Abstract:Embryonic stem cells(ESCs) are pluripotent cells derived from the inner cell mass of blastocyst-stage embryos, possessing permanent self-renewal and indefinite proliferative capacity in vitro. And ESCs could differentiate into the hematopoietic cell fate. Therefore ESCs may provide an alternative for hematopoietic stem cell transplantation and blood cells transfusion. Furthermore, ESCs differentiation could also provide a powerful model system to better understand the hematopoietic development and the mechanism involved. The current status for efforts to differentiate ESCs into hematopoietic lineages were reviewed.

Abstract:Echinoderms are deuterostome, and occupy the top taxonomic position of invertebrates, where is the evolutionary linkage between invertebrates and vertebrates. This is a critical phylogenetic vantage point to infer both the early evolution of bilaterian immunity and the underpinnings of the vertebrate adaptive immune system. The available published literatures on echinoderm immunological mechanisms are compiled and discussed here to understand its evolution process and the hot spots or directions in future investigations. Echinoderms have an innate immune system like vertebrates but their adaptive attributes have not been observed. Its immune responses are based on coelomocytes activity working in parallel with a variety of humoral factors that react directly with invading pathogens. Comparative studies of immune functions in echinoderms have demonstrated that there is a complement system in echinoderms that appears to have alternative pathway and lectin pathway but lack classical pathway and terminal pathway. The sea urchin innate immune system has a number of large gene families. Further investigations are needed to understand the unknown immune-associated genes, proteins, immune signaling pathways and effector molecules, and to know the origin, the underlying mechanisms and evolution of innate immune system.

Abstract:Cdc7 protein, a serine-threonine kinase, acts as a regulator in the initiation of DNA replication by phosphorylation, and is also implicated in S phase checkpoint response via the interaction with ATR/Chk1 pathway. A human homologue of Cdc7 (huCdc7) is ubiquitously expressed in human tissues, while is overexpressed and activated in a high level in many tumor cell lines. Recent studies show huCdc7 kinase play an important role in the development and chemical resistance of cancer, and huCdc7 has been accepted as a new biomarker of tumor. More recently, it has been reported that PHA-767491, an efficient inhibitor of huCdc7, suppresses tumor growth without the damage of normal cells. Thus, the insight into huCdc7 kinase and its inhabitors would provide a novel strategy for tumor treatment.

Abstract:The process of protein synthesis is terminated by one of the three stop codons which are recognized by classⅠ polypeptide release factors. Subsequently, it could promote the hydrolysis of the ester bond of peptidy-tRNA, resulting in release of the nascent polypeptide. Recent results from cryoelectron microscopy, crystallography, NMR, molecular dynamic and biochemical experiments have shed considerable light on the function and structure of the classⅠ release factors. The progress in these aspects were summarized.

Abstract:To establish a conditional knocking down system by RNA interfering, the cassette of LoxP-neo-LoxP was inserted into the pBS/U6/CHIPi construct. The destination vector is named pLoxP/CHIPi, which generates an siRNA targeting CHIP (C-terminal hsc70 interacting protein) gene, dependent on expression of the CRE recombinase. In this conditional knocking down model, it is demonstrated that the siRNA of CHIP could effectively decrease not only the mRNA level but also the exogenous or endogenous protein level in mammalian cells after CRE recombinase was expressed, as testing by RT-PCR and Western blot analysis. Luciferase assay showed that transfection of pLoxP/CHIPi with Cre released the CHIP-mediated inhibition of TGF-β signaling. Significantly, pLoxP/CHIPi facilitated the TGF-β signal transduction in the presence of CRE. These results demonstrate that the conditional knocking down system was successfully constructed, and was applicable for further investigation of the negatively regulatory effect of CHIP on TGF-βsignal pathway. The study also provide a powerful tool for further study on the molecular mechanisms and path-physiologic basis related to CHIP-mediated TGF-β signal pathway.

