Abstract:Post-translational modification of proteins is a key step in the process of regulating their biological functions and it's an important molecular basis in the dynamic response and interaction of proteins, meanwhile, it can be considered as important targets to regulate the cell signaling networks. At present, post-translational modification of protein has become a critical area in the research of proteins. In prokarytes, post-translational modification of proteins can play a crucial role in the life activities, such as cell signal transduction, metabolism, protein degradation, pathogenicity of pathogenic microorganisms and so on. The types, mechanisms and functions of the classical post-translational modification of proteins in prokaryotes were summarized and the recently discovered global acetylation in Salmonella enterica, as well as the ubiquitin-like modification in Mycobacterium tuberculosis, were also introduced.

Abstract:Tanslesion DNA synthesis (TLS) is one mode of DNA damage tolerance in cells upon genotoxic agent treatments, which utilizes specialized low-fidelity DNA polymerases to traverse replication-blocking lesions. TLS can be classified into two categories: error-prone TLS and error-free TLS. Error-prone TLS is one of the fundamental mechanisms for genome mutagenesis. In addition, recent studies suggest that TLS is closely related to tumor chemoresistance. So far, multiple TLS polymerases have been identified. The known major TLS polymerases belong to Y-family DNA polymerases, which include Polκ. The general properties of TLS and the current understanding of Polκ in mammals were summarized.

Abstract:Hundreds of proteins with Kelch motif have been identified in diverse organisms including viruses, plants, fungi and mammals. Although most of the Kelch proteins are not functionally characterized at present, some of the known Kelch proteins seemed to function diversely in vivo. In mammals, nearly 41 Kelch proteins have been reported and proved to mediate the protein-protein interactions, protein degradation, and gene expression and signal transmission and so on. It was proposed that the crystal structure of Kelch domain of Keap 1 could be a preferable model for researching Kelch proteins in mammals. And it was also divided these mammal Kelch proteins into three groups according to their domain organizations and paid much attention to summarize their known function in vivo.

Abstract:Oncolytic viruses like measles virus, reovirus, adenovirus are promising alternatives in tumor treatment. Vesicular stomatitis virus(VSV) is a potent reagent for tumor virotherapy and has been tried in many types of tumor models. The selectivity of VSV replication in tumor cells has been regarded due to the defective of IFN signaling in tumor cells compared with normal cells. It has been reported that 80% of the tumor cells are typeⅠ IFN sinaling defective, which makes oncolytic VSV a promising method for tumor therapy. Recent analysis has demonstrated that defective control of mRNA translation initiation also plays a crucial role in cell permissiveness to VSV. Translation control downstream of PKR activation, frequently dysregulated in many transformed cells, can cooperate with attenuated IFN antiviral activity to facilitated VSV oncolysis. The problem with VSV oncolysis included that cleaning of viruses by host immune system, so the virus can not replicate effectively in tumors, the other is that the viruses can not spread effectively in the tumor tissue because of the tumor microenviroment, but one of the biggest problem is its safety. It was reported that when animals were injected with high dose of wild type VSV, neurotoxicity like hind limb paralysis occurred. There are kinds of strategies to improve safety for oncolytic viruses. Development of tumor antigen targeting VSV is an ideal alternative to improve safety and efficacy of the vector. Adult T cell leukemia (ATL) is a kind of human CD4 T cell tumor caused by HTLV-1, with phenotype of CD4+, CD3+, CD25+ and also CCR5+, CXCR4+. All of the present treatments only arrive partial success, which is underlying the urgency for new therapeutic drugs. Based on these rationales, it was hypothesized that the HIV gp160 pseudotyped VSV (VSV-ΔG-gp160G ) could be targeted at ATL cells, because entry of HIV-1 into human CD4 T cell depends on recognition of human CD4 and some co-receptors like CCR5 or CXCR4 and cause fusion between viral and cellular membranes. In the studies, with gp160 cytoplasmic tail replaced with that of VSV G, the HIV-1 envelope protein could be successfully incorporated into VSV. In one step replication curve, VSV-ΔG-gp160G can arrive at the highest titer at around 24 h post infection and the VSV-GFP titer did that at around 12 h post infection, and the titer of VSV-GFP was higher than that of VSV-gp160G, all these indicated that VSV-ΔG-gp160G has been attenuated after pseudotyping. To testify if VSV-ΔG-gp160G could kill ATL tumor cells efficiently, in particular, specifically. HTLV-1 transformed ATL tumor cells and non-ATL cells were infected with VSV-ΔG-gp160G or VSV-GFP for control respectively. The data indicated that the novel VSV could kill CD4 positive ATL cells selectively and potently, but not replicated in CD4 negative non ATL cells. This virus will be promising in treating adult T cell leukemia and lymphoma as well.

