Nucleotide analogs and molecular modeling studies reveal key interactions involved in substrate recognition by the yeast RNA triphosphatase

RNA triphosphatases (RTPases) are involved in the addition of the distinctive cap structure found at the 5′ ends of eukaryotic mRNAs. Fungi, protozoa and some DNA viruses possess an RTPase that belongs to the triphosphate tunnel metalloenzyme family of enzymes that can also hydrolyze nucleoside triphosphates. Previous crystallization studies revealed that the phosphohydrolase catalytic core is located in a hydrophilic tunnel composed of antiparallel β-strands. However, all past efforts to obtain structural information on the interaction between RTPases and their substrates were unsuccessful. In the present study, we used computational molecular docking to model the binding of a nucleotide substrate into the yeast RTPase active site. In order to confirm the docking model and to gain additional insights into the molecular determinants involved in substrate recognition, we also evaluated both the phosphohydrolysis and the inhibitory potential of an important number of nucleotide analogs. Our study highlights the importance of specific amino acids for the binding of the sugar, base and triphosphate moieties of the nucleotide substrate, and reveals both the structural flexibility and complexity of the active site. These data illustrate the functional features required for the interaction of an RTPase with a ligand and pave the way to the use of nucleotide analogs as potential inhibitors of RTPases of pathogenic importance.     

Structural analysis of the lipopolysaccharide derived core oligosaccharides of Actinobacillus pleuropneumoniae serotypes 1, 2, 5a and the genome strain 5b

The structures of the core oligosaccharides of the lipopolysaccharides (LPS) from Actinobacillus pleuropneumoniae serotypes 1, 2, 5a and 5b were elucidated. The LPS's were subjected to a variety of degradative procedures. The structures of the purified products were established by monosaccharide and methylation analyses, NMR spectroscopy and mass spectrometry. The following structures for the core oligosaccharides were determined on the basis of the combined data from these experiments. [carbohydrate formula see text] For serotype 1: R is (1S)-GalaNAc-(1-->4,6)-alpha-Gal II-(1-->3)-beta-Gal I-(1-->, and R' is H For serotype 2: R is beta-Glc III-(1-->, and R' is D-alpha-D-Hep V-(1--> For serotypes 5a and 5b: R is H and R' is D-alpha-D-Hep V-(1--> All oligosaccharides elaborated a conserved inner core structure, as illustrated. All sugars were in the pyranose ring form apart from the open-chain N-acetylgalactosamine, the identification of which in the serotype 1 LPS was of interest.     

Characterization of the metal ion binding properties of the hepatitis C virus RNA polymerase

The hepatitis C virus nonstructural 5B protein (NS5B) protein has been shown to require either magnesium or manganese for its RNA-dependent RNA polymerase activity. As a first step toward elucidating the nature and the role(s) of the metal ions in the reaction chemistry, we have utilized endogenous tryptophan fluorescence to quantitate the interactions of magnesium and manganese ions with this protein. The association of either Mg(2+) or Mn(2+) ions with the enzyme resulted in a decrease in the intensity of the tryptophan emission spectrum. This decrease was used to determine the apparent dissociation constants for both ions. The apparent K(d) values for the binding of Mg(2+) and Mn(2+) ions to the free enzyme were 3.1 and 0.3 mm, respectively. Dual ligand titration experiments demonstrated that both ions bind to a single common site, for which they compete. The kinetics of real time metal ion binding to the NS5B protein were also investigated. Based on the results of our fluorescence and near-UV circular dichroism experiments, we show that NS5B undergoes conformational changes upon the binding of metal ions. However, this process does not significantly stimulate the binding to the RNA or NTP substrates. We envisage that the ion-induced conformational change is a prerequisite for catalytic activity by both correctly positioning the side chains of the residues located in the active site of the enzyme and also contributing to the stabilization of the intermediate transition state.     

