ABSTRACT
Objective To determine the expression of human protection of telomeres 1 (hPOT1) and its relationship with Helicobacter pylori (HP) infection in gastric carcinoma.Methods The expressions of hPOT1 protein and hPOT mRNA were detected in 53 gastric carcinoma specimens (observed group) and 20 normal gastric mucosa tissues (control group) by SP immunohistochemical method and in situ hybridization (ISH), respectively.HP infection was examined by Warthin-Starry method in observed group.Results The positive expression rate of hPOT1 protein was 84.91％ (45/53) in observed group, higher than that in control group [30.00 ％ (6/20)] (P ＜ 0.01).The positive expression rate of hPOT1 mRNA was 58.49 ％ (31/53) in observed group, higher than that in control group [10.00 ％ (2/20)] (P ＜ 0.05).The positive co-expression rate of hPOT1 protein and mRNA was 56.60 ％ (30/53), both had positive relationship in gastric carcinoma (r =0.394, P ＜ 0.05).The rate of HP infection in 53 cases of gastric carcinoma was 52.83 ％ (28/53).The positive expression rates of hPOTI protein and mRNA in observed group with HP infection were significantly higher than those in observed group without HP infection[protein: 96.43 ％ (27/28) vs 72.00 ％ (18/25), P ＜0.05;mRNA: 85.75 ％ (24/28) vs 28.00 ％ (7/25), P ＜ 0.01].Conclusions hPOT1 may be associated with occurrence of gastric carcinoma.Combined detection of hPOT1 protein and mRNA can be used for the diagnosis of gastric carcinoma.HP infection may be associated with abnormal expression of hPOT1 in occurrence of gastric carcinoma.
ABSTRACT
HIV-1 integrase (IN) is a key enzyme for the viral replication. The protein-protein interaction (PPI) between HIV-1 IN and a cellular cofactor lens epithelium-derived growth factor (LEDGF/p75) is a validated target for anti-HIV drug discovery. In order to build the platform for screening inhibitor against PPI between IN and LEDGF/p75, the vector containing the LEDGF/p75 protein cDNA was constructed and expressed in Escherichia coli and the function of the LEDGF/p75 protein was assayed. The LGDGF/p75 encoding gene optimized according to the preference codon usage of E. coli, was synthesized and cloned into the expression vector pGEX-4T-1 to form a recombined plasmid, then transformed into host cell E. coli BL21 (DE3). The recombined clones were identified and confirmed by BamH I/Sal I digestion and sequencing, the successfully recombined plasmid in the host cell was induced by IPTG and the condition of the expression was optimized. The expressed protein was purified by the Ni2+ affinity chromatography column and SDS-PAGE was used to analyze the molecular weight and specificity. In addition, ELISA assay was used to analyze the function of the recombinant protein. The recombinant LGDGF/p75 was soluble, and expressed highly and stably in E. coli. The protein was proved to enhance HIV-1 IN strand transfer activity in vitro by ELISA. It will be helpful to build the platform of screening inhibitors against PPI between IN and LEDGF/p75.
ABSTRACT
The drug resistant mutations in human immunodefieiency virus type 1 (HIV-1) are a major impediment to successful highly active antiretrovirai therapy (HAART) and new drug design. In order to understand the drug resistance mechanism of HIV-1 integrase (IN) mutually existed for multiple drug-resistant strains to the most potent IN inhibitors diketo acids (DKAs), three S-1360-resistant HIV-1 strains were selected and molecular docking and molecular dynamics (MD) simulations were performed to obtain the inhibitor binding modes. Based on the binding modes, compelling differences between the wild-type and the 3 mutants for IN have been observed. The results showed that: 1) In the mutants, the inhibitor is close to the funetional loop 3 region but far away from the DNA binding site. Different binding sites lead to the decrease in susceptibility to S-1360 in mutants compared to the wild-type IN. 2) The fluctuations in the region of residues 138～166 are important to the biological function of IN. 2 hydrogen-bonds between S-1360 with residues N155 and K159 restrict the flexibility of the region. Drug resistant mutations result in a lack of the interaction, consequently, the less susceptible to S-1360. 3) In the 3 mutant IN complexes, the benzyl ring of S-1360 is far from the viral DNA binding site, thus, S-1360 can not prevent the end of the viral DNA from exposure to human DNA. 4) After T66I mutation, the long side chain of I occupied the active pocket in the 3 mutants, consequently, the inhibitor could not move into the same binding site or have the same orientation. All the above contribute to drug resistance. These results will be useful for the rational inhibitor modify and design.