Disruption of YPS1 and PEP4 genes reduces proteolytic degradation of secreted HSA/PTH in Pichia pastoris GS115

Human serum albumin (HSA) and human parathyroid hormone (1-34) [PTH (1-34)] fusion protein [HSA/PTH (1-34)] is a promising long-acting form of PTH (1-34) for osteoporosis treatment. Secretory expression of intact HSA/PTH (1-34) in Pichia pastoris GS115 was accompanied by two degradation fragments, with molecular weights around 66 kDa, in addition to the well-known ~45 kDa HSA-truncated fragment, resulting in a low yield of intact protein. In this study, two internal cleavage sites were identified in the PTH (1-34) portion of the fusion protein by Western Blot analysis. To minimize proteolytic cleavages, several protease genes including PEP4 (encoding proteinase A), PRB1 (proteinase B) and seven YPSs genes (yapsin family members) were knocked out respectively by disruption of the individual genes and the selective combinations. Reduced degradation was observed by single disruption of either PEP4 gene or YPS1 gene, and the lowest level of degradation was observed in a pep4△yps1△ double disruptant. After 72 h of induction, more than 80 % of the HSA/PTH (1-34) secreted by the pep4△yps1△ double disruptant remained intact, in comparison to only 30 % with the wild-type strain.     

The cell agglutination agent, phytohemagglutinin-L, improves the efficiency of somatic nuclear transfer cloning in cattle (Bos taurus)

One of the several factors that contribute to the low efficiency of mammalian somatic cloning is poor fusion between the small somatic donor cell and the large recipient oocyte. This study was designed to test phytohemagglutinin (PHA) agglutination activity on fusion rate, and subsequent developmental potential of cloned bovine embryos. The toxicity of PHA was established by examining its effects on the development of parthenogenetic bovine oocytes treated with different doses (Experiment 1), and for different durations (Experiment 2). The effective dose and duration of PHA treatment (150 microg/mL, 20 min incubation) was selected and used to compare membrane fusion efficiency and embryo development following somatic cell nuclear transfer (Experiment 3). Cloning with somatic donor fibroblasts versus cumulus cells was also compared, both with and without PHA treatment (150 microg/mL, 20 min). Fusion rate of nuclear donor fibroblasts, after phytohemagglutinin treatment, was increased from 33 to 61% (P < 0.05), and from 59 to 88% (P < 0.05) with cumulus cell nuclear donors. The nuclear transfer (NT) efficiency per oocyte used was improved following PHA treatment, for both fibroblast (13% versus 22%) as well as cumulus cells (17% versus 34%; P < 0.05). The cloned embryos, both with and without PHA treatment, were subjected to vitrification and embryo transfer testing, and resulted in similar survival (approximately 90% hatching) and pregnancy rates (17-25%). Three calves were born following vitrification and embryo transfer of these embryos; two from the PHA-treated group, and one from non-PHA control group. We concluded that PHA treatment significantly improved the fusion efficiency of somatic NT in cattle, and therefore, increased the development of cloned blastocysts. Furthermore, within a determined range of dose and duration, PHA had no detrimental effect on embryo survival post-vitrification, nor on pregnancy or calving rates following embryo transfer.     

Response of rice (Oryza sativa) with root surface iron plaque under aluminium stress

