观赏桃ISSR-PCR反应体系的优化

采用改良CTAB法从观赏桃满天红叶片中提取基因组DNA,通过单因素实验探讨了模板DNA、Mg~(2+)、dNTPs和Taq DNA酶等条件对观赏桃ISSR-PCR扩增结果的影响,建立了ISSR-PCR扩增的最佳体系:2.5μl反应体系中包含10×Buffer 2.5μl,模板DNA 40ng,Mg~(2+)浓度2.5mmol/L,引物浓度04 μmol/L,dNTPs浓度0.4mmol/L,Taq DNA酶0.5U.利用所建立的体系对红叶桃、菊花桃和春艳等13份材料进行检验,其结果表明优化后的体系适合观赏桃的ISSR-PCR反应.     

Development and Application of a Real-time PCR Method for Pharmacokinetic and Biodistribution Studies of Recombinant Adenovirus

A replication-deficient recombinant adenovirus (Ad5-LFA-3/IgG₁) that encodes secreted LFA-3/IgG₁ was constructed for gene therapy treatment of psoriasis. The purpose of this study was to develop a real-time PCR method for pharmacokinetic and biodistribution studies of Ad5-LFA-3/IgG₁ within the circulation and organs. This method showed good specificity, sensitivity and reproducibility over a wide dynamic range of concentrations. Quantitative measurement of recombinant adenoviral DNA suggested that the level of Ad5-LFA-3/IgG₁ DNA in circulating blood peaked within 10 min following intravenous injection in rhesus macaques. Following this peak, the adenoviral DNA level dropped significantly to a very low level. Real-time PCR revealed that Ad5-LFA-3/IgG₁ DNA was enriched in the spleen, lung and liver after injection of the adenovirus into rats through the tail vein. The adenoviral DNA was barely detected in other tissues. These data provide important information for clinical trials of Ad5-LFA-3/IgG₁ and confirm the utility of the real-time PCR assay for monitoring gene therapy trials.     

Metabolic impact of redox cofactor perturbations in Saccharomyces cerevisiae

Redox cofactors play a pivotal role in coupling catabolism with anabolism and energy generation during metabolism. There exists a delicate balance in the intracellular level of these cofactors to ascertain an optimal metabolic output. Therefore, cofactors are emerging to be attractive targets to induce widespread changes in metabolism. We present a detailed analysis of the impact of perturbations in redox cofactors in the cytosol or mitochondria on glucose and energy metabolism in Saccharomyces cerevisiae to aid metabolic engineering decisions that involve cofactor engineering. We enhanced NADH oxidation by introducing NADH oxidase or alternative oxidase, its ATP-mediated conversion to NADPH using NADH kinase as well as the interconversion of NADH and NADPH independent of ATP by the soluble, non-proton-translocating bacterial transhydrogenase. Decreasing cytosolic NADH level lowered glycerol production, while decreasing mitochondrial NADH lowered ethanol production. However, when these reactions were coupled with NADPH production, the metabolic changes were more moderated. The direct consequence of these perturbations could be seen in the shift of the intracellular concentrations of the cofactors. The changes in product profile and intracellular metabolite levels were closely linked to the ATP requirement for biomass synthesis and the efficiency of oxidative phosphorylation, as estimated from a simple stoichiometric model. The results presented here will provide valuable insights for a quantitative understanding and prediction of cellular response to redox-based perturbations for metabolic engineering applications.     

Consumption of aphids by spiders and the effect of additional prey: evidence from microcosm experiments

Spiders are common generalist predators in agroecosystems and have been suggested to lower herbivore abundance in crops. It is not clear, however, if spiders can effectively suppress pest populations, and if so, by what mechanisms. In a microcosm experiment, we examined the consumption of the bird cherry-oat aphid, Rhopalosiphum padi L. (Homoptera: Aphididae), a pest species in wheat fields, by three spider species that differ in their hunting methods. We then tested the effect of additional prey type on the ability of erigonid spiders to reduce aphids. In a 48-h experiment Mermessus denticulatus (Banks) (Araneae: Linyphiidae; Erigoninae) consumed more aphids than did Enoplognatha gemina Bosmans and Van Keer (Araneae: Theridiidae) and Bathyphantes cf. extricatus (O·P.-Cambridge) (Araneae: Linyphiidae; Linyphiinae). This difference may be due to the ability of erigonids to forage actively on the vegetation in addition to using their webs to catch prey. In a 7-week experiment, we provided springtails (Collembola) in high and low densities as additional prey to mated erigonids, prior to aphid introduction. Spiders in the low-density springtail treatment built more webs on the vegetation, and caused a 50% reduction in aphid populations. There were significantly fewer aphids in the low-density springtail treatment, but not in the high-density treatment, in comparison to the control (high-density springtails without spiders). The results suggest that additional prey density affects predatory interactions between M. denticulatus and R. padi and that erigonids, which occur in high densities in wheat fields in the Negev desert, may be involved in aphid suppression in these agroecosystems.

