Characterization of the 3a protein of SARS-associated coronavirus in infected vero E6 cells and SARS patients

Proteomics was used to identify a protein encoded by ORF 3a in a SARS-associated coronavirus (SARS-CoV). Immuno-blotting revealed that interchain disulfide bonds might be formed between this protein and the spike protein. ELISA indicated that sera from SARS patients have significant positive reactions with synthesized peptides derived from the 3a protein. These results are concordant with that of a spike protein-derived peptide. A tendency exists for co-mutation between the 3a protein and the spike protein of SARS-CoV isolates, suggesting that the function of the 3a protein correlates with the spike protein. Taken together, the 3a protein might be tightly correlated to the spike protein in the SARS-CoV functions. The 3a protein may serve as a new clinical marker or drug target for SARS treatment.     

Disruption of Kaposi's sarcoma-associated herpesvirus latent nuclear antigen leads to abortive episome persistence

Latent nuclear antigen (LNA) is implicated in Kaposi's sarcoma-associated herpesvirus (KSHV) episome persistence. LNA colocalizes with KSHV episomes on chromosomes in metaphase, and it maintains the stability and replication of KSHV terminal repeat-containing plasmids. In this study, we examined the function of LNA in episome persistence in the context of full-length KSHV genome by mutagenesis analysis. We generated a KSHV mutant, BAC36-DeltaLNA, with LNA disrupted by transposon-based mutagenesis with a KSHV BAC clone, BAC36, as a template. Immunofluorescence antibody staining revealed that the insertion of a transposon cassette into LNA disrupted its expression but had no effect on the expression of two adjacent genes, the vCyclin and vFLIP genes. Using a green fluorescent protein (GFP) cassette as a tracking marker for the KSHV episome, we found 8.7-fold-fewer GFP-positive cells in BAC36-DeltaLNA cultures than in wild-type BAC36 cultures at the early stage following episome delivery into 293 cells by transfection, which could be partially rescued by cotransfection with a LNA expression plasmid but not a control plasmid. Cells harboring BAC36-DeltaLNA with or without transient complementation rapidly lost episomes and became virus-free after 2 weeks of culture based on GFP expression and Gardella gel analysis and quantitative PCR assays for detecting KSHV genomes. In contrast, BAC36 episomes were stably maintained during the same period. Stable cultures with close to 100% of cells harboring KSHV episomes were readily established by hygromycin selection for BAC36 but not for BAC36-DeltaLNA. These results conclusively indicate that LNA is essential for the establishment and persistence of KSHV episomes in mammalian cells.     

Small molecules blocking the entry of severe acute respiratory syndrome coronavirus into host cells

Severe acute respiratory syndrome coronavirus (SARS-CoV) is the pathogen of SARS, which caused a global panic in 2003. We describe here the screening of Chinese herbal medicine-based, novel small molecules that bind avidly with the surface spike protein of SARS-CoV and thus can interfere with the entry of the virus to its host cells. We achieved this by using a two-step screening method consisting of frontal affinity chromatography-mass spectrometry coupled with a viral infection assay based on a human immunodeficiency virus (HIV)-luc/SARS pseudotyped virus. Two small molecules, tetra-O-galloyl-beta-D-glucose (TGG) and luteolin, were identified, whose anti-SARS-CoV activities were confirmed by using a wild-type SARS-CoV infection system. TGG exhibits prominent anti-SARS-CoV activity with a 50% effective concentration of 4.5 microM and a selective index of 240.0. The two-step screening method described here yielded several small molecules that can be used for developing new classes of anti-SARS-CoV drugs and is potentially useful for the high-throughput screening of drugs inhibiting the entry of HIV, hepatitis C virus, and other insidious viruses into their host cells.     

AFLP analysis of genetic diversity and relationship among some Chinese domestic ducks and wild ducks

The amplified fragment length polymorphic(AFLP)technique was used to analyze the genome DNA polymorphism among 8 breeds of domestic ducks and 2 species of wild ducks.Nine of the 17 selected primers pairs gave reproducible polymorphic DNA amplification bands.The amplified bands ranged from 44 to 83 per primer pair.Of the 513 AFLP markers obtained.498 were polymorphic.The proportion of polymorphic loci was 97.1%.The genetic distance(D)and similarity coefficients(GS)were calculated based on the polymorphic data.Between domestic ducks D ranged from 0.331 to 0.589,while between domestic ducks and the wild ducks,it ranged from 0.298 to 0.520(vs.Anas Platyrhynchos)and from 0.316 to 0.522(vs.A.Poecilorhyncha),respectively.The variance analysis showed no significant difference between the two groups of data,which indicated that both mallard and spot-billed ducks made contributions to domestic duck evolution.A dendrogram was constructed according to the D value.     

