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A J Morris

Publications and source records attributed to A J Morris.

At least 109 records · Page 6Linked to original sources
PubMed1998

Viral kinetics can predict early response to alpha-interferon in chronic hepatitis C.

AIMS/BACKGROUND: Response rates to alpha-interferon therapy for chronic hepatitis C are poor. An early indication of efficacy would reduce the need for prolonged therapy, leading to significant cost savings. It was established that a change in quantitative hepatitis C virus RNA (HCV-RNA) titre at 4 weeks could predict the outcome of alpha-interferon therapy in chronic hepatitis C. METHODS: Serum HCV-RNA titres were quantified using branched chain DNA (bDNA) assay in 26 patients who responded to alpha-interferon (serum HCV-RNA negative after 12 weeks therapy) and 11 age and sex matched non-responders. Quantitative bDNA and qualitative RT-PCR assays for HCV-RNA were measured pretreatment and at 4 weeks. The change in quantitative HCV-RNA titre between pre-therapy and after 4 weeks was compared in the two groups. RESULTS: Seventeen of the 37 patients had become RT-PCR negative at 4 weeks (early responders) and had an undetectable HCV-RNA titre on bDNA assay. Nine patients were RT-PCR positive at 4 weeks but negative by 12 weeks (delayed responders), and of these, 8 had an undetectable viral titre at 4 weeks on bDNA assay. The patient with a detectable HCV titre did become RT-PCR negative after 12 weeks, but subsequently became RT-PCR positive again at 24 weeks. All the non-responders had a detectable bDNA titre (> 0.2 Meq/ml) at 4 weeks. CONCLUSIONS: Change in quantitative HCV-RNA titre measured by bDNA assay at 4 weeks predicts response to alpha interferon. If HCV-RNA remains detectable on bDNA assay at 4 weeks, no sustained response to treatment is found and alpha-interferon can be discontinued.

Hepacivirus↗
PubMed1998

Ligand-independent GLUT4 translocation induced by guanosine 5'-O-(3-thiotriphosphate) involves tyrosine phosphorylation.

To delineate the signaling pathway leading to glucose transport protein (GLUT4) translocation, we examined the effect of microinjection of the nonhydrolyzable GTP analog, guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS), into 3T3-L1 adipocytes. Thirty minutes after the injection of 5 mM GTPgammaS, 40% of injected cells displayed surface GLUT4 staining indicative of GLUT4 translocation compared with 55% for insulin-treated cells and 10% in control IgG-injected cells. Treatment of the cells with the phosphatidylinositol 3-kinase inhibitor wortmannin or coinjection of GST-p85 SH2 fusion protein had no effect on GTPgammaS-mediated GLUT4 translocation. On the other hand, coinjection of antiphosphotyrosine antibodies (PY20) blocked GTPgammaS-induced GLUT4 translocation by 65%. Furthermore, microinjection of GTPgammaS led to the appearance of tyrosine-phosphorylated proteins around the periphery of the plasma membrane, as observed by immunostaining with PY20. Treatment of the cells with insulin caused a similar phosphotyrosine-staining pattern. Electroporation of GTPgammaS stimulated 2-deoxy-D-glucose transport to 70% of the extent of insulin stimulation. In addition, immunoblotting with phosphotyrosine antibodies after electroporation of GTPgammaS revealed increased tyrosine phosphorylation of several proteins, including 70- to 80-kDa and 120- to 130-kDa species. These results suggest that GTPgammaS acts upon a signaling pathway either downstream of or parallel to activation of phosphatidylinositol 3-kinase and that this pathway involves tyrosine-phosphorylated protein(s).

3T3 Cells↗
PubMed1997

Different domains of mammalian ADP-ribosylation factor 1 mediate interaction with selected target proteins.

Mammalian ADP-ribosylation factor 1 (mARF1) is a small GTP-binding protein that is activated by a Golgi guanine nucleotide exchange factor. Once bound to the Golgi membranes in the GTP form, mARF1 initiates the recruitment of the adaptor protein 1 (AP-1) complex and coatomer (COPI) onto these membranes and activates phospholipase D1 (PLD1). To map the domains of mARF1 that are important for these activities, we constructed chimeras between mARF1 and Saccharomyces cerevisiae ARF2, which functions poorly in all of these processes except COPI recruitment. The carboxyl half of mARF1 (amino acids 95-181) was essential for activation by the Golgi guanine nucleotide exchange factor, whereas a separate domain (residues 35-94) was required to effectively activate PLD1 and to promote efficient AP-1 recruitment. Since residues 35-94 of mARF1 are critical for optimal activity in both PLD1 activation and AP-1 recruitment, we hypothesize that this region of ARF contains residues that interact with effector molecules.

