFP-Mel on the proliferation of hepatocarcinoma cells were determined by X-gal staining and MTT assay respectively. The tumorigenicity of hepatocarcinoma cells transfected by Ad-rAFP-Mel and the antitumor effect of Ad-rAFP-Mel on the transplanted tumors in nude mice were detected in vivo. RESULTS: The mRNA of the melittin gene was transcripted in HepG2 hepatocellular carcinoma cells transducted by Ad-rAFP-Mel. The efficiency of adenovirus mediated gene transfered to BEL-7402 hepatocarcinoma cells was 100% when the multiplicities of infection (MOI) of Ad-rAFP-Mel was 10 in vitro and was high in vivo as well. The inhibitive rates of Ad-rAFP-Mel and Ad-rAFP for BEL7402 cells were 66.2%+/-2.7% and 2.9%+/-2.3% (t = 30.83) by MTT assay. The inhibitive rates of Ad-CMV-Mel for BEL7402, SMMC7721 and L02 cells were 58.9%+/-9.6%, 65.9%+/-3.8%, 31.7%+/-1.2%, respectively, and those of the Ad-rAFP-Mel were 6.2%+/-2.7%, 16.1%+/-6.6%, 7.5%+/-3.3%, respectively (t = 1.27; t = 11.31, and t = 12.12, vs. Ad-CMV-Mel group in same cells). The tumorigenicity rates of hepatocarcinoma cells transfected by Ad-rAFP-Mel were decreased. A significant antineoplastic effect was detectd on transplanted tumor in nude mice by intratumoral injection of Ad-rAFP-Mel. CONCLUSION: Ad-rAFP-Mel can inhibit specifically the proliferation of AFP-producing human hepatocarcinoma cells in vitro and in vivo. It suggests that animal toxin gene can be used as an interesting antitumor gene.

[The induced apoptosis of recombinant adenovirus carrying melittin gene for hepatocellular carcinoma cell].

OBJECTIVE: To observe the induced apoptosis of recombinant adenovirus carrying melittin gene (Ad-rAFP-Mel) for hepatocellular carcinoma cell line (BEL-7402). METHODS: The morphological observe, DNA electrophoresis, TUNEL and Flow cytometry assay were used to study the apoptosis of BEL-7042 cell line transfected by Ad-rAFP-Mel. RESULTS: The morphological changes and apoptosis of BEL-7402 transfected by Ad-rAFP-Mel were confirmed with microscopy and DNA electrophoresis, TUNEL, Flow cytometry assay. The DNA ladder could be demonstrated on DNA electrophoresis in Ad-rAFP-Mel group. The apoptosis rates of BEL-7402 cells in Ad-rAFP-Mel, Ad-rAFP, and control groups were (21.5+/-2.4)%, (10.5+/-4.4)% and (3.0+/-1.4)% respectively by TUNEL assay (F = 38.0, P < 0.05) and were (7.3+/-0.5)%, (3.9+/-0.1)% and (0.8+/-0.1)% respectively by flow cytometry assay (F = 415.1, P < 0.05). CONCLUSION: It seems that melittin inducing apoptosis might be one of the antitumor mechanisms.

Impact of infection with recombinant adenovirus carrying melittin gene on CD54 expression in HepG2 cells.

OBJECTIVE: To observe the effects of melittin gene transfection on the biological behavior of hepatocarcinoma cells. METHODS: Melittin gene was recombined into a shuttle plasmid under the control by alpha-fetoprotein (AFP) transcription regulatory element (rAFP), and the gene of interest was then recombined into an adenoviral backbone plasmid through a simplified efficient bacterial homologous recombination system. The recombinant adenoviral plasmids were subsequently linearized with Pac I and transfected into 293 cells mediated by lipofectin to generate the desired recombinant adenoviruses. After infections of the hepatocarcinoma cells with the resultant viruses carrying melittin gene was achieved, melittin gene transcription was verified by way of RT-PCR, and CD54 expression was measured by flow cytometry. RESULTS: Recombinant adenoviral vectors containing melittin gene were successfully constructed, and melittin gene transcription was approved by RT-PCR. Flow cytometry results showed that melittin gene transfection inhibited CD54 expression in the hepatocarcinoma cells. CONCLUSION: Melittin gene transfection may result in changes of the biological behavior of hepatocarcinoma cells, thus decreasing the malignancy of the tumor cells.