Studies on the Interaction between Single-strand Oligonucleotide and Ginsenosides by Electrospray Ionization Mass Spectrometry

Oligonucleotide from SARS virus was selected as a target molecule in the paper. The noncovalent complexes of ginsenosides with the target molecule were investigated by electrospray ionization mass spectrometry. The effects of experimental conditions were examined firstly on the formation of noncovalent complexes. Based on the optimized experimental conditions, the interaction of different ginsenosides with the target molecule was researched, finding that the interaction orders are relative with the structure of aglycons, the length and terminal sugar types of saccharide chains in the ginsenosides. There are certain rules for the interaction between the ginsenosides and DNA target molecule. For different type ginsenosides, the interaction intensity takes the orders 20-S-protopanaxatriol > 20-S-protopanaxadiol, and panaxatriol ginsenosides > panaxadiol ginsenosides. For the ginsenosides with the same type aglycone, tri-saccharide chain > di-saccharide chain > tetra-saccharide chain and single-saccharide chain > panaxatriol. For the ginsenosides with the same tetra-saccharide chain, the ginsenosides with smaller molecule masses > the ginsenosides with larger molecule masses.

DNA与生物碱类化合物非共价作用的负离子电喷雾质谱研究(Ⅰ)

目前 ,临床上使用的许多抗病毒药物均是通过与 DNA,RNA发生相互作用破坏其结构 ,进而影响基因调控与表达的功能 ,表现出抗病毒活性 [1,2 ] .因此 ,核酸与药物分子相互作用的研究对阐述抗病毒药物的作用机理 ,以及对药物的体外筛选都具有重要的意义 .电喷雾电离质谱作为一种软电离手段 ,可将溶液中生物分子与药物分子的非共价复合物转为气相进行分析 ,再现其生理状态 ,使其成为分子水平上进行药物筛选的最佳方法 [3～ 6 ] 和在分子水平上筛选中药抗病毒活性成分的理想工具 .本文选择合成了与 SARS病毒相关的 DNA片段作为抗病毒药物筛选的靶分子 ,用电喷雾质谱技术 ,通过对靶分子与 5种生物碱的非共价复合作用 ,探讨了生物质谱方法用于药物筛选的可行性 .1　实验部分1 .1　材料及样品制备　 DNA分子系人工合成 ,由 1 5个碱基组成 ,分子量为 470 4 ,结构为 GGTAA-GATGGAGAGC( 1 5 - mer) ;腺苷 ( Adenosine,Mw=2 67)、鸟苷 ( Guanosine,Mw=2 83)和胞苷 ( Cyti-dine,Mw=2 4 3)购自 Sigma公司 ;小檗碱、...

核酸靶分子与苦参中生物碱成分的非共价作用研究

本文选择合成了与SARS病毒相关的DNA片段作为抗病毒药物筛选的靶分子.利用生物质谱技术,通过对该核酸分子与常见中药苦参中的两种主要生物碱成分的非共价复合作用的研究,探讨该方法作为药物筛选方法的可行性.

Population-Dynamics And Production Of Euphausiids. IV Euphausia-Krohni Nematoscelis-Megalops And Thysanoessa-Gregaria And 8 Rare Species In The North-Atlantic Ocean

Seasonal changes in the abundance, size and occurrence of furciliae of Euphausia krohni (Brandt), Nematoscelis megalops (G. O. Sars) and Thysanoessa gregaria G. O. Sars are described from samples taken at 10 m depth with the Continuous Plankton Recorder (CPR) over a period of 2 yr (January 1966 to December 1967) in the North Atlantic Ocean. E. krohni and T. gregaria were found to breed through most of the year but N. megalops bred only in spring and summer. Annual mean biomass was calculated directly from the data and production was estimated from published P:B ratios. The seasonal occurrences of E. brevis Hansen, E. hemigibba Hansen, E. mutica Hansen, E. tenera Hansen, Stylocheiron longicorne G. O. Sars, S. maximum Hansen, Thysanopoda acutifrons Holt and Tattershall and T. aequalis Hansen in the samples are described.

Continuous Plankton Records - Geographical Variations In Numerical Abundance Biomass And Production Of Euphausiids In The North-Atlantic Ocean And The North-Sea

Geographical variations in the numbers, biomass and production of euphausiids and the contribution of common species to the total are described from samples taken during 1966 and 1967 in the North Atlantic Ocean and the North Sea by the Continuous Plankton Recorder at 10 m depth. Euphausiids were most abundant in the central and western North Atlantic Ocean and the Norwegian Sea. Thysanoessa longicaudata (Krøyer) was numerically dominant. Biomass was greatest in the Norwegian Sea and the north-eastern North Sea where Meganyctiphanes norvegica (M. Sars) accounted for 81 and 59%, respectively, of the total biomass. Production was highest off Nova Scotia and in Iberian coastal waters; the dominant species were T. raschi (M. Sars) in the former area and Nyctiphanes couchi (Bell) in the latter. The mean P:B ratios were correlated with temperature.