ADP-Ribosylation Factors↗
PubMed1997

Membrane-binding properties of phospholipase C-beta1 and phospholipaseC-beta2: role of the C-terminus and effects of polyphosphoinositides, G-proteins and Ca2+.

We have studied the binding of two G-protein-regulated phospholipase C (PLC) enzymes, PLCs-beta1 and -beta2, to membrane surfaces using sucrose-loaded bilayer phospholipid vesicles of varying compositions. Neither enzyme binds appreciably to pure phosphatidylcholine vesicles at lipid concentrations up to 10(-3) M. PLC-beta1 and PLC-beta2 bind vesicles composed of phosphatidylcholine, phosphatidylserine and phosphatidylethanolamine (molar ratio 1:1:1) with an approximate Kd of 10(-5) M. Inclusion of 2% PtdIns(4,5)P2 in these vesicles had no effect on the affinity of this interaction. As reported by others, removal of the C-terminus of PLC-beta1 and PLC-beta2 produces catalytically active fragments. The affinity of these truncated proteins for phospholipid vesicles is dramatically reduced suggesting that this region of the proteins contains residues important for membrane binding. Inclusion of G-protein alpha- and betagamma-subunit activators in the phospholipid vesicles does not increase the binding of PLC-beta1 or PLC-beta2, and the magnitude of G-protein-mediated PLC activation observed at low phospholipid concentrations (10(-6) M) is comparable to that observed at concentrations at which the enzymes are predominantly membrane-bound (10(-3) M). PLC-beta1 and -beta2 contain C2 domains but Ca2+ does not enhance binding to the vesicles. Our results indicate that binding of these enzymes to membranes involves the C-temini of the proteins and suggest that activation of these enzymes by G-proteins results from a regulated interaction between the membrane-bound proteins rather than G-protein-dependent recruitment of soluble enzymes to a substrate-containing phospholipid surface.

Animals↗
PubMed1997

Measurement of phospholipase D activity.

Phosphodiesteric cleavage of phosphatidylcholine by members of a growing family of phospholipases D produces choline and phosphatidic acid. These enzymes can also catalyse a transphosphatidylation reaction in which the aliphatic chain of a primary alcohol is transferred to the phosphatidyl moiety of the phosphatidic acid product. PLD enzymes are found in a variety of organisms including bacteria, yeast, plants, and vertebrates. In mammalian systems, biochemical and cell biological approaches have identified phosphatidic acid as a mediator (or progenitor of mediators) that play important roles in the transduction of extracellular signals. Phosphatidic acid or its metabolites may be regulators of key cellular processes such as the control of intracellular protein trafficking, secretion, and alterations in cell morphology and motility. This review discusses methods for the determination of PLD activity both in vitro and in intact cells.

Animals↗
PubMed1997

Correlation models for childhood epidemics.

One of the simplest set of equations for the description of epidemics (the SEIR equations) has been much studied, and produces reasonable approximations to the dynamics of communicable disease. However, it has long been recognized that spatial and social structure are important if we are to understand the long-term persistence and detailed behaviour of disease. We will introduce three pair models which attempt to capture the underlying heterogeneous structure by studying the connections and correlations between individuals. Although modelling the correlations necessarily leads to more complex equations, this pair formulation naturally incorporates the local dynamical behaviour generating more realistic persistence. In common with other studies on childhood diseases we will focus our attention on measles, for which the case returns are particularly well documented and long running.

Age Distribution↗
PubMed1997

Nosocomial infection in Auckland Healthcare hospitals.

AIM: To determine the prevalence of nosocomial infection in Auckland Healthcare hospitals. BACKGROUND: Nosocomial infections cause patient morbidity and prolong hospital stay. Reporting surveillance results to staff has been shown to reduce nosocomial infection rates. METHOD: Point prevalence study for