Evidence from the anti-idiotypic network that the acetylcholine receptor is a rabies virus receptor.

We have developed idiotype-anti-idiotype monoclonal antibodies that provide evidence for rabies virus binding to the acetylcholine receptor (AChR). Hybridoma cell lines 7.12 and 7.25 resulted after fusion of NS-1 myeloma cells with spleen cells from a BALB/c mouse immunized with rabies virus strain CVS. Antibody 7.12 reacted with viral glycoprotein and neutralized virus infectivity in vivo. It also neutralized infectivity in vitro when PC12 cells, which express neuronal AChR, but not CER cells or neuroblastoma cells (clone N18), which have no AChR, were used. Antibody 7.25 reacted with nucleocapsid protein. Anti-idiotypic monoclonal antibody B9 was produced from fusion of NS-1 cells with spleen cells from a mouse immunized with 7.12 Fab. In an enzyme-linked immunosorbent assay and immunoprecipitation, B9 reacted with 7.12, polyclonal rabies virus immune dog serum, and purified AChR. The binding of B9 to 7.12 and immune dog serum was inhibited by AChR. B9 also inhibited the binding of 7.12 to rabies virus both in vitro and in vivo. Indirect immunofluorescence revealed that B9 reacted at neuromuscular junctions of mouse tissue. B9 also reacted in indirect immunofluorescence with distinct neurons in mouse and monkey brain tissue as well as with PC12 cells. B9 staining of neuronal elements in brain tissue of rabies virus-infected mice was greatly reduced. Rabies virus inhibited the binding of B9 to PC12 cells. Mice immunized with B9 developed low-titer rabies virus-neutralizing antibody. These mice were protected from lethal intramuscular rabies virus challenge. In contrast, anti-idiotypic antibody raised against nucleocapsid antibody 7.25 did not react with AChR.     

Design, synthesis and biological evaluation of novel acrylamide analogues as inhibitors of BCR-ABL kinase

A series of acrylamide analogues were designed and synthesized from Imatinib and Nilotinib as novel BCR-ABL inhibitors by application of the principle of nonclassical electronic isostere. All new compounds were evaluated for their inhibitory effects on the activity of BCR-ABL kinase and the proliferation of K562 leukemia cancer cells in vitro. The acrylamide analogues in which the substituent in C ring was trifluoromethyl group were identified as highly potent BCR-ABL kinase inhibitors. Compound 13f exhibited an IC(50) value as low as 20.6nM in ABL kinase inhibition and an IC(50) value of 32.3nM for antiproliferative activity, about 10.5-fold and 12-fold lower than those of Imatinib respectively. These results suggest that compound 13f is a promising candidate as a novel BCR-ABL kinase inhibitor for further development.     

Calcium sensing receptor regulates cardiomyocyte function through nuclear calcium

Nuclear Ca2+ plays a pivotal role in the regulation of gene expression. IP3 (inositol-1,4,5-trisphosphate) is an important regulator of nuclear Ca2+. We hypothesized that the CaR (calcium sensing receptor) stimulates nuclear Ca2+ release through IICR (IP3-induced calcium release) from perinuclear stores. Spontaneous Ca2+ oscillations and the spark frequency of nuclear Ca2+ were measured simultaneously in NRVMs (neonatal rat ventricular myocytes) using confocal imaging. CaR-induced nuclear Ca2+ release through IICR was abolished by inhibition of CaR and IP3Rs (IP3 receptors). However, no effect on the inhibition of RyRs (ryanodine receptors) was detected. The results suggest that CaR specifically modulates nuclear Ca2+ signalling through the IP3R pathway. Interestingly, nuclear Ca2+ was released from perinuclear stores by CaR activator-induced cardiomyocyte hypertrophy through the Ca2+-dependent phosphatase CaN (calcineurin)/NFAT (nuclear factor of activated T-cells) pathway. We have also demonstrated that the activation of the CaR increased the NRVM protein content, enlarged cell size and stimulated CaN expression and NFAT nuclear translocation in NRVMs. Thus, CaR enhances the nuclear Ca2+ transient in NRVMs by increasing fractional Ca2+ release from perinuclear stores, which is involved in cardiac hypertrophy through the CaN/NFAT pathway.     