Abstract:α-Fetoprotein (AFP) has a property to maintain the growth of hepetocellular cancinoma cells(HCC) in vivo, but the critical functional step of AFP is still obscurity. In order to explore AFP influences on the transduction of PTEN/AKT signal in HCC, expression of PTEN in human hepatoma cells and the effect of all trans retinoic acid (ATRA) on PTEN expression in these cancer cells were detected by Western blotting. AFP interacted with PTEN was analyzed by co-immunoprecipitation (Co-IP). Laser confocal microscopy was used to observe co-localization of AFP and PTEN. Short small RNA interfering (RNAi) was applied to knockdown the expression of AFP in Bel 7402 cells, and the phosphorylation of protein kinase B(AKT) was detected by Western blotting. Growth of Bel 7402 and HLE cells were assayed by MTT. Results showed that Bel 7402 cells expressed PTEN, and the expression of PTEN was induced by ATRA(160 μmol/L) mildly. Co-IP indicated that AFP has a property to interact with PTEN. PTEN co-localization with AFP was observed in cytoplasm of Bel 7402 cells. Constructed RNAi vector could knockdown the expression of AFP, expression of PTEN was promoted and phosphorylation of AKT was decreased when the expression of AFP was interfered or the cells were treated with ATRA(160 μmol/L). AFP-expressed vector pcDNA3.1-afp was transfected into human hepatoma HLE cells (AFP-non production). Co-IP analysis indicated that AFP interacted with PTEN, and expression of p-AKT(Ser473) was promoted in the tumor cells. It was also proved that pcDNA3.1-afp was able to reduce the role of Ly294002 in inhibiting the activity of AKT in HLE cells. These data provide the first evidence that AFP has a capacity to both interact with and inhibit activity of PTEN, which is also the pivotal events in the process that AFP activated the transduction of PI3K/AKT signal in hepatoma cells. Cytoplasmic AFP plays importance role on the resistance to ATRA induced apoptosis of HCC.

Abstract:A novel type of stress proteins has been identified in mammals to defend environmental stresses and maintain tissue integrity. Cornulin (CRNN) that contains S100 EF-hand Ca²⁺-binding motif is a stress protein highly expressed in the human esophageal squamous epithelial cells. It is downregulated in esophageal squamous cell carcinoma and functions as a modifier of deoxycholic acid (DCA) mediated cell injury. The S100 domain may be central to the function of CRNN. To further characterize the S100 domain of CRNN, the S100 domain in Escherichia coli, was cloned, expressed, purified and demonstrated that it was properly folded and suitable for biochemical and biophysical studies. More importantly, by nuclear magnetic resonance, gel-filtration, analytical ultracentrifugation, electrospray ionization-mass spectrometry, and Cross-linking analyses, a Ca²⁺-dependent multimeric property of S100 domain was identified. Furthermore, in response to DCA and ethanol challenge, the multimers have stronger protective effects on cells than dimers do. These data indicate that the S100 domain is a key domain in CRNN, which functions as a survival factor through multimerization. This work helps to further understand the feature of S100 domain and its association with cell injury.

Abstract:To investigate the function of Laminin G domain (LG domain) of STGC3 in human nasopharyngeal carcinoma cell line CNE2. Recombinant plasmids pcDNA3.1(+)-STGC3 and pcDNA3.1(+)-STGC3^Δ1～42AA were respectively transfected into CNE2 cells by liposome-mediated transfection. Therefore, the CNE2/pcDNA3.1(+)- STGC3 and CNE2/pcDNA3.1(+)-STGC3^Δ1～42AA cell lines of stable expression STGC3 were established. Here, it is reported that deletion of LG domain in this STGC3 reduces the tumor suppression activity of it, as demonstrated by drawing growth curve, experimenting plate clone formation as well as detecting cell cycle distribution. The results showed: the ability of STGC3^Δ1～42AA suppressing CNE2 cell proliferation was obviously decreased to compare with wild type STGC3 (n=4, P < 0.05). These results indicate that the LG domain is necessary for the tumor suppression activity of the STGC3.

