Purification, characterization and cDNA cloning of a trypsin from the hepatopancreas of snakehead (Channa argus)

A trypsin was purified from the hepatopancreas of snakehead (Channa argus) by ammonium sulfate fractionation and a series of column chromatographies including DEAE-Sepharose, Sephacryl S-200 HR and Hi-Trap Capto-Q. The molecular mass of the purified trypsin was about 22 kDa, as estimated by SDS-PAGE. The optimum pH and temperature of the purified trypsin were 9.0 and 40 °C, respectively. The trypsin was stable in the pH range of 7.5-9.5 and below 45 °C. The enzymatic activity was strongly inhibited by serine proteinase inhibitors, such as MBTI, Pefabloc SC, PMSF, LBTI and benzamidine. Peptide mass fingerprinting (PMF) of the purified protein obtained 2 peptide fragments with 25 amino acid residues and were 100% identical to the trypsinogen from pufferfish (Takifugu rubripes). The activation energy (Ea) of this enzyme was 24.65 kJ·M^− 1. Apparent K_m was 1.02 μM and k_cat was 148 S^− 1 for fluorogenic substrate Boc-Phe-Ser-Arg-MCA. A trypsinogen gene encoding 247 amino acid residues was further cloned on the basis of the sequence obtained from PMF and the conserved site peptide of trypsinogen together with 5′-RACE and 3′-RACE. The deduced amino acid sequence contains a signal peptide of 15 residues and an activation peptide of 9 amino acid residues with a mature protein of 223 residues. The catalytic triad His-64, Asp-107, Ser-201 and 12 Cys residues which may form 6 disulfide bonds were conserved. Compared with the PMF data, only 2 amino acid residues difference were identified, suggesting the cloned trypsinogen is quite possibly the precursor of the purified trypsin.     

Expression of phytoene synthase gene (Psy) is enhanced during fruit ripening of Cara Cara navel orange (Citrus sinensis Osbeck)

Citrus is an important fruit crop as regards accumulation of carotenoids. In plant carotenoid biosynthesis, phytoene synthase gene (Psy) plays a key role in catalyzing the head-to-head condensation of geranylgeranyl diphosphate molecules to produce colorless phytoene. In the present paper, we reported the phytoene contents determination and characterization of Psy during fruit ripening of “Washington” navel orange and its red-fleshed mutant “Cara Cara”. Results showed that phytoene was exclusively accumulated in peel and pulp of “Cara Cara”. Although phytoene was observed accumulating with fruit ripening of “Cara Cara”, the contents in pulp were 10 times higher than those in peel. The isolated two Psy cDNAs were both 1520 bp in full length, containing 436 deduced amino acid residues, with a different amino acid at 412th. Genomic hybridization results showed that one or two copies might be present in “Cara Cara” and “Washington” genomes. During “Cara Cara” and “Washington” fruit coloration, expression of Psy was observed to be up-regulated, as revealed by tissue specific profiles in the flavedo, albedo, segment membrane and juice sacs. However, Psy expression in albedo of “Cara Cara” was higher than that in “Washington”, as evidenced by phytoene accumulation in the peel.     

Expression profile of long non-coding RNAs during the differentiation of human umbilical cord derived mesenchymal stem cells into cardiomyocyte-like cells

We aimed to investigate the differentially expressed long non-coding RNAs (lncRNAs) during the differentiation of human umbilical cord derived mesenchymal stem cells (hUCMSCs) into cardiomyocyte-like cells induced by 5-aza. hUCMSCs were isolated and purified from umbilical cords. After treated with 10 μmol/L 5-Aza for 24 h, hUCMSCs wereas continued to be cultured for 14 days. Comparison of cardiac specific genes and the expression profile of lncRNAs on hUCMSCs between day 14 and day 0 was performed using immunofluorescence staining, immunohistochemistry, Western blot assay, RT-PCR and lncRNA microarray. Results show that well-organized sarcomeric structure and more cTnI and MLC2a staining were seen in hUCMSCs of day 14 after 5-aza-induced compared to those in day 0. Expression of Desmin, Nkx2.5, cTnI and MLC2a of hUCMSCs was much higher on day 14 compared with day 0 (P?<?0.01). 41 differentially expressed lncRNAs were found on day 14 hUCMSCs compared those of day 0 were identified. Among them, 25 upregulated and 16 downregulated. Four out of the five upregulated lncRNAs (P?=?0.00035, 0.014, 0.016 and 0.005 for uc010vei.1, X72487, BC064139, AK092074) and four out of the five downregulated lncRNAs (P?=?0.038, 0.0014, 0.00026 and 0.004 for X85157, uc007keu.1, AK309872, NR_029399) showed significantly different expressions in further validation using RT-PCR. Our results illustrated that there was a dysregulation of the lncRNA profile during the differentiation of hUCMSCs into cardiomyocyte-like cells, which will provide the foundation for further study of the biological functions and mechanism of lncRNAs in the differentiation of hUCMSCs into cardiomyocyte-like cells.     

