A low-barrier hydrogen bond between histidine of secreted phospholipase A2 and a transition state analog inhibitor

This work describes in-depth NMR characterization of a unique low-barrier hydrogen bond (LBHB) between an active site residue from the enzyme and a bound inhibitor: the complex between secreted phospholipase A(2) (sPLA(2), from bee venom and bovine pancreas) and a transition-state analog inhibitor HK32. A downfield proton NMR resonance, at 17-18 ppm, was observed in the complex but not in the free enzyme. On the basis of site-specific mutagenesis and specific 15N-decoupling, this downfield resonance was assigned to the active site H48, which is part of the catalytic dyad D99-H48. These results led to a hypothesis that the downfield resonance represents the proton (H(epsilon 2) of H48) involved in the H-bonding between D99 and H48, in analogy with serine proteases. However, this was shown not to be the case by use of the bovine enzyme labeled with specific [15N(epsilon 2)]His. Instead, the downfield resonance arises from H(delta1) of H48, which forms a hydrogen bond with a non-bridging phosphonate oxygen of the inhibitor. Further studies showed that this proton displays a fractionation factor of 0.62(+/-0.06), and an exchange rate protection factor of >100 at 285 K and >40 at 298 K, which are characteristic of a LBHB. The pK(a) of the imidazole ring of H48 was shown to be shifted from 5.7 for the free enzyme to an apparent value of 9.0 in the presence of the inhibitor. These properties are very similar to those of the Asp em leader His LBHBs in serine proteases. Possible structural bases and functional consequences for the different locations of the LBHB between these two types of enzymes are discussed. The results also underscore the importance of using specific isotope labeling, rather than extrapolation of NMR results from other enzyme systems, to assign the downfield proton resonance to a specific hydrogen bond. Although our studies did not permit the strength of the LBHB to be accurately measured, the data do not provide support for an unusually strong hydrogen bond strength (i.e. >10 kcal/mol).     

Towards Antiviral shRNAs Based on the AgoshRNA Design

RNA interference (RNAi) can be induced by intracellular expression of a short hairpin RNA (shRNA). Processing of the shRNA requires the RNaseIII-like Dicer enzyme to remove the loop and to release the biologically active small interfering RNA (siRNA). Dicer is also involved in microRNA (miRNA) processing to liberate the mature miRNA duplex, but recent studies indicate that miR-451 is not processed by Dicer. Instead, this miRNA is processed by the Argonaute 2 (Ago2) protein, which also executes the subsequent cleavage of a complementary mRNA target. Interestingly, shRNAs that structurally resemble miR-451 can also be processed by Ago2 instead of Dicer. The key determinant of these “AgoshRNA” molecules is a relatively short basepaired stem, which avoids Dicer recognition and consequently allows alternative processing by Ago2. AgoshRNA processing yields a single active RNA strand, whereas standard shRNAs produce a duplex with guide and passenger strands and the latter may cause adverse off-target effects. In this study, we converted previously tested active anti-HIV-1 shRNA molecules into AgoshRNA. We tested several designs that could potentially improve AgoshRNA activity, including extension of the complementarity between the guide strand and the mRNA target and reduction of the thermodynamic stability of the hairpins. We demonstrate that active AgoshRNAs can be generated. However, the RNAi activity is reduced compared to the matching shRNAs. Despite reduced RNAi activity, comparison of an active AgoshRNA and the matching shRNA in a sensitive cell toxicity assay revealed that the AgoshRNA is much less toxic.     

Design of extended short hairpin RNAs for HIV-1 inhibition

RNA interference (RNAi) targeted towards viral mRNAs is widely used to block virus replication in mammalian cells. The specific antiviral RNAi response can be induced via transfection of synthetic small interfering RNAs (siRNAs) or via intracellular expression of short hairpin RNAs (shRNAs). For HIV-1, both approaches resulted in profound inhibition of virus replication. However, the therapeutic use of a single siRNA/shRNA appears limited due to the rapid emergence of RNAi-resistant escape viruses. These variants contain deletions or point mutations within the target sequence that abolish the antiviral effect. To avoid escape from RNAi, the virus should be simultaneously targeted with multiple shRNAs. Alternatively, long hairpin RNAs can be used from which multiple effective siRNAs may be produced. In this study, we constructed extended shRNAs (e-shRNAs) that encode two effective siRNAs against conserved HIV-1 sequences. Activity assays and RNA processing analyses indicate that the positioning of the two siRNAs within the hairpin stem is critical for the generation of two functional siRNAs. E-shRNAs that are efficiently processed into two effective siRNAs showed better inhibition of virus production than the poorly processed e-shRNAs, without inducing the interferon response. These results provide building principles for the design of multi-siRNA hairpin constructs.     

