Objective To construct an eukaryotic expression plasmid containing gene coding for the hemagglutinin-neuraminidase(HN)of newcastle disease virus(NDV)oncolytic strain Italien,and then to express the protein in eukaryotic cell.Method The HN cDNA was synthesized from viral RNA by RT-PCR,and the eukaryotic expression vector of HN gene(named pcDNA3.1-HN)was constructed.The vector pcDNA3.1-HN was transfected into CHO-K1 cell by liposome,and G418 was used to select stable clones expressing HN gene.The expression of HN protein was visualized by Western blot and Immunofluorescence microscopy.Results Restriction analysis and DNA sequencing proved that HN gene was correctly cloned into expression vector.Western blot analysis and immunofluorescence showed that the HN was expressed in CHO-K1 cells.Conclusion The HN cDNA of NDV was successfully cloned into eukaryotic vector which showed good expression of HN protein in CHO-K1 cells.

Occupational Exposure ; prevention & control ; Risk Assessment ; methods

Smoke water and distillation liquid were used to treat the seeds of Trichosathes kirilowii and to study the effects of smoke water and distillation liquid on the seed germination and seedling growth of T. kirilowii. The results showed that germination rate, germination index and germination vigor of T. kirilowii all were significantly improved with the treatment of SW and DL treatment. The activity of α-amylase were significantly increased with the treatment of SW and DL at 1:2,000. SW and DL treatment showed no significant effects on the activity of SOD. The activity of POD were markedly enhanced under the treatment of SW (1:000) and DL (1:2,000). CAT activity were increased with the treatment of SW and DL at 1:2,000 while were inhibited by SW and DL at 1:500. Seedling height and root length were increased with the treatment of SW and DL (1:1,000, 1:2,000). SW and DL treaments improved the content of chlorophyll, and moreover with the concentration of SW and DL, the stimulatory were also increased. This work demonstrated that smoke water and diatillation liquid at 1:2,000 could stimulate the seed germination and seedling growth of T. kirilowii, and it provided the references for the study of seed germination technology.
Agriculture ; instrumentation ; methods ; Distillation ; Germination ; Seedlings ; growth & development ; metabolism ; Seeds ; growth & development ; metabolism ; Smoke ; analysis ; Trichosanthes ; growth & development ; metabolism ; Water ; chemistry ; metabolism

Stability is the important feature of the control system. Stability exists extensively in the biological systems such as different cell types during the process of individual development, new biological properties formed under the environment and/or diet factors, drug addition, long-term memory formation, and so on. The underlying mechanisms generally include epigenetically modification, the emergence of certain substance with long half life, the formation of positive feedback loop, and the activation of stem cells. Introduction of stability into researches of Chinese medicine syndrome is essentially to explore the mechanisms of quantitative changes into the qualitative alternation. It is also helpful for us to choose an appropriate time window of animal models for Chinese syndrome and to consider what are the core effects of Chinese materia medica in improving syndrome. The stability not only meets the features of Chinese medicine theories, but also agrees with the progress of modern systems theory and biology. We hope it could promote studies on Chinese medicine syndrome.
Allostasis ; Humans ; Medicine, Chinese Traditional ; Research

Trichosanthes kirilowii has been widely cultivated as its medicinal use, health care and food value. Drought resistance of seedlings is an important feature in breeding. Seeds of two T. kirilowii strains were used to research the difference of surface ultrastructure characteristic and drought resistance. Scanning electron microscope was used to identify the surface ultrastructure characteristic of seeds and PEG was used to simulate drought stress. The seeds germination rate, MDA content, chlorophyll content and the antioxidant enzymes activity were measured under the drought stress. The results showed that the seed surface colour of KXY-001 was lighter than that of KXY-005. The testa cobwebbing of KXY-001 was more intensive than that of KXY-005. The germination rate of KXY-001 was higher than that of KXY-005 under drought stress. The MDA content was increased and the chlorophyll content was decreased with the increasing of drought degree. The SOD activity of KXY-001 was higher than that of KXY-005, while the activity of POD and CAT was also increased firstly and decreased later. Surface reticulate of seeds and hilar traits can be used as identification points to identify the investigated strains. SOD and POD are activated to resist drought in T. kirilowii seedlings and the drought resistance of KXY-001 is superior than that of KXY-005.
Adaptation, Physiological ; drug effects ; Catalase ; metabolism ; Chlorophyll ; metabolism ; Droughts ; Germination ; Malondialdehyde ; metabolism ; Microscopy, Electron, Scanning ; Peroxidase ; metabolism ; Polyethylene Glycols ; pharmacology ; Seedlings ; metabolism ; Seeds ; growth & development ; metabolism ; ultrastructure ; Species Specificity ; Superoxide Dismutase ; metabolism ; Trichosanthes ; classification ; growth & development ; metabolism

