A suicidal DNA vaccine co-expressing two major membrane-associated proteins of porcine reproductive and respiratory syndrome virus antigens induce protective responses

We constructed a suicidal DNA vaccine pSFV-ORF5m/ORF6 co-expressing GP5m (a modified GP5) and M proteins of porcine reproductive and respiratory syndrome virus (PRRSV). In mice immunization, specific immune responses were elicited by the suicidal DNA vaccine pSFV-ORF5m/ORF6. The immunogenicity and protective efficiency was then evaluated in piglets immunized with pSFV-ORF5m/ORF6 before virus challenge: PRRSV-specific neutralizing antibodies and lymphocyte proliferative responses were developed. Post-PRRSV challenge, these immune responses were further boosted and partial protection was obtained.     

Lycopene accumulation affects the biosynthesis of some carotenoid-related volatiles independent of ethylene in tomato

For elucidating the regulatory mechanism of ethylene on carotenoid-related volatiles (open chain) compounds and the relationship between lycopene and carotenoid-related volatiles,transgenic tomato fruits in which ACC synthase was suppressed were used.The transgenic tomato fruit showed a significant reduction of lycopene and aroma volatiles with low ethylene production.6-methyl-5-hepten-2-one,6-methyl-5-hepten-2-ol and geranylacetone,which were suspected to be lycopene degradation products,were lower than those in wild type tomato fruits.In order to identify whether lycopene accumulation effects the biosynthesis of some carotenoid-related volatiles independent of ethylene in tomato or not,the capability of both wild type and transgenic tomato fruits discs to convert lycopene into carotenoid-related volatiles was evaluated.The data showed that external lycopene could convert into 6-methyl-5-hepten-2-one and 6-methyl-5-hepten-2-ol in vivo,Indicating that the strong inhibition of ethylene production had no effect on enzymes in the biosynthesis pathway of some carotenoid-related volatiles.Therefore,in ACS-suppression transgenic tomato fruits,the low levels of 6-methyl-5-hepten-2-one,6-methyl-5-hepten-2-ol was due to decreased lycopene accumulation,not ethylene production.Ethylene only affected the accumulation of lycopene,and then indirectly influenceed the level of lycopene-related volatiles.     

Cloning, expression and characterization of recombinant elastase from Pseudomonas aeruginosa in Picha pastoris

The gene lasB from Pseudomonas aeruginosa, which encoded elastase, was cloned and firstly successfully expressed in Pichia pastoris stain KM71 under the control of AOX promoter. The effects on the recombinant elastase activities of different pH, different temperatures and different metal ions were assayed. The full-length gene (1497 bp) encodes a preproenzyme including an N-terminal signal peptide (23 aa), a propeptide (197 aa) and mature elastase (301 aa). The recombinant elastase was secreted into culture supernatants using signal sequence from lasB and showed a single band at about 34 kDa by SDS-PAGE. The recombinant elastase expression hit the highest level of approximately 450 mg/L and the specific elastolytic activity of the recombinant elastase was 130 U/ml, which was approximately 26-fold higher than that of elastase obtained from P. aeruginosa. The optimal temperature and pH of the recombinant elastase was 28 degrees C and 7.4, respectively. The enzyme possessed high resistance to heat, and can be activated by Ca(2+). These enzyme properties suggested that it could be produced in an industrial scale and has the potential to be a commercial enzyme.     

Synthesis, characterization and bioactivity of two novel trinuclear Cu(II)/Ni(II) complexes with pentadentate ligand N-2-methyl-acryloyl-salicylhydrazide

Two new trinuclear complexes, Cu₃L₂(py)₂ (1) and Ni₃L₂(py)₄ (2), have been synthesized and characterized, where L³⁻ is N-2-methyl-acryloyl-salicylhydrazidate. Central metal ion and two terminal metal ions in the two complexes are combined by two bridging deprotonated L³⁻ ligands, forming a bent trinuclear structure unit with an M-N-N-M-N-N-M core. The bent angles in complexes 1 and 2 are 167.6(1)° and 75.4(1)°, respectively. Three nickel ions in compound 2 exhibit alternating square-planar and octahedral geometries, while three copper ions in compound 1 follow square-planar mode. The studies in solution integrity and stability of compounds 1 and 2 show they are soluble and stable in DMF. UV-Vis titrations demonstrate compound 1 is stable in DMF even in the presence of excess metal ions. Antibacterial screening data indicate the two compounds all have stronger antimicrobial activities against the tested microorganisms than ligand. The trinuclear copper compound 1 is more active than monocopper compounds in the previous study, and the trinuclear nickel compound 2 is less active than tetranuclear nickel compound in the previous study.     