Population-Dynamics And Production Of Euphausiids. III Meganyctiphanes-Norvegica And Nyctiphanes-Couchi In The North-Atlantic Ocean And The North-Sea

Seasonal changes in abundance, size and aspects of the population structure of Meganyctiphanes norvegica (M. Sars) and Nyctiphanes couchi (Bell) are described from samples taken with the “Continuous Plankton Recorder” at 10 m depth over a 2 yr period (1966 and 1967) in the North Atlantic Ocean and the North Sea. M. norvegica lived for a maximum of just over 2 yr, and adults of both year-classes spawned during a limited breeding season in the spring or summer. N. couchi spawned over a prolonged breeding season, giving rise to a complex of cohorts with overlapping size ranges. It was concluded that 3 or 4 cohorts were spawned in each year and that the maximum life span was probably greater than 1 yr, although maturity may be attained in less than a year. Estimated annual production at 10 m depth for M. norvegica ranged from 0.80 to 18.74 mg m-3yr-1 and for N. couchi from 0.67 to 8.23 mg m-3yr-1. P:B ratios ranged from 1.3:1 to 6.3:1 for M. norvegica and 4.0:1 to 5.5:1 for N. couchi.

Population-Dynamics And Production Of Euphausiids .2. Thysanoessa-Inermis And Thysanoessa-Raschi In The North-Sea And American Coastal Waters

Results from the Continuous Plankton Recorder (CPR) survey for 1966 and 1967 are used to describe seasonal changes in abundance, size and aspects of the population structure of Thysanoessa inermis (Krøyer) and T. raschi (M. Sars) at a depth of 10 m in the North Sea and in American coastal waters from the Grand Banks to the Gulf of Maine. Production and dry weight were estimated from these data. Two year-groups were usually present in the breeding population, the proportion surviving into a second year being higher in American waters than in the North Sea. Annual production for each species was within the range 0.69 to 4.66 mg m-3 and the ratio between production and biomass (P:B) was between 1.3 and 4.2; values outside these ranges were obtained only for American coastal waters in 1967, when the frequency of sampling was low.

Major genetic marker of nidoviruses encodes a replicative endoribonuclease

Coronaviruses are important pathogens that cause acute respiratory diseases in humans. Replication of the 30-kb positive-strand RNA genome of coronaviruses and discontinuous synthesis of an extensive set of subgenome-length RNAs (transcription) are mediated by the replicase-transcriptase, a barely characterized protein complex that comprises several cellular proteins and up to 16 viral subunits. The coronavirus replicase-transcriptase was recently predicted to contain RNA-processing enzymes that are extremely rare or absent in other RNA viruses. Here, we established and characterized the activity of one of these enzymes, replicative nidoviral uridylate-specific endoribonuclease (NendoU). It is considered a major genetic marker that discriminates nidoviruses (Coronaviridae, Arteriviridae, and Roniviridae) from all other RNA virus families. Bacterially expressed forms of NendoU of severe acute respiratory syndrome coronavirus and human coronavirus 229E were revealed to cleave single-stranded and double-stranded RNA in a Mn2+-dependent manner. Single-stranded RNA was cleaved less specifically and effectively, suggesting that double-stranded RNA is the biologically relevant NendoU substrate. Double-stranded RNA substrates were cleaved upstream and downstream of uridylates at GUU or GU sequences to produce molecules with 2'-3' cyclic phosphate ends. 2'-O-ribose-methylated RNA substrates proved to be resistant to cleavage by NendoU, indicating a functional link with the 2'-O-ribose methyltransferase located adjacent to NendoU in the coronavirus replicative polyprotein. A mutagenesis study verified potential active-site residues and allowed us to inactivate NendoU in the full-length human coronavirus 229E clone. Substitution of D6408 by Ala was shown to abolish viral RNA synthesis, demonstrating that NendoU has critical functions in viral replication and transcription.

Characterization of White bream virus reveals a novel genetic cluster of nidoviruses.