Proposed GSSP for the base of Cambrian Stage 7, coinciding with the first appearance of Lejopyge laevigata, Hunan, China

The Luoyixi section, exposed in a roadcut along the Youshui River (Fengtan Reservoir), Guzhang County, Hunan Province, China, is proposed as the stratotype for the base of an unnamed stage boundary (base of the Cambrian stage provisionally termed Stage 7). The proposed position of the GSSP is 121.3 m above the base of the Huaqiao Formation, at a horizon coinciding with the first appearance of the cosmopolitan agnostoid trilobite Lejopyge laevigata. The section fulfills all the requirements for a GSSP, and the horizon can be constrained not only with the primary stratigraphic marker (L. laevigata) but also with secondary biostratigraphic, sequence-stratigraphic, and chemostratigraphic correlation tools. The first appearance of L. laevigata is one of the most readily recognizable levels in the Cambrian, and can be correlated with precision to all paleocontinents.     

Characterization of cheW genes of Leptospira interrogans and their effects in Escherichia coli

The motility and chemotaxis systems are critical for the virulence of leptospires. In this study, the phylogenetic profiles method was used to predict the interaction of chemotaxis proteins. It was shown that CheW1 links to CheA1, CheY, CheB and CheW2, CheW3 links to CheA2, MCP （LA2426）, CheB3 and CheD1; and CheW2 links only to CheW1. The similarity analysis demonstrated that CheW2 of Leptospira interrogans strain Lai had poor homology with Chew of Escherichia coli in the region of residues 30-50. In order to verify the function of these proteins, the putative cheW genes were cloned into pQE31 vector and expressed in wild-type E. coli strain RP437 or chew defective strain RP4606. The swarming results indicated that CheW1 and CheW3 could restore swarming of RP4606 while CheW2 could not. Overexpression of CheW1 and CheW3 in RP437 inhibited the swarming of RP437, whereas the inhibitory effect of CheW2 was much lower. Therefore, we presumed that CheW1 and CheW3 might have the function of CheW while CheW2 does not. The existence of multiple copies of chemotaxis homologue genes suggested that L. interrogans strain Lai might have a more complex chemosensory pathway.     

Transient Receptor Potential Vanilloid-1 (TRPV1) and Ankyrin-1 (TRPA1) Participate in Visceral Hyperalgesia in Chronic Water Avoidance Stress Rat Model

Stressfull life events have powerful influences on visceral perception of certain IBS patients. In the present study, we aimed to examine the involvement of TRPV1 and TRPA1 in the stress-induced visceral hyperalgesia. Rats were exposed to 1-h water avoidance stress (WAS) daily for 10 consecutive days. The abdominal withdrawal reflex (AWR) to colorectal distension was assessed at the end of the 10-day period. Western-blotting analysis was applied to investigate the alterations of TRPV1 and TRPA1 in the colonic afferent dorsal root ganglia (DRG). Compared with control rats, the WAS-treated rats demonstrated a significant increase in the AWR with the pressure ≥40 mm Hg (P < 0.05). Meanwhile, in the WAS-treated rats, western-blotting analysis showed significant upregulation of TRPV1 and TRPA1 in the colonic afferent DRG. The results indicate that WAS could induce the upregulation of TRPV1 and TRPA1 in the colonic afferent DRG, and both receptors may be candidate molecules involved in the stress-induced visceral hyperalgesia in rats.     

生物碱的提取方法研究进展

生物碱是广泛存在于自然界天然植物中的碱性含氮有机化合物。大多数生物碱具有显著的生理活性,是许多药用植物的有效成分,提取与纯化是生物碱制备的关键环节。综述了生物碱制备的传统方法及新技术的应用进展,分析了它们的原理及优缺点,探讨了生物碱制备的发展前景。     

A mixed two-stage method for detecting interactions in genomewide association studies

Genomewide association studies (GWAS) are being conducted to unravel the genetic etiology of complex diseases, in which complex epistasis may play an important role. One-stage method in which interactions are tested using all samples at one time may be computationally problematic, may have low power as the number of markers tested increases and may not be cost-efficient. A common two-stage method may be a reasonable and powerful approach for detecting interacting genes using all samples in both two stages. In this study, we introduce an alternative two-stage method, in which some promising markers are selected using a proportion of samples in the first stage and interactions are then tested using the remaining samples in the second stage. This two-stage method is called mixed two-stage method. We then investigate the power of both one-stage method and mixed two-stage method to detect interacting disease loci for a range of two-locus epistatic models in a case-control study design. Our results suggest that mixed two-stage method may be more powerful than one-stage method if we choose about 30% of samples for single-locus tests in the first stage, and identify less than and equal to 1% of markers for follow-up interaction tests. In addition, we compare both two-stage methods and find that our two-stage method will lose power because we only use part of samples in both two stages.