上海地区番茄黄化曲叶病毒病的鉴定及嫁接接种法研究

番茄黄化曲叶病毒(tomato yellow leafcurl virus,TYLCV)是一种由烟粉虱(Bemisia tabaci)和嫁接传播的双生病毒,在热带、亚热带地区给番茄生产造成严重威胁.根据番茄黄化曲叶病毒的保守序列设计一对引物,运用PCR技术从上海地区的感病番茄中扩增出一条575bp的特异带,而健康植株无此带.测序表明该序列与番茄黄化曲叶病毒具有极高的同源性(97%～99%).将健康接穗嫁接到感染番茄黄化曲叶病毒的番茄砧木上,间隔15 d和30 d,分别提取接穗的DNA,并用PCR法检测病毒,发现嫁接15 d后在部分接穗中检测到TYLCV病毒,嫁接30 d后在所有的接穗中均检测到病毒,因此,嫁接法可以作为番茄黄化曲叶病毒病的接种鉴定方法.     

Twenty-one novel tri- and tetranucleotide microsatellite loci for the Amur tiger (Panthera tigris altaica)

Amur tiger is the largest subspecies of tiger in the world and his conservation has also received much attention. In this study, we isolated and characterized twenty-one tri- and tetranucleotide microsatellite markers from this species. The number of alleles for each locus ranged from two to nine in a group of 60 individuals and the observed and expected heterozygosities were 0.333–0.917 and 0.302–0.822, respectively. The overall discrimination power and exclusion probabilities in parentage and paternity testing for these markers were 1.00, 0.9947 and 0.9999, respectively, indicating high-resolution power of microsatellite markers.     

Human functional genetic studies are biased against the medically most relevant primate-specific genes

Background  

Many functional, structural and evolutionary features of human genes have been observed to correlate with expression breadth and/or gene age. Here, we systematically explore these correlations.     

Non-peptide calcitonin gene-related peptide receptor antagonists from a benzodiazepinone lead

High-throughput screening of the Merck sample collection identified benzodiazepinone tetralin-spirohydantoin 1 as a CGRP receptor antagonist with micromolar activity. Comparing the structure of 1 with those of earlier peptide-based antagonists such as BIBN 4096 BS, a key hydrogen bond donor-acceptor pharmacophore was hypothesized. Subsequent structure activity studies supported this hypothesis and led to benzodiazepinone piperidinyldihydroquinazolinone 7, CGRP receptor K(i)=44nM and IC(50)=38nM. Compound 7 was orally bioavailabile in rats and is a lead in the development of orally bioavailable CGRP antagonists for the treatment of migraine.     

Optimization of CCR4 antagonists: side-chain exploration

The design, synthesis, and activity of novel and selective small molecule antagonists of the CC chemokine receptor-4 (CCR4) are presented. Compound 8c was efficacious in a murine allergic inflammation model (ED(50) 30 mg/kg).     

Bicyclic and tricyclic thiophenes as protein tyrosine phosphatase 1B inhibitors

A novel pyridothiophene inhibitor of PTP1B was discovered by rational screening of phosphotyrosine mimics at high micromolar concentrations. The potency of this lead compound has been improved significantly by medicinal chemistry guided by X-ray crystallography and molecular modeling. Excellent consistency has been observed between structure-activity relationships and structural information from PTP1B-inhibitor complexes.     

Chelerythrine and sanguinarine dock at distinct sites on BclXL that are not the classic BH3 binding cleft

The ratio of the levels of pro-survival and pro-apoptotic members of the Bcl-2 protein family is thought to be an important regulatory factor for determining the sensitivity of the mammalian cells to apoptotic stimuli. High levels of expression of pro-survival members such as Bcl(XL) in human cancers were frequently found to be a good prognostic indicator predicting poor response to chemotherapy. The pro-survival members of the Bcl-2 family mediate their effects through heterodimerization with the BH3 region of the pro-apoptotic members. Structural analyses of the binding complex of the BH3 peptide and Bcl(XL) showed that a hydrophobic groove termed the BH3 binding cleft is the docking site for the BH3 region. Chemical mimetics of the BH3 region such as BH3I-1 that target the BH3 binding cleft indeed exhibit pro-apoptotic activities. Chelerythrine (CHE) and sanguinarine (SAN) are natural benzophenanthridine alkaloids that are structurally homologous to each other. CHE was previously identified as an inhibitor of Bcl(XL) function from a high-throughput screen of natural products, but its mode of interaction with Bcl(XL) is not known. By determining the effect of site-directed mutagenesis on ligand binding and using saturation transfer difference (STD) NMR experiments, we have verified locations of these docked ligands. Surprisingly, CHE and SAN bind separately at the BH groove and BH1 region of Bcl(XL) respectively, different from the BH3 binding cleft where other known inhibitors of Bcl(XL) target. Interestingly, certain residues on the flexible loop between helices alpha1 and alpha2 of Bcl(XL) are also perturbed upon CHE, but not SAN or BH3I-1 binding. Although CHE and SAN are similarly effective as BH3I-1 in displacing bound BH3 peptide, they are much more effective in inducing apoptosis, raising the possibility that CHE and SAN might be able to antagonize other pro-survival mechanisms in addition to the one that involves BH3 region binding.     