Initial binding of the broad spectrum antiviral nucleoside ribavirin to the hepatitis C virus RNA polymerase

Ribavirin is a broad spectrum antiviral nucleoside that displays activity against a variety of RNA and DNA viruses. Ribavirin is currently used in combination with interferon-alpha for the treatment of hepatitis C virus (HCV) infection and was recently shown to be directly incorporated by the HCV RNA polymerase into RNA products. This capacity ultimately leads to increased mutation rates and drastically reduces the viral fitness. As a first step toward elucidating the nature of the specific interaction between ribavirin and the HCV polymerase, we have utilized fluorescence spectroscopy to monitor precisely the binding of ribavirin triphosphate (RTP) to the viral polymerase. This spectroscopic approach allowed us to clearly separate the RTP binding activity from the concomitant catalytic steps. We report here the first detailed study of the binding kinetics and thermodynamic parameters involved in the interaction between RTP and an RNA polymerase. We demonstrate that RTP binds to the same active site as nucleotides. Furthermore, we provide evidence that the HCV polymerase cannot only bind to RTP but also to nonphosphorylated ribavirin, albeit with less affinity. By using various combinations of template-primers, we also demonstrate that base pairing is not involved in the initial binding of RTP to the HCV polymerase. Based on the results of circular dichroism and denaturation studies, we show that the RNA polymerase undergoes subtle conformational changes upon the binding of RTP, although the interaction does not significantly modify the stability of the protein. Finally, although metal ions are required for catalytic activity, they are not required for the initial binding of RTP to the polymerase. Such quantitative analyses are of primary importance for the rational design of new ribavirin analogues of potential therapeutic value and provide crucial insights on the interaction between RTP and the HCV RNA polymerase.     

The amyloid-β1–42-oligomer interacting peptide D-AIP possesses favorable biostability,pharmacokinetics, and brain region distribution

We have previously developed a unique 8-amino acid Aβ42 oligomer-Interacting Peptide (AIP) as a novel anti-amyloid strategy for the treatment of Alzheimer’s disease. Our lead candidate has successfully progressed from test tubes (i.e., in vitro characterization of protease-resistant D-AIP) to transgenic flies (i.e., in vivo rescue of human Aβ42-mediated toxicity via D-AIP-supplemented food). In the present study, we examined D-AIP in terms of its stability in multiple biological matrices (i.e., ex-vivo mouse plasma, whole blood, and liver S9 fractions) using MALDI mass spectrometry, pharmacokinetics using a rapid and sensitive LC-MS method, and blood brain barrier (BBB) penetrance in WT C57LB/6 mice. D-AIP was found to be relatively stable over 3 h at 37 °C in all matrices tested. Finally, label-free MALDI imaging showed that orally administered D-AIP can readily penetrate the intact BBB in both male and female WT mice. Based upon the favorable stability, pharmacokinetics, and BBB penetration outcomes for orally administered D-AIP in WT mice, we then examined the effect of D-AIP on amyloid “seeding” in vitro (i.e., freshly monomerized versus preaggregated Aβ42). Complementary biophysical assays (ThT, TEM, and MALDI-TOF MS) showed that D-AIP can directly interact with synthetic Aβ42 aggregates to disrupt primary and/or secondary seeding events. Taken together, the unique mechanistic and desired therapeutic potential of our lead D-AIP candidate warrants further investigation, that is, testing of D-AIP efficacy on the altered amyloid/tau pathology in transgenic mouse models of Alzheimer’s disease.     

Effects of bioaugmentation strategies in UASB reactors with a methanogenic consortium for removal of phenolic compounds

The removal of phenol, ortho- (o-) and para- (p-)cresol was studied with two series of UASB reactors using unacclimatized granular sludges bioaugmented with a consortium enriched against these substances. The parameters studied were the amount of inoculum added to the sludges and the method of immobilization of the inoculum. Two methods were used, adsorption to the biomass or encapsulation within calcium alginate beads. In the bioaugmentation by adsorption experiment, and with a 10% inoculum, complete phenol removal was obtained after 36 d, while 178 d were required in the control reactor. For p-cresol, 95% removal was obtained in the bioaugmented reactor on day 48 while 60 d were required to achieve 90% removal in the control reactor. For o-cresol, the removals were only marginally better with the bioaugmented reactors. Tests performed with the reactors biomass under non-limiting substrate concentrations showed that the specific activities of the bioaugmented biomasses were larger than the original biomass for phenol, and p-cresol even after 276 of operations, showing that the inoculum bacteria successfully colonized the sludge granules. Immobilization of the inoculum by encapsulation in calcium alginate beads, was performed with 10% of the inoculum. Results showed that the best activities were obtained when the consortium was encapsulated alone and the beads added to the sludges. This reactor presented excellent activity and the highest removal of the various phenolic compounds a few days after start-up. After 90 d, a high-phenolic compounds removal was still observed, demonstrating the effectiveness of the encapsulation technique for the start-up and maintenance of high-removal activities.     