A conceptual model for optimizing vaccine coverage to reduce vector-borne infections in the presence of antibody-dependent enhancement

Background

Many vector-borne diseases co-circulate, as the viruses from the same family are also transmitted by the same vector species. For example, Zika and dengue viruses belong to the same Flavivirus family and are primarily transmitted by a common mosquito species Aedes aegypti. Zika outbreaks have also commonly occurred in dengue-endemic areas, and co-circulation and co-infection of both viruses have been reported. As recent immunological cross-reactivity studies have confirmed that convalescent plasma following dengue infection can enhance Zika infection, and as global efforts of developing dengue and Zika vaccines are intensified, it is important to examine whether and how vaccination against one disease in a large population may affect infection dynamics of another disease due to antibody-dependent enhancement.

Methods

Through a conceptual co-infection dynamics model parametrized by reported dengue and Zika epidemic and immunological cross-reactivity characteristics, we evaluate impact of a hypothetical dengue vaccination program on Zika infection dynamics in a single season when only one particular dengue serotype is involved.

Results

We show that an appropriately designed and optimized dengue vaccination program can not only help control the dengue spread but also, counter-intuitively, reduce Zika infections. We identify optimal dengue vaccination coverages for controlling dengue and simultaneously reducing Zika infections, as well as the critical coverages exceeding which dengue vaccination will increase Zika infections.

Conclusion

This study based on a conceptual model shows the promise of an integrative vector-borne disease control strategy involving optimal vaccination programs, in regions where different viruses or different serotypes of the same virus co-circulate, and convalescent plasma following infection from one virus (serotype) can enhance infection against another virus (serotype). The conceptual model provides a first step towards well-designed regional and global vector-borne disease immunization programs.

     

Strong-light photoinhibition treatment accelerates the changes of protein secondary structures in triton-treated photosystem I and photosystem II complexes

Changes in the protein secondary structure and electron transport activity of the Triton X-100-treated photosystem I (PSI) and photosystem II (PSII) complexes after strong illumination treatment were studied using Fourier transform-infrared (FT-IR) spectroscopy and an oxygen electrode. Short periods of photoinhibitory treatment led to obvious decreases in the rates of PSI-mediated electron transport activity and PSII-mediated oxygen evolution in the native or Triton-treated PSI and PSII complexes. In the native PSI and PSII complexes, the protein secondary structures had little changes after the photoinhibitory treatment. However, in both Triton-treated PSI and PSII complexes, short photoinhibition times caused significant loss of -helical content and increase of -sheet structure, similar to the conformational changes in samples of Triton-treated PSI and PSII complexes after long periods of dark incubation. Our results demonstrate that strong-light treatment to the Triton-treated PSI and PSII complexes accelerates destruction of the transmembrane structure of proteins in the two photosynthetic membranes.     

The over-expression of Chrysanthemum crassum CcSOS1 improves the salinity tolerance of chrysanthemum

Soil salinity represents a major constraint on plant growth. Here, we report that the over-expression of the Chrysanthemum crassum plasma membrane Na⁺/H⁺ antiporter gene CcSOS1, driven by the CaMV 35S promoter, improved the salinity tolerance of chrysanthemum ‘Jinba’. In salinity-stressed transgenic plants, both the proportion of the leaf area suffering damage and the electrical conductivity of the leaf were lower in the transgenic lines than in salinity-stressed wild type plants. After a 6 day exposure to 200 mM NaCl, the leaf content of both chlorophyll (a+b) and proline was higher in the transgenic than in the wild type plants. The activity of both superoxide dismutase and peroxidase was higher in the transgenic than in the wild type plants throughout the period of NaCl stress. The transgenic plants had a stronger control over the ingress of Na⁺ into the plant, particularly with respect to the youngest leaves, and so maintained a more favorable K⁺/Na⁺ ratio. The result suggests that a possible strategy for improving the salinity tolerance of chrysanthemum could target the restriction of Na⁺ accumulation. This study is the first to report the transgenic expression of a Na⁺ efflux carrier in chrysanthemum.     

