Identification and characterization of the rhizosphere phosphate-solubilizing bacterium <Emphasis Type="Italic">Pseudomonas frederiksbergensis</Emphasis> JW-SD2 and its plant growth-promoting effects on poplar seedlings

The rhizospheric bacterium JW-SD2 was identified as Pseudomonas frederiksbergensis based on phenotypic features, the Biolog Identification System and 16S rRNA sequence analysis. The phosphate-solubilizing activity, acidification in culture media, growth rate and organic acid secretion of JW-SD2 were investigated during 192 h of cultivation. The phosphate solubilized by JW-SD2 reached 7.75 mM. The decrease of pH and increase of titratable acidity were closely correlated (Pearson’s r?=??0.953 and 0.969, respectively) with the phosphate-solubilizing activity. High concentrations of gluconic, 2-ketogluconic, pyruvic, maleic and malic acids were detected before 96 h of culture, when the strain displayed a high level of phosphate-solubilizing activity, indicating that these organic acids were efficient components in phosphate solubilization. However, acetic acid did not affect phosphate solubilization as shown by a remarkable increase at 144 h of culture when the phosphate-solubilizing activity decreased. The phosphate-solubilizing ability of JW-SD2 was significantly (P?<?0.01) affected by environmental factors. Over a broad ranges of temperature (20?35 °C), pH (4?9), salinity (0?3.0 %), and volume of medium (1/5?3/5 of flask volume), the phosphate solubilized by JW-SD2 remained above 4.00 mM, demonstrating good potential in adapting to a changing environment. The inoculation experiments indicated that JW-SD2 could significantly (P?<?0.05) promote growth of poplar (Populus euramericana cv. NL-895) in both sterilized and non-sterilized soils. The effects of plant growth promotion were greater in non-sterilized than in sterilized soil. During the 150 days of the trial, the effects of plant growth promotion by JW-SD2 first increased then decreased over time, suggesting that, in field applications, the periodic supplementation of the strain into the rhizosphere should be considered.     

人干扰素α-2b基因在烟草中的表达研究

应用PCR的技术从质粒pAIFN中扩增人干扰素α-2b(Human interferon α-2b,HuIFN α-2b)编码基因,将其连接到pBI121双元载体构建植物真核表达载体pBIFN;用冻融法将该载体转染根癌农杆菌LBA4404;并用叶盘浸染法转化烟草叶片,经转化的烟草叶片的组织培养,诱导愈伤获得再生植株。通过应用PCR,RT-PCR,Wes-tern blot和WISH/VSV方法检测获得的烟草再生植株,结果表明HuIFN α-2b基因已成功整合进烟草核基因组并表达出具有活性的HuIFN α-2b蛋白。本文对HuIFN α-2b基因在烟草核系统中的表达进行了研究,为进一步在烟草叶绿体系统中该基因的表达研究奠定了基础。     

Accuracy and precision of gait events derived from motion capture in horses during walk and trot

This study aimed to create an evidence base for detection of stance-phase timings from motion capture in horses. The objective was to compare the accuracy (bias) and precision (SD) for five published algorithms for the detection of hoof-on and hoof-off using force plates as the reference standard.     

Negative Interference by Rheumatoid Factor of Plasma B-Type Natriuretic Peptide in Chemiluminescent Microparticle Immunoassays

Background

The chemiluminescent microparticle immunoassay (CMIA) is widely used for the quantitative determination of B-type natriuretic peptide (BNP) in human ethylenediaminetetraacetic acid plasma. Rheumatoid factor (RF) is usually thought to result in a positive interference in immunoassays, but it is not clear whether its presence in plasma can lead to interferences in the CMIA of BNP.

Methods

The estimation of BNP recovery was carried out by diluting high-concentration BNP samples with RF-positive or RF-negative plasma at a ratio of 1∶9. The diluted samples were then tested using the ARCHITECT i2000 System and ARCHITECT BNP Reagent Kits and the recovery was then calculated.

