ABSTRACT
<p><b>OBJECTIVE</b>To screen and clone hepatocyte protein interacting with hepatitis C virus NS5ATP4A protein for studying its biological functions.</p><p><b>METHODS</b>Bait plasmids of hepatitis C virus NS5ATP4A were constructed. After verifying that hepatitis C virus NS5ATP4A protein could be steadily expressed in AH109 yeast strain, yeast-two hybrid assay was performed by mating AH109 with Y187 which pre-transformed with liver cDNA library plasmids pACT2, and the diploidy yeast cells were plated on quadruple dropout (QDO) medium and assayed for X-a-gal activity. Nineteen yeast colonies which grew on QDO and had a-gal activity were obtained, and then the library plasmids were extracted and sequenced.</p><p><b>RESULTS</b>Seven genes were screened out and one of them was a formerly unknown gene. They were associated with RNA synthesis, protein translation, cell cycling and tumor immunity.</p><p><b>CONCLUSION</b>NS5ATP4A binding proteins were successfully screened, which offers new clues for further studying the signal transduction pathway of NS5ATP4A and the pathogenic mechanism of HCV.</p>
Subject(s)
Humans , Base Sequence , Cloning, Molecular , Gene Library , Genome, Viral , Hepacivirus , Metabolism , Hepatocytes , Metabolism , Molecular Sequence Data , Protein Binding , Sequence Homology , Two-Hybrid System Techniques , Viral Nonstructural Proteins , MetabolismABSTRACT
HIV-TAT protein transduction domain (PTD) is a new kind of peptide that is responsible for transduction of proteins through the plasma membrane with high efficiency. Linked covalently to proteins, peptides, nucleic acid, it could transduce into nearly all kinds of cells and tissues with high efficiency and without any damages. In this study, we constructed the TAT-EDAG and TAT-GFP prokaryotic expression vectors and expressed soluble TAT-EDAG and TAT-GFP fusions in E. coli BL21 (DE3) successfully. By using the Ni-NTA-agrose purification system under the native condition we got the purified fusion proteins whose purification were higher than 90%. After desalting we found the TAT-GFP could transduced successfully into the mouse fibroblast cells and the TAT-EDAG could transduced into HL-60 cells in vitro. It will be useful to amplify the HSCs in vitro in the next step.
Subject(s)
Animals , Humans , Mice , Escherichia coli , Genetics , Fibroblasts , Metabolism , HL-60 Cells , Nuclear Proteins , Genetics , Recombinant Fusion Proteins , Transduction, Genetic , tat Gene Products, Human Immunodeficiency Virus , GeneticsABSTRACT
@#ObjectiveTo explore the mental problems of landless farmer for improving their mental health.MethodsThe questionnaire was used in a survey of 322 landless farmers in east and west China and the impacts of demographic and psychosocial factors on mental problems of landless farmers were also analyzed.ResultsThe landless farmers in China had little symptoms of mental problems, but the status of mental health for landless farmers in west China was worse that those in east China. The different mental symptoms of landless farmers were affected by demographic and psychosocial factors in different degrees.ConclusionThe study reveals that special attention and concern might be attached to the employment and the vocation training of landless farmers, especially to the landless farmers in west China.
ABSTRACT
Myostatin, a member of the TGF-beta family, negatively regulates skeletal muscle development. Mutation of myostatin activity leads to increases muscle growth and carcass lean yield. The bovine myostatin mutation cDNA was amplified by polymerase chain reaction, and then sub-cloned into the expression vector pET-30a( + ) to form the expression plasmid pET30a (+)-action/ Myostatin. The recombinant plasmid was transformed into E. coli BL21. The overexpression product of pET30a (+)-action/ Myostatin was been showed in vitro. Sheep skeletal muscle cell were cultured with the purified myostatin mutation C-terminal peptide. The results of this study suggest that had a powerful activity to stimulate the hyperplasia and proliferation of sheep muscle cells and shows high biochemical activity.
Subject(s)
Animals , Cattle , Cell Proliferation , Cells, Cultured , Cloning, Molecular , Genetic Vectors , Genetics , Muscle Development , Genetics , Physiology , Muscle, Skeletal , Cell Biology , Metabolism , Mutation , Myostatin , Genetics , Metabolism , Peptides , Genetics , Metabolism , SheepABSTRACT
The epithelial sodium channel (ENaC) constitutes the rate-limiting step for sodium absorption across airway epithelia, which in turn regulates airway surface liquid (ASL) volume and the efficiency of mucociliary clearance. This role in ASL volume regulation suggests that ENaC activity is influenced by local factors rather than systemic signals indicative of total body volume homeostasis. Based on reports that ENaC may be regulated by extracellular serine protease activity in Xenopus and mouse renal epithelia, we sought to identify proteases that serve similar functions in human airway epithelia. Homology screening of a human airway epithelial cDNA library identified two trypsin-like serine proteases (prostasin and TMPRSS2) that, as revealed by in situ hybridization, are expressed in airway epithelia. Functional studies in the Xenopus oocyte expression system demonstrated that prostasin increased ENaC currents 60--80%, whereas TMPRSS2 markedly decreased ENaC currents and protein levels. Studies of primary nasal epithelial cultures in Ussing chambers revealed that inhibition of endogenous serine protease activity with aprotinin markedly decreased ENaC-mediated currents and sensitized the epithelia to subsequent channel activation by exogenous trypsin. These data, therefore, suggest that protease-mediated regulation of sodium absorption is a function of human airway epithelia, and prostasin is a likely candidate for this activity.
