Argonaute2, a link between genetic and biochemical analyses of RNAi.

Double-stranded RNA induces potent and specific gene silencing through a process referred to as RNA interference (RNAi) or posttranscriptional gene silencing (PTGS). RNAi is mediated by RNA-induced silencing complex (RISC), a sequence-specific, multicomponent nuclease that destroys messenger RNAs homologous to the silencing trigger. RISC is known to contain short RNAs ( approximately 22 nucleotides) derived from the double-stranded RNA trigger, but the protein components of this activity are unknown. Here, we report the biochemical purification of the RNAi effector nuclease from cultured Drosophila cells. The active fraction contains a ribonucleoprotein complex of approximately 500 kilodaltons. Protein microsequencing reveals that one constituent of this complex is a member of the Argonaute family of proteins, which are essential for gene silencing in Caenorhabditis elegans, Neurospora, and Arabidopsis. This observation begins the process of forging links between genetic analysis of RNAi from diverse organisms and the biochemical model of RNAi that is emerging from Drosophila in vitro systems.

Potent direct inhibition of mammalian phospholipase D isoenzymes by calphostin-c.

Calphostin-c inhibits protein kinase C (PKC) isoenzymes by covalent modification of the lipid binding regulatory domain. Exposure of cells to calphostin-c elicits PKC independent effects including disruption of intracellular transport, growth inhibition, and stimulation of apoptosis suggesting actions at additional targets. Phospholipase D (PLD) enzymes are targets for activation by PKC. We have investigated the PKC isoenzyme selectivity for activation of two mammalian PLD enzymes, PLD1 and PLD2, by PKC. We examined the sensitivity of this process to widely used PKC inhibitors and report the surprising finding that calphostin-c is a potent direct inhibitor of PLD1 and PLD2. In vitro, calphostin-c inhibits activity of both PLD1 and PLD2 with an IC(50) of approximately 100 nM. Inhibition is not overcome by protein and lipid activators of these enzymes and does not involve blockade of phosphatidylinositol 4,5-bisphosphate-dependent PLD binding to substrate containing liposomes. Studies using a series of deletion and point mutants of the enzymes suggest that calphostin-c targets the PLD catalytic domain. Inhibition of PLD by calphostin-c in vitro involves stable and apparently irreversible modification of the enzyme. Activity of both PLD1 and PLD2 can be inhibited by calphostin-c treatment of intact cells in a manner that is independent of upstream actions of PKC. Our results suggest that inhibition of PLD1 and PLD2 may explain some of the PKC-independent effects of calphostin-c observed when the compound is applied to intact cells.

Post-transcriptional gene silencing by double-stranded RNA.

Imagine being able to knock out your favourite gene with only a day's work. Not just in one model system, but in virtually any organism: plants, flies, mice or cultured cells. This sort of experimental dream might one day become reality as we learn to harness the power of RNA interference, the process by which double-stranded RNA induces the silencing of homologous endogenous genes. How this phenomenon works is slowly becoming clear, and might help us to develop an effortless tool to probe gene function in cells and animals.

Role for a bidentate ribonuclease in the initiation step of RNA interference.

RNA interference (RNAi) is the mechanism through which double-stranded RNAs silence cognate genes. In plants, this can occur at both the transcriptional and the post-transcriptional levels; however, in animals, only post-transcriptional RNAi has been reported to date. In both plants and animals, RNAi is characterized by the presence of RNAs of about 22 nucleotides in length that are homologous to the gene that is being suppressed. These 22-nucleotide sequences serve as guide sequences that instruct a multicomponent nuclease, RISC, to destroy specific messenger RNAs. Here we identify an enzyme, Dicer, which can produce putative guide RNAs. Dicer is a member of the RNase III family of nucleases that specifically cleave double-stranded RNAs, and is evolutionarily conserved in worms, flies, plants, fungi and mammals. The enzyme has a distinctive structure, which includes a helicase domain and dual RNase III motifs. Dicer also contains a region of homology to the RDE1/QDE2/ARGONAUTE family that has been genetically linked to RNAi.

An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells.

In a diverse group of organisms that includes Caenorhabditis elegans, Drosophila, planaria, hydra, trypanosomes, fungi and plants, the introduction of double-stranded RNAs inhibits gene expression in a sequence-specific manner. These responses, called RNA interference or post-transcriptional gene silencing, may provide anti-viral defence, modulate transposition or regulate gene expression. We have taken a biochemical approach towards elucidating the mechanisms underlying this genetic phenomenon. Here we show that 'loss-of-function' phenotypes can be created in cultured Drosophila cells by transfection with specific double-stranded RNAs. This coincides with a marked reduction in the level of cognate cellular messenger RNAs. Extracts of transfected cells contain a nuclease activity that specifically degrades exogenous transcripts homologous to transfected double-stranded RNA. This enzyme contains an essential RNA component. After partial purification, the sequence-specific nuclease co-fractionates with a discrete, approximately 25-nucleotide RNA species which may confer specificity to the enzyme through homology to the substrate mRNAs.

