Tetralones and flavonoids from Pyrola calliantha

Two new tetralones, pyrolones A (1) and B (2), and a new flavonol glycoside, 2'-O-(4-hydroxybenzoyl)hyperin (3), were isolated from Pyrola calliantha (whole plant), together with six structurally related compounds, including 2'-O-galloylhyperin (4), hyperin (5), formononetin (6), quercetin 3-O-alpha-L-arabinopyranoside (7), quercetin 3-O-alpha-L-arabinofuranoside (8), and kaempferol 3-O-beta-D-galactopyranoside (9). The structures and absolute configurations of the new compounds were elucidated on the basis of spectroscopic (UV, ORD, CD, NMR) and mass-spectrometric (HR-ESI-MS) analyses.     

Viral small interfering RNAs target host genes to mediate disease symptoms in plants

The Cucumber mosaic virus (CMV) Y-satellite RNA (Y-Sat) has a small non-protein-coding RNA genome that induces yellowing symptoms in infected Nicotiana tabacum (tobacco). How this RNA pathogen induces such symptoms has been a longstanding question. We show that the yellowing symptoms are a result of small interfering RNA (siRNA)-directed RNA silencing of the chlorophyll biosynthetic gene, CHLI. The CHLI mRNA contains a 22-nucleotide (nt) complementary sequence to the Y-Sat genome, and in Y-Sat-infected plants, CHLI expression is dramatically down-regulated. Small RNA sequencing and 5' RACE analyses confirmed that this 22-nt sequence was targeted for mRNA cleavage by Y-Sat-derived siRNAs. Transformation of tobacco with a RNA interference (RNAi) vector targeting CHLI induced Y-Sat-like symptoms. In addition, the symptoms of Y-Sat infection can be completely prevented by transforming tobacco with a silencing-resistant variant of the CHLI gene. These results suggest that siRNA-directed silencing of CHLI is solely responsible for the Y-Sat-induced symptoms. Furthermore, we demonstrate that two Nicotiana species, which do not develop yellowing symptoms upon Y-Sat infection, contain a single nucleotide polymorphism within the siRNA-targeted CHLI sequence. This suggests that the previously observed species specificity of Y-Sat-induced symptoms is due to natural sequence variation in the CHLI gene, preventing CHLI silencing in species with a mismatch to the Y-Sat siRNA. Taken together, these findings provide the first demonstration of small RNA-mediated viral disease symptom production and offer an explanation of the species specificity of the viral disease.     

Self-incompatibility: Smi silences through a novel sRNA pathway

Self-incompatibility in Brassicaceae is determined by the interaction between S-Locus Protein 11 (SP11) on the pollen and S-receptor kinase (SRK) in the stigma. Pollen from heterozygotes generally displays products of both SP11 alleles, but in some heterozygotes SP11 expression is monoallelic, with one allele (SP11(R)) being silenced by promoter methylation. An exciting development in understanding the mechanism behind monoallelic silencing came recently when Y. Tarutani et al. [Nature 2010;466:983-986] identified a 24-nucleotide sRNA (termed Smi) derived from a non-coding gene within the dominant S-haplotype, and suggested that Smi directs promoter methylation. We propose that rather than having a direct effect on DNA methylation, Smi is the first step in a novel cis-acting siRNA pathway that directs widespread monoallelic SP11(R) promoter methylation.     

