Activity of recombinant dengue 2 virus NS3 protease in the presence of a truncated NS2B co-factor, small peptide substrates, and inhibitors

Recombinant forms of the dengue 2 virus NS3 protease linked to a 40-residue co-factor, corresponding to part of NS2B, have been expressed in Escherichia coli and shown to be active against para-nitroanilide substrates comprising the P6-P1 residues of four substrate cleavage sequences. The enzyme is inactive alone or after the addition of a putative 13-residue co-factor peptide but is active when fused to the 40-residue co-factor, by either a cleavable or a noncleavable glycine linker. The NS4B/NS5 cleavage site was processed most readily, with optimal processing conditions being pH 9, I = 10 mm, 1 mm CHAPS, 20% glycerol. A longer 10-residue peptide corresponding to the NS2B/NS3 cleavage site (P6-P4') was a poorer substrate than the hexapeptide (P6-P1) para-nitroanilide substrate under these conditions, suggesting that the prime side substrate residues did not contribute significantly to protease binding. We also report the first inhibitors of a co-factor-complexed, catalytically active flavivirus NS3 protease. Aprotinin was the only standard serine protease inhibitor to be active, whereas a number of peptide substrate analogues were found to be competitive inhibitors at micromolar concentrations.

Bracken (Pteridium aquilinum)-induced DNA adducts in mouse tissues are different from the adduct induced by the activated form of the bracken carcinogen ptaquiloside

Following treatment with bracken fern (Pteridium aquilinum) extract and bracken spores a number of DNA adducts were detected by P-32-postlabeling. Three of these adducts have been described previously (Povey et al., Br. J. Cancer (1996) 74, 1342-1348) and in this study, using a slightly different protocol, four new adducts, with higher chromatographic mobility, were detected at levels ranging from 50 to 230% of those previously described, When DNA was treated in vitro with activated ptaquiloside (APT) and analysed by butanol extraction or nuclease P1 treatment, only one adduct was detected by P-32-postlabeling, This adduct was not present in the DNA from mice treated with bracken fern or spores, suggesting either that bracken contains genotoxins other than ptaquiloside or that the metabolism of ptaquiloside produces genotoxins not reflected by activated ptaquiloside. However, as the ATP-derived adduct has been detected previously in ileal DNA of bracken-fed calves, species-specific differences in the metabolism of bracken genotoxins may exist, thereby leading to differences in their biological outcomes. (C) 2001 Academic Press.

No evidence for inhibition of ENaC through CFTR-mediated release of ATP

Both purinergic stimulation and activation of cystic fibrosis transmembrane conductance regulator (CFTR) increases Cl- secretion and inhibit amiloride-sensitive Na+ transport. CFTR has been suggested to conduct adenosine 5'-triphosphate (ATP) or to control ATP release to the luminal side of epithelial tissues. Therefore, a possible mechanism on how CFTR controls the activity of epithelial Na+ channels (ENaC) could be by release of ATP or uridine 5'-triphosphate (UTP), which would then bind to P2Y receptors and inhibit ENaC. We examined this question in native tissues from airways and colon and in Xenopus oocytes. Inhibition of amiloride-sensitive transport by both CFTR and extracellular nucleotides was observed in colon and trachea. However, nucleotides did not inhibit ENaC in Xenopus oocytes, even after coexpression of P2Y(2) receptors. Using different tools such as hexokinase, the P2Y inhibitor suramin or the Cl- channel blocker 4,4'diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), we did not detect any role of a putative ATP secretion in activation of Cl- transport or inhibition of amiloride sensitive short circuit currents by CFTR. In addition, N-2,2'-O-dibutyrylguanosine 3',5-cyclic monophosphate (cGMP) and protein kinase G (PKG)-dependent phosphorylation or the nucleoside diphosphate kinase (NDPK) do not seem to play a role for the inhibition of ENaC by CFTR, which, however, requires the presence of extracellular Cl-. (C) 2002 Elsevier Science B.V. All rights reserved.

Adenosine triphosphate acts as both a competitive antagonist and a positive allosteric modulator at recombinant N-methyl-D-aspartate receptors

ATP and glutamate are fast excitatory neurotransmitters in the central nervous system acting primarily on ionotropic P2X and glutamate [N-methyl-D-aspartate (NMDA) and non-NMDA] receptors, respectively. Both neurotransmitters regulate synaptic plasticity and long-term potentiation in hippocampal neurons. NMDA receptors are responsible primarily for the modulatory action of glutamate, but the mechanism underlying the modulatory effect of ATP remains uncertain. In the present study, the effect of ATP on recombinant NR1a + 2A, NR1a + 2B, and NR1a + 2C NMDA receptors expressed in Xenopus laevis oocytes was investigated. ATP inhibited NR1a + 2A and NR1a + 2B receptor currents evoked by low concentrations of glutamate but potentiated currents evoked by saturating glutamate concentrations. In contrast, ATP potentiated NR1a + 2C receptor currents evoked by nonsaturating glutamate concentrations. ATP shifted the glutamate concentration-response curve to the right, indicating a competitive interaction at the agonist binding site. ATP inhibition and potentiation of glutamate-evoked currents was voltage-independent, indicating that ATP acts outside the membrane electric field. Other nucleotides, including ADP, GTP, CTP, and UTP, inhibited glutamate-evoked currents with different potencies, revealing that the inhibition is dependent on both the phosphate chain and nucleotide ring structure. At high concentrations, glutamate outcompetes ATP at the agonist binding site, revealing a potentiation of the current. This effect must be caused by ATP binding at a separate site, where it acts as a positive allosteric modulator of channel gating. A simple model of the NMDA receptor, with ATP acting both as a competitive antagonist at the glutamate binding site and as a positive allosteric modulator at a separate site, reproduced the main features of the data.