HPTLC determination of vasicine and vasicinone in Adhatoda vasica

A simple high-performance thin-layer chromatographic (HPTLC) method has been developed for the simultaneous determination of the pharmacologically important quinazoline alkaloids vasicine and vasicinone in Adhatoda vasica. The assay combines the separation and quantification of the analytes on silica gel 60 GF254 HPTLC plates with visualisation under UV and scanning at 270 and 281 nm. Using this technique, the alkaloidal content of different parts of the title plant have been determined.     

Probing the shRNA characteristics that hinder Dicer recognition and consequently allow Ago-mediated processing and AgoshRNA activity

Recent evidence indicates the presence of alternative pathways for microRNA (miRNA) and short hairpin (shRNA) processing. Specifically, some of these molecules are refractory to Dicer-mediated processing, which allows alternative processing routes via the Ago2 endonuclease. The resulting RNA molecules differ in size and sequence and will thus trigger the silencing of different target RNAs. It is, therefore, important to understand these processing routes in mechanistic detail such that one can design exclusive RNA reagents for a specific processing route. The exact sh/miRNA properties that determine this routing toward Dicer or Ago2 are incompletely understood. The size of the base-paired stem seems an important determinant, but other RNA elements may contribute as well. In this study, we document the importance of a weak G-U or U-G base pair at the top of the hairpin stem.     

Litterfall,leaf decomposition and litter colonization of Tessaria integrifolia (compositae) in the Parana river floodplain

Litterfall and leaf decomposition rates were measured in Choui Island, 45 km downstream from the confluence of the Paraná and Paraguay rivers. The material was collected biweekly from April 1985 through September 1986. Decomposition was measured in situ by the litter bag technique.Annual litterfall of Tessaria integrifolia gallery forest measured in the period April 1985 to March 1986 was 8.15 t ha^-1. Leaf litterfall was seasonal, i.e. significantly less leaf litter was shed during the high water phase than during the low water phase. The half life of the T. integrifolia litter over 38 days of decomposition was 20 days. At the beginning of the experiment, 15 and 38 days subsamples of remaining detritus were analyzed in order to determine changes in the nutrient content. After 38 days of incubation, the order of nutrient disappearance was Ca > K > N > Mg > Na > P.The number of invertebrates per g remaining litter of Tessaria integrifolia increased between incubations days 7 and 31. Collector-gatherers were more abundant after 38 days incubation; there were no shredders colonizing the leaf litter bags.     

Macroinvertebrates on Eichhornia crassipes roots in two lakes of the Paraná River floodplain

Changes in the abundance, biomass and in the relativeproportions of functional feeding groups ofmacroinvertebrates were studied for 17 months duringlow and high water periods in two floodplain lakeswith indirect connections to the Paraná River.At low water, Eichhornia crassipes root clusterswere colonized by comparatively low densities ofmacroinvertebrates, especially in the lake moredistant to the river. Collector-gatherers andpredators were the most abundant functional feedinggroups, but different taxa of these groups dominatedin the two lakes during low water.At high water, macroinvertebrates densities increasedas a result of increased flow through the roots. Inthis period, collector-filterers dominated at bothlakes and constituted over 34% of themacroinvertebrates. Four independent variables(conductivity, hydrological periods, dissolved oxygenand site) explain 87% of the variability inmacroinvertebrates density expressed as the number ofindividuals per m2. No significant relationshipwere found between macroinvertebrate biomass and anyof the independent variables.     

