慢生型大豆根瘤菌GX201与固氮相关的putA、lrp基因的研究

该研究运用分子生物学方法，克隆并测定了慢生型大豆根瘤菌的putA、lrp基因序列，通过BLAST软件分析发现，lrp基因存在putA基因的上游且转录方向相反.将含有gus报告基因的转座子Tn5gusA5转座到以pLAFR3为载体的重组质粒pGXN300后，利用pLAFR3与pPHIJI质粒的不相容性建立了一套适合于慢生型大豆根瘤菌的转座子诱变体系，应用该体系诱变慢生型大豆根瘤菌GX201，成功地构建了putA、lrp基因的突变体GX20108和GX20120.根据已测定的慢生型大豆根瘤菌的putA、lrp基因序列，设计一对核苷酸引物，PCR扩增putA基因的调控调区，研究lrp基因与putA基因的相互作用机制，离体试验证明putA基因的启动子区存在一个与lrp基因相关蛋白结合区域.

利用Tn5gusA5对大豆慢生根瘤菌lrp基因的诱变

通过对重组质粒ｐＧＸＮ３００中的 ２．３ｋｂ ＥｃｏＲＩ片段测序分析发现，其上有一完整的ｌｒｐ基因和部分 ｐｕｔＡ基因，与 Ｋｉｎｇ ＮＤ等［１］报道的 Ｂ．ｊａｐｏｎｉｃｕｍ的ｌｒｐ基因ＤＮＡ序列有 ８８％同源性。利用 Ｔｎ５ ｇｕｓＡ５定位 诱变方法，对质粒ｐＧＸＮ３００进行插入诱变，得到２．３ｋｂ　ＥｃｏＲＩ片段上有Ｔｎ５ｇｕｓＡ５插入位点的质粒ｐＧＸＮ３００－ Ｔ３８，将ｐＧＸＮ３００－Ｔ３８转移到大豆馒生根瘤苗（Ｂ．ｊａｐｏｎｉｃｕｍ）ＧＸ２０１中，得到的ＧＸ２０１转移接合子与不相容 质粒ｐＰＨ１ＪＩ发生同源双交换。通过抗性及ｇｕｓＡ活性检测，筛选到一ｌｒｐ基因突变株。Ｓｏｕｔｈｅｍ杂交分析证 明这突变株的 Ｔｎ５ ｇｕｓＡ５插入确实是同源交换而不是转座产生，表明 Ｔｎ５ ｇｕｓＡ５ 诱变可以应用于大豆慢生根 瘤菌中的突变林筛选。

慢生大豆根瘤菌与竞争结瘤相关的lrp基因的克隆

通过对重组质粒 pGXN30 0中的 2 .3kbEcoRI片段测序分析发现 ,其中含有一完整的lrp基因和部分 putA基因 ,与King等报道的B .japonicum的lrp基因DNA序列有 88%同源性。应用Tn5gusA5定位诱变的方法获得了gusA基因表达的根瘤菌lrp基因突变体GX2 0 10 8。植株试验表明 ,突变株结瘤时间明显推迟 ,竞争结瘤能力也显著下降

LRP-1 variation is not associated with risk of Alzheimer's disease

Alzheimer's disease (AD) is characterised by the extensive deposition of amyloid beta (Aß) within the parenchyma and vasculature of the brain. It is hypothesised that a dysfunction in Aß degradation and/or its removal from the brain may result in accumulation as plaques. Low density lipoprotein receptor-related protein-1 (LRP-1) is a multifunctional receptor shown to be involved in cholesterol metabolism but also the removal of Aß from the brain. Its ability to transport Aß from the brain to the periphery has made it an attractive candidate for involvement in Alzheimer's disease (AD). We have assessed the frequencies of 9 tag- SNPs and the commonly studied synonymous SNP within exon 3 (rs1799986) in a multi-centre AD/control cohort and performed haplotype analysis. We found no evidence from a combined total of 412 controls and 1057 AD patients to support the involvement of LRP-1 variation, including the most commonly studied variant in rs1799986 in conferring genetic susceptibility to increased risk of AD.

Effect of neoadjuvant chemotherapy on low-density lipoprotein (LDL) receptor and LDL receptor-related protein 1 (LRP-1) receptor in locally advanced breast cancer

Low-density lipoprotein (LDL) receptors are overexpressed in most neoplastic cell lines and provide a mechanism for the internalization and concentration of drug-laden nanoemulsions that bind to these receptors. The aim of the present study was to determine whether the administration of standard chemotherapeutic schemes can alter the expression of LDL and LDL receptor-related protein 1 (LRP-1) receptors in breast carcinoma. Fragments of tumoral and normal breast tissue from 16 consecutive volunteer women with breast cancer in stage II or III were obtained from biopsies before the beginning of neoadjuvant chemotherapy and after chemotherapy, from fragments excised during mastectomy. Tissues were analyzed by immunohistochemistry for both receptors. Because complete response to treatment was achieved in 4 patients, only the tumors from 12 were analyzed. Before chemotherapy, there was overexpression of LDL receptor in the tumoral tissue compared to normal breast tissue in 8 of these patients. LRP-1 receptor overexpression was observed in tumors of 4 patients. After chemotherapy, expression of both receptors decreased in the tumors of 6 patients, increased in 4 and was unchanged in 2. Nonetheless, even when chemotherapy reduced receptors expression, the expression was still above normal. The fact that chemotherapy does not impair LDL receptors expression supports the use of drug carrier systems that target neoplastic cells by the LDL receptor endocytic pathway in patients on conventional chemotherapy.

