673 research outputs found

    Components required for in vitro cleavage and polyadenylation of eukaryotic mRNA

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    We have studied in vitro cleavage/polyadenylation of precursor RNA containing herpes simplex virus type 2 poly A site sequences and have analysed four RNA/protein complexes which form during in vitro reactions. Two complexes, A and B, form extremely rapidly and are then progressively replaced by a third complex, C which is produced following cleavage and polyadenylation of precursor RNA. Substitution of ATP with cordycepin triphosphate prevents polyadenylation and the formation of complex C however a fourth complex, D, results which contains cleaved RNA. A precursor RNA lacking GU-rich downstream sequences required for efficient cleavage/ polyadenylation fails to form complex B and produces a markedly reduced amount of complex A. As these GU-rich sequences are required for efficient cleavage, this establishes a relationship between complex B formation and cleavage/polyadenylation of precursor RNA in vitro. The components required for in vitro RNA processing have been separated by fractionation of the nuclear extract on Q-Sepharose and Biorex 70 columns. A Q-Sepharose fraction forms complex B but does not process RNA. Addition of a Biorex 70 fraction restores cleavage activity at the poly A site but this fraction does not appear to contribute to complex formation. Moreover, in the absence of polyethylene glycol, precursor RNA is not cleaved and polyadenylated, however, complexes A and B readily form. Thus, while complex B is necessary for in vitro cleavage and polyadenylation, it may not contain all the components required for this processing

    2,2,2-Tris(pyrazol-1-yl)ethanol

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    The title compound TPE, C11H12N6O, was prepared by slow evaporation from diethyl ether. In the crystal, there is a hydrogen bond between the alcohol H atom and an N in the pyrazole ring of a neighboring mol­ecule

    Metal Ion Complexes of N,N′-Bis(2-Pyridylmethyl)-trans-1,2-Diaminocyclohexane-N,N′-Diacetic Acid, H2bpcd: Lanthanide(III)–bpcd2– Cationic Complexes

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    The synthesis and characterization of N,N′-bis(2-pyridylmethyl)-trans-1,2-diaminocyclohexane-N,N′-diacetic acid (H2bpcd) cationic complexes of La(III), Nd(III), and Sm(III) are reported. The Ln(III)–bpcd2– complex ions, where bpcd2– stands for N,N′-bis(2-pyridylmethyl)-trans-1,2-diaminocyclohexane-N,N′-diacetate, were isolated as PF6– salts. These salts were characterized by elemental analysis, X-ray crystallography, IR, and 1H and 13C NMR spectroscopy. Binuclear [La2(bpcd)2(H2O)2]2+ crystallized from an aqueous solution in the monoclinic P21/c space group as a cocrystallate with Na2bpcd and NaPF6, nominally Na2.34[La1.22(C22H26N4O4)2(H2O)2][PF6]2·2H2O, with a = 11.3343(6) Å, b = 17.7090(9) Å, c = 15.0567(8) Å, β = 110.632(3)°, and Z = 4 (Z′ = 2). La is eight-coordinate with distorted dodecahedral coordination geometry provided by a N4O4 donor atom set. In addition to four N atoms from the bpcd2– ligand, La’s coordination sphere includes O atoms from a water molecule and three acetate groups (one O atom from singly bound acetate and two O atoms from acetate groups that bridge the La centers). The 1H and 13C assignments for H2bpcd and the metal–bpcd2– complexes were made on the basis of 2D COSY and HSQC experiments, which established 1H–1H and 1H–13C correlations. The NMR spectral data were used to establish the symmetry of the cationic complexes present in aqueous solution. The data indicate that the La(III)–bpcd2– and Sm(III)–bpcd2– complexes are present in solution as a single species with C2 symmetry. The 1H NMR spectrum of [Nd(bpcd)]PF6 in D2O consists of eight considerably line-broadened, paramagnetic-shifted singlets. The ab initio quantum mechanical calculations at the PCM/MP2/SDD//HF/SDD level, which were established previously for determining isomerization energies for octahedral M(III)–bpad2– complex ions, were used to determine the relative free energies of the geometric isomers possible for eight- and nine-coordinate La(III)–bpcd2– cationic aqua complexes in aqueous solution, i.e., [La(bpcd)(H2O)2]+ and La(bpcd)(H2O)3]+

    Dimerization-driven interaction of hepatitis C virus core protein with NS3 helicase