Echovirus 6, Human ; genetics ; isolation & purification ; Enterovirus Infections ; virology ; Genes, Viral ; Humans ; Sequence Analysis

Objective To explore the effect of Wenyang Huazhuo Tongluo recipe on pulmonary microvascular injury in mice with scleroderma based on mitophagy.Methods Fifty mice were randomly divided into blank control group(0.9％NaCl,by gavage),control group(0.9％NaCl,by gavage),model group,Wenyang Huazhuo Tongluo recipe group(47mg·kg-1·d-1 Wenyang Huazhuo Tongluo recipe by gavage),positive control group(10 mg·kg-1·d-1 KC7F2 dissolved in phosphate buffer solution intraperitoneal injection),continuous administration for 4 weeks.The expression levels of in vitro membrane translocation enzyme 20(TOMM20),hypoxia inducible factor-1α(HIF-1α),B cell lymphoma-2/adenovirus E1B-19 kDa interacting protein 3(BNIP3),PTEN inducible muscle enzyme protein 1(PINK1)and E3 ubiquitin ligase(Parkin)in lung tissue were detected by immunohistochemistry(IHC).Western blot(WB)was used to detect the expression levels of mitophagy-related proteins(TOMM20,LC3B)and HIF-1α/BNIP3/PINK1/Parkin pathway proteins in pulmonary microvascular endothelial cells.Results The relative content of HIF-1α in microvascular endothelial cells of lung tissue in the control group,model group,Wenyang Huazhuo Tongluo recipe group and positive control group were 0.17±0.02,0.98±0.01,0.66±0.03 and 0.48±0.01;the relative content of BNIP3 were 0.40±0.02,0.74±0.01,0.56±0.01 and 0.60±0.02;the relative content of PINK1 were 0.26±0.04,0.88±0.01,0.65±0.02 and 0.67±0.02;the relative contents of Parkin were 0.33±0.02,0.89±0.01,0.65±0.02 and 0.77±0.02;the relative contents of TOMM20 were 1.10±0.02,0.58±0.01,1.02±0.01 and 0.98±0.03;the relative contents of LC3B-Ⅰ/LC3B-Ⅱ were 0.24±0.01,0.80±0.01,0.53±0.02 and 0.70±0.02,respectively.The content of HIF-1α,BNIP3,PINK1,Parkin and LC3B-Ⅰ/LC3B-Ⅱ in model group was higher than those in control group.Wenyang Huazhuo Tongluo recipe can effectively reduce its content.The content of TOMM20 in the model group was lower than that in control group,and Wenyang Huazhuo Tongluo recipe can effectively increase its content.Conclusion Wenyang Huazhuo Tongluo recipe may inhibit mitophagy and improve SSc pulmonary microvascular injury by increasing TOMM20 and inhibiting the protein expression of LC3B and HIF-1α/BNIP3/PINK1/Parkin signaling pathway.

This study was purposed to construct a fusion DNA vaccine containing WT1 multi-epitope and stimulating epitope of mycobacterium tuberculosis heat shock protein 70 and to detect its expression and immunogenicity. On the basis of published data, a multi-epitope gene (Multi-WT1) containing three HLA *0201-restricted CTL epitopes: one HLA*2402-restricted CTL epitope, two Th epitopes and one universal Th Pan-DR epitope (PADRE) was constructed. DNA-coding sequence was modified by Computer-Aided Design (CAD) to optimize proteasome-mediated epitope processing through the introduction of different amino acid spacer sequences. The synthetic nucleotide sequence was then inserted into an eukaryotic vector to construct the plasmid pcDNA3.1-WT1.For enhancing CTL activity, HSP70 fragment including stimulatory domain P407-426 was amplified by PCR from mycobacterial HSP70 gene and cloned into pcDNA3.1(+). Then Multi-WT1 was fused to the N-terminal of pcDNA3.1-mHSP70(407-426) to make the multi-epitope fusion gene vaccine pcDNA3.1-WT1-mHSP70(407-426). HEK-293T cells were transfected with this vaccine and the expressed product was identified by RT-PCR. Enzyme-linked immunospot assay (ELISPOT) was used to evaluate the immunological responses elicited by vaccine. The results showed that the most of WT1 epitopes could be correctly cleaved which was confirmed by software Net Chop 3.1 and PAPROCIanalysis. RT-PCR showed correct expression of target gene in HEK293T cells and ELISPOT showed specific T-cell responses. It is concluded that the eukaryotic expression vector PcDNA3.1-WT1-mHSP70(407-426) fusion gene has been successfully constructed and the immunity response is also elicited, which is a good candidate for further research of DNA vaccine.
Bacterial Proteins ; genetics ; immunology ; Epitopes ; genetics ; immunology ; Genetic Vectors ; HSP70 Heat-Shock Proteins ; genetics ; immunology ; Humans ; Immunodominant Epitopes ; Vaccines, DNA ; genetics ; immunology ; WT1 Proteins ; genetics ; immunology