Loss of receptor regulation by a phospholipase D1 mutant unresponsive to protein kinase C.

Activation of phosphatidylcholine-specific phospholipase D (PLD) constitutes an important part of the cellular response to agonist signaling. PLD1 is stimulated in vitro in a direct and synergistic manner by protein kinase C (PKC), ADP-ribosylation factor (ARF) and Rho family members. However, the direct and specific role of each of these effectors in agonist-stimulated PLD activation is poorly understood. We have used transposon mutagenesis to generate a library of PLD1 alleles containing random pentapeptide insertions. Forty-five alleles were characterized to identify functionally important regions. Use of an allele unresponsive to PKC, but otherwise seemingly normal, to examine coupling of PLD1 to a subset of G-protein-coupled receptors demonstrates for the first time direct stimulation of PLD1 in vivo by PKC and reveals that this direct stimulation is unexpectedly critical for PLD1 activation.

Characterisation of penicillin G uptake in human small intestinal brush border membrane vesicles.

BACKGROUND: Many beta lactams are well absorbed by the small intestine, although the reasons for this are poorly understood. AIMS: To characterise the uptake of penicillin G into human small intestinal brush border membrane vesicles (BBMV) and to compare the uptake characteristics to those of rabbit BBMV. METHODS AND RESULTS: Uptake of penicillin G was studied in human BBMV. Penicillin G was actively transported into the lumen of BBMV via an H+ dependent, Na+ independent uptake system. The carrier mediated process was saturable and adhered to Michaelis-Menten kinetics (Vmax 52 nmol penicillin G per mg protein per 30 seconds, Km 13.9 mM). These results are similar to those previously reported in rabbit BBMV. CONCLUSIONS: It is suggested that penicillin G can be used as a model molecule for peptide and beta lactam transport studies as it is cheap and readily available in isotopically labelled form. Furthermore, rabbit BBMV may be used as an acceptable substitute for human BBMV for the study of penicillin transport.

Phospholipase D stimulates release of nascent secretory vesicles from the trans-Golgi network.

Phospholipase D (PLD) is a phospholipid hydrolyzing enzyme whose activation has been implicated in mediating signal transduction pathways, cell growth, and membrane trafficking in mammalian cells. Several laboratories have demonstrated that small GTP-binding proteins including ADP-ribosylation factor (ARF) can stimulate PLD activity in vitro and an ARF-activated PLD activity has been found in Golgi membranes. Since ARF-1 has also been shown to enhance release of nascent secretory vesicles from the TGN of endocrine cells, we hypothesized that this reaction occurred via PLD activation. Using a permeabilized cell system derived from growth hormone and prolactin-secreting pituitary GH3 cells, we demonstrate that immunoaffinity-purified human PLD1 stimulated nascent secretory vesicle budding from the TGN approximately twofold. In contrast, a similarly purified but enzymatically inactive mutant form of PLD1, designated Lys898Arg, had no effect on vesicle budding when added to the permeabilized cells. The release of nascent secretory vesicles from the TGN was sensitive to 1% 1-butanol, a concentration that inhibited PLD-catalyzed formation of phosphatidic acid. Furthermore, ARF-1 stimulated endogenous PLD activity in Golgi membranes approximately threefold and this activation correlated with its enhancement of vesicle budding. Our results suggest that ARF regulation of PLD activity plays an important role in the release of nascent secretory vesicles from the TGN.

Mutagenesis of phospholipase D defines a superfamily including a trans-Golgi viral protein required for poxvirus pathogenicity.

Phospholipase D (PLD) genes are members of a superfamily that is defined by several highly conserved motifs. PLD in mammals has been proposed to play a role in membrane vesicular trafficking and signal transduction. Using site-directed mutagenesis, 25 point mutants have been made in human PLD1 (hPLD1) and characterized. We find that a motif (HxKxxxxD) and a serine/threonine conserved in all members of the PLD superfamily are critical for PLD biochemical activity, suggesting a possible catalytic mechanism. Functional analysis of catalytically inactive point mutants for yeast PLD demonstrates that the meiotic phenotype ensuing from PLD deficiency in yeast derives from a loss of enzymatic activity. Finally, mutation of an HxKxxxxD motif found in a vaccinia viral protein expressed in the Golgi complex results in loss of efficient vaccinia virus cell-to-cell spreading, implicating the viral protein as a member of the superfamily and suggesting that it encodes a lipid modifying or binding activity. The results suggest that vaccinia virus and hPLD1 may act through analogous mechanisms to effect viral cellular egress and vesicular trafficking, respectively.