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    Hepatitis C virus (HCV) infects over 130 million people causing a worldwide epidemic of liver cirrhosis and hepatocellular-carcinoma. Because current HCV treatments are only partially effective, molecular mechanisms involved in HCV propagation are actively being pursued as possible drug targets. Here, we report on a new macromolecular interaction between the HCV capsid core protein and the helicase portion of HCV non-structural protein 3 (NS3h), confirmed by four different biochemical methods. The protease portion of NS3 is not required. Interaction between the two proteins could be disrupted by two types of specific inhibitors of core dimerization, the small molecule SL201 and core106, a C-terminally truncated core protein. Cross-linking experiments suggest that the physical interaction with NS3h is probably driven by core oligomerization. Moreover, SL201 blocks the production of infectious virus, but not the production of a subgenomic HCV replicon by hepatoma cells. Time-of-addition experiments confirm that SL201 has no effect on entry of the virus. These data underline the essential role of core as a key organizer of HCV particle assembly, confirm the importance of oligomerization, reveal the interaction with viral helicase and support a new molecular understanding of the formation of the viral particle at the level of the lipid droplets, before its migration to the site of release and budding

    Bidirectional lipid droplet velocities are controlled by differential binding strengths of HCV Core DII protein

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    Host cell lipid droplets (LD) are essential in the hepatitis C virus (HCV) life cycle and are targeted by the viral capsid core protein. Core-coated LDs accumulate in the perinuclear region and facilitate viral particle assembly, but it is unclear how mobility of these LDs is directed by core. Herein we used two-photon fluorescence, differential interference contrast imaging, and coherent anti-Stokes Raman scattering microscopies, to reveal novel core-mediated changes to LD dynamics. Expression of core protein’s lipid binding domain II (DII-core) induced slower LD speeds, but did not affect directionality of movement on microtubules. Modulating the LD binding strength of DII-core further impacted LD mobility, revealing the temporal effects of LD-bound DII-core. These results for DII-core coated LDs support a model for core-mediated LD localization that involves core slowing down the rate of movement of LDs until localization at the perinuclear region is accomplished where LD movement ceases. The guided localization of LDs by HCV core protein not only is essential to the viral life cycle but also poses an interesting target for the development of antiviral strategies against HCV

    Predicting Outcome in dogs with Primary Immune-Mediated Hemolytic Anemia: Results of a Multicenter Case Registry

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    BACKGROUND: Outcome prediction in dogs with immune‐mediated hemolytic anemia (IMHA) is challenging and few prognostic indicators have been consistently identified. OBJECTIVES: An online case registry was initiated to: prospectively survey canine IMHA presentation and management in the British Isles; evaluate 2 previously reported illness severity scores, Canine Hemolytic Anemia Score (CHAOS) and Tokyo and to identify independent prognostic markers. ANIMALS: Data from 276 dogs with primary IMHA across 10 referral centers were collected between 2008 and 2012. METHODS: Outcome prediction by previously reported illness‐severity scores was tested using univariate logistic regression. Independent predictors of death in hospital or by 30‐days after admission were identified using multivariable logistic regression. RESULTS: Purebreds represented 89.1% dogs (n = 246). Immunosuppressive medications were administered to 88.4% dogs (n = 244), 76.1% (n = 210) received antithrombotics and 74.3% (n = 205) received packed red blood cells. Seventy‐four per cent of dogs (n = 205) were discharged from hospital and 67.7% (n = 187) were alive 30‐days after admission. Two dogs were lost to follow‐up at 30‐days. In univariate analyses CHAOS was associated with death in hospital and death within 30‐days. Tokyo score was not associated with either outcome measure. A model containing SIRS‐classification, ASA classification, ALT, bilirubin, urea and creatinine predicting outcome at discharge was accurate in 82% of cases. ASA classification, bilirubin, urea and creatinine were independently associated with death in hospital or by 30‐days. CONCLUSIONS AND CLINICAL IMPORTANCE: Markers of kidney function, bilirubin concentration and ASA classification are independently associated with outcome in dogs with IMHA. Validation of this score in an unrelated population is now warranted

    Full genome sequence and sfRNA interferon antagonist activity of Zika virus from Recife, Brazil

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    Background: The outbreak of Zika virus (ZIKV) in the Americas has transformed a previously obscure mosquito-transmitted arbovirus of the Flaviviridae family into a major public health concern. Little is currently known about the evolution and biology of ZIKV and the factors that contribute to the associated pathogenesis. Determining genomic sequences of clinical viral isolates and characterization of elements within these are an important prerequisite to advance our understanding of viral replicative processes and virus-host interactions. Methodology/Principal findings: We obtained a ZIKV isolate from a patient who presented with classical ZIKV-associated symptoms, and used high throughput sequencing and other molecular biology approaches to determine its full genome sequence, including non-coding regions. Genome regions were characterized and compared to the sequences of other isolates where available. Furthermore, we identified a subgenomic flavivirus RNA (sfRNA) in ZIKV-infected cells that has antagonist activity against RIG-I induced type I interferon induction, with a lesser effect on MDA-5 mediated action. Conclusions/Significance: The full-length genome sequence including non-coding regions of a South American ZIKV isolate from a patient with classical symptoms will support efforts to develop genetic tools for this virus. Detection of sfRNA that counteracts interferon responses is likely to be important for further understanding of pathogenesis and virus-host interactions
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