A Novel Small Molecule Inhibitor of Influenza A Viruses that Targets Polymerase Function and Indirectly Induces Interferon

Influenza viruses continue to pose a major public health threat worldwide and options for antiviral therapy are limited by the emergence of drug-resistant virus strains. The antiviral cytokine, interferon (IFN) is an essential mediator of the innate immune response and influenza viruses, like many viruses, have evolved strategies to evade this response, resulting in increased replication and enhanced pathogenicity. A cell-based assay that monitors IFN production was developed and applied in a high-throughput compound screen to identify molecules that restore the IFN response to influenza virus infected cells. We report the identification of compound ASN2, which induces IFN only in the presence of influenza virus infection. ASN2 preferentially inhibits the growth of influenza A viruses, including the 1918 H1N1, 1968 H3N2 and 2009 H1N1 pandemic strains and avian H5N1 virus. In vivo, ASN2 partially protects mice challenged with a lethal dose of influenza A virus. Surprisingly, we found that the antiviral activity of ASN2 is not dependent on IFN production and signaling. Rather, its IFN-inducing property appears to be an indirect effect resulting from ASN2-mediated inhibition of viral polymerase function, and subsequent loss of the expression of the viral IFN antagonist, NS1. Moreover, we identified a single amino acid mutation at position 499 of the influenza virus PB1 protein that confers resistance to ASN2, suggesting that PB1 is the direct target. This two-pronged antiviral mechanism, consisting of direct inhibition of virus replication and simultaneous activation of the host innate immune response, is a unique property not previously described for any single antiviral molecule.     

Global analysis of protein phosphorylation networks in insulin signaling by sequential enrichment of phosphoproteins and phosphopeptides

Although the important role of protein phosphorylation in insulin signaling networks is well recognized, its analysis in vivo has not been pursued in a systematic fashion through proteome-wide studies. Here we undertake a global analysis of insulin-induced changes in the rat liver cytoplasmic and endosomal phosphoproteome by sequential enrichment of phosphoproteins and phosphopeptides. After subcellular fractionation proteins were denatured and loaded onto iminodiacetic acid-modified Sepharose with immobilized Al3? ions (IMAC-Al resin). Retained phosphoproteins were eluted with 50 mM phosphate and proteolytically digested. The digest was then loaded onto an IMAC-Al resin and phosphopeptides were eluted with 50 mM phosphate, and resolved by 2-dimensional liquid chromatography, which combined offline weak anion exchange and online reverse phase separations. The peptides were identified by tandem mass spectrometry, which also detected the phosphorylation sites. Non-phosphorylated peptides found in the flow-through of the IMAC-Al columns were also analyzed providing complementary information for protein identification. In this study we enriched phosphopeptides to ~85% purity and identified 1456 phosphopeptides from 604 liver phosphoproteins. Eighty-nine phosphosites including 45 novel ones in 83 proteins involved in vesicular transport, metabolism, cell motility and structure, gene expression and various signaling pathways were changed in response to insulin treatment. Together these findings could provide potential new markers for evaluating insulin action and resistance in obesity and diabetes.     

Screening of alpha-1-antitrypsin gene by denaturing gradient gel electrophoresis (DGGE)

Alpha-1-antitrypsin (AAT) is a serine protease inhibitor whose deficiency could cause emphysema and liver disease and, as recently described, could be a risk factor for lung cancer development. Alpha-1-antitrypsin inhibits a variety of proteases but its primary target is neutrophil elastase, an extracellular endopeptidase capable of degrading most protein components of the extracellular matrix. Inhibition of neutrophil elastase by AAT has an important role in maintaining the integrity of connective tissue. The gene encoding for AAT spans over 12.2 kb, consists of seven exons and is highly polymorphic. Therefore several methods for mutation screening of alpha-1-antitrypsin gene have been developed. Method described here is based on denaturing gradient gel electrophoresis (DGGE). This method is highly efficient and reliable and allows rapid analysis of entire coding region of alpha-1-antitrypsin gene, including splice junction sites. Previously described DGGE based analysis of AAT gene included overnight electrophoresis of individually amplified fragments. The optimization of the method described in this paper is directed towards the shortening of the duration of electrophoresis and amplification of fragments in multiplex reaction in order to make the analysis less time-consuming and therefore more efficient.     

Design and characterization of alcalase–chitosan conjugates as potential biocatalysts

Bioprocess and Biosystems Engineering - In this study, alcalase (protease from Bacillus licheniformis) immobilization by adsorption, enzyme crosslinking and covalent enzyme binding to activated...     