Abstract:To investigate the hepatic development association with hematopoiesis, a high proliferative potential colony forming cells (HPP-CFC) model of mouse fetal liver was set up. Some differentiational assays based on individual HPP colonies were performed. Under the condition of combinations of hematopoietic and hepatic factors, some individual HPP colonies were induced into hematopoietic and hepatic cells, which were examined with transmission electron microscope (TEM), nested RT-PCR and immunofluorescence staining. The results showed that induced HPP colonies cells with a specific ultrastructure similar to hepatic epithelial cells, expressed hepatic markers including albumin (ALB), α-fetoprotein (AFP), cytokeratins (CK8, CK18) at different extent of percentage. These cells also expressed mesenchymal marker α-SMA and primary endothelial cell marker Flk-1. The MACS results suggested that the fetal liver-derived HPP-CFCs are all from CD45⁺ cells, while CD45^- cells have no capacity to form hematopoietic colony at all. The FACS sorted CD49f⁺/Sca-1⁺ cells have no difference of hepatic differentiation potential compared with whole fetal liver cells. The clonality was confirmed by cell mixing assay. Taken together, the HPP-CFC may represent a novel clonal model for hepatic differentiation from the blood cells in the mouse feta liver and will shed light on the associations underlying the hepatic and hematopoietic development.

Abstract:A serum inhibited gene Si1 (GenBank acession number: AY050169) was previously cloned and identified by differential expression of genes in U251 cells. For the further study of biological function of Si1, prediction procedure was performed to predict its subcelluar-localization. Relative experiments were carried out at the same time. The expression of EGFP/Si1 recombinant in HeLa cells showed Si1 protein located in nuclear which corroborated the prediction results of PsortⅡ, Proloc, Cello version2, Subnuclear compartments prediction system, NUCLEO and NUCPRED. According to the PredictNLS prediction, twelve different fragments of EGFP/Si1 recombinants were constructed to identify precise NLS regulation sequence. Findings proved that the real NLS regulation sequence was not the same as the software predicted(1 206 bp～1 239 bp on Si1 ORF), but located on 1 395 bp～1 594 bp of Si1. A tumor relatived mutation/EGFP recombinant localization result showed though the mutation site (1 639 bp on Si1 ORF) does not located in NLS regulation sequence, it did affect wildtype Si1 protein divert to nuclear and may affect its natural function in cell, perhaps it is the main reason for highly mutation rate of Si1 in tumor.

Abstract:In order to identify the radiosensitivity-related genes，microarray technology was used for candidate genes screening and it was identified that IER5 (Immidiate early response 5) could be up-regulated after radiation. Biological relationship between IER5 and radiation was investigated, and its biological function was examined in human cervical carcinoma treated with radiation. RNAi technology was employed to knock down IER5 gene in HeLa cells and establish the stable transfected cell lines, IER5-siRNA-HeLa. It was found that there was more S/G2/M phases cells with larger size in IER5-siRNA-HeLa cells than that in the control cells when treated with irradiation. The results indicated that IER5 might be involved in cell growth and proliferation. Most importantly, IER5 knockdown increased the radioresistance in cells, which indicated IER5 might be a potential target for the radiotherapeutical treatment in the cervical carcinoma.

Abstract:Keratinocyte growth factor 2 (KGF-2) is a member of the FGF family that is mainly synthesized by mesenchymal cells and acts predominantly on epithelial cells in a paracrine manner. It is known to play an important role in fetal limb and lung development; skin wound healing and prostatic epithelial cell growth. The KGF-2 coding sequence were isolated from human kidney cDNA library, revealing that the Kgf-2 gene is also expressed in the kidney apparatus. Purified from prokaryotic E. coli cells, the effects of the recombinant KGF-2 protein in cultured keratinocyte were analyzed by using MTT assay and in situ TUNEL assay. Interestingly, results revealed that KGF-2 promoted keratinocyte cell growth by stimulating cell proliferation and attenuating cell apoptosis. These findings supported a few evidences that KGF-2 could contribute to alveolar epithelial cells against apoptosis. Cell migration assays for the first time revealed that KGF-2 could stimulate keratinocyte cell migration in vitro. In addition, in the pilot animal test, recombinant KGF-2 incorporated within the hydrogel dressing exhibited significantly stimulatory effect on cutaneous wound healing. These combined effects implicate a potential therapeutic application of human recombinant KGF-2 in the future.