Expression of OsAMT1 (1.1-1.3) in rice varieties differing in nitrogen accumulation

OsAMT is a high-affinity ammonium transporter responsible for NH ₄ ⁺ uptake by rice plants. To investigate the expression patterns of OsAMT in different genotypes in relation to nitrogen accumulation, we measured the expression of OsAMT1.1, OsAMT1.2, and OsAMT1.3 using Real-Time PCR (RT-PCR) in GD (higher N accumulation) and NG (lower N accumulation) seedlings of the Oryza sativa L. cultivar treated with 0.1 mM NH₄NO₃ and 2 mM NH₄NO₃. We found that the expression level of OsAMT1.1 was significantly higher than those of OsAMT1.2 and OsAMT1.3 in the roots treated with 0.1 mM NH₄NO₃, suggesting that OsAMT1.1 contributed the most to N accumulation among the three genes. In GD root, OsAMT1.1 had significantly higher expression levels when it was up-regulated by 0.1 mM NH₄NO₃ than when down-regulated by 2 mM NH₄NO₃. OsAMT1.1 was mainly found in GD roots treated with 0.1 mM NH₄NO₃. We conclude that the OsAMT1.1 in GD roots, which was significantly up-regulated by low N and down-regulated by high N, was the dominating factor in determining the higher N acquisition in GD than in NG at 0.1 mM NH₄NO₃.     

选择性环氧合酶-2抑制剂联合奥曲肽诱导人胃癌细胞的凋亡

目的探讨选择性环氧合酶-2抑制剂NS-398与奥曲肽联合应用对人胃癌细胞株BGC-823生长、凋亡的影响。方法体外培养BGC-823细胞,分别用NS-398(100μmol/L)与奥曲肽(1μmol/L)单独及联合处理不同时间后,倒置显微镜观察细胞形态学变化;观察生长曲线的变化;流式细胞仪检测细胞凋亡率;实时定量(Real-time)PCR检测COX-2mRNA的表达;Western blot法检测Caspase-3蛋白表达。结果倒置显微镜下,对照组BGC-823细胞生长良好,药物处理后,细胞变小、变圆,悬浮,联合组细胞形态学改变显著强于单纯用药组;药物作用后,细胞生长受抑制,出现负增长,联合组作用明显强于单纯用药组;流式细胞仪检测表明联合用药组诱导BGC-823细胞的凋亡率明显高于单一用药组和对照组(P0.01);各处理组均使BGC-823细胞COX-2mRNA表达下调(P0.05);药物处理后细胞Caspase-3蛋白表达明显增加。结论 NS-398、奥曲肽联合可协同抑制BGC-823细胞生长、增殖,其机制可能与下调COX-2mRNA表达、诱导肿瘤细胞凋亡相关。     

吉林珲春自然保护区东北虎和东北豹及其有蹄类猎物的多度与分布

珲春国家级自然保护区是东北虎(Panthera tigris altaica)、东北豹(Panthera pardus orientalis)等濒危物种在中国的核心分布区。为了探究该区域野生动物的多度水平和空间分布, 了解人类干扰情况, 我们运用相对多度指数(relative abundance index, RAI)分析了2013年4-6月设置于此的83个红外相机位点的监测数据。红外相机的总捕获天数6,060 d, 共捕获10科18种野生哺乳动物, 其中鼬科4种, 猫科动物3种, 犬科、鹿科和松鼠科各2种, 猪科、熊科、麝科、猬科和兔科各1种。研究期间共拍摄到东北虎11只个体, 东北豹13只个体。从相对多度指数来看, 东北虎的相对多度(0.84)远高于东北豹(0.48), 它们的有蹄类猎物中梅花鹿(Cervus nippon)的相对多度最高(2.18), 其次为狍(Capreolus pygargus)(1.53)和野猪(Sus scrofa)(0.92)。人类活动和放牧的相对多度水平(分别为40.64和2.76)显著高于野生动物。在空间分布上, 东北虎和梅花鹿主要在保护区的核心区分布, 且与保护区社区共管区的多度水平差异显著, 而东北豹在不同功能区之间的分布差异不显著, 狍在保护区北部的多度水平较高, 但各功能区之间差异不显著, 野猪在社区共管区的多度水平显著高于核心区。可见, 核心区频繁的人类活动和放牧活动对野生动物的保护产生了影响, 未来应加强关于人类干扰对虎、豹种群及其有蹄类猎物的影响评估。     

