Saponins from soy bean and mung bean inhibit the antigen specific activation of helper T cells by blocking cell cycle progression

Treatment of helper T (Th) cells with saponins from soy bean and mung bean prevented their activation by inhibiting cell proliferation and cytokine secretion. However, the saponins did not affect the expression of major histocompatibility complex class II (A^b) and co-stimulatory molecule (CD86) on professional antigen-presenting cells. Instead, the saponins directly inhibited Th cell proliferation by blocking the G₁ to S phase cell cycle transition. Moreover, blocking of the cell cycle by the saponins was achieved by decreased expression of cyclin D1 and cyclin E, and constitutive expression of p27^KIP1. Saponins also increased stability of p27^KIP1 in Th cells after antigenic stimulation.     

Anaplasma phagocytophilum AnkA binds to granulocyte DNA and nuclear proteins

Human granulocytic anaplasmosis (HGA) is caused by the obligate intracellular bacterium Anaplasma phagocytophilum. The bacterium infects, survives, propagates in, and alters neutrophil phenotype, indicating unique survival mechanisms. AnkA is the only known A. phagocytophilum component that gains access beyond neutrophil vacuoles and is transported to the infected host cell nucleus. The ability of native and recombinant AnkA to bind DNA and nuclear proteins from host HL-60 cells was assessed by the use of immunoprecipitation after cis-diamminedichloroplatinum (cis-DDP) DNA-protein crosslinking, by probing uninfected HL-60 cell nuclear lysates for AnkA binding, and by recovery and sequence analysis of immunoprecipitated DNA. AnkA binds HL-60 cell DNA as well as nuclear proteins of approximately 86, 53 and 25 kDa, whereas recombinant A. phagocytophilum Msp2 or control proteins do not. DNA immunoprecipitation reveals AnkA binding to a variety of target genes in the human genome, including genes that encode proteins with ATPase, tyrosine phosphatase and NADH dehydrogenase-like functions. These data indicate that AnkA could exert some effect on cells through binding to protein:DNA complexes in neutrophil nuclei. Whether AnkA binding leads to neutrophil functional alterations, and how such alterations might occur will depend upon definitive identification of binding partners and associated metabolic and biochemical pathways.     

Immunoregulatory role of nitric oxide in Kilham rat virus-induced autoimmune diabetes in DR-BB rats

Macrophages play a critical role in the pathogenesis of Kilham rat virus (KRV)-induced autoimmune diabetes in diabetes-resistant BioBreeding (DR-BB) rats. This investigation was initiated to determine the role of macrophage-derived soluble mediators, particularly NO, in the pathogenesis of KRV-induced diabetes in DR-BB rats. We found that the expression of inducible NO synthase (iNOS), an enzyme responsible for NO production, was significantly increased during the early phase of KRV infection. Inhibition of iNOS by aminoguanidine (AG) treatment resulted in the prevention of diabetes in KRV-infected animals. The expression of IL-1beta, TNF-alpha, and IL-12 was significantly decreased in the spleen of AG-treated, KRV-infected DR-BB rats compared with PBS-treated, KRV-infected control rats. Subsequent experiments revealed that AG treatment exerted its preventive effect in KRV-infected rats by maintaining the finely tuned immune balance normally disrupted by KRV, evidenced by a significant decrease in the expression of IFN-gamma, but not IL-4, and a decrease in Th1-type chemokine receptors CCR5, CXCR3, and CXCR4. We also found that iNOS inhibition by AG decreased the KRV-induced expression of MHC class II molecules and IL-2R alpha-chain, resulting in the suppression of T cell activation, evidenced by the decreased cytolytic activity of CD8(+) T cells. We conclude that NO plays a critical immunoregulatory role by up-regulating macrophage-derived proinflammatory cytokines, up-regulating the Th1 immune response, and activating T cells, leading to type 1 diabetes after KRV infection, whereas suppression of NO production by AG treatment prevents KRV-induced autoimmune diabetes in DR-BB rats.     