Functional maturation of isolated neural progenitor cells from the adult rat hippocampus

Although neural progenitor cells (NPCs) may provide a source of new neurons to alleviate neural trauma, little is known about their electrical properties as they differentiate. We have previously shown that single NPCs from the adult rat hippocampus can be cloned in the presence of heparan sulphate chains purified from the hippocampus, and that these cells can be pushed into a proliferative phenotype with the mitogen FGF2 [Chipperfield, H., Bedi, K.S., Cool, S.M. & Nurcombe, V. (2002) Int. J. Dev. Biol., 46, 661-670]. In this study, the active and passive electrical properties of both undifferentiated and differentiated adult hippocampal NPCs, from 0 to 12 days in vitro as single-cell preparations, were investigated. Sparsely plated, undifferentiated NPCs had a resting membrane potential of approximate to -90 mV and were electrically inexcitable. In > 70%, ATP and benzoylbenzoyl-ATP evoked an inward current and membrane depolarization, whereas acetylcholine, noradrenaline, glutamate and GABA had no detectable effect. In Fura-2-loaded undifferentiated NPCs, ATP and benzoylbenzoyl-ATP evoked a transient increase in the intracellular free Ca2+ concentration, which was dependent on extracellular Ca2+ and was inhibited reversibly by pyridoxalphosphate-6-azophenyl-2'-4'-disulphonic acid (PPADS), a P2 receptor antagonist. After differentiation, NPC-derived neurons became electrically excitable, expressing voltage-dependent TTX-sensitive Na+ channels, low- and high-voltage-activated Ca2+ channels and delayed-rectifier K+ channels. Differentiated cells also possessed functional glutamate, GABA, glycine and purinergic (P2X) receptors. Appearance of voltage-dependent and ligand-gated ion channels appears to be an important early step in the differentiation of NPCs.

Articulatory function in hypokinetic dysarthria: An electropalatographic examination of two cases

The present study employed electropalatography (EPG) and a nonspeech measure of lingual function to examine, in detail, the articulatory production deficits of two individuals with Parkinson disease (PD) and hypokinetic dysarthria. Participants read 10 repetitions of CV words contained within the carrier phrase I saw a _ today while wearing an EPG artificial palate. Target consonants included the alveolar stop /t/, lateral approximant /l/, and the alveolar fricative /s/ in the /a/ vowel environment. The results of the two participants were compared to an age-matched control group. Examination of the perceptual features of articulatory production, lingual strength, fine force control and endurance, tongue-palate contact patterns, and segment durations were conducted. Results of the study revealed quite different articulatory deficits in the two participants. Specifically, the articulation of Participant One (P1) was characterized by a fast rate of speech, undershooting of articulatory targets, and reduced duration of consonant closures. In contrast, Participant Two (P2) demonstrated tongue-palate contact patterns indicative of impaired lingual control in the presence of both normal and increased articulatory segment durations. Potential reasons for the differing articulatory deficits were hypothesized. The current study demonstrated that assessment with EPG identified potential causes of consonant imprecision in two individuals with hypokinetic dysarthria. Directions for speech pathology intervention, salient from the results of the study, were also noted.

Site-directed mutagenesis and kinetic studies of the West Nile virus NS3 protease identify key enzyme-substrate interactions

The flavivirus West Nile virus (WNV) has spread rapidly throughout the world in recent years causing fever, meningitis, encephalitis, and fatalities. Because the viral protease NS2B/NS3 is essential for replication, it is attracting attention as a potential therapeutic target, although there are currently no antiviral inhibitors for any flavivirus. This paper focuses on elucidating interactions between a hexapeptide substrate (Ae-KPGLKR-p-nitroanilide) and residues at S1 and S2 in the active site of WNV protease by comparing the catalytic activities of selected mutant recombinant proteases in vitro. Homology modeling enabled the predictions of key mutations in VWNV NS3 protease at S1 (V115A/F, D129A/ E/N, S135A, Y150A/F, S160A, and S163A) and S2 (N152A) that might influence substrate recognition and catalytic efficiency. Key conclusions are that the substrate P1 Arg strongly interacts with S1 residues Asp-129, Tyr-150, and Ser-163 and, to a lesser extent, Ser-160, and P2 Lys makes an essential interaction with Asn-152 at S2. The inferred substrate-enzyme interactions provide a basis for rational protease inhibitor design and optimization. High sequence conservation within flavivirus proteases means that this study may also be relevant to design of protease inhibitors for other flavivirus proteases.