BACKGROUND AND AIMS: Rice (Oryza sativa) is an aquatic plant with a characteristic of forming iron plaque on its root surfaces. It is considered to be the most Al-tolerant species among the cereal crops. The objective of this study was to determine the effects of root surface iron plaque on Al translocation, accumulation and the change of physiological responses under Al stress in rice in the presence of iron plaque. METHODS: The japonica variety rice, Koshihikari, was used in this study and was grown hydroponically in a growth chamber. Iron plaque was induced by exposing the rice roots to 30 mg L(-1) ferrous iron either as Fe(II)-EDTA in nutrient solution (6 d, Method I) or as FeSO(4) in water solution (12 h, Method II). Organic acid in root exudates was retained in the anion-exchange resin and eluted with 2 m HCl, then analysed by high-performance liquid chromatography (HPLC) after proper pre-treatment. Fe and Al in iron plaque were extracted with DCB (dithionite-citrate-bicarbonate) solution. KEY RESULTS AND CONCLUSIONS: Both methods (I and II) could induce the formation of iron plaque on rice root surfaces. The amounts of DCB-extractable Fe and Al on root surfaces were much higher in the presence of iron plaque than in the absence of iron plaque. Al contents in root tips were significantly decreased with iron plaque; translocation of Al from roots to shoots was significantly reduced with iron plaque. Al-induced secretion of citrate was observed and iron plaque could greatly depress this citrate secretion. These results suggested that iron plaque on rice root surfaces can be a sink to sequester Al onto the root surfaces and Fe ions can pre-saturate Al-binding sites in root tips, which protects the rice root tips from suffering Al stress to a certain extent.     

Analysis of the downstream region of nodD3 P1 promoter by deletion and complementation tests in Sinorhizobium meliloti

Rhizobia specifically interacts with the host, leguminous plants, leading to the formation of nitrogen-fixing root nodules. The Rhizobium genes essential for nodule formation are called nodulation genes (nod or nol). The expression of nod genes requires the presence of host signals, generally flavonoids, and the product of regulatory nodD gene, NodD[1,2]. The expression of nod genes results in the synthesis of Nod factors, which serve as the signal molecules to elicit root cor-tical cells di…     

三七总RNA提取方法的对比研究

比较利用改进的异硫氰酸胍一步法、异硫氰酸胍高盐法、CTAB法和Thomas’RNA提取法等4种方法提取三七根茎2个部位总RNA的可行性。结果表明,改进的异硫氰酸胍一步法和异硫氰酸胍高盐法能有效地抑制酚类物质、多糖及皂苷等次级代谢产物对总RNA的影响,可从三七根茎中获得质量高、完整性好的总RNA。RT—PCR分析显示提取的总RNA具有反转录活性。这2种方法具有快速、简单、有效的特点。     

红豆杉生物工程的研究进展

自从紫杉醇(taxol,又称paclitaxel,结构见图1)被批准作为植源性抗癌药应用以来,由于其天然资源的限制,使得近几年来对寻找及扩大紫杉醇药源途径的研究取得了极大的进展。这些途径大致包括:(1)筛选高产量红豆杉(Taxus)栽培品种,(2)化学合成,(3)生物技术,(4)微生物生产。在这些研究领域特别是生物技术研究领域中,由于目前对紫杉醇的生物合成及关键酶研究所取得的进展,已使人们相信通过基因工程手段作为最佳生产紫杉醇药源途径之一,在不久的将来将会变为现实。本文报道的就是有关紫杉醇在生物工程方面的研究进展概况。1　研究简史红豆杉体外培…     

Characterization and function analysis of a cold-induced AmCIP gene encoding a dehydrin-like protein in Ammopiptanthus mongolicus.

A cDNA clone was isolated after difference screening from cotyledons of two-week cold-treated Ammopiptanthus mongolicus. The full-length cDNA sequence [designated as AmCIP (A. mongolicus cold-induced protein) gene] was 806 bp long and contained a 465 bp open reading frame (ORF) encoding a 16.6 kD protein of 154 amino acids. Bioinformatic analyses indicated that CIP belongs to dehydrin family with the features of high hydrophilicity, a helix K-segment, a long Gly-rich region and a phosphorylatable tract of Ser as well as deficiency in Cys and Trp. The expression of CIP gene increased after two weeks of cold treatment and more expression was detected in radicle than in cotyledon. And PCR amplification of the AmCIP gene from genome of A. mongolicus revealed this gene has no intron. Function prediction suggested this protein seems to protect the stabilization of membrane structure and prevent macromolecular coagulation or sequestrate calcium ions by association or disassociation with membrane under low temperature conditions.     