RalA interacts directly with the Arf-responsive, PIP2-dependent phospholipase D1.

RalA GTPase associates with a phospholipase D (PLD) that is activated in v-Src- and v-Ras-transformed cells. Two mammalian PLDs were recently cloned: PLD1, which is activated by Arf family GTPases and dependent upon phosphatidylinositol-4,5-bisphosphate (PIP2), and PLD2, which is also dependent upon PIP2, but not stimulated by Arf. Another PLD has been described that is stimulated by oleate. Evidence is provided that the RalA-assiciated PLD is PLD1. First, the PLD precipitated by RalA from murine fibroblasts was stimulated by Arf, dependent upon PIP2, and inhibited by oleate. Second, immobilized RalA precipitated PLD1 from sf9 insect cells overexpressing PLD1. Third, a series of RalA mutants precipitated PLD activity from both PLD1-expressing insect cells and murine fibroblasts with the same efficiency. And finally, immobilized RalA precipitated PLD1 from a purified PLD1 preparation. These data argue that RalA associates directly with the Arf-responsive, PIP2-dependent PLD1.

Phospholipase D2, a distinct phospholipase D isoform with novel regulatory properties that provokes cytoskeletal reorganization.

BACKGROUND: Activation of phospholipase D (PLD) is an important but poorly understood component of receptor-mediated signal transduction responses and regulated secretion. We recently reported the cloning of the human gene encoding PLD1; this enzyme has low basal activity and is activated by protein kinase C and the small GTP-binding proteins, ADP-ribosylation factor (ARF), Rho, Rac and Cdc42. Biochemical and cell biological studies suggest, however, that additional and distinct PLD activities exist in cells, so a search was carried out for novel mammalian genes related to PLD1. RESULTS: We have cloned the gene for a second PLD family member and characterized the protein product, which appears to be regulated differently from PLD1: PLD2 is constitutively active and may be modulated in vivo by inhibition. Unexpectedly, PLD2 localizes primarily to the plasma membrane, in contrast to PLD1 which localizes solely to peri-nuclear regions (the endoplasmic reticulum, Golgi apparatus and late endosomes), where PLD activity has been shown to promote ARF-mediated coated-vesicle formation. PLD2 provokes cortical reorganization and undergoes redistribution in serum-stimulated cells, suggesting that it may have a role in signal-induced cytoskeletal regulation and/or endocytosis. CONCLUSIONS: PLD2 is a newly identified mammalian PLD isoform with novel regulatory properties. Our findings suggest that regulated secretion and morphological reorganization, the two most frequently proposed biological roles for PLD, are likely to be effected separately by PLD1 and PLD2.

Characterization of two alternately spliced forms of phospholipase D1. Activation of the purified enzymes by phosphatidylinositol 4,5-bisphosphate, ADP-ribosylation factor, and Rho family monomeric GTP-binding proteins and protein kinase C-alpha.

We previously reported the cloning of a cDNA encoding human phosphatidylcholine-specific phospholipase D1 (PLD1), an ADP-ribosylation factor (ARF)-activated phosphatidylcholine-specific phospholipase D (Hammond, S. M., Tsung, S., Autschuller, Y., Rudge, S. A., Rose, K., Engebrecht, J., Morris, A. J., and Frohman, M. A. (1995) J. Biol. Chem. 270, 29640-29643). We have now identified an evolutionarily conserved shorter splice variant of PLD1 lacking 38 amino acids (residues 585-624) that arises from regulated splicing of an alternate exon. Both forms of PLD1 (PLD1a and 1b) have been expressed in Sf9 cells using baculovirus vectors and purified to homogeneity by detergent extraction and immunoaffinity chromatography. PLD1a and 1b have very similar properties. PLD1a and 1b activity is Mg2+dependent but insensitive to changes in free Ca2+ concentration. Phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate activate PLD1a and 1b but a range of other acidic phospholipids are ineffective. PLD1a and 1b are highly responsive to activation by GTP-gammaS-liganded ADP-ribosylation factor-1 (ARF-1) and can also be activated to a lesser extent by three purified RHO family monomeric GTP-binding proteins, RHO A, RAC-1, and CDC42. Activation of PLD1a and 1b by the RHO family monomeric GTP-binding proteins is GTP-dependent and synergistic with ARF-1. Purified protein kinase C-alpha activates PLD1a and 1b in a manner that is stimulated by phorbol esters and does not require ATP. Activation of PLD1a and 1b by protein kinase C-alpha is synergistic with ARF and with the RHO family monomeric GTP-binding proteins, suggesting that these three classes of regulators interact with different sites on the enzyme.

A psychometric analysis of a short device for assessing depression in elderly people.

