Ethanol extract of fermented soybean, Chungkookjang, inhibits the apoptosis of mouse spleen, and thymus cells

Apoptosis is a step of the cell cycle which is important in the regulation of immune cell populations. Chungkookjang is a Korean traditional fermented soybean containing microorganisms, enzymes, and bioactive compounds which was used in the treatment of mouse spleen as well as thymus cells (CH1-fermented soybean containing barley, wormwood, and sea tangle; CH2-fermented soybean) and was found to exhibit substantially reduced small DNA fragmentation. An MTT assay showed that the treatment of CH1 and CH2 into the mouse splenocytes and thymocytes sharply increased their survival. Moreover, a FACS analysis also showed that CH1 and CH2 are effective at suppressing the apoptosis of splenocytes and thymocytes. The fermented soybean isoflavone concentrations, which are implicated in lowering breast and prostate cancers, lowering the risk of cardiovascular diseases, and improving bone health, were determined using Capillary Electrophoresis-Electrochemical Detection (CE-ED). The amount of Daidzein in fermented soybean significantly increased by 44-fold dramatically, compared with those in unfermented soybean. In this study, we demonstrated that ethanol extracts of Chungkookjang promote the survival of the mouse spleen and thymus cells in culture by suppressing their apoptotic death. Future studies should investigate which genes are related to apoptosis of the immune cells.     

Defining the N-linked glycosylation site of Hantaan virus envelope glycoproteins essential for cell fusion

The Hantaan virus (HTNV) is an enveloped virus that is capable of inducing low pH-dependent cell fusion. We molecularly cloned the viral glycoprotein (GP) and nucleocapsid (NP) cDNA of HTNV and expressed them in Vero E6 cells under the control of a CMV promoter. The viral gene expression was assessed using an indirect immunofluorescence assay and immunoprecipitation. The transfected Vero E6 cells expressing GPs, but not those expressing NP, fused and formed a syncytium following exposure to a low pH. Monoclonal antibodies (MAbs) against envelope GPs inhibited cell fusion, whereas MAbs against NP did not. We also investigated the N-linked glycosylation of HTNV GPs and its role in cell fusion. The envelope GPs of HTNV are modified by N-linked glycosylation at five sites: four sites on G1 (N134, N235, N347, and N399) and one site on G2 (N928). Site-directed mutagenesis was used to construct eight GP gene mutants, including five single N-glycosylation site mutants and three double-site mutants, which were then expressed in Vero E6 cells. The oligosaccharide chain on residue N928 of G2 was found to be crucial for cell fusion after exposure to a low pH. These results suggest that G2 is likely to be the fusion protein of HTNV.     

O2-binding albumin thin films: solid membranes of poly(ethylene glycol)-conjugated human serum albumin incorporating iron porphyrin

Poly(ethylene glycol) (PEG)-conjugated human serum albumin (HSA) incorporating the tetrakis(alpha,alpha,alpha,alpha-o-amidophenyl)porphinatoiron(II) derivative (FeP) [PEG(HSA-FeP)] is a unique plasma protein-based O2 carrier as a red blood cell substitute. The aqueous solution of PEG(HSA-FeP) [mw of PEG: 2-kDa (PEG2) or 5-kDa (PEG5)] was evaporated on a glass surface to produce a red-colored solid membrane. Scanning electron microscopy observations revealed that the PEG2(HSA-FeP) membrane consisted of two parts: (i) a surface layer made of a fibrous component (10 microm thickness), and (ii) a bottom layer of an amorphous phase (5 microm thickness). The condensed solution provided a thick membrane (70 microm), which also has the amorphous bottom layer. On the other hand, the PEG5(HSA-FeP) produced homogeneous membrane made of the fibrous component. The FeP active sites in the solid membrane formed very stable O2-adduct complexes at 37 degrees C with a half-lifetime of 40 h. The O2-binding affinity of the PEG2(HSA-FeP) membrane (P1/2 = 40 Torr, 25 degrees C) was 4-fold lower than that in aqueous solution, which is kinetically due to the low association rate constant. The membrane was soluble again in water and organic solvents (ethanol and chloroform) without deformation of the secondary structure of the protein. The addition of hyaluronic acid gave a free-standing flexible thin film, and it can also bind and release O2 as well. These O2-carrying albumin membranes with a micrometer-thickness would be of significant medical importance for a variety of clinical treatments.     

