Acta Biochimica et Biophysica Sinica - current issue

The anti-viral protein of trichosanthin penetrates into human immunodeficiency virus type 1

Zhao, W., Feng, D., Sun, S., Han, T., Sui, S. — Mon, 25 Jan 2010 07:33:58 PST

Trichosanthin (TCS) is a type I ribosome-inactivating protein with potent inhibitory activity against human immunodeficiency virus type 1, and has been clinically applied in acquired immunodeficiency syndrome (AIDS) therapy. Previous studies revealed that TCS recognized human immunodeficiency virus type 1 (HIV-1) particles. Here, we investigated the physical relationship between TCS and HIV-1 particles, and demonstrated that TCS penetrates into viral particles, where it is protected from various protease digestion. The penetration of TCS exerts no obvious effect on viral integrity. FYY140–142, D176, and K177 were identified as key amino acid residues for the membrane-translocation process. Moreover, TCS penetrated into HIV-1 virions showed potent anti-viral activity. Overall, the observations suggest that the penetration of TCS into HIV-1 particles may be important for eliminating the virus.

Investigation into the regulation mechanisms of TRAIL apoptosis pathway by mathematical modeling

Zhang, T., Wu, M., Chen, Q., Sun, Z. — Mon, 25 Jan 2010 07:33:58 PST

TNF-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in tumor cell lines, suggesting its great potential in cancer therapy. Many components in TRAIL-mediated signaling pathway have been identified, but how they interact with each other to determine the network dynamics and final apoptosis remains elusive. Here we constructed a mathematical model of this pathway, which integrated current available information from related experimental literatures, to make a systematic and quantitative description of the cellular responses to TRAIL stimulation. By applying sensitivity analysis, we identified the key components and reactions that have the highest impact on the network dynamics, and then analyzed the regulatory role of several key players in this pathway. To elucidate the function of TRAIL decoy receptors, we compared the competitive ligand binding hypothesis versus the pre-ligand binding hypothesis. Our results show that the pre-ligand binding hypothesis is more suitable for explaining the fact that over-expression of decoy receptors can inhibit apoptosis potently. These results, together with our investigation on other downstream proteins and feedback loops in this pathway, provide insights into the molecular mechanisms of the TRAIL-mediated apoptosis pathway.

Human amniotic epithelial cell feeder layers maintain mouse embryonic stem cell pluripotency via epigenetic regulation of the c-Myc promoter

Mon, 25 Jan 2010 07:33:59 PST

Mouse embryonic stem cells (ESCs) are typically cultured on a feeder layer of mouse embryonic fibroblasts (MEFs), with leukemia inhibitory factor (LIF) added to maintain them in an undifferentiated state. We have previously shown that human amniotic epithelial cells (hAECs) can be used as feeder cells to maintain mouse ESC pluripotency, but the mechanism for this is unknown. In the present study, we found that CpG islands 5' of the c-Myc gene remain hypomethylated in mouse ESCs cultured on hAECs. In addition, levels of acetylation of histone H3 and trimethylation of histone H3K4 in the c-Myc gene promoter were higher in ES cells cultured on hAECs than those in ES cells cultured on MEFs. These data suggested that hAECs can alter mouse ESC gene expression via epigenetic modification of c-Myc, providing a possible mechanism for the hAEC-induced maintenance of ESCs in an undifferentiated state.

Generation of sp3111 transgenic RNAi mice via permanent integration of small hairpin RNAs in repopulating spermatogonial cells in vivo

Shi, W., Shi, T., Chen, Z., Lin, J., Jia, X., Wang, J., Shi, H. — Mon, 25 Jan 2010 07:33:59 PST

The spermatid-specific gene, sp3111, is a new member of the four-transmembrane gene family. However, its reproductive biological function remains elusive. The aim of this study was to establish an sp3111-knockdown male mouse model for further studying the effect of suppression of sp3111 gene expression on its reproductive functions. Recombinant vectors of pSUPER-sp3111-shRNA, containing the transcribed functional small hairpin RNA sequence against different regions of mouse sp3111 mRNA, were constructed and identified. Then, the linearized recombinant vectors were injected into mature mouse testes and transfected to spermatogonial cells by electroporation to generate sp3111 transgenic RNAi mice. These electroporated male mice were mated with wild-type females 30 days after electroporation and their progenies were examined both by fluorescence microscopy and PCR. It was found that the suppression efficiency of RNAi on the testis expression of sp3111 mRNA in vivo was more than 30% and could be transmitted stably to F3 generation, and compared with the wild-type male mice, the mean number of offspring of the sp3111 RNAi male mice was reduced from 11 ± 1.3 to 6 ± 1.4. In conclusion, the intra-testicular microinjection technique could also be used to develop the transgenic RNAi mice, and sp3111 transgenic RNAi mouse model may provide the possibility to further study the sp3111 gene reproduction function in vivo.