Results

When the RF level ranged from 48 to 1420 IU/mL, the average recovery of BNP was 79.29% and 91.61% in the RF-positive and RF-negative plasma samples, respectively, and was thus significantly lower in the group of RF-positive plasma samples than in the group of RF-negative plasma samples. At a dilution of 1∶16, the measured BNP level increased by >36% in six of the seven RF-positive plasma samples. The recovery of BNP increased significantly in the RF-positive plasma samples after pretreatment with IgG-sensitive latex particles. In addition, The BNP recovery was not significantly related to the plasma RF at concentrations ranging from 48 to 2720 IU/mL.

Conclusions

Measurement of BNP by CMIA is susceptible to interference from RF leading to predominantly (but not exclusively) lower results. Pretreatment of samples with blocking reagents is advisable prior to the initiation of denying patient''s necessary treatment.     

靶向铜绿假单胞菌(Pseudomonas aeruginosa)粘肽合成酶PBP3的抗菌先导化合物的虚拟筛选及活性研究

【目的】开发出与铜绿假单胞菌粘肽合成酶PBP3有高亲和力,具有全新结构的先导化合物。【方法】以铜绿假单胞菌粘肽合成酶PBP3为靶点,通过分子对接软件DOCK6.5,对含有104万个小分子化合物的数据库进行了大规模虚拟筛选,选取结构相对简单的中选化合物进行合成,得到先导化合物,并验证其抑菌活性。【结果】通过grid score进行第一轮初筛,筛选出grid score分值小于–30 kcal/mol的6万个化合物,接着以amber score进行第二轮筛选,筛出amber score分值小于–20 kcal/mol的化合物约200个。最终,经过观察分析,从中挑选出4种打分高并且结构新颖的小分子化合物作为先导化合物。合成出的先导化合物及其衍生物对铜绿假单胞菌等常见微生物的最小抑菌浓度(MIC)在175–275μg/m L之间,对革兰氏阴性菌和阳性菌均有效,MIC值比作为阳性对照的磺胺嘧啶更低,说明先导化合物具有较好的抗菌活性。【结论】这些先导化合物可以进一步开发为新型抗菌药物,用于解决铜绿假单胞菌的耐药性问题。     

Induction of multiple cytotoxic T lymphocyte responses in mice by a multiepitope DNA vaccine against dengue virus serotype 1

Dengue virus (DENV) is still a major threat to human health in most tropical and subtropical countries and regions. In the present study, a multi‐epitope DNA vaccine that encodes 15 immunogenic and conserved HLA‐A*0201‐, HLA‐A*1101‐, HLA‐A*2402‐restricted CTL epitopes from DENV serotype 1 (DENV‐1) was constructed based on the eukaryotic expressing plasmid pcDNA^TM3.1/myc‐His(?) A. Immunization of HLA‐A*0201, HLA‐A*1101 and HLA‐A*2402 transgenic mice with the recombinant plasmid pcDNA^TM3.1/myc‐His(?) A‐DENV‐1‐Meg resulted in significantly greater IFN‐γ‐secreting T‐cell responses against most (14/15) CTL epitopes than occurred in mice immunized with the empty plasmid pcDNA^TM3.1/myc‐His(?) A. Additionally, the epitope‐specific T cells directed to some epitopes secreted not only IFN‐γ but also IL‐6 and/or TNF‐α. Finally, the induced epitope‐specific T cells also efficiently lysed epitope‐pulsed splenocytes and DENV‐1‐infected splenic monocytes. The present study confirms the immunogenicity of multi‐epitope DENV vaccine, suggesting that it may contribute to the development of a universal DENV vaccine.