Why new business development projects fail : coping with the differences of technological versus market knowledge

Managing through projects has become important for generating new knowledge to cope with technological and market discontinuities. This paper examines how the fit between the creation of technological and market knowledge and important project management characteristics, i.e. project autonomy and completion criteria, influences the success of new business development (NBD) projects. In-depth longitudinal case research on NBD projects commercialised from 1993 to 2003 in the consumer electronics industry highlights that project management characteristics focusing only on the creation of technological knowledge contributed to the failure of those NBD projects that required new market knowledge as well. The findings indicate that senior management support and engaging in an alliance with partners possessing complementary market knowledge can offset this misalignment of the organisation of NBD projects.

How boards strategise : a strategy as practice view

While board involvement in strategy is seen as increasingly important, our understanding of how boards fulfil this role is limited. This article draws on indepth qualitative research with directors and senior managers to develop a Strategy as Practice view on how boards "do" strategy. Two different but complementary strategising practices - Procedural Strategising and Interactive Strategising - are identified and elaborated in terms of their underlying micro-activities. The internal boardroom factors that affect the relative emphasis on these strategies practices - the strategic stance of the board, board power and perceive legitimacy of each practice - are also identified and discussed. These findings are then integrated into a typology of board strategising. A key implication of this paper is that boards need to consciously choose the nature and extent of their involvement in strategy.

Dynamic capabilities and performance : strategy, structure and environment

Dynamic capabilities are widely considered to incorporate those processes that enable organizations to sustain superior performance over time. In this paper, we argue theoretically and demonstrate empirically that these effects are contingent on organizational structure and the competitive intensity in the market. Results from partial least square structural equation modeling (PLS-SEM) analyses indicate that organic organizational structures facilitate the impact of dynamic capabilities on organizational performance. Furthermore, we find that the performance effects of dynamic capabilities are contingent on the competitive intensity faced by firms. Our findings demonstrate the performance effects of internal alignment between organizational structure and dynamic capabilities, as well as the external fit of dynamic capabilities with competitive intensity. We outline the advantages of PLS-SEM for modeling latent constructs, such as dynamic capabilities, and conclude with managerial implications.

Polymer nanocarrier system for endosome escape and timed release of siRNA with complete gene silencing and cell death in cancer cells

An influenza virus-inspired polymer mimic nanocarrier was used to deliver siRNA for specific and near complete gene knockdown of an osteoscarcom cell line (U-2SO). The polymer was synthesized by single-electron transfer living radical polymerization (SET-LRP) at room temperature to avoid complexities of transfer to monomer or polymer. It was the only LRP method that allowed good block copolymer formation with a narrow molecular weight distribution. At nitrogen to phosphorus (N/P) ratios of equal to or greater than 20 (greater than a polymer concentration of 13.8 μg/mL) with polo-like kinase 1 (PLK1) siRNA gave specific and near complete (>98%) cell death. The polymer further degrades to a benign polymer that showed no toxicity even at polymer concentrations of 200 μg/mL (or N/P ratio of 300), suggesting that our polymer nanocarrier can be used as a very effective siRNA delivery system and in a multiple dose administration. This work demonstrates that with a well-designed delivery device, siRNA can specifically kill cells without the inclusion of an additional clinically used highly toxic cochemotherapeutic agent. Our work also showed that this excellent delivery is sensitive for the study of off-target knockdown of siRNA.

Comparative analysis of FimB and FimE recombinase activity

FimB and FimE are site-specific recombinases, part of the λ integrase family, and invert a 314 bp DNA switch that controls the expression of type 1 fimbriae in Escherichia coli. FimB and FimE differ in their activity towards the fim switch, with FimB catalysing inversion in both directions in comparison to the higher-frequency but unidirectional on-to-off recombination catalysed by FimE. Previous work has demonstrated that FimB, but not FimE, recombination is completely inhibited in vitro and in vivo by a regulator, PapB, expressed from a distinct fimbrial locus. The aim of this work was to investigate differences between FimB and FimE activity by exploiting the differential inhibition demonstrated by PapB. The research focused on genetic changes to the fim switch that alter recombinase binding and its structural context. FimB and FimE still recombined a switch in which the majority of fimS DNA was replaced with a larger region of non-fim DNA. This demonstrated a minimal requirement for FimB and FimE recombination of the Fim binding sites and associated inverted repeats. With the original leucine-responsive regulatory protein (Lrp) and integration host factor (IHF)-dependent structure removed, PapB was now able to inhibit both recombinases. The relative affinities of FimB and FimE were determined for the four ‘half sites’. This analysis, along with the effect of extensive swaps and duplications of the half sites on recombination frequency, demonstrated that FimB recruitment and therefore subsequent activity was dependent on a single half site and its context, whereas FimE recombination was less stringent, being able to interact initially with two half sites with equally high affinity. While increasing FimB recombination frequencies failed to overcome PapB repression, mutations made in recombinase binding sites resulted in inhibition of FimE recombination by PapB. Overall, the data support a model in which the recombinases differ in loading order and co-operative interactions. PapB exploits this difference and FimE becomes susceptible when its normal loading is restricted or changed.