The order Nidovirales comprises viruses from the families Coronaviridae (genera Coronavirus and Torovirus), Roniviridae (genus Okavirus), and Arteriviridae (genus Arterivirus). In this study, we characterized White bream virus (WBV), a bacilliform plus-strand RNA virus isolated from fish. Analysis of the nucleotide sequence, organization, and expression of the 26.6-kb genome provided conclusive evidence for a phylogenetic relationship between WBV and nidoviruses. The polycistronic genome of WBV contains five open reading frames (ORFs), called ORF1a, -1b, -2, -3, and -4. In WBV-infected cells, three subgenomic RNAs expressing the structural proteins S, M, and N were identified. The subgenomic RNAs were revealed to share a 42-nucleotide, 5' leader sequence that is identical to the 5'-terminal genome sequence. The data suggest that a conserved nonanucleotide sequence, CA(G/A)CACUAC, located downstream of the leader and upstream of the structural protein genes acts as the core transcription-regulating sequence element in WBV. Like other nidoviruses with large genomes (>26 kb), WBV encodes in its ORF1b an extensive set of enzymes, including putative polymerase, helicase, ribose methyltransferase, exoribonuclease, and endoribonuclease activities. ORF1a encodes several membrane domains, a putative ADP-ribose 1"-phosphatase, and a chymotrypsin-like serine protease whose activity was established in this study. Comparative sequence analysis revealed that WBV represents a separate cluster of nidoviruses that significantly diverged from toroviruses and, even more, from coronaviruses, roniviruses, and arteriviruses. The study adds to the amazing diversity of nidoviruses and appeals for a more extensive characterization of nonmammalian nidoviruses to better understand the evolution of these largest known RNA viruses.

A complex zinc finger controls the enzymatic activities of nidovirus helicases.

Nidoviruses (Coronaviridae, Arteriviridae, and Roniviridae) encode a nonstructural protein, called nsp10 in arteriviruses and nsp13 in coronaviruses, that is comprised of a C-terminal superfamily 1 helicase domain and an N-terminal, putative zinc-binding domain (ZBD). Previously, mutations in the equine arteritis virus (EAV) nsp10 ZBD were shown to block arterivirus reproduction by disrupting RNA synthesis and possibly virion biogenesis. Here, we characterized the ATPase and helicase activities of bacterially expressed mutant forms of nsp10 and its human coronavirus 229E ortholog, nsp13, and correlated these in vitro activities with specific virus phenotypes. Replacement of conserved Cys or His residues with Ala proved to be more deleterious than Cys-for-His or His-for-Cys replacements. Furthermore, denaturation-renaturation experiments revealed that, during protein refolding, Zn2+ is essential for the rescue of the enzymatic activities of nidovirus helicases. Taken together, the data strongly support the zinc-binding function of the N-terminal domain of nidovirus helicases. nsp10 ATPase/helicase deficiency resulting from single-residue substitutions in the ZBD or deletion of the entire domain could not be complemented in trans by wild-type ZBD, suggesting a critical function of the ZBD in cis. Consistently, no viral RNA synthesis was detected after transfection of EAV full-length RNAs encoding ATPase/helicase-deficient nsp10 into susceptible cells. In contrast, diverse phenotypes were observed for mutants with enzymatically active nsp10, which in a number of cases correlated with the activities measured in vitro. Collectively, our data suggest that the ZBD is critically involved in nidovirus replication and transcription by modulating the enzymatic activities of the helicase domain and other, yet unknown, mechanisms.

Biochemical characterization of arterivirus nonstructural protein 11 reveals the nidovirus-wide conservation of a replicative endoribonuclease

Nidoviruses (arteriviruses, coronaviruses, and roniviruses) are a phylogenetically compact but diverse group of positive-strand RNA viruses that includes important human and animal pathogens. Nidovirus RNA synthesis is mediated by a cytoplasmic membrane-associated replication/transcription complex that includes up to 16 viral nonstructural proteins (nsps), which carry common enzymatic activities, like the viral RNA polymerase, but also unusual and poorly understood RNA-processing functions. Of these, a conserved endoribonuclease (NendoU) is a major genetic marker that is unique to nidoviruses. NendoU activity was previously verified in vitro for the coronavirus nsp15, but not for any of its distantly related orthologs from other nidovirus lineages, like the arterivirus nsp11. Here, we show that the bacterially expressed nsp11 proteins of two arteriviruses, equine arteritis virus and porcine respiratory and reproductive syndrome virus, possess pyrimidine-specific endoribonuclease activity. RNA cleavage was independent of divalent cations in vitro and was greatly reduced by replacement of residues previously implicated in catalysis. Comparative characterization of the NendoU activity in arteriviruses and severe acute respiratory syndrome coronavirus revealed common and distinct features of their substrate requirements and reaction mechanism. Our data provide the first biochemical evidence of endoribonuclease activity associated with arterivirus nsp11 and support the conclusion that this remarkable RNA-processing enzyme, whose substrate in the infected cell remains to be identified, distinguishes nidoviruses from all other RNA viruses.

Ribose 2'-O-methylation provides a molecular signature for the distinction of self and non-self mRNA dependent on the RNA sensor Mda5.