Simultaneous expression of glutaryl-7-aminocephalosporanic acid acylase gene and lysis genes of phage λ in a recombinant E. coli

Glutaryl-7-aminocephalosporanic acid acylase (GLA), recommended for use in the form of immobilized-enzyme, is one of the two key enzymes in the two-step synthesis of 7-aminocephalosporanic acid. For simplifying the process of cell disruption and immobilization, the lysis genes of phage λ (S⁻RRz) with the S amber mutation were designed to introduce into the over-expression system of GLA. A novel recombinant strain, E. coli TB1/pMKC-AS, simultaneously containing the maltose binding protein gene (malE), the lysis genes (S⁻RRz) and the target GLA gene (Acy) in a same operon, was successfully constructed. Under neutral pH conditions, cell growth and GLA activity of TB1/pMKC-AS was not affected by the presence of the lysis genes, however, autolysis phenomenon was observed under weak alkaline conditions. Through pH control and fed-batch culture, the GLA activity of TB1/pMKC-AS reached as high as 6810 U/L with 24.8 g/L dry cell density (OD₆₀₀ = 67.9) in a 5 L fermentor. In contrast to the cells of E. coli TB1/pMKC-Acy without the lysis genes, the mild EDTA/Tris buffer (pH 8.0) can cause the lysis of the cells of TB1/pMKC-AS containing the lysis genes. Correspondingly, a mild pH 9.0/42 °C incubation method was developed for conveniently degrading the recombinant cells of TB1/pMKC-AS, based on the expression of the lysis genes. Further experiments showed that the cell lysate after the mild incubation disruption can be directly immobilized by 10% polyacrylamide to make the immobilized enzymes. In comparison with the immobilized GLA from TB1/pMKC-Acy, the immobilized cell lysate of TB1/pMKC-AS has the similar characteristics of catalysis stability, implying a great potential for industrial application of the lysis genes-assisted cell disruption.     

Facilitation promotes invasions in plant‐associated microbial communities

While several studies have established a positive correlation between community diversity and invasion resistance, it is less clear how species interactions within resident communities shape this process. Here, we experimentally tested how antagonistic and facilitative pairwise interactions within resident model microbial communities predict invasion by the plant–pathogenic bacterium Ralstonia solanacearum. We found that facilitative resident community interactions promoted and antagonistic interactions suppressed invasions both in the lab and in the tomato plant rhizosphere. Crucially, pairwise interactions reliably explained observed invasion outcomes also in multispecies communities, and mechanistically, this was linked to direct inhibition of the invader by antagonistic communities (antibiosis), and to a lesser degree by resource competition between members of the resident community and the invader. Together, our findings suggest that the type and strength of pairwise interactions can reliably predict the outcome of invasions in more complex multispecies communities.     

LC-MS/MS determination of helicid in human plasma and its application in pharmacokinetic studies

Helicid is a traditional Chinese medicine used to treat headache and insomnia with definite effects. To facilitate pharmacokinetic studies of helicid in man, a sensitive and specific LC-MS/MS method for the quantitative detection of helicid in human plasma was developed and validated. The method involved the addition of bergeninum as the internal standard (IS), protein precipitation, HPLC separation, and quantification by MS/MS system using negative electrospray ionization in the multiple reaction monitoring mode (MRM). The precursor→product ion transitions were monitored at m/z 282.8→120.9 for helicid and m/z 326.9→192.2 for the IS, respectively. The lower limit of quantification (LLOQ) was 0.2 μg/L. The calibration curves for helicid was linear over a concentration range of 0.2-20 μg/L. The intra- and inter-batch analyses of QC samples at 0.4, 2, 20 μg/L indicated good precision (%R.S.D. between 2.69 and 5.47%) and accuracy (between 96.15 and 105.05%). The helicid was stable in human plasma stored at room temperature for at least 24h, 4°C for at least 24h, -20°C for at least 1 month, and for routine three freeze-thaw cycles. This accurate and specific assay provides a useful method for evaluating the pharmacokinetic profile of helicid in humans.