转基因低水平混杂问题——政策与内涵

转基因技术近年来在世界范围内快速发展,但由于不同国家在转基因审批上的不同步,以及各国设立严格的转基因低水平混杂阈值,导致正常的农产品贸易由于无意混入少量转基因成分而发生贸易摩擦,甚至导致贸易中断.从转基因低水平混杂(LLP)的含义出发分析其特殊性,分析了世界主要国家的LLP政策以及严格的LLP政策对贸易产生的负面影响.根据研究结果指出:在当前转基因作物采用率不断提高,以及转基因新品种研发加速背景下,转基因低水平混杂在技术上是不可避免的.因此,有必要在全球范围内建立转基因安全互信机制,各国需要尽量减少审批不同步时滞,设置合理的转基因低水平阈值,降低对正常贸易的负面影响.研究结果对中国转基因LLP政策和有关标准制定有一定借鉴意义.     

长期施肥对双季稻种植下土壤有机碳库和固碳量的影响

研究了长期施用化肥和猪粪(PM)、稻草(RS)对双季稻集约化种植下30年期间(1981-2010年)土壤有机碳(SOC)及其组分的影响.结果表明：化肥平衡施用处理(NPK)的SOC、颗粒有机C(POC)和KMnO₄氧化C(KMnO₄C)组分高于化肥非平衡施用处理(NP和NK);猪粪、稻草与化肥(NK+PM、NP+RS和NPK+RS)长期配合施用处理的SOC、POC和KMnO₄ C组分显著增加.连续种植30年60季水稻后,猪粪与NK配施处理0～45 cm土层的SOC(84.71 t C·hm^-2)、POC(8.94 t C·hm^-2)和KMnO₄ C(21.09 t C·hm^-2)数量最高,其次是NPK+RS处理;NK+PM处理(485 kg C·hm^-2·a^-1)的固C量最高,其次是NPK+RS处理(375 kg C·hm^-2·a^-1).化肥与猪粪、稻草配施处理SOC的固C效率(CSE)明显高于单施化肥处理;施肥处理POC的固C效率(0.4%～1.2%)低于KMnO₄C(3.0%～8.3%).采用腐殖化常数值(h)和Jenkinson方程的衰减常数(k)可以预测不同处理2010年的SOC储量,通过Jenkinson方程可以计算维持1981年的SOC储量水平所需要的C投入量(A_E).双季稻种植下,长期连续施用NK+PM、NP+RS和NPK+RS处理的SOC含量增加是由于年C输入量高于A_E所致.在南方亚热带双季稻种植区,化肥与猪粪、稻草长期配施将促进水稻土有机碳的固定.     

Role of recombinant plasmid pEGFP-N1-IGF-1 transfection in alleviating osteoporosis in ovariectomized rats

Decreased levels of serum insulin-like growth factor-1 (IGF-1) have been proven to cause osteoporosis. Gene transfer of IGF-1 offers an attractive technology to treat skeletal metabolic disorders including osteoporosis, but the viral vectors are limited by their high antigenicity and immune response. Our purpose was to investigate the expression of a non-invasive vector, recombinant plasmid enhanced green fluorescent protein-N1 (pEGFP-N1) that transferred IGF-1 gene into ovariectomized (OVX) rats in vivo and evaluate the effect of this therapy on osteoporosis. OVX or sham operations were performed in 60 female, 7-month-old unmated SD rats. 12 weeks after OVX operation, the vectors were transfected to the 10-month-old rats and experimental data were detected from 48 h to 7 week after transfection. Our results showed that remarkable expression of fluorescence and serum IGF-1 was observed in the rats transfected by recombinant plasmids, indicating that IGF-1 gene was successfully transferred to OVX rats by injecting the vector through hydrodynamic method via the tail vein. The bone metabolism index including serum alkaline phosphatase, the histomorphometric parameters of lumbar vertebra including trabecular area percentage, trabecular thickness, trabecular number and trabecular separation, and the bone mineral density (BMD) and biomechanical parameters of lumbar vertebra including BMD, maximum condensing force, crushing strength in OVX rats transfected by pEGFP-N1-IGF-1 were improved remarkably compared with OVX+pEGFP-N1 rats, indicating that the transfection of recombinant plasmid pEGFP-N1-IGF-1 played a significant role in alleviating osteoporosis in rats induced by OVX. This encouraged a potential approach of IGF-1 gene therapy to the treatment of osteoporosis.     