The 5' cap structures of higher eukaryote mRNAs have ribose 2'-O-methylation. Likewise, many viruses that replicate in the cytoplasm of eukaryotes have evolved 2'-O-methyltransferases to autonomously modify their mRNAs. However, a defined biological role for 2'-O-methylation of mRNA remains elusive. Here we show that 2'-O-methylation of viral mRNA was critically involved in subverting the induction of type I interferon. We demonstrate that human and mouse coronavirus mutants lacking 2'-O-methyltransferase activity induced higher expression of type I interferon and were highly sensitive to type I interferon. Notably, the induction of type I interferon by viruses deficient in 2'-O-methyltransferase was dependent on the cytoplasmic RNA sensor Mda5. This link between Mda5-mediated sensing of viral RNA and 2'-O-methylation of mRNA suggests that RNA modifications such as 2'-O-methylation provide a molecular signature for the discrimination of self and non-self mRNA.

Elucidating the molecular physiopathology of acute respiratory distress syndrome in severe acute respiratory syndrome patients

Acute respiratory distress syndrome (ARDS) is a severe form of acute lung injury. It is a response to various diseases of variable etiology, including SARS-CoV infection. To date, a comprehensive study of the genomic physiopathology of ARDS (and SARS) is lacking, primarily due to the difficulty of finding suitable materials to study the disease process at a tissue level (instead of blood, sputa or swaps). Hereby we attempt to provide such study by analyzing autopsy lung samples from patient who died of SARS and showed different degrees of severity of the pulmonary involvement. We performed real-time quantitative PCR analysis of 107 genes with functional roles in inflammation, coagulation, fibrosis and apoptosis: some key genes were confirmed at a protein expression level by immunohistochemistry and correlated to the degree of morphological severity present in the individual samples analyzed. Significant expression levels were identified for ANPEP (a receptor for CoV), as well as inhibition of the STAT1 pathway, IFNs production and CXCL10 (a T-cell recruiter). Other genes unassociated to date with ARDS/SARS include C1Qb, C5R1, CASP3, CASP9, CD14, CD68, FGF7, HLA-DRA, ICF1, IRF3, MALAT-1, MSR1, NFIL3, SLPI, USP33, CLC, GBP1 and TACI. As a result, we proposed to therapeutically target some of these genes with compounds such as ANPEP inhibitors, SLPI and dexamethasone. Ultimately, this study may serve as a model for future, tissue-based analyses of fibroinflammatory conditions affecting the lung. (C) 2009 Elsevier B.V. All rights reserved.

Morbillivirus cross-species infection:is there a risk for humans?

The World Health Organisation (WHO) has set regional elimination goals for Measles (MV) eradication to be achieved by 2020 or earlier. A major question is whether an opportunity for veterinary virus infection of humans may arise when MV is eradicated and if vaccination is discontinued. Lessons have been learned from animal to human virus transmission i.e. human immunodeficiency virus (HIV) and more recently from severe acute respiratory syndrome (SARS) and avian influenza virus infections. We are therefore alerted to the risk of zoonosis from the veterinary morbilliviruses. In this review the evidence from viral genomics, animal studies and cell culture experiments will be explored to evaluate the possibility of cross infection of humans with these viruses.

Slopes of Avian Species-Area Relationships, Human Population Density, and Environmental Factors

There is increasing interest in how humans influence spatial patterns in biodiversity. One of the most frequently noted and marked of these patterns is the increase in species richness with area, the species-area relationship (SAR). SARs are used for a number of conservation purposes, including predicting extinction rates, setting conservation targets, and identifying biodiversity hotspots. Such applications can be improved by a detailed understanding of the factors promoting spatial variation in the slope of SARs, which is currently the subject of a vigorous debate. Moreover, very few studies have considered the anthropogenic influences on the slopes of SARs; this is particularly surprising given that in much of the world areas with high human population density are typically those with a high number of species, which generates conservation conflicts. Here we determine correlates of spatial variation in the slopes of species-area relationships, using the British avifauna as a case study. Whilst we focus on human population density, a widely used index of human activities, we also take into account (1) the rate of increase in habitat heterogeneity with increasing area, which is frequently proposed to drive SARs, (2) environmental energy availability, which may influence SARs by affecting species occupancy patterns, and (3) species richness. We consider environmental variables measured at both local (10 km x 10 km) and regional (290 km x 290 km) spatial grains, but find that the former consistently provides a better fit to the data. In our case study, the effect of species richness on the slope SARs appears to be scale dependent, being negative at local scales but positive at regional scales. In univariate tests, the slope of the SAR correlates negatively with human population density and environmental energy availability, and positively with the rate of increase in habitat heterogeneity. We conducted two sets of multiple regression analyses, with and without species richness as a predictor. When species richness is included it exerts a dominant effect, but when it is excluded temperature has the dominant effect on the slope of the SAR, and the effects of other predictors are marginal.