Selenium Deficiency Influences the Gene Expressions of Heat Shock Proteins and Nitric Oxide Levels in Neutrophils of Broilers

The aim of the present study was to investigate the effects of selenium (Se) deficiency on the expressions of heat shock proteins (Hsp90, 70, 60, 40, and 27) and nitric oxide (NO) levels in neutrophils of broilers. One hundred eighty 1-day-old broilers were randomly assigned into two groups and were fed on a low-Se diet (0.008 mg/kg Se) or a control diet (0.2 mg/kg Se), respectively. Then, the messenger RNA (mRNA) levels of Hsp90, 70, 60, 40, and 27, induced nitric oxide synthase (iNOS), and NO levels were examined. The results showed that Se deficiency increased the mRNA levels of Hsps and iNOS and induced higher level of NO in chicken neutrophils (P?iNOS had the biggest correlation with Hsp60, which indicated that Hsp60 might play an important function in inhibiting the production of NO, and the correlation coefficient between Hsp60 and Hsp70 was over 0.9, which indicated that they might have a synergistic effect. These results suggested that the level of NO and Hsp expression levels in neutrophils can be influenced by Se deficiency. And Hsp40 might play the crucial protective role in neutrophils induced by Se deficiency.     

Metabolic reprogramming of cancer-associated fibroblasts by TGF-β drives tumor growth: Connecting TGF-β signaling with “Warburg-like” cancer metabolism and L-lactate production

We have previously shown that a loss of stromal Cav-1 is a biomarker of poor prognosis in breast cancers. Mechanistically, a loss of Cav-1 induces the metabolic reprogramming of stromal cells, with increased autophagy/mitophagy, mitochondrial dysfunction and aerobic glycolysis. As a consequence, Cav-1-low CAFs generate nutrients (such as L-lactate) and chemical building blocks that fuel mitochondrial metabolism and the anabolic growth of adjacent breast cancer cells. It is also known that a loss of Cav-1 is associated with hyperactive TGF-β signaling. However, it remains unknown whether hyperactivation of the TGF-β signaling pathway contributes to the metabolic reprogramming of Cav-1-low CAFs. To address these issues, we overexpressed TGF-β ligands and the TGF-β receptor I (TGFβ-RI) in stromal fibroblasts and breast cancer cells. Here, we show that the role of TGF-β in tumorigenesis is compartment-specific, and that TGF-β promotes tumorigenesis by shifting cancer-associated fibroblasts toward catabolic metabolism. Importantly, the tumor-promoting effects of TGF-β are independent of the cell type generating TGF-β. Thus, stromal-derived TGF-β activates signaling in stromal cells in an autocrine fashion, leading to fibroblast activation, as judged by increased expression of myofibroblast markers, and metabolic reprogramming, with a shift toward catabolic metabolism and oxidative stress. We also show that TGF-β-activated fibroblasts promote the mitochondrial activity of adjacent cancer cells, and in a xenograft model, enhancing the growth of breast cancer cells, independently of angiogenesis. Conversely, activation of the TGF-β pathway in cancer cells does not influence tumor growth, but cancer cell-derived-TGF-β ligands affect stromal cells in a paracrine fashion, leading to fibroblast activation and enhanced tumor growth. In conclusion, ligand-dependent or cell-autonomous activation of the TGF-β pathway in stromal cells induces their metabolic reprogramming, with increased oxidative stress, autophagy/mitophagy and glycolysis, and downregulation of Cav-1. These metabolic alterations can spread among neighboring fibroblasts and greatly sustain the growth of breast cancer cells. Our data provide novel insights into the role of the TGF-β pathway in breast tumorigenesis, and establish a clear causative link between the tumor-promoting effects of TGF-β signaling and the metabolic reprogramming of the tumor microenvironment.