Abstract:Analysis of regular elements in promoter region is the base for elucidating the mechanism of gene transcription initiation. The TATA and the TATA-less promoters of plant RNA polymeraseⅡ gene are chosen from the PlanPromDB. The GC bias，position structure conservation，nucleotide content and conservative motifs of sequences，position distribution of TATA box and conservation of correlation position are analyzed. Many specific regulars for the two types of promoters are found. These features can offer some help for revealing the transcription regulation of plant gene. A new prediction algorithm based on position-correlation weight matrix (PCWM) is proposed. The better discrimination results for two sort plant promoters are obtained by using score function. It is confirmed that the performance of position-correlation weight matrix (PCWM) is superior to single-base position weight matrix (PWM).

Abstract:The hypoxanthine-guanine phosphoribosyl transferase (HGPRT) gene mutation is responsible for gouty arthritis, kidney stone, and Lesch-Nyhan Syndrome (LNS). It has been reported that the expression of HGPRT is decreased or even absent in these diseases. Rabbits are an ideal model for studying the pathology of these diseases. Therefore, the development of an HGPRT-knockdown rabbit model will be highly beneficial in such studies. Stable HGPRT-knockdown transgenic fibroblast lines were generated by transfecting rabbit fibroblasts with RNA interference (RNAi) plasmids. Polymerase chain reaction (PCR) analyses indicated that the average positive rate was 83.3%. The mRNA and protein levels of HGPRT in the transgenic fibroblast lines were significantly lower than that in the control. Transgenic rabbit blastocysts were derived after performing nuclear transfer. The results show that RNAi can be used to stably knock down expression of the HGPRT in rabbit fibroblasts and further improvements in related technologies will facilitate the use of this method for the generation of HGPRT-knockdown rabbits.

Abstract:To identify the proteins associated with nasopharyngeal carcinoma (NPC) oncogenesis, a two-dimensional electrophoresis and mass spectrometry was used to screen for differential proteins between NPC and adjacent normal nasopharyngeal epithelial tissue (ANNET). As a result, 21 differential proteins were identified, and Raf kinase inhibitor protein (RKIP) was one of the nine downregulated proteins in NPC compared to ANNET. To investigate for the role and mechanisms of RKIP in the metastasis of NPC, Western blot and immunohistochemistry was respectively used to detect RKIP expression in 5-8F and 6-10B NPC cell lines with the different metastatic potentials, as well as in the normal nasopharyngeal epithelial tissue (NNET), primary NPC and NPC metastasis. Furthermore, 5-8F and 6-10B cells were stably transfected with plasmids that expressed sense and antisense RKIP cDNA, respectively, or with empty vector to establish the stable transfected cell lines. The effects of RKIP expression on in vitro cell invasion, and the activity of NF-κB signaling pathway were analyzed in the transfected cell lines. The results showed that RKIP was significantly downregulated in 5-8F compared with 6-10B, in NPC compared with NNET, and not detectable in NPC metastasis. Overexpressed RKIP in 5-8F could decrease its in vitro cell invasion, whereas downregulated RKIP in 6-10B could increase its in vitro cell invasion. Overexpressed RKIP in 5-8F could decrease phosphorylated-IκB-α level and tansactivation activity of NF-κB, whereas downregulated RKIP in 6-10B could increase phosphorylated-IκB-α level and tansactivation activity of NF-κB. Taken together, the results suggest that RKIP may be a NPC cell metastasis suppressor, and decreased RKIP expression is associated with the increased metastasis capability of NPC cells possibly through the activation of NF-κB signaling pathway.

Abstract:Not only calmodulin (CaM) with Ca²⁺ regulates the activity of many enzymes and proteins, but also free-CaM (no Ca²⁺ bound) and Ca²⁺-independent CaM-binding proteins play roles in plant and animal cells. There is no in vivo method to identify the interaction between free-CaM and Ca²⁺-independent CaM-binding protein (CaMBP). Using site-directed mutagenesis by polymerase chain reaction (PCR), 5 mutant Arabidopsis calmodulin isoform 2 (AtCaM2) genes, mCaM2₁, mCaM2₁₂, mCaM2₁₂₃, mCaM2₁₂₄ and mCaM2₁₂₃₄ were obtained. The mutant mCaM2 encoded glutamine in place of glutamate (E32Q; E68Q; E105Q; E141Q) in one or more EF-hand Ca²⁺-binding motifs of AtCaM2. The recombinant mCaM2 proteins were produced in Escherichia coli, and subsequently separated on SDS-PAGE in the presence of Ca²⁺ or EGTA, their electrophoresis mobilities were related with that of mutant EF-hand motifs. ⁴⁵Ca²⁺ overlay analysis indicated that the more glutamate replaced by glutamine, the lower affinity with Ca²⁺ in the mCaM2 proteins. The mCaM2₁₂₃₄ mutant protein (E32Q; E68Q; E105Q;E141Q) was unable to bind Ca²⁺. Using yeast two-hybrid technique with mCaM2₁₂₃₄ as bait, it was possible to see interaction in Arabidopsis of AtCaM2 with IQD26, a calcium-independent CaM-binding protein. Site-directed mutation of AtCaM2 will aid the research of Ca²⁺, CaM and Ca²⁺-independent CaMBPs in plant biological processes.