Caspases regulate VAMP-8 expression and phagocytosis in dendritic cells

During an inflammation and upon encountering pathogens, immature dendritic cells (DC) undergo a maturation process to become highly efficient in presenting antigens. This transition from immature to mature state is accompanied by various physiological, functional and morphological changes including reduction of caspase activity and inhibition of phagocytosis in the mature DC. Caspases are cysteine proteases which play essential roles in apoptosis, necrosis and inflammation. Here, we demonstrate that VAMP-8, (a SNARE protein of the early/late endosomes) which has been shown previously to inhibit phagocytosis in DC, is a substrate of caspases. Furthermore, we identified two putative conserved caspase recognition/cleavage sites on the VAMP-8 protein. Consistent with the up-regulation of VAMP-8 expression upon treatment with caspase inhibitor (CI), immature DC treated with CI exhibits lower phagocytosis activity. Thus, our results highlight the role of caspases in regulating VAMP-8 expression and subsequently phagocytosis during maturation of DC.     

Biological and Molecular Characterization of a Crucifer Tobamovirus Infecting Oilseed Rape

In China, the tobamovirus that infects oilseed rape has been misdiagnosed as Tobacco mosaic virus (TMV) based on its morphological similarity and serological relatedness. Recently, a tobamovirus has been isolated from oilseed rape in China, which we named Youcai mosaic virus Br (YoMV-Br), according to its biological and molecular characteristics. It had strong infectivity to Cruciferae but less to Solanaceae, Leguminosae, and Cucurbitaceae, and its virion morphology was consistent with that of the tobamoviruses. At high concentrations, it serologically cross reacted with TMV antiserum. The 3′ terminal sequence (2,283 nucleotides) of YoMV-Br was determined, including the 3′ noncoding region, the CP and MP genes, and the C-terminal part of the replicase gene. Between the MP and CP genes, 77 nucleotides overlapped. Compared with homologous regions of 21 recognized species of Tobamovirus, YoMV-Br had a much higher identity to crucifer species than to other tobamoviruses. Phylogenetic analysis demonstrated that YoMV-Br was closely related to the YoMV cluster of tobamoviruses and distantly to TMV, so that they likely belong to different strains of the same species.     

Ultrasound stimulates NF‐κB activation and iNOS expression via the Ras/Raf/MEK/ERK signaling pathway in cultured preosteoblasts

It has been shown that ultrasound (US) stimulation accelerates fracture healing in the animal models and non‐operatively clinical uses. Nitric oxide (NO) is a crucial early mediator in mechanically induced bone formation. Here we found that US‐mediated inducible nitric oxide synthase (iNOS) expression was attenuated by Ras inhibitor (manumycin A), Raf‐1 inhibitor (GW5074), MEK inhibitor (PD98059), NF‐κB inhibitor (PDTC), and IκB protease inhibitor (TPCK). US‐induced Ras activation was inhibited by manumycin A. Raf‐1 phosphorylation at Ser³³⁸ by US was inhibited by manumycin A and GW5074. US‐induced MEK and ERK activation was inhibited by manumycin A, GW5074, and PD98059. Stimulation of preosteoblasts with US activated IκB kinase α/β (IKK α/β), IκBαphosphorylation, p65 phosphorylation at Ser²⁷⁶, p65, and p50 translocation from the cytosol to the nucleus, and κB‐luciferase activity. US‐mediated an increase of IKK α/β, IκBα, and p65 phosphorylation, κB‐luciferase activity and p65 and p50 binding to the NF‐κB element was inhibited by manumycin A, GW5074, and PD98059. Our results suggest that US increased iNOS expression in preosteoblasts via the Ras/Raf‐1/MEK/ERK/IKKαβ and NF‐κB signaling pathways. J. Cell. Physiol. 220: 196–203, 2009. © 2009 Wiley‐Liss, Inc.     

Effect of Interdomain Linker Length on an Antagonistic Folding-Unfolding Equilibrium between Two Protein Domains

Fusion of one protein domain with another is a common event in both evolution and protein engineering experiments. When insertion is at an internal site (e.g., a surface loop or turn), as opposed to one of the termini, conformational strain can be introduced into both domains. Strain is manifested by an antagonistic folding-unfolding equilibrium between the two domains, which we previously showed can be parameterized by a coupling free-energy term (ΔG_X). The extent of strain is predicted to depend primarily on the ratio of the N-to-C distance of the guest protein to the distance between ends of the surface loop in the host protein. Here, we test that hypothesis by inserting ubiquitin (Ub) into the bacterial ribonuclease barnase (Bn), using peptide linkers from zero to 10 amino acids each. ΔG_X values are determined by measuring the extent to which Co²⁺ binding to an engineered site on the Ub domain destabilizes the Bn domain. All-atom, unforced Langevin dynamics simulations are employed to gain structural insight into the mechanism of mechanically induced unfolding. Experimental and computational results find that the two domains are structurally and energetically uncoupled when linkers are long and that ΔG_X increases with decreasing linker length. When the linkers are fewer than two amino acids, strain is so great that one domain unfolds the other. However, the protein is able to refold as dimers and higher-order oligomers. The likely mechanism is a three-dimensional domain swap of the Bn domain, which relieves conformational strain. The simulations suggest that an effective route to mechanical unfolding begins with disruption of the hydrophobic core of Bn near the Ub insertion site.