Scaffold stiffness affects the contractile function of three-dimensional engineered cardiac constructs

We investigated the effects of the initial stiffness of a three-dimensional elastomer scaffold--highly porous poly(glycerol sebacate)--on functional assembly of cardiomyocytes cultured with perfusion for 8 days. The polymer elasticity varied with the extent of polymer cross-links, resulting in three different stiffness groups, with compressive modulus of 2.35 ± 0.03 (low), 5.28 ± 0.36 (medium), and 5.99 ± 0.40 (high) kPa. Laminin coating improved the efficiency of cell seeding (from 59 ± 15 to 90 ± 21%), resulting in markedly increased final cell density, construct contractility, and matrix deposition, likely because of enhanced cell interaction and spreading on scaffold surfaces. Compact tissue was formed in the low and medium stiffness groups, but not in the high stiffness group. In particular, the low stiffness group exhibited the greatest contraction amplitude in response to electric field pacing, and had the highest compressive modulus at the end of culture. A mathematical model was developed to establish a correlation between the contractile amplitude and the cell distribution within the scaffold. Taken together, our findings suggest that the contractile function of engineered cardiac constructs positively correlates with low compressive stiffness of the scaffold.     

Effects of leachates of the invasive plant, Ageratina adenophora (Sprengel) on soil microbial community

The invasive plant Ageratina adenophora (Sprengel) changed soil microbial communities in the invaded area to facilitate its growth and inhibit native plants. However, little is known about the driving forces underlying the alteration of soil biota. Leachates from root and aerial part (stem and leaves) of A. adenophora were mixed into soil to imitate field invasion processes for evaluation of its impact on invasion of soil microbial community. The results indicated that soil microbial community was significantly changed when the soil taken from the newly-invaded area was treated with A. adenophora root and aerial part leachates for 3 and 5 weeks, respectively. The biota of newly invaded soil treated with concentration of 100 mg/mL A.adenophora leachates was much closer to that of heavily invaded soil, but was significantly different from that of control soil (newly invaded soil without treatment). A.adenophora leachates promoted growth of the seven dominant rhizosphere bacterial species in the invaded soil. The effect of A.adenophora leachates on soil biota and dominant rhizosphere bacteria was positively correlated with the concentration of leachates, however, the effect of root leachates was stronger than the aerial part leachates. It is assumed that A.adenophora change soil microbial community via nutritional and chemical communication, which helps it in better colonization of the invaded soil.     

Profiling of Substrate Specificity of SARS-CoV 3CLpro

Background

The 3C-like protease (3CL^pro) of severe acute respiratory syndrome-coronavirus is required for autoprocessing of the polyprotein, and is a potential target for treating coronaviral infection.

Methodology/Principal Findings

To obtain a thorough understanding of substrate specificity of the protease, a substrate library of 198 variants was created by performing saturation mutagenesis on the autocleavage sequence at P5 to P3'' positions. The substrate sequences were inserted between cyan and yellow fluorescent proteins so that the cleavage rates were monitored by in vitro fluorescence resonance energy transfer. The relative cleavage rate for different substrate sequences was correlated with various structural properties. P5 and P3 positions prefer residues with high β-sheet propensity; P4 prefers small hydrophobic residues; P2 prefers hydrophobic residues without β-branch. Gln is the best residue at P1 position, but observable cleavage can be detected with His and Met substitutions. P1'' position prefers small residues, while P2'' and P3'' positions have no strong preference on residue substitutions. Noteworthy, solvent exposed sites such as P5, P3 and P3'' positions favour positively charged residues over negatively charged one, suggesting that electrostatic interactions may play a role in catalysis. A super-active substrate, which combined the preferred residues at P5 to P1 positions, was found to have 2.8 fold higher activity than the wild-type sequence.

Conclusions/Significance

Our results demonstrated a strong structure-activity relationship between the 3CL^pro and its substrate. The substrate specificity profiled in this study may provide insights into a rational design of peptidomimetic inhibitors.