Characterization of Nef-CXCR4 interactions important for apoptosis induction

The HIV-1 Nef protein was analyzed for apoptotic structural motifs that interact with the CXCR4 receptor and induce apoptosis in CD4(+) lymphocytes. Two apoptotic motifs were identified. One centered on Nef amino acids (aa) 50 to 60, with the overlapping 20-mer peptides retaining about 82% of the activity of the full Nef protein. The second centered on aa 170 to 180, with the overlapping 20-mer peptides retaining about 30% of the activity of the full protein. Significant apoptotic abilities were observed for 11-mer motif peptides spanning aa 50 to 60 and aa 170 to 180, with a scrambled version of the 11-mer motif peptide corresponding to aa 50 to 60 showing no apoptotic ability. Hallmarks of apoptosis, such as the formation of DNA ladders and caspase activation, that were observed with the full-length protein were equally evident upon exposure of cells to these motif peptides. A CXCR4 antibody and the endogenous ligand SDF-1alpha were effective in blocking Nef peptide-induced apoptosis as well as the physical binding of a fluorescently tagged Nef protein, while CCR5 antibodies were ineffective. The CXCR4-negative cell line MDA-MB-468 was resistant to the apoptotic peptides and became sensitive to the apoptotic peptides upon transfection with a CXCR4-expressing vector. A fluorescently tagged motif peptide and Nef protein displayed physical binding to CXCR4-transfected MDA-MB-468 cells, but not to CCR5-transfected cells. The removal of the apoptotic motif sequences from the full-length protein completely eliminated the ability of Nef to induce apoptosis. However, these modified Nef proteins still retained the ability to enhance viral infectivity. Thus, specific sequences in the Nef protein appear to be necessary for Nef protein-induced apoptosis as well as for physical interaction with CXCR4 receptors.     

RNA interference-inducing hairpin RNAs in plants act through the viral defence pathway

RNA interference (RNAi) is widely used to silence genes in plants and animals. It operates through the degradation of target mRNA by endonuclease complexes guided by approximately 21 nucleotide (nt) short interfering RNAs (siRNAs). A similar process regulates the expression of some developmental genes through approximately 21 nt microRNAs. Plants have four types of Dicer-like (DCL) enzyme, each producing small RNAs with different functions. Here, we show that DCL2, DCL3 and DCL4 in Arabidopsis process both replicating viral RNAs and RNAi-inducing hairpin RNAs (hpRNAs) into 22-, 24- and 21 nt siRNAs, respectively, and that loss of both DCL2 and DCL4 activities is required to negate RNAi and to release the plant's repression of viral replication. We also show that hpRNAs, similar to viral infection, can engender long-distance silencing signals and that hpRNA-induced silencing is suppressed by the expression of a virus-derived suppressor protein. These findings indicate that hpRNA-mediated RNAi in plants operates through the viral defence pathway.     

RNA silencing and plant viral diseases

RNA silencing plays a critical role in plant resistance against viruses, with multiple silencing factors participating in antiviral defense. Both RNA and DNA viruses are targeted by the small RNA-directed RNA degradation pathway, with DNA viruses being also targeted by RNA-directed DNA methylation. To evade RNA silencing, plant viruses have evolved a variety of counter-defense mechanisms such as expressing RNA-silencing suppressors or adopting silencing-resistant RNA structures. This constant defense-counter defense arms race is likely to have played a major role in defining viral host specificity and in shaping viral and possibly host genomes. Recent studies have provided evidence that RNA silencing also plays a direct role in viral disease induction in plants, with viral RNA-silencing suppressors and viral siRNAs as potentially the dominant players in viral pathogenicity. However, questions remain as to whether RNA silencing is the principal mediator of viral pathogenicity or if other RNA-silencing-independent mechanisms also account for viral disease induction. RNA silencing has been exploited as a powerful tool for engineering virus resistance in plants as well as in animals. Further understanding of the role of RNA silencing in plant-virus interactions and viral symptom induction is likely to result in novel anti-viral strategies in both plants and animals.     