An Improved Method of Gene Synthesis Based on DNA Works Software and Overlap Extension PCR

A bottleneck in recent gene synthesis technologies is the high cost of oligonucleotide synthesis and post-synthesis sequencing. In this article, a simple and rapid method for low-cost gene synthesis technology was developed based on DNAWorks program and an improved single-step overlap extension PCR (OE-PCR). This method enables any DNA sequence to be synthesized with few errors, then any mutated sites could be corrected by site-specific mutagenesis technology or PCR amplification-assembly method, which can amplify different DNA fragments of target gene followed by assembly into an entire gene through their overlapped region. Eventually, full-length DNA sequence without error was obtained via this novel method. Our method is simple, rapid and low-cost, and also easily amenable to automation based on a DNAWorks design program and defined set of OE-PCR reaction conditions suitable for different genes. Using this method, several genes including Manganese peroxidase gene (Mnp) of Phanerochaete chrysosporium (P. chrysosporium), Laccase gene (Lac) of Trametes versicolor (T. versicolor) and Cip1 peroxidase gene (cip 1) of Coprinus cinereus (C. cinereus) with sizes ranging from 1.0 kb to 1.5 kb have been synthesized successfully. Bingxue Dong and Runqian Mao contributed equally to this work.     

Anti-proliferation and apoptosis induced by a novel intestinal metabolite of ginseng saponin in human hepatocellular carcinoma cells

20-O-(beta-D-glucopyranosyl)-20(S)-protopanaxadiol (IH-901), a novel intestinal bacterial metabolite of ginseng protopanaxadiol saponins, is reported to induce apoptosis in a variety of cancer cells. We purified the compound and measured its in vitro anti-tumor activity. IH-901 inhibited cell growth of human hepatocellular carcinoma SMMC7721 cells in a dose- and time-dependent manner. We also found that IH-901 induced apoptotic cell death concurrent with cell cycle arrest in G0-G1 phase in SMMC7721 cells. At the molecular level, we show that IH-901 upregulates cytochrome c, p53, and Bax expression, and downregulates pro-caspase-3 and pro-caspase-9 expressions in a dose-dependent manner, while the levels of Bcl-2 and Bcl-X(L) were unchanged in IH-901-treated SMMC7721 cells. These results provide significant insight into the anticarcinogenic action of IH-901.     

观赏用的红肉苹果的组织培养

1植物名称红肉苹果[Malus pumila var.niedzwet- zkyana(Dieck)Schneid]。2材料类别春梢幼嫩茎段。3培养条件MS为基本培养基,(1)芽诱导培养基:MS 6-BA 0.2mg·L~(-1)(单位下同) NAA 0.2;(2)芽增殖培养基:MS 6-BA 0.5 NAA 0.5:(3)生根培养基:1/2MS IBA 0.1 NAA 0.1。以上培养基中均加入5.5 g·L~(-1)琼脂粉和30g·L~(-1)蔗糖,pH 5.8;     

基于叶绿体微卫星研究鄂报春谱系遗传结构

鄂报春Primula obconica作为一种广泛栽培的园艺植物,其野生居群的遗传多样性及遗传结构的研究还少见报道。本文通过叶绿体微卫星分析了17个鄂报春野生居群(共278个个体),共发现4个多态性位点(16个等位基因),得到14个单倍型。结果表明鄂报春具有很高的总基因多样性(HT=0.971)和极低的居群内基因多样性(HS=0.028);分子方差分析(AMOVA)显示98%的变异存在于居群间。这些结果说明早期的生境片断化及有限的种子传播能力是造成当前遗传结构的重要原因。Nst值显著大于Gst值,表明关系相近的单倍型会出现在相同的地区内,同时最小生成树(MST)的分析结果证实了这样的结论。我们在最小生成树的两个组中推断出一些古老单倍型,并推测在冰期时湖北和我国的西南地区可能是该物种的避难所。