     

泥鳅多糖清除活性氧和保护DNA链的作用

采用化学发光法和分光光度法在多种化学模拟体系中研究了泥鳅多糖清除活性氧的作用 ,并用化学发光法观察了泥鳅多糖对·OH导致DNA链损伤的抑制作用。结果表明 ,泥鳅多糖能够有效地清除O·-2 、·OH、H2 O2 等活性氧 ,对DNA链具有良好的保护作用     

DNA damage evaluated by gammaH2AX foci formation by a selective group of chemical/physical stressors

It has been reported that the phosphorylated form of histone variant H2AX (gammaH2AX) plays an important role in the recruitment of DNA repair and checkpoint proteins to sites of DNA damage, particularly at double strand breaks (DSBs). Using gammaH2AX foci formation as an indicator for DNA damage, several chemicals/stress factors were chosen to assess their ability to induce gammaH2AX foci in a 24h time frame in a human amnion FL cell line. Two direct-acting genotoxins, methyl methanesulfonate (MMS) and N-ethyl-N-nitrosourea (ENU), can induce gammaH2AX foci formation in a time- and dose-dependent manner. Similarly, an indirect-acting genotoxin, benzo[a]pyrene (BP), also induced the formation of gammaH2AX foci in a time- and dose-dependent manner. Another indirect genotoxin, 2-acetyl-aminofluorene (AAF), did not induce gammaH2AX foci formation in FL cells; however, AAF can induce gammaH2AX foci formation in Chinese hamster CHL cells. Neutral comet assays also revealed the induction of DNA strand breaks by these agents. In contrast, epigenetic carcinogens azathioprine and cyclosporine A, as well as non-carcinogen dimethyl sulfoxide, did not induce gammaH2AX foci formation in FL cells. In addition, heat shock and hypertonic saline did not induce gammaH2AX foci. Cell survival analyses indicated that the induction of gammaH2AX is not correlated with the cytotoxic effects of these agents/factors. Taken together, these results suggest that gammaH2AX foci formation could be used for evaluating DNA damage; however, the different cell types used may play an important role in determining gammaH2AX foci formation induced by a specific agent.     

Lipopolysaccharide-mediated reactive oxygen species and signal transduction in the regulation of interleukin-1 gene expression

Lipopolysaccharide (LPS) stimulates macrophages to release inflammatory cytokines, interleukin-1 beta (IL-1), and tumor necrosis factor (TNF). LPS-induced TNF suppresses scavenger receptor functions in macrophages (van Lenten, B. J., and Fogelman, A. M. (1992) J. Immunol. 148, 112-116), which is regulated by TNF-mediated protein kinases (Hsu, H. Y., and Twu, Y. C. (2000) J. Biol. Chem. 275, 41035-41048). To examine the molecular mechanism for LPS induction of IL-1 in macrophages, we demonstrated that LPS quickly stimulated reactive oxygen species (ROS), and 3 h later induced prointerleukin-1 beta (pro-IL-1, precursor of IL-1) production and IL-1 secretion. LPS stimulated pro-IL-1 message/protein between 3 and 10 h; however, there was a 40% reduction of pro-IL-1 in preincubation of the antioxidant, N-acetylcysteine (NAC). Moreover, NAC moderated LPS-induced IL-1 secretion partially via interleukin 1-converting enzyme. The maximal activity of LPS-induced ERK, JNK, and p38 was 12- (30 min), 5- (30 min), and 16-fold (15 min), respectively. In contrast, NAC reduced ERK activity to 60% and decreased p38 activity to the basal level, but JNK activity was induced 2-fold. Furthermore, the pharmacological antagonists LY294002, SB203580, curcumin, calphostin C, and PD98059 revealed the diverse roles of LPS-mediated protein kinases in pro-IL-1. On the other hand, NAC and diphenyleneiodonium chloride partially inhibited LPS-induced Rac activity and protein-tyrosine kinase (PTK), indicating that LPS-mediated ROS and NADPH oxidase correspond to Rac activation and IL-1 expression. Our findings establish for the first time that LPS-mediated PTK/phosphatidylinositol 3-kinase/Rac/p38 pathways play a more important role than pathways of PTK/PKC/MEK/ERK and of PTK/phosphatidylinositol 3-kinase/Rac/JNK in the regulation of pro-IL-1/IL-1. The findings also further elucidate the critical role of LPS-mediated ROS in signal transduction pathways. Our results suggest that understanding LPS-transduced signals in IL-1 induction upon the antibacterial action of macrophages should provide a therapeutic strategy for aberrant inflammatory responses leading to severe cellular injury or concurrent multiorgan septic damage.