OBJECTIVETo construct an acid-sensitive mutant of Streptococcus mutans (S. mutans) by transposon mutagenesis and to find a new gene related to the acid tolerance of S. mutans.

METHODSThe transposon Tn917 was delivered into S. mutans UA159 by the temperature-sensitive plasmid pTV1-OK bearing Tn917 and transposition of Tn917 was induced after incubation at non-permissive temperature (42 degrees C). Transposants harboring Tn917 in the chromosome were screened for the selection of mutant that had diminished growth at low pH. Southern analysis was performed with EcoRI (no cut within Tn917) digests of S. mutans UA159 and the selected aid-sensitive mutant, with DIG-labeled probe of 4.3 kb KpnI fragment of pTV1-OK containing Tn917. Genetic backcross experiment was performed by transforming the genome of the mutant to another S. mutans strain MT8148 to determine the linkage of Tn917 insertion to the change of phenotype (acid-sensitivity). Comparison of the abilities to grow at low pH, the glycolytic pH drop and killing pH values were done between the acid-sensitive mutant and the parent strain. The asymmetric PCR method was used to obtain the fragment flanking Tn917 and the PCR products were cloned to pMD18-T vector for sequencing.

RESULTSOne mutant that showed no growth at pH 5.0 was isolated from 2 316 transposants and was named as b23. Southern analysis and genetic backcross experiment confirmed the linkage between single Tn917 insertion into the chromosome and the phenotypic change (acid sensitive). b23 was less acid tolerant than UA159 in that it showed poorer growth at low pH and higher glycolytic pH minimum and higher killing pH. BLAST results indicated that Tn917 inserted into the genome of S. mutans UA159 at the site of 996 123 bp.

CONCLUSIONAn acid-sensitive mutant of S. mutans was successfully constructed and a new gene that is responsible for the acid tolerance in S. mutans UA159 was revealed.

Objective To evaluate the applicability of inhalation risk assessment model provided by US Environmental Protection Agency (USEPA ) in occupational health risk assessment in paper -making,chemical and electroplating industries.Methods A total of 1 1 factories in paper-making,chemical and electroplating industries were investigated. The risk assessment model of USEPA was used to assess occupational health risk levels,including cancer and non-cancer risks.These results were compared with occupational exposure limits,classification of occupational hazards and related literatures.Results The hazard quotient (HQ)of nasal lesions of the olfactory mucosa caused by hydrogen sulfide in paper-making industry was 36.3.The HQs of hyperplasia of nasal mucosa larynx and trachea caused by hydrogen chloride and cerebellar lesions caused by methyl chloride in organosilicon synthesis factory were 8.2 and 1.4 respectively.The HQs of decreased pulmonary function or increased severity of rhinitis and pneumonia caused by ammonia in pesticide factory were 1.2 and 1 .5 respectively.The HQ of impaired motor coordination caused by xylenes in chemical solvent factory was 2.4. The HQs of hyperplasia of nasal mucosa larynx and trachea caused by hydrogen chloride and nasal septum atrophy caused by hexavalent chromium in electroplating industry were 2.3-75.3 and 1 92.5 -25 675 respectively .The risk levels of paper-making,chemical and electroplating industries were all identified as high on the basis of evidence showing that HQs were all higher than 1 .The risk of lung cancer caused by hexavalent chromium in electroplating industry was 0.001 -0.1 647.The results of risk assessment were consistent with the reported literature.Conclusion Inhalation risk assessment model provided by USEPA can be used to assess the occupational health risks of paper -making, chemical and electroplating industries and have better identification ability.