Abstract:To investigate the effects of porcine Ghrelin (pGhrelin), a newly discovered peptide on porcine preadipocyte Caspase-3 activity and gene expression, piglet subcutaneous preadipocytes treated with 0, 1, 10 and 100 nmol/L pGhrelin were cultured for 48 h, and preadipocyte morphology was observed under an inverted biological microscope, the effects of preadipocyte prolifaration was mesured by MTT, preadipocyte Caspase-3 activity was detected with spectrophotometric method, Caspase-3 gene expression level was determined using real-time fluorescent quantitative RT-PCR. 10 nmol/L pGhrelin significantly decreased preadipocyte Caspase-3 activity and mRNA expression level (P < 0.05), 100 nmol/L pGhrelin significantly promoted porcine preadipocyte proliferation compared to control group (P < 0.01). pGhrelin downregulated Caspase-3 activity and expression mRNA of porcine preadipocyte, induced preadipocyte proliferation and inhibited apoptosis. Caspase-3 dependent apoptosis regulation signalling was supposed to be involved in the apoptosis mechanism.

Abstract:LRRC4, leucine-rich repeat C4 protein, is a new member of leucine-rich repeat (LRR) superfamily. It is a novel tumor suppressor. LRRC4 inactivation is commonly found in glioma cell lines and primary glioma biopsies. Previous study did not find any genetic alteration of LRRC4 in primary glioma. In order to explore an alternative mechanism underlying inactivation of LRRC4 in glioma, glioma cell lines SF126 and SF767 were treated by methylase inhibitor 5-Aza-2′-deoxycytidine (5-Aza-CdR). Methylation-specific PCR was used to examine the methylation status changes of LRRC4 promoter in SF126 and SF767 cell lines. LRRC4 mRNA expression in SF126 and SF767 cell lines treated by 5-Aza-CdR was detected by RT-PCR. In addition, the effect on glioma cell lines cell growth and cell cycle of 5-Aza-CdR were assayed by MTT and FCM. The results indicated that LRRC4 promter was completely methylated in SF126 and SF767 cells. However, LRRC4 promoter aberrant hypermethylation can be reversed and LRRC4 expression can be induced by 5-Aza-CdR. Moreover, 5-Aza-CdR displayed a growth inhitory effect on SF126 and SF767 cells in a dose- and time-dependent manner after exposure to 5-Aza-CdR at different concentration for different time. FCM analysis showed that SF126 and SF767 cells cell cycles were blocked at G0/G1 phase after 5-Aza-CdR treatment for three days. Taken together, glioma cell lines SF126 and SF767 cell growth could be inhibited and cell cycles could be blocked by 5-Aza-CdR; promoter hypermethylation is the important mechanism of LRRC4 inacctivation in glioma cell lines; methylation can be reversed and LRRC4 expression can be induced by 5-Aza-CdR. All this suggest that LRRC4 may serve as a demethylation therapeutic target in glioma.