Abstract:3, 5-Hydroxy -6, 7, 3′, 4′-tetramethoxyflavone (HTMF) isolated from Laggera pterodonta is known to have an antiproliferative effects in vitro on human cancer. However, the exact mechanisms retain unclear. HTMF was investigated for its antiproliferative effects on human nasopharygeal carcinoma CNE cells. 3-(4, 5-Dimethylthiazol-2-yl)- 2, 5-diphenyl tetrazolium bromide (MTT) assay was used to deserve the inhibitory effect of HTMF. The changes of the cell and nuclear morphological characteristics were observed under the inverted and fluorescence microscope. The cell apoptosis was displayed by Hoechst 33258 staining and flow cytometry (FCM). The expression of Caspase3 and Caspase9 was detected by Western blotting. The mitochondrial membrane potential were analyzed by FCM and laser confocal microscope with Jc-1 fluorescence staining. MTT assay results show that HTMF significantly inhibited the growth of CNE cells in dose and time dependent manners. The IC₅₀ values of HTMF were 69.02, 28.31 and 3.95 mg/L at 24, 48 and 72 h treatments, respectively. The apoptosis percentage in CNE cells is significantly increased compaired with control group. HTMF with 0, 5.0, 10.0, 20.0 and 40.0 mg/L at 48 h and 40.0 mg/L at 0, 6, 12, 24 and 48 h treatments, respectively increased the expression of Caspase3 and Caspase9 and degraded mitochondrial membrane potential in dose and time dependent manners. The mechanistic investigation revealed that HTMF has high inhibitory effects on the proliferation of CNE cells and induced the apoptosis of CNE cells by the decrease in mitochondrial membrane potential and increasing the expression of Caspase3 and Caspase9.

Abstract:As a major complication of diabetes, diabetic nephropathy (DN) has become a major cause for end stage renal failure. Increasing evidences demonstrated that DN is a kind of inflammation disease. However, the exact mechanism is not completely elucidated. In an effort to investigate the possible role of C3aR in the development of DN, it was found unexpectedly a kind of C3aR highly expressing infiltrates in the renal tissue of patients with DN. To identify the cells and evaluate the possible role of the cells in the development of DN, forty five cases of patients with DN were selected and sections of their renal biopsy were analyzed by histological, immunohistochemical, double immunofluorescence labeling, immunoelectronicmicroscopic methods and toluidine blue staining while another 15 sections of renal biopsy from normal transplantation donor were used as normal controls. The number of C3aR highly positive cells in each section was counted and the density of the cell was calculated, and the correlation between the density of the cell and the development of DN was analyzed. Light microscopic and electron-microscopic analysis revealed that the cells were similar morphologically to mast cell. Double immunofluorescence labeling analysis demonstrated that the cells were positive in CD68 and tryptase staining while negative in CD45 staining, which is also in accordance with that of mast cells. Toluidine blue staining analysis further proved that the cells were a kind of mast cells. Only very small number of the C3aR highly positive mast cells was found in the renal biopsy of normal control group. However, the number of the cells increased with the development of DN, and correlated linearly with the urine protein level and serum creatinine. These results strongly suggested that mast cells are involved in the development of DN. C3aR might play an important role in the recruitment and activation of renal mast cell and thus contribute to the development of DN.

Abstract:Gateway technology has been demonstrated to be easy and successful in construction of expression vectors for genes of interest. However, the present Gateway plant destination vectors do not contain any sequence for targeting expressed proteins of interest to chloroplasts. The XmnⅠ restriction site was converted into a HindⅢ site in the Gateway entry vector pENTR-2B to generate pENTR*-2B and a Gateway entry vetcor designated as pENTR*-PrbcS-*T-GFP was constructed by subcloning the tomato Rubisco small subunit 3C promoter (PrbcS) and its transit peptide sequence (*T) as well as a GFP (green fluorescent protein) reporter gene into pENTR*-2B. These results demonstrated that the pENTR*-PrbcS-*T-GFP could be used to generate the plant expression vector via Gateway technology for targeting the expressed GFP into the chloroplasts of transgenic plant leaves. Similar results were also obtained for GUS (β-glucuronidase) reporter gene when it was used to replace the GFP gene in pENTR*-PrbcS-*T-GFP. These results indicated that pENTR*-PrbcS-*T-GFP can be generally applied to generate an entry vector for a target gene by replacement of the GFP with the target gene and the plant expression vector that serves to localize the expressed target protein in chloroplasts can be achieved rapidly via Gateway technology.

Abstract:Screening of protein crystallization conditions remains the major limiting step for three-dimensional structure determination, therefore developing more efficient screening kits is essential. Up to now the methods used for designing the screening kits include the incomplete factorial design, the sparse matrix sampling and the systematic screening. Based on these three methods, researchers have developed a series of crystallization screening kits which are proved applicable in practical protein crystallization. The development in designing the screening kits and the applications of the screening kits since 20th century were reviewed, and the future prospect in this field was discussed.