Novel biosynthetic functions of lipopolysaccharide rfaJ homologs from Helicobacter pylori

Activity screening and insertional inactivation of lipopolysaccharide (LPS) biosynthetic genes in Helicobacter pylori have led to the successful characterization of two key enzymes encoded by HP0159 (JHP0147) and HP1105 (JHP1032) open reading frames (ORFs) which are members of the large and diverse carbohydrate active enzymes (CAZY) GT-8 (rfaJ) family of glycosyltransferases. Activity screening of a genomic library led to the identification of the enzyme involved in the biosynthesis of the type 2 N-acetyl-lactosamine O-chain backbone, the beta-1,3-N-acetyl-glucosaminyl transferase. In addition, the activity screening approach led to the identification and characterization of a key core biosynthetic enzyme responsible for the biosynthesis of the alpha-1,6-glucan polymer. This alpha-1,6-glucosyltransferase protein is encoded by the HP0159 ORF. Both enzymes play an integral part in the biosynthesis of LPS, and insertional inactivation leads to the production of a truncated LPS molecule on the bacterial cell surface. The LPS structures were determined by mass spectrometry and chemical analyses. The linkage specificity of each glycosyltransferase was determined by nuclear magnetic resonance (NMR) analysis of model compounds synthesized in vitro. A cryogenic probe was used to structurally characterize nanomole amounts of the product of the HP1105 (JHP1032) enzyme. In contrast to the HP0159 enzyme, which displays the GT-8-predicted retaining stereochemistry for the reaction product, HP1105 (JHP1032) is the first member of this GT-8 family to have been shown to have an inverting stereochemistry in its reaction products.     

Viral diseases of the giant fresh water prawn Macrobrachium rosenbergii: a review

The giant freshwater prawn Macrobrachium rosenbergii is cultivated essentially in Southern and South-eastern Asian countries such as continental China, India, Thailand and Taiwan. To date, only two viral agents have been reported from this prawn. The first (HPV-type virus) was observed by chance 25 years ago in hypertrophied nuclei of hepatopancreatic epithelial cells and is closely related to members of the Parvoviridae family. The second, a nodavirus named MrNV, is always associated with a non-autonomous satellite-like virus (XSV), and is the origin of so-called white tail disease (WTD) responsible for mass mortalities and important economic losses in hatcheries and farms for over a decade. After isolation and purification of these two particles, they were physico-chemically characterized and their genome sequenced. The MrNV genome is formed with two single linear ss-RNA molecules, 3202 and 1250 nucleotides long, respectively. Each RNA segment contains only one ORF, ORF1 coding for the RNA-dependant RNA polymerase located on the long segment and ORF2 coding for the structural protein CP-43 located on the small one. The XSV genome (linear ss-RNA), 796 nucleotides long, contains a single ORF coding for the XSV coat protein CP-17. The XSV does not contain any RdRp gene and consequently needs the MrNV polymerase to replicate.     

Viral diseases in commercially exploited crabs: a review

Viruses and viral diseases of crabs were observed and investigated earlier than the first observation of viruses in shrimp. In fact, crabs were used as biological models to investigate crustacean virology at the beginning of shrimp aquaculture development. More than 30 viruses have been reported in crabs, including those related to the known virus families Reoviridae, Bunyaviridae, Roniviridae and a group of Bacilliform enveloped nuclear viruses. This review reports data on several important viral diseases of crabs, particularly those associated with pathology of organs and tissues of commercially and ecologically significant host species.