Copper-induced germline apoptosis in Caenorhabditis elegans: The independent roles of DNA damage response signaling and the dependent roles of MAPK cascades

Excess copper is toxic to life. Copper has been shown to induce apoptosis in various cell lines and tissues. However, due to the lack of appropriate gene knockout animal models, data concerning the underlying pathways of copper-induced apoptosis are insufficient, especially with regards to in vivo systems. The nematode Caenorhabditis elegans is a good model to study basic biological processes, including stress responses and apoptosis. In the present study, we investigated copper-induced germline apoptosis in the C. elegans strains carrying mutated alleles of homologs to known mammalian genes that are involved in apoptosis regulation. We show here that exposing C. elegans to copper causes dose- and time-dependent germline apoptosis. The knockout of checkpoint genes hus-1, clk-2, the Bcl-2 homolog ced-9, and the BH3-only domain egl-1 did not prevent cells of the germline from copper-induced apoptosis. The loss-of-function of the tumor suppressor gene, p53/cep-1, caused a significant increase in germline apoptosis with exposure to copper, and the depletion of p53 antagonist ABL1 significantly enhanced apoptosis. The knockout of the caspase gene ced-3 and the Apaf-1 homolog ced-4 abrogated both copper-induced and physiological germline apoptosis. Germline apoptosis stopped increase in the strains lin-45(ku51), mek-2(n1989), mpk-1(ku1) under copper stresses, respectively. Copper-induced apoptosis was blocked in the loss-of-function alleles of both JNK and p38 MAPK cascades excepting pmk-3, one of the three p38 MAPK components. Together, the results of this study suggest that caspase and Apaf-1 are required for copper-induced germline apoptosis while DNA damage response genes are not essential, and that the Raf-MEK-ERK, ASK1/2-MKK7-JNK, ASK1/2-MKK3/6-p38 signaling pathways are indispensable in mediating this apoptotic response.     

Efficient Generation of Myostatin (MSTN) Biallelic Mutations in Cattle Using Zinc Finger Nucleases

Genetically engineered zinc-finger nucleases (ZFNs) are useful for marker-free gene targeting using a one-step approach. We used ZFNs to efficiently disrupt bovine myostatin (MSTN), which was identified previously as the gene responsible for double muscling in cattle. The mutation efficiency of bovine somatic cells was approximately 20%, and the biallelic mutation efficiency was 8.3%. To evaluate the function of the mutated MSTN locus before somatic cell nuclear transfer, MSTN mRNA and protein expression was examined in four mutant cell colonies. We generated marker-gene-free cloned cattle, in which the MSTN biallelic mutations consisted of a 6-bp deletion in one of the alleles and a 117-bp deletion and 9-bp insertion in the other allele, resulting in at least four distinct mRNA splice variants. In the MSTN mutant cattle, the total amount of MSTN protein with the C-terminal domain was reduced by approximately 50%, and hypertrophied muscle fibers of the quadriceps and the double-muscled phenotype appeared at one month of age. Our proof-of-concept study is the first to produce MSTN mutations in cattle, and may allow the development of genetically modified strains of double-muscled cattle.     

利用蛋白表达谱鉴定毛壳属真菌2 个种的子囊果毛形态变异性

【目的】毛壳属真菌的子囊果毛形态曾是重要的分类性状,但因容易发生变异,在现代毛壳属的分类中它们的分类学价值受到质疑。印度毛壳(Chaetomium indicum)和绳生毛壳(Chaetomium funicola)是2个依据子囊果毛形态差异定义的物种,本研究旨在从蛋白表达谱水平认识这2个种的差异及其子囊果毛的变异性,同时探讨蛋白表达谱在真菌分类中的应用价值。【方法】通过形态学观察获得印度毛壳和绳生毛壳典型菌株和子囊果毛形态变异菌株,利用双向电泳技术对典型菌株和变异菌株的蛋白表达谱进行分析和比较。【结果】双向电泳(2DE)图谱特征反映出印度毛壳和绳生毛壳之间的蛋白表达谱差异明显。根据Neighbor-joining(NJ)算法生成的系统发育树进一步显示印度毛壳和绳生毛壳分别聚在2个不同分支,即同一种的典型菌株和变异菌株聚在一起。【结论】依据子囊果毛形态特征和种特异性的蛋白表达谱特征对印度毛壳和绳生毛壳的分类结果是一致的,表明子囊果毛特征仍然是毛壳属真菌分类的重要指标。     

百部内生放线菌的分离、分类及次级代谢潜力

【目的】以对叶百部块根为材料分离内生放线菌,并对分离菌株进行分类、抗菌活性和次级代谢产物合成基因研究。【方法】样品经过严格的表面消毒,选用4种培养基分离百部内生放线菌;分离菌株通过形态观察和16S rRNA序列分析进行分类鉴定;采用琼脂移块法测试分离菌株的抗菌活性;通过PCR检测分离菌株的PKS/NPRS和卤化酶基因;使用HPLC-UV/VIS-ESI-MS/MS分析发酵产物。【结果】从6个样品中获得18株内生放线菌,分属链霉菌属(Streptomyces)、小单孢菌属(Micromonospora)、假诺卡氏菌属(Pseudonocardia)和甲基杆菌属(Methylobacterium)。分离菌株绝大部分具有抗菌活性和次级代谢产物合成基因,其中13株对耐药金黄色葡萄球菌和/或绿脓杆菌有拮抗活性,17株具有PKS/NRPS基因,8株菌具有卤化酶基因,且卤化酶阳性代表菌株的发酵产物具有抗细菌活性和卤代化合物特征。【结论】百部作为一种传统中药,其内生放线菌以链霉菌和小单孢菌为主,在次级代谢产物合成方面具有很好的潜力,可作为一类重要微生物资源进行活性产物开发。