Mapping QTLs with digenic epistasis under multiple environments and predicting heterosis based on QTL effects

Mixed linear model approach was proposed for mapping QTLs with the digenic epistasis and QTL by environment (QE) interaction as well as additive and dominant effects. Monte Carlo simulations indicated that the proposed method could provide unbiased estimations for both positions and genetic main effects of QTLs, as well as unbiased predictions for QE interaction effects. A method was suggested for predicting heterosis based on individual QTL effects. The immortalized F₂ (IF₂) population constructed by random mating among RI or DH lines is appropriate for mapping QTLs with epistasis and their QE interaction. Based on the models and methodology proposed, we developed a QTL mapping software, QTLMapper 2.0 on the basis of QTLmapper 1.0, which is suitable for analyzing populations of DH, RIL, F₂ and IF₂. Data of thousand grain weight of IF₂ population with 240 lines derived from elite hybrid rice Shanyou 63 were analyzed as a worked example.     

家蚕对浓核病毒中国株(BmDNV-3)抗性及感性品系中肠的差异蛋白质分析

【目的】通过比较家蚕Bombyx mori抗性及感性品系的中肠蛋白质表达谱,获得家蚕对家蚕浓核病毒中国株（BmDNV-3）抗性相关的蛋白。【方法】利用双向电泳(2-DE)对感性品种华八35和抗性品种秋丰接种病毒后48 h的蛋白质表达谱进行比较分析,并对其中的差异蛋白进行MALDI-TOF-TOF质谱分析,通过NCBInr和MSDB数据库进行蛋白点的鉴定和功能分析。【结果】获得重复性较好的差异蛋白点16个,其中质谱鉴定出5种蛋白,它们分别是糖基转移酶（glycosyltransferase）、糖基转移酶-S（GlcAT-S）、21.5 kD小热休克蛋白（21.5 kD small heat shock protein）、V-ATP酶（vacuolar ATP synthase）和精氨酸激酶（arginine kinase）。这5种蛋白在抗性品系秋丰中的表达量均高于感性品系华八35。【结论】糖基转移酶和糖基转移酶-S仅在抗性品系中存在,提示它们可能是与抗性有关的蛋白。此外,增强的应激反应和能量代谢也可能与家蚕对BmDNV-3的抗性产生相关。     

山羊KAP6-1.2基因的克隆表达及分析

目的：研究角蛋白关联蛋白KAP6-1.2对不同品种羊绒和羊毛在核苷酸和氨基酸水平上的影响及在山羊皮肤中的定位表达。方法：以阿尔巴斯白绒山羊KAP6-1.2 cDNA设计特异引物,扩增5种山羊的KAP6-1.2基因的编码区,克隆并测序。制备KAP6-1.2 cRNA探针,通过组织切片原位杂交在皮肤中进行定位表达分析。结果：6种山羊的KAP6-1.2在核苷酸和氨基酸水平上高度同源,仅吐根堡奶山羊的编码区有2个碱基与其他5种山羊不同,导致编码的两个氨基酸残基也发生了改变。原位杂交结果显示,KAP6-1.2 mRNA在10月份皮肤、胚胎125d皮肤、胚胎115d皮肤初级和次级毛囊的皮质层均有强烈的表达。结论：KAP6-1.2的核苷酸序列和氨基酸序列在不同地区、不同品种山羊中高度保守,在胚胎和成年山羊皮肤的初级和次级毛囊的皮质层均有强烈的表达。     

High-throughput antibody generation using multiplexed immunization and immunogen array analysis

In this work, the authors developed a new screening approach using multiplexed immunization and immunogen array analysis to improve the efficiency of antibody screening for high-throughput antibody generation. The immunogen array is based on a 96-well format in which different immunogens and negative as well as positive controls are immobilized in each well, thus making it possible to screen hundreds of antibody candidates simultaneously. To demonstrate this approach, a model of 4 mixed immunogens immunization was employed. In total, 675 antibody candidates were screened before and after established antibody hybridomas in parallel with immunogen arrays and enzyme-linked immunosorbent assay. The signal intensity, specificity, and cross-reactivity of produced antibody candidates were analyzed using a hierarchical cluster algorithm to track the characteristics of antibody candidates during antibody generation, which might reduce the number of false-positive and false-negative binding of antibodies. Moreover, 4 monoclonal antibodies that were produced successfully recognized their corresponding target antigens.