The Schwab-Gilleard Depression Scale is a short self-report index for assessing depression in elderly people. The aim of this study is to investigate the psychometric properties of this device with particular reference to its reliability, criterion validity and ability to discriminate depression from dementia. The total sample comprised 166 elderly people living in Part III residential homes in south-west London. For 111 of these, the Schwab-Gilleard Depression Scale (SGDS), the Geriatric Depression Scale (GDS), the Short-Comprehensive Assessment and Referral Evaluation (CARE), and the Clifton Assessment Procedures for the Elderly (CAPE) were completed. The GDS and CAPE only were completed with the remaining 55 participants. Psychiatric diagnoses were available from the medical notes on each resident. In addition, a subsample of 49 had been assessed using the SGDS and CARE 14 months previously. These data were available for test-retest analyses. The self-rating SGDS was found to be a reliable and valid measure of depression and is recommended for use as a screening tool for depression in elderly people in residential homes.

Characterisation of penicillin-G uptake in rabbit small-intestinal brush-border membrane vesicles.

Uptake of penicillin-G has been studied in rabbit intestinal brush-border membrane vesicles (BBMV). Penicillin-G was transported into the lumen of BBMV via an H+-dependent, Na+-independent uptake system. This was a saturable carrier-mediated process, which adhered to Michaelis-Menten kinetics, having a pH optimum of 4.5 and resulting in a net-negative charge transfer. Vmax was 59 nmol penicillin-G (mg protein)-1 (30s)-1 and Km 22.7 mM. Ampicillin, penicillin-V, cefadroxil, cephalexin, cephalothin, cephradine, L-carnosine, glycyl-L-alanine, glycyl-L-tyrosine and glycylglycylglycine inhibited the uptake of penicillin-G. However, glycylsarcosine stimulated uptake by 92%. Countertransport experiments suggested that this effect took place at the active site of the transporter. Penicillin-G uptake appeared to be mediated via a common transport system shared by penicillins, cephalosporins and peptides.

Human ADP-ribosylation factor-activated phosphatidylcholine-specific phospholipase D defines a new and highly conserved gene family.

Activation of phosphatidylcholine-specific phospholipase D (PLD) has been implicated as a critical step in numerous cellular pathways, including signal transduction, membrane trafficking, and the regulation of mitosis. We report here the identification of the first human PLD cDNA, which defines a new and highly conserved gene family. Characterization of recombinant human PLD1 reveals that it is membrane-associated, selective for phosphatidylcholine, stimulated by phosphatidylinositol 4,5-bisphosphate, activated by the monomeric G-protein ADP-ribosylation factor-1, and inhibited by oleate. PLD1 likely encodes the gene product responsible for the most widely studied endogenous PLD activity.

In vitro activity of the tribactam GV104326 against gram-positive, gram-negative, and anaerobic bacteria.

GV104326 is the first member of a new class of antibiotics (tribactams) selected for development. It combines a particularly broad spectrum (including gram-negative and gram-positive aerobes and anaerobes) with high potency, resistance to beta-lactamases, and complete stability to dehydropeptidases. Comparative MICs were determined for GV104326 against 415 recent clinical isolates (including beta-lactamase producers), using representative antibacterial agents (imipenem, amoxicillin-clavulanic acid, cefpirome, ciprofloxacin, gentamicin, and erythromycin). GV104326 was particularly active against gram-positive bacteria; in general, its in vitro activity was equivalent to that of imipenem, equivalent to or better than that of amoxicillin-clavulanic acid, and superior to that of cefpirome, ciprofloxacin, and erythromycin. Against gram-negative bacteria, GV104326 possessed activity similar to that of imipenem and cefpirome against enterobacteria and Haemophilus spp. but its activity was superior to that of amoxicillin-clavulanic acid. GV104326 showed excellent antianaerobe activity. GV104326 was stable to all clinically relevant beta-lactamases and was rapidly lethal to susceptible bacteria. In Escherichia coli, GV104326 bound predominantly to PBPs 1a and 2 and at low concentrations osmotically stable round forms were observed. GV104326 showed an affinity for PBPs 2 and 4 of Staphylococcus aureus.

Inhibitors of lipopolysaccharide biosynthesis impair the virulence potential of Escherichia coli.

Inhibition of 3-deoxy-manno-octulosonate cytidylytransferase (CMP-KDO transferase; EC 2.7.7.38) by 8-amino-2,6-anhydro-3,8-dideoxy-D-glycero-D-talo-octonic acid (NH2dKDO) halts the growth of Gram-negative bacteria by depriving the cells of the 3-deoxy-D-manno-2-octulosonate required for the biosynthesis of the core region of the lipopolysaccharide components of the outer membrane. Low levels of this inhibitor increase the vulnerability of Escherichia coli to hydrophobic antibiotics, detergents, the complement-mediated antibacterial activity of serum, phagocytosis, and enhance the rate at which bacteria are cleared from the mouse bloodstream.