Proliferation and differentiation potential of mouse adult hepatic progenitor cells cultured in vitro

Song, L., Wang, H., Gao, X., Shen, K., Niu, W., Qin, X. — Mon, 25 Jan 2010 07:33:59 PST

This study aimed to isolate the stem cells or progenitors, if exist, from normal adult mouse liver and investigate their potential of proliferation and differentiation. Hepatocytes were isolated by modified two-step liver perfusion method and centrifugation, and then cultured in modified serum-containing DMEM for observation more than 60 days. Immunofluorescence technique was applied to check the hepatocytes and to examine the formation of colonies with albumin, -fetoprotein (AFP) and cytokeratin 19 (CK19). Results showed that some hepatocytes that were strongly positive for hepatocyte specific markers albumin on Day 1 in culture, could be activated at Days 2–3, followed by rapid proliferation and formation of colonies. The colonies could expand continually for more than 60 days. On Day 5, all the cells in the colony expressed hepatic stem cell (HSC) markers AFP. With the time of culture, some cells in colonies lost ability to divide at Days 13–15, and differentiated into cells which had a large cytoplasm and some two nuclei, similar to the appearance of mature hepatocytes morphologically. These differentiated cells demonstrated strong expression of albumin. Around Day 30, some big cells appeared in colonies and expressed bile duct cell marker CK19. Therefore, this subpopulation of mouse hepatocytes could acquire some characteristics of immature hepatocytes and showed the profile of hepatic progenitor cells with a high proliferating ability and bi-potential of differentiation. They were isolated from normal adult mouse, hence, named adult hepatic progenitor cells (AHPCs). Mouse AHPCs may be used as an HSC model for hepatocytes transplantation and hepatopathy study.

Production of an anti-idiotypic antibody single chain variable fragment vaccine against Edwardsiella tarda

Qin, H., Jin, X., Huang, W., Liu, Y. — Mon, 25 Jan 2010 07:33:59 PST

Edwardsiella tarda is the pathogen responsible for edwardsiellosis, a serious infectious disease of freshwater and marine fish species, and currently recognized to be the species pathogenic for human. An anti-idiotypic monoclonal antibody (mAb), 1E11, has been developed. It mimics the protective epitope of E. tarda and can prevent fish from infection of E. tarda. In this study, the correct variable heavy (V_H) and variable light (V_L) genes were obtained from 1E11 by using bioinformatics methods, and a 15 amino acid (Gly₄Ser)₃ linker was used to hold the two V domains together for the construction of V_L–linker–V_H form of single chain variable fragment (scFv) gene. Then, the scFv was subcloned into the vector pET-28a, expressed in the Escherichia coli BL21 cells, and identified by SDS–PAGE and western blotting. Red drum (Sciaenops ocellatus L.) weighing about 50 g was subjected to challenge with different E. tarda strains after 4 weeks followed by vaccination, the mortality rates and relative percentage survival were recorded and calculated, and the survival rate of fish in the scFv subgroups was obviously higher than that of control subgroups (P < 0.01). Enzyme-linked immunosorbent assay results show that after 4 weeks of post-vaccination, the level of specific antibody in fish sera of scFv groups was significantly higher than control groups. This study indicates that the recombinant antibody scFv was successfully developed, and it may serve as an effective vaccine candidate against E. tarda.

Therapeutic potential of siRNA-mediated combined knockdown of the IAP genes (Livin, XIAP, and Survivin) on human bladder cancer T24 cells

Mon, 25 Jan 2010 07:33:59 PST

Livin, X-linked inhibitor of apoptosis (XIAP), and Survivin are three well-known inhibitors of apoptosis almost exclusively over-expressed in cancer cells and are considered potent targets for cancer treatment. In the present study, we found that Livin, XIAP, and Survivin were simultaneously expressed in bladder cancer cells. We speculated that Livin, XIAP, and Survivin might have synergistic effects on cell growth and apoptosis. Our results confirmed that combined knockdown of all these three genes can synergistically inhibit the proliferation and transformation ability of high-grade bladder cancer T24 cells and promote the cell apoptotic sensitivity to chemotherapy. Furthermore, combined knockdown of Livin, XIAP, and Survivin can markedly increase the abundance of active caspase-3, active caspase-7, active caspase-9, and cytosolic Smac. Our findings imply that combined silencing of Livin, XIAP, and Survivin may be a potent multi-targeted gene therapy for bladder cancer.