Genetic determinants of bone density and fracture risk: State of the art and future directions

Context: Osteoporosis is a common, highly heritable condition that causes substantial morbidity and mortality, the etiopathogenesis of which is poorly understood. Genetic studies are making increasingly rapid progress in identifying the genes involved. Evidence Acquisition and Synthesis: In this review, we will summarize the current understanding of the genetics of osteoporosis based on publications from PubMed from the year 1987 onward. Conclusions: Most genes involved in osteoporosis identified to date encode components of known pathways involved in bone synthesis or resorption, but as the field progresses, new pathways are being identified. Only a small proportion of the total genetic variation involved in osteoporosis has been identified, and new approaches will be required to identify most of the remaining genes.

Withania somnifera reverses Alzheimer's disease pathology by enhancing low-density lipoprotein receptor-related protein in liver

A 30-d course of oral administration of a semipurified extract of the root of Withania somnifera consisting predominantly of withanolides and withanosides reversed behavioral deficits, plaque pathology, accumulation of beta-amyloid peptides (A beta) and oligomers in the brains of middle-aged and old APP/PS1 Alzheimer's disease transgenic mice. It was similarly effective in reversing behavioral deficits and plaque load in APPSwInd mice (line J20). The temporal sequence involved an increase in plasma A beta and a decrease in brain A beta monomer after 7 d, indicating increased transport of A beta from the brain to the periphery. Enhanced expression of low-density lipoprotein receptor-related protein (LRP) in brain microvessels and the A beta-degrading protease neprilysin (NEP) occurred 14-21 d after a substantial decrease in brain A beta levels. However, significant increase in liver LRP and NEP occurred much earlier, at 7 d, and were accompanied by a rise in plasma sLRP, a peripheral sink for brain A beta. In WT mice, the extract induced liver, but not brain, LRP and NEP and decreased plasma and brain A beta, indicating that increase in liver LRP and sLRP occurring independent of A beta concentration could result in clearance of A beta. Selective down-regulation of liver LRP, but not NEP, abrogated the therapeutic effects of the extract. The remarkable therapeutic effect of W. somnifera mediated through up-regulation of liver LRP indicates that targeting the periphery offers a unique mechanism for A beta clearance and reverses the behavioral deficits and pathology seen in Alzheimer's disease models.

Wnt and FGF signaling in C. elegans vulval cell lineage polarity

The interpretation of extracellular cues leading to the polarization of intracellular components and asymmetric cell divisions is a fundamental part of metazoan organogenesis. The C. elegans vulva, with its invariant cell lineage and interaction of multiple cell signaling pathways, provides an excellent model for the study of cell polarity within an organized epithelial tissue. Herein I discuss the interaction of Wnt and FGF signaling in controlling vulval cell lineage polarity with emphasis on the posterior-most cell that forms the vulva, P7.p.

The mirror symmetry of the C. elegans vulva is achieved by the opposite division orientation of the vulval precursor cells (VPCs) flanking the axis of symmetry. Opposing Wnt signals control the division patterns of the VPCs by controlling the localization of SYS-1/ β-catenin toward the direction of the Wnt gradient. Multiple Wnt signals, expressed at the axis of symmetry, promote the wild-type, anterior-facing, P7.p orientation, whereas Wnts EGL-20 and CWN-1 from the tail and posterior body wall muscle, respectively, promote the daughter cells of P7.p to face the posterior. EGL-20 acts through a member of the LDL receptor superfamily, LRP-2, along with Ror/CAM-1 and Van Gogh/VANG-1. All three transmembrane proteins control orientation through the localization of the SYS-1.

The Fibroblast Growth Factor (FGF) pathway acts in concert with LIN-17/Frizzled to regulate the localization of SYS-1. The source of the FGF ligand is the 1° VPC, P6.p, which controls the polarity of the neighboring 2° VPC, P7.p, by signaling through the sex myoblasts (SMs), activating the FGF pathway. The Wnt, cwn-1, is expressed in the posterior body wall muscle of the worm as well as the SMs, making it the only Wnt expressed on the posterior and anterior sides of P7.p at the time of the polarity decision. Both sources of cwn-1 act instructively to influence P7.p polarity in the direction of the Wnt gradient. The FGF pathway leads to the regulation of cwn-1 transcripts in the SMs. These results illustrate the first evidence of the interaction between FGF and Wnt in C. elegans development and vulval cell lineage polarity as well as highlight the promiscuous nature of Wnt signaling within C. elegans.