Flagellin of Pseudomonas aeruginosa inhibits Na+ transport in airway epithelia

Pseudomonas aeruginosa causes severe life-threatening airway infections that are a frequent cause for hospitalization of cystic fibrosis (CF) patients. These Gram-negative pathogens possess flagella that contain the protein flagellin as a major structural component. Flagellin binds to the host cell glycolipid asialoGM1 (ASGM1), which appears enriched in luminal membranes of respiratory epithelial cells. We demonstrate that in mouse airways, luminal exposure to flagellin leads to inhibition of Na+ absorption by the epithelial Na+ channel ENaC, but does not directly induce a secretory response. Inhibition of ENaC was observed in tracheas of wild-type mice and was attenuated in mice homozygous for the frequent cystic fibrosis conductance regulator (CFTR) mutation G551D. Similar to flagellin, anti-ASGM1 antibody also inhibited ENaC. The inhibitory effects of flagellin on ENaC were attenuated by blockers of the purinergic signaling pathway, although an increase in the intracellular Ca2+ concentration by recombinant or purified flagellin or whole flagella was not observed. Because an inhibitor of the mitogen-activated protein kinase (MAPK) pathway also attenuated the effects of flagellin on Na+ absorption, we conclude that flagellin exclusively inhibits ENaC, probably due to release of ATP and activation of purinergic receptors of the P2Y subtype. Stimulation of these receptors activates the MAPK pathway, thereby leading to inhibition of ENaC. Thus, P. aeruginosa reduces Na+ absorption, which could enhance local mucociliary clearance, a mechanism that seem to be attenuated in CF.

Synaptogenesis in the mushroom body calyx during metamorphosis in the honeybee Apis mellifera: An electron microscopic study

The goals of this study are to determine relationships between synaptogenesis and morphogenesis within the mushroom body calyx of the honeybee Apis mellifera and to find out how the microglomerular structure characteristic for the mature calyx is established during metamorphosis. We show that synaptogenesis in the mushroom body calycal neuropile starts in early metamorphosis (stages P1-P3), before the microglomerular structure of the neuropile is established. The initial step of synaptogenesis is characterized by the rare occurrence of distinct synaptic contacts. A massive synaptogenesis starts at stage P5, which coincides with the formation of microglomeruli, structural units of the calyx that are composed of centrally located presynaptic boutons surrounded by spiny postsynaptic endings. Microglomeruli are assembled either via accumulation of fine postsynaptic processes around preexisting presynaptic boutons or via ingrowth of thin neurites of presynaptic neurons into premicroglomeruli, tightly packed groups of spiny endings. During late pupal stages (P8-P9), addition of new synapses and microglomeruli is likely to continue. Most of the synaptic appositions formed there are made by boutons (putative extrinsic mushroom body neurons) into small postsynaptic profiles that do not exhibit presynaptic specializations (putative intrinsic mushroom body neurons). Synapses between presynaptic boutons characteristic of the adult calyx first appear at stage P8 but remain rare toward the end of metamorphosis. Our observations are consistent with the hypothesis that most of the synapses established during metamorphosis provide the structural basis for afferent information flow to calyces, whereas maturation of local synaptic circuitry is likely to occur after adult emergence.

Conservation and accessibility of an inner core lipopolysaccharide epitope of Neisseria meningitidis

We investigated the conservation and antibody accessibility of inner core epitopes of Neisseria meningitidis lipopolysaccharide (LPS) because of their potential as vaccine candidates. An immunoglobulin G3 murine monoclonal antibody (MAb), designated MAb B5, was obtained by immunizing mice with a galE mutant of N. meningitidis H44/76 (B.15.P1.7,16 immunotype L3). We have shown that MAb B5 can bind to the core LPS of wild-type encapsulated MC58 (B.15.P1.7,16 immunotype L3) organisms in vitro and ex vivo. An inner core structure recognized by MAb B5 is conserved and accessible in 26 of 34 (76%) of group B and 78 of 112 (70%) of groups A, C, W, X, Y, and Z strains. N. meningitidis strains which possess this epitope are immunotypes in which phosphoethanolamine (PEtn) is linked to the 3-position of the beta-chain heptose (HepII) of the inner core. In contrast, N. neningitidis strains lacking reactivity with MAb B5 have an alternative core structure in which PEtn is linked to an exocyclic position (i.e., position 6 or 7) of HepII (immunotypes L2, L4, and L6) or is absent (immunotype L5). We conclude that MAb B5 defines one or more of the major inner core glycoforms of N. meningitidis LPS. These findings support the possibility that immunogens capable of eliciting functional antibodies specific to inner core structures could be the basis of a vaccine against invasive infections caused by N. meningitidis.