Abstract:Non-secreted macrophage colony-stimulating factor(M-CSF) plays an important role in genesis and progression of tumors. To explore the regulation of cytoplasmic M-CSF on the proliferation of HeLa cells, pCMV/myc/cyto-M-CSF vectors were transfected into HeLa cells. After comfirmed by RT-PCR, Western blot and immunocytochemistry, the effect of cytoplasmic M-CSF on the proliferation of HeLa cells were analyzed by MTT and antisense oligonucleotides. The doubling time was counted according to cell growth curves. The mRNA expression of cyclinE, cyclinD1/2/3, CDK2/4/6 were assayed by RT-PCR. The results from RT-PCR, Western blot and immunocytochemistry showed that both M-CSF mRNA and protein were expressed at a higher level and localized to the cytoplasm in M-CSF-transfected HeLa cells, compared with either pCMV/myc/cyto-transfected HeLa cells or untransfected HeLa cells. M-CSF-transfected HeLa cells had shorter doubling time and more significantly augmented reproductive activity than either pCMV/myc/cyto-transfected HeLa cells or untransfected HeLa cells. M-CSF specific antisense oligonucleotides significantly inhibited the proliferation of the M-CSF-transfected cells, but had little effect on the other two groups. Furthermore, cytoplasmic M-CSF up-regulated the mRNA expression of cyclinD1, cyclinD3, CDK2 and CDK6(P < 0.05). So it was concluded that cytoplasmic M-CSF accelerates the proliferation of HeLa cells by up-regulating the mRNA expression of cyclinD1/D3 and CDK2/6.

Abstract:Baculovirus-mediated gene transfer into mammalian cells has been used to develop non-replicative vector vaccines against a number of diseases in several animal models. A baculovirus pseudotyped with the glycoprotein of vesicular stomatitis virus was used as vector to construct the recombinant baculovirus expressing classical swine fever virus (CSFV) E2 protein under the control of ie1 promoter from white spot syndrome virus. The E2 gene was shown to be efficiently expressed in both insect and mammalian cells. Intramuscular injection of mice with the recombinant baculovirus resulted in the production of high-level CSFV-specific antibodies. Specific lymphoproliferative responses to the CSFV stimulation were induced in the splenocytes of the immunized mice as demonstrated by CFSE staining assay and WST-8 assay. The results indicates that the pseudotyped baculovirus- delivered gene can be a potential non-replicative vaccine against CSFV infection.

Abstract:The modern large-scale pyrosequencing technology is a revolution of DNA sequencing. One of the key points in this technology is to get an ATP sulfurylase immobilized on the surface of magnetic beads and with a high activity. Biotinylated ATP sulfurylase can be immobilized on magnetic beads coated with streptavidin through the specific conjunction between biotin and streptavidin, but using chemical modification method to biotinylate ATPS will affect the activity of the enzyme. ATP sulfurylase fused with the carboxyl terminal 87 residues of Escherichia coli biotin carboxyl carrier protein (BCCP87) was expressed in E. coli using fusion expression strategy. Results from Western blot analysis and SDS-PAGE analysis showed that the fusion protein could be biotinylated in vivo, and the molecular mass of the fusion protein was about 64 ku. The biotinylated ATP sulfurylase could be immobilized on the surface of magnetic beads coated with strepavidin, and the immobilized ATPS could be used for quantification of PPi and pyrosequencing. An effective enzyme for the large-scale chip-based pyrosequencing system was supplied.

Abstract:A reporter system for ФC31 integrase was developed in NIH3T3 cells. The reporter plasmid coding green fluorescent protein (GFP) coupled with red fluorescent protein (RFP) was co-transfected with the plasmid coding ФC31 integrase, to show the activity of integrase in the cells. Fluorescence activated cell sorter (FACS) was used to measure the proportion of the cells containing red and green fluorescence. The increment of green cells was positively related to the increase in the transfection with plasmid coding ФC31 integrase. Approximately 90% of green cells were observed under a ratio of [plasmid-ФC31-integrase]/[reporter plasmid] at 10∶1. This suggests that the ФC31 integrase reporter system provides a probe for the function of ФC31 integrase in cells.

Abstract:Cre-LoxP homologous recombinant system was used to disrupt eag gene in B. anthracis AP422. To construct the recombinant vector, homologous regions and Spc^r cassette with two loxP sites were amplified from the corresponding templates. The produced shuttle vector was then transformed into B. anthracis AP422. Under the pressure of temperature and antibiotic, homologous recombination occurred between the vector and genome of the host bacterium and the recombinants were selected by agar media with spectinomycin (Spc) and X-gal. To remove the Spc^r cassette, a plasmid with Cre recombinase was introduced into the recombinant to delete the relevant DNA fragment, resulting in a single loxP site within the targeted genomic segment. The markerless mutant strains were detected by genome PCR, RT-PCR, total proteins SDS-PAGE analysis and Western blot. The results showed that the eag gene was successfully deleted.