Comparative profiling of genes and miRNAs expressed in the newborn, young adult, and aged human epididymides

Mon, 25 Jan 2010 07:33:59 PST

To understand roles of transcriptional factors and miRNAs in regulating gene expression in the epididymis from postnatal development through aging, systematic profiling of genes and miRNAs expressed in the newborn, young adult, and aged human epididymides was performed by cDNA array and miRNA array analysis, respectively. The newborn human epididymis expressed the fewest mRNAs but the largest number of miRNAs, whereas the adult and aged epididymides expressed the most mRNAs but the fewest miRNAs, a negative correlation between mRNAs and miRNA during aging. By integrative analysis, a set of miRNA targets were predicted based on the miRNA and cDNA arrays. In the newborn epididymis, 127 miRNAs were exclusively or preferentially expressed but only 3 and 2 miRNAs showed an age-enriched expression pattern in the adult and aged epididymides, respectively. This study provides a basic database as well as new insights and foundations for further studies on the complex regulation of gene expression in the epididymis.

Identification and expression analysis of genes in response to high-salinity and drought stresses in Brassica napus

Chen, L., Ren, F., Zhong, H., Jiang, W., Li, X. — Mon, 25 Jan 2010 07:33:59 PST

High salinity and drought are the major abiotic stresses that adversely affect plant growth and agricultural productivity. To investigate genes that are involved in response to abiotic stresses in Brassica napus, a comprehensive survey of genes induced by high-salinity and drought stresses was done by macroarray analysis. In total, 536 clones were identified to be putative high-salinity- or drought-responsive genes. Among them, 172 and 288 clones are detected to be putative high-salinity- and drought-inducible genes, whereas 141 and 189 are candidates for high-salinity- and drought-suppressed genes, respectively. The functional classification of these genes are indicated that belonged to gene families encoding metabolic enzymes, regulatory factors, components of signal transduction, hormone responses, some abiotic stresses-related proteins, and other processes related to growth and development of B. napus. From the up-regulated candidate genes, some interested genes were further demonstrated to be high-salinity- or/and drought-induced expression by real-time quantitative RT-PCR analysis. The experimental results also revealed that some genes may function in abscisic acid-dependent signaling pathway related to drought or salinity stress. Collectively, the data presented in this study will facilitate the understanding of molecular mechanism of B. napus in response to high-salinity and drought stresses, and also provide us the basis of effective genetic engineering strategies for improving stress tolerance of B. napus.

Purification and characterization of hatching enzyme from brine shrimp Artemia salina

Fan, T., Wang, J., Yuan, W., Zhong, Q., Shi, Y., Cong, R. — Mon, 25 Jan 2010 07:33:59 PST

By using Artemia chorion as a specific substrate, the hatching enzyme from Artemia salina (AHE) was purified by gel-filtration and ion-exchange chromatography, and characterized biochemically and enzymatically in this study. It was found that the AHE had a molecular weight of 82.2 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and often contained 73.3 kDa molecules in preparation. The AHE had obvious choriolytic activity, which was optimal at pH 7.0 and a temperature of 40°C. The K_m value of the AHE for dimethyl casein was 8.20 mg/ml. The AHE activity was almost completely inhibited by soybean trypsin inhibitor and p-amidinophenyl methane sulfonyl fluoride hydrochloride, greatly inhibited by N-tosyl-l-lysyl chloromethyl ketone, phenylmethanesulfonyl fluoride, and lima bean trypsin inhibitor, slightly inhibited by pepstatin, N-tosyl-l-phenylalanyl chloromethyl ketone, leupeptin, N-ethylmaleimide, and iodoacetamide, and not inhibited by chymostatin and bestatin. All these results imply that AHE is most probably a trypsin-type serine protease. Besides of these, AHE was also sensitive to EDTA and Zn²⁺. Combined with the results that the EDTA-pre-treated HE activity could be perfectly recovered by Zn²⁺, it is indicated that AHE might be also a kind of Zn-metalloprotease.