Effect of storage conditions on virulence of Fusarium avenaceum and Alternaria alternata on apple fruits

To overcome difficulty in phytopathogenic fungi control during storage of apple fruits, the effect of different storage conditions on the occurrence and development of Fusarium avenaceum and Alternaria alternata infections on apple cultivar “Cripps Pink” was investigated during and after storage. Inhibitory effects of wild oregano essential oil on apple fruit rots caused by F. avenaceum and A. alternata were also tested as possible rot control measure. Artificially inoculated apple fruits were kept in cold storage with normal (NA) and controlled (CA) atmosphere for 95 days and at room temperature only. The obtained results indicated that different storage conditions significantly affect necrosis development on apple fruits caused by F. avenaceum and A. alternata after storage, as well as during shelf life.     

Expression and characterization of recombinant vitamin K-dependent gamma-glutamyl carboxylase from an invertebrate, Conus textile.

The marine snail Conus is the sole invertebrate wherein both the vitamin K-dependent carboxylase and its product, gamma-carboxyglutamic acid, have been identified. To examine its biosynthesis of gamma-carboxyglutamic acid, we studied the carboxylase from Conus venom ducts. The carboxylase cDNA from Conus textile has an ORF that encodes a 811-amino-acid protein which exhibits sequence similarity to the vertebrate carboxylases, with 41% identity and approximately 60% sequence similarity to the bovine carboxylase. Expression of this cDNA in COS cells or insect cells yielded vitamin K-dependent carboxylase activity and vitamin K-dependent epoxidase activity. The recombinant carboxylase has a molecular mass of approximately 130 kDa. The recombinant Conus carboxylase carboxylated Phe-Leu-Glu-Glu-Leu and the 28-residue peptides based on residues -18 to +10 of human proprothrombin and proFactor IX with Km values of 420 micro m, 1.7 micro m and 6 micro m, respectively; the Km for vitamin K is 52 micro m. The Km values for peptides based on the sequence of the conotoxin epsilon-TxIX and two precursor analogs containing 12 or 29 amino acids of the propeptide region are 565 micro m, 75 micro m and 74 micro m, respectively. The recombinant Conus carboxylase, in the absence of endogenous substrates, is stimulated up to fivefold by vertebrate propeptides but not by Conus propeptides. These results suggest two propeptide-binding sites in the carboxylase, one that binds the Conus and vertebrate propeptides and is required for substrate binding, and the other that binds only the vertebrate propeptide and is required for enzyme stimulation. The marked functional and structural similarities between the Conus carboxylase and vertebrate vitamin K-dependent gamma-carboxylases argue for conservation of a vitamin K-dependent carboxylase across animal species and the importance of gamma-carboxyglutamic acid synthesis in diverse biological systems.     

Absorption and linear dichroism spectroscopy at room and low temperatures of single crystals of the B800–850 light-harvesting complex from Rhodopseudomonas acidophila strain 10050

The absorbance, polarized absorbance and linear dichroism spectra of single crystals of the B800–850 light-harvesting complex from Rhodopseudomonas acidophila strain 10050 taken at room (298 K) and low (85 K) temperatures are presented. The spectra are compared and contrasted with random phase solution spectra from the same complex. The single crystal spectra display a spectral narrowing at low temperatures in the BChl Q_x (550–650 nm) and carotenoid (450–550 nm) regions similar to that observed from the random phase solution. The single crystal absorption spectra in the BChl Q_y (750–900 nm) region are broader than the solution spectra and remain broad as the temperature is lowered. It is suggested that this broadening is the result of specific exciton interactions between the BChl chromophore Q_y transition dipoles and is a molecular feature which occurs only in the crystalline complex.     

Isothiocyanates: cholinesterase inhibiting,antioxidant, and anti-inflammatory activity

Finding a new type of cholinesterase inhibitor that would overcome the brain availability and pharmacokinetic parameters or hepatotoxic liability has been a focus of investigations dealing with the treatment of Alzheimer’s disease. Isothiocyanates have not been previously investigated as potential cholinesterase inhibitors. These compounds can be naturally produced from their glucosinolate precursors, secondary metabolites widely distributed in our daily Brassica vegetables. Among 11 tested compounds, phenyl isothiocyanate and its derivatives showed the most promising inhibitory activity. 2-Methoxyphenyl ITC showed best inhibition on acetylcholinesterase with IC₅₀ of 0.57?mM, while 3-methoxyphenyl ITC showed the best inhibition on butyrylcholinesterase having 49.2% at 1.14?mM. Assessment of the antioxidant efficacy using different methods led to a similar conclusion. The anti-inflammatory activity was also tested using human COX-2 enzyme, ranking phenyl isothiocyanate, and 3-methoxyphenyl isothiocyanate as most active, with ～99% inhibition at 50?μM.