OBJECTIVETo examine the role of domain II of hepatitis C virus (HCV) 5' noncoding region (5' NCR) in its translation initiation activity.

METHODSThe fragment of HCV 5' NCR with deletions of 5'-proximal 118 nucleotides were amplified with PCR, then was used to substitute for the full length HCV 5' NCR of plasmid pCMVNCRluc, a firefly luciferase (Fluc) eukaryotic expression plasmid regulated by HCV 5' NCR, to generate recombinant plasmid pCNl-d3. pCMVNCRluc, pCNl-d2 (modified pCMVNCRluc with deletions of HCV 5' NCR ntl-43) and pCNl-d3 were transfected into HepG2 cells with liposome transfection protocol, respectively, and the relative luciferase activity was measured to analyze the regulatory effect of the truncated 5'NCR on Flue gene expression. Meanwhile the Flue mRNA levels were detected by RT-PCR.

RESULTSThe recombinant plasmid was successfully constructed. The Flue mRNA levels of the 3 plasmids were not significantly different (P > 0.05), and there were also no significant difference between the relative luciferase activity of plasmids pCNl-d2 and pCMVNCRluc (P > 0.05). However, that of pCNl-d3 was decreased significantly (P < 0.01, compared with pCNl-d2 or pCMVNCRluc).

CONCLUSIONStructural domain II of HCV 5' NCR plays an important role in its translation initiation activity.

5' Untranslated Regions ; Hep G2 Cells ; Hepacivirus ; chemistry ; genetics ; metabolism ; Hepatitis C ; Humans ; Nucleic Acid Conformation ; Peptide Chain Initiation, Translational ; RNA, Viral ; chemistry ; genetics

Major depressive disorder is characterized by cellular and molecular alterations resulting in the depressive behavioral phenotypes. Preclinical and clinical studies have demonstrated the deficits, including cell atrophy and loss, in limbic and cortical regions of patients with depression, which is restored with antidepressants by reestablishing proper molecular changes. These findings have implicated the involvement of relevant intracellular signaling pathways in the pathogenetic and therapeutic mechanisms of depressive disorders. This review summarizes the current knowledge of the signal transduction mechanisms related to depressive disorders, including cyclic-AMP, mitogen-activated protein kinase, Akt, and protein translation initiation signaling cascades. Understanding molecular components of signaling pathways regulating neurobiology of depressive disorders may provide the novel targets for the development of more efficacious treatment modalities.
Antidepressive Agents ; Atrophy ; Cyclic AMP ; Depression ; Depressive Disorder ; Depressive Disorder, Major ; Humans ; Neurobiology ; Peptide Chain Initiation, Translational ; Phenotype ; Protein Kinases ; Signal Transduction

OBJECTIVETo increase prokaryotic expression level of IFN-alpha 1C gene through the quantitative theory of translational control and the translational enhancer sequence.

METHODSStepwise polymerase chain reaction (PCR) was used to alter the 5 terminal cDNA sequence of IFN-alpha 1C in three different grades of base mutation. In this way, the free energy (Delta G) of the secondary structure in translational initiation region (TIR) was decreased gradually. An expression plasmid (pBVE) was constructed to contain the translational enhancer cDNA sequence by modifying pBV220 upstream of the SD region.

RESULTSThe expression levels of three kinds of IFN-alpha 1C modified gene were all increased. Furthermore, it presented an increasing trend with decreasing in delta G varying from -50,241.6 to -22,190.0 J/mol. The highest expression was 2.43 x 10(8) U/L, covering twelve times more than its original cDNA. IFN-alpha 1C gene and its modified cDNA was inserted into pBVE as reporting genes. E.Coli cells harbouring pBVE/IFN-alpha 1Cs cDNA produced two to five times more IFN than cells harbouring pBV220/IFN-alpha 1Cs.

CONCLUSIONSpBVE containing translational enhancer is a high level prokaryotic expression vector. The theory of quantitative translational control can effectively be used to enhance the IFN-alpha 1C gene expression level in E.coli.

DNA, Circular ; genetics ; Enhancer Elements, Genetic ; Escherichia coli ; genetics ; Gene Expression ; Gene Expression Regulation ; Genetic Vectors ; Interferon Type I ; genetics ; Peptide Chain Initiation, Translational ; genetics ; Polymerase Chain Reaction ; methods

To improve the expression level and catalytic efficiency of (R)-carbonyl reductase from Candida parapsilosis in Escherichia coli, we optimized the mRNA secondary structure of (R)-carbonyl reductase gene in translation initiation region (from +1 to +78), and constructed the corresponding variant. The formation of hairpin structure was significantly reduced and the Gibbs free energy was dramatically decreased from -9.5 kcal/mol to -5.0 kcal/mol after optimization. As a result, the expression level of (R)-carbonyl reductase in the variant was increased by 4-5 times and its specific activity in cell-free extract was enhanced by 61.9% compared to the wild-type strain. When using the whole cells as catalyst and 2-hydroxyacetophenone as substrate with a high concentration of 5.0 g/L, the variant showed excellent performance to give (R)-1-phenyl-1, 2-ethanediol with optical purity of 93.1% enantiomeric excess and a yield of 81.8%, which were increased by 27.5% and 40.5% respectively than those of the wild-type. In conclusion, the optimization of mRNA secondary structure in translation initiation region can overcome the steric hindrance of translation startup, promote translation smoothly to acquire high expression of target protein, and favor protein folding correctly to efficiently improve the enzyme specific activity and biotransformation function.
Alcohol Oxidoreductases ; biosynthesis ; chemistry ; genetics ; Base Sequence ; Biocatalysis ; Candida ; enzymology ; Catalysis ; Escherichia coli ; genetics ; metabolism ; Molecular Sequence Data ; Mutant Proteins ; genetics ; Nucleic Acid Conformation ; Peptide Chain Initiation, Translational ; RNA, Messenger ; chemistry ; Recombinant Proteins ; biosynthesis ; genetics ; Stereoisomerism