两种血吸虫病DNA疫苗的候选抗原基因研究

目的：以日本血吸虫基因SjFABP和SjGST原核表达产物检测二价DNA疫苗pVIVO2-SjFABP-SjGST在体内诱发的特异性抗体。方法：克隆日本血吸虫抗原基因SjFABP和sjGST,构建重组原核表达载体pET30a-SjFABP、pET30a-SjGST及真核表达载体pVIVO2-SjFABP-SjGST;将pET30a-SjFABP和pET30a-sjGST进行原核表达,并将表达产物用镍亲和柱分离纯化;采用Western印迹对日本血吸虫DNA疫苗pVIVO2-SjFABP-SjGST免疫4周后的BALB／c小鼠血清进行特异性抗体检测。结果：克隆了日本血吸虫抗原基因SjFABP（399bp）和町GST（657bp）,并构建了pET30a-SjFABP、pET30a-SjGST及pVIVO2-SjFABP-SjGST重组质粒;经Western印迹检测,pET30a-SjFABP及pET30a-SjGST原核表达的抗原蛋白均能够与经日本血吸虫二价DNA疫苗pVIVO2-SjFABP-SjGST免疫的小鼠的血清产生特异性免疫反应。结论：日本血吸虫町尉即和町GST基因的原核表达系统成功建立;原核表达的抗原蛋白具有免疫原性;以原核表达产物可检测日本血吸虫DNA疫苗pVIVO2-SiFABP-SiGST在体内诱发的特异性抗体。     

Small molecule antagonizes autoinhibition and activates AMP-activated protein kinase in cells

AMP-activated protein kinase (AMPK) serves as an energy sensor and is considered a promising drug target for treatment of type II diabetes and obesity. A previous report has shown that mammalian AMPK alpha1 catalytic subunit including autoinhibitory domain was inactive. To test the hypothesis that small molecules can activate AMPK through antagonizing the autoinhibition in alpha subunits, we screened a chemical library with inactive human alpha1(394) (alpha1, residues 1-394) and found a novel small-molecule activator, PT1, which dose-dependently activated AMPK alpha1(394), alpha1(335), alpha2(398), and even heterotrimer alpha1beta1gamma1. Based on PT1-docked AMPK alpha1 subunit structure model and different mutations, we found PT1 might interact with Glu-96 and Lys-156 residues near the autoinhibitory domain and directly relieve autoinhibition. Further studies using L6 myotubes showed that the phosphorylation of AMPK and its downstream substrate, acetyl-CoA carboxylase, were dose-dependently and time-dependently increased by PT1 with-out an increase in cellular AMP:ATP ratio. Moreover, in HeLa cells deficient in LKB1, PT1 enhanced AMPK phosphorylation, which can be inhibited by the calcium/calmodulin-dependent protein kinase kinases inhibitor STO-609 and AMPK inhibitor compound C. PT1 also lowered hepatic lipid content in a dose-dependent manner through AMPK activation in HepG2 cells, and this effect was diminished by compound C. Taken together, these data indicate that this small-molecule activator may directly activate AMPK via antagonizing the autoinhibition in vitro and in cells. This compound highlights the effort to discover novel AMPK activators and can be a useful tool for elucidating the mechanism responsible for conformational change and autoinhibitory regulation of AMPK.     

六种同域分布雌雄异株榕树挥发物多样性与系统发育一致性

植物花挥发物是维持昆虫-植物传粉互作的重要化学信号,其组成的多样性对于理解传粉昆虫与植物相互作用的形成、维持和进化具有重要意义。榕树与其传粉榕小蜂是一种专性的互惠共生关系,榕树为榕小蜂提供繁殖场所,榕小蜂为榕树传粉。而榕果雌花期（接收期）释放的特异性挥发物是维持双方相互作用的关键媒介。本研究以西双版纳地区同域分布的6种雌雄异株榕树为研究对象,用固相微萃取和气相色谱质谱联用仪对雌花期榕果的挥发物组成进行了分析。共发现77种化合物,包含脂肪酸类衍生物6种、单萜类化合物16种、倍半萜类衍生物50种和芳香族化合物5种,不同化合物对挥发物距离差异性的贡献率依次是：对甲基苯甲醚（4-methylanisole,2.15）、β-罗勒烯（β-Ocimene,0.84）和喜沙木烯（Prezizaene,0.73）。不同物种间挥发物组成有显著的差异,且挥发物的差异与榕树系统发育距离具有正相关性,但同一物种不同性别间的挥发物组成上没有显著的差异。本研究中发现雌雄异株榕树挥发物组成具有多样性高、种内性别间相似、种间差异大,系统发育一致性的特点,上述特点在榕树物种分化以及维持雌雄异株榕树繁殖的化学通讯机制中可能发挥着关键作用。