Identification of the Serratia marcescens hemolysin determinant by cloning into Escherichia coli.

A cosmid bank of Serratia marcescens was established from which DNA fragments were cloned into the plasmid pBR322, which conferred the chromosomally encoded hemolytic activity to Escherichia coli K-12. By transposon mutagenesis with Tn1000 and Tn5 IS50L::phoA (TnphoA), the coding region was assigned to a DNA fragment, designated hly, comprising approximately 7 kilobases. Two proteins with molecular weights of 61,000 (61K protein) and 160,000 (160K protein) were expressed by the pBR322 derivatives and by a plasmid which contained the hly genes under the control of a phage T7 promoter and the T7 RNA polymerase. When strongly overexpressed the 160K protein was released by E. coli cells into the extracellular medium concomitant with hemolytic activity. The genes encoding the 61K and the 160K proteins were transcribed in the same direction. Mutants expressing a 160K protein truncated at the carboxy-terminal end were partially hemolytic. Hemolysis was progressively inhibited by saccharides with increasing molecular weights from maltotriose (Mr 504) to maltoheptaose (Mr 1,152) and was totally abolished by dextran 4 (Mr 4,000). This result and the observed influx of [14C]sucrose into erythrocytes in the presence of hemolytic E. coli transformants under osmotically protective conditions suggest the formation of defined transmembrane channels by the hemolysin.     

Precise and imprecise somatic excision of the transposon Tc1 in the nematode C. elegans.

Eleven chromosomal products of somatic excision of Tc1 transposable elements have been cloned and sequenced. The cloning method did not involve genetic reversion; therefore the products analyzed should be representative. Six empty religated target sites were from excision of one Tc1 element inserted near actin genes on linkage group V; five were from a second Tc1 element inserted elsewhere on the same linkage group. All six products from the first element were identical in sequence to an empty target site from a second strain, indicating excision had been precise. Two of the products from the second element were also precise, whereas the other three contained four extra nucleotides at the point of excision, indicating an imprecise excision. The four nucleotides are the same in all cases and could represent two terminal nucleotides of the transposon plus a two-nucleotide target site duplication. The difference in the ratio of precise to imprecise excision at the two insertion sites suggests a possible chromosomal position effect on the pathway of Tc1 somatic excision.     

重楼属植物甾体皂甙的高效液相色谱分析

应用高效液相层析技术,对重楼属十八个种植物的甾体皂甙进行了定性、定量分析。植物用甲醇提取,抽出物经DIAION柱,以90％甲醇洗出总甙。在HPLC上,用ODS柱先将总甙以用醇:水(9:1)洗脱分为三馏段,每馏再在ODS柱或Rp-8柱上以甲醇:水(8:2;7:3.5)洗脱,使各个皂甙成分完全分离。被分离的每个色谱峰与已知重楼皂甙的保留时间进行比较并配合HPLC的加入法,TLC分析及用HPLC制备少量样品做MS测定来加以定性鉴定。定量采用内标及校正曲线法。     

东亚飞蝗行为和形态型变的判定指标

通过田间、室外罩笼、室内行为测试等一系列实验,研究了东亚飞蝗群居型和散居型之间的行为和形态差异。确立了东亚飞蝗不同生态型个体的形态和行为指标．结果表明,雌雄散居型蝗蝻每分钟的跳跃次数均在1．4以下,转向次数分别在1．3和1．4以下;雌雄群居型蝗蝻每分钟的跳跃次数均在1．6以上,转向次数分别在1．6和1．5以上．群居型蝗虫的跳跃次数、转向次数显著高于散居型蝗虫。所以跳跃次数、转向次数可作为东亚飞蝗行为型变判定指标．在同型不同性别的蝗虫之间行为型变指标没有显著差异．F／C值可作为4龄以上东亚飞蝗的形态型变判定指标。而E／F值可作为东亚飞蝗成虫的形态型变判定指标．两型的F／C值都随龄期的增长而增加,且同龄期雄虫F／C均大于雌虫F／C．F／C、E／F值在不同型态和同型不同性别间均存在极显著差异．因此,确定两型形态型变标准时应将雌、雄虫分开,即雌性和雄性散居型第4、5龄及成虫的F／C值分别大于2．5、2．8、3．3和2．6、2．9、3．5;雌性和雄性群居型第4、5龄及成虫的F／C值分别小于2．5、2．7、3．1和2．5、2．8、3．3．成虫的E／F值也可以作为成蝗形态型变的判断指标．