Biodiversity matters in a changing world

It is now widely accepted that the climate of our planet is changing, but it is still hard to predict the consequences of these changes on ecosystems. The impact is worst at the poles, with scientists concerned that impacts at lower latitudes will follow suit. Canada has a great responsibility and potential for studying the effects of climate changes on the ecological dynamics, given its geographical location and its scientific leadership in this field. The 5th annual meeting of the Canadian Society for Ecology and Evolution was held in the International Year of Biodiversity, to share recent advances in a wide variety of disciplines ranging from molecular biology to behavioural ecology, and to integrate them into a general view that will help us preserve biodiversity and limit the impact of climate change on ecosystems.     

RNAi-mediated inhibition of HIV-1 by targeting partially complementary viral sequences

Potent antiviral RNAi can be induced by intracellular expression of short hairpin RNAs (shRNAs) and artificial microRNAs (miRNAs). Expression of shRNA and miRNA results in target mRNA degradation (perfect base pairing) or translational repression (partial base pairing). Although efficient inhibition can be obtained, error-prone viruses such as human immunodeficiency virus type 1 (HIV-1) can escape from RNAi-mediated inhibition by mutating the target sequence. Recently, artificial miRNAs have been shown to be potent RNAi inducers due to their efficient processing by the RNAi machinery. Furthermore, miRNAs may be more proficient in suppressing imperfect targets than shRNAs. In this study, we tested the knockdown efficiency of miRNAs and shRNAs against wild-type and RNAi-escape HIV-1 variants with one or two mutations in the target sequence. ShRNAs and miRNAs can significantly inhibit the production of HIV-1 variants with mutated target sequences in the open reading frame. More pronounced mutation-tolerance was measured for targets in the 3′ untranslated region (3′ UTR). Partially complementary sequences within the 3′ UTR of the HIV-1 RNA genome efficiently act as target sites for miRNAs and shRNAs. These data suggest that targeting imperfect target sites by antiviral miRNAs or shRNAs provides an alternative RNAi approach for inhibition of pathogenic viruses.     

Genetic and metabolic effects of gluconasturtiin, a glucosinolate derived from cruciferae

It is thought that induction of detoxifying phase-II drug metabolizing enzymes or inhibition of bioactivating phase-I by phytoalexins could protect against mutagens and neoplasia. In the search for potential naturally occurring molecular chemoprevention agents, particular attention has been devoted to isothiocyanates, which are breakdown products-via myrosinase-of glucosinolates such as gluconasturtiin (GNST), a natural constituent of cruciferae. Here, we first investigated the ability of GNST to modulate metabolizing enzymes in male Swiss Albino CD1 mice injected by gavage (24 mg/kg or 48 mg/kg b.w.) with GNST either in single or repeated (daily for four consecutive days) dose. Using selected probes to various cytochrome P450 (CYP) isoforms, a marked and generalized decrease of CYP content, NADPH-(CYP)-c-reductase and various CYP-linked monooxygenases (measuring CYP1A1, CYP2B1/2, CYP3A1/2, CYP1A2 and CYP2E1), was observed in hepatic, renal and pulmonary subcellular preparations (up to approximately 66% loss, liver). Similar behavior was recorded using the regio- and stereo-selective hydroxylation of testosterone as multibiomarker (CYP2A1 and CYP2B9, up to approximately 96% loss), as well as with the phase-II marker glutathione S-transferase (up to approximately 50% loss, liver). We also performed genotoxicity investigations, using the diploid D7 strain of yeast Saccharomyces cerevisiae as a biological test system. GNST was able to significantly induce point reverse mutation in growing cells without myrosinase, thus suggesting either a direct GNST or a CYP-linked metabolite role in the genotoxic response. On the contrary, in suspension test, the addition of myrosinase significantly increased mitotic gene conversion, probably due to the formation of GNST-derived phenylethyl isothiocyanate (PEITC) breakdown product. Taken together, our data suggest that GNST exerts a dual effect: while strongly inhibiting the microsomal (bioactivating) metabolism, GNST also possesses genotoxic activity. This concomitant mutagenic activity underlines the necessity of overall toxicological characterization of this (or any other molecule) prior to mass chemopreventive use.