Subject(s)
Insulin-Like Growth Factor I/chemistry , Muscle, Skeletal/cytology , Muscle, Skeletal/metabolism , Peptide Fragments/pharmacology , Proteolysis/drug effects , Signal Transduction/drug effects , Animals , Gene Expression Regulation/drug effects , Male , Muscle Proteins/metabolism , Muscle, Skeletal/drug effects , Muscle, Skeletal/physiology , Phosphorylation/drug effects , Physical Conditioning, Animal , Protein Serine-Threonine Kinases/metabolism , Rats , Rats, Wistar , SKP Cullin F-Box Protein Ligases/metabolism , Ubiquitin-Protein Ligases/metabolismABSTRACT
AIM: To obtain human recombinant 70 kDa heat shock protein (Hsp70) in baculovirus expression system and to study its antiviral activity. MATERIALS AND METHODS: Baculovirus expression system was used to obtain recombinant HSP70. Plasmid pFastBacHTb-Hsp70 containing sequence coding HSP70 gene with insertion of 6 histidine residues in protein reading frame was constructed. Competent cells MAX Efficiency DH 10 Bac were transfected with pFastBacHTb-Hsp70 plasmid with following extraction of recombinant bacmid Bac-Hsp70. In order to obtain baculovirus expressing HSP70, Sf-9 cells were transfected with Bac-Hsp70 bacmid. Hsp70 extraction and purification was performed with column metal-chelating affinity chromatography using Ni2+ ions. Protective efficacy of recombinant human HSP70 was estimated using model of Venezuelan equine encephalitis (VEE) in mice. RESULTS: Recombinant bacmid Bac-Hsp70 was constructed based on Bac-to-Bac expression system. Baculovirus expressing human HSP70 have been produced after transfection of Sf-9 cells with Bac-Hsp70 bacmid. Cultivation of recombinant baculovirus in Sf-9 cells and application of metal-chelating affinity chromatography allowed to extract purified fraction of HSP70. Experiments on mice infected with VEE virus demonstrated significant protection from death after administration of HSP70 in dose 15 mcg/mice. CONCLUSION: Application of baculovirus expression system and insect cell line for accumulation of recombinant baculoviruses in combination with Ni(2+)-mediated metal-chelating affinity chromatography allowed to obtain highly purified human recombinant HSP70 with marked antiviral activity.
Subject(s)
Antiviral Agents/pharmacology , Baculoviridae , Encephalitis Virus, Venezuelan Equine , Encephalomyelitis, Venezuelan Equine/prevention & control , HSP70 Heat-Shock Proteins/pharmacology , Animals , Cell Line , HSP70 Heat-Shock Proteins/biosynthesis , HSP70 Heat-Shock Proteins/genetics , Humans , Male , Mice , Mice, Inbred BALB C , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Recombinant Proteins/pharmacologyABSTRACT
In experiments on animals study of pathogenicity of 9 clinical strains of Burkholderia cepacia isolated from patients with chronic lung diseases was performed. Preliminary identification of studied strains by means of biochemical and genetic methods allowed to establish their belonging to B. cepacia species. It was determined that 6 of 9 strains are epidemiologically significant. Experiments showed that bacteria of studied strains are not able to cause infectious process in white mice and hamadryas baboons. Conclusion about appropriateness of development and use of other biological models was made.
Subject(s)
Burkholderia Infections/microbiology , Burkholderia cepacia/isolation & purification , Burkholderia cepacia/pathogenicity , Lung Diseases/microbiology , Animals , Burkholderia cepacia/classification , Chronic Disease , Humans , Mice , Papio hamadryas , VirulenceABSTRACT
Physiological evidences and several association studies suggest that the DRD2 gene is implicated in the pathogenesis of schizophrenia. The DRD2 capital ES, Cyrillic939capital TE, Cyrillic (rs6275) single nucleotide polymorphism was genotyped in 272 patients and 362 healthy controls. An association between this polymorphism and schizophrenia was found (p=0,02), the frequency of the TT genotype being higher in patients compared to the controls (c2=7,2; small er, Cyrillic=0,007; OR=1,96, 95% CI=1,2-3,2). The results obtained replicate the data of a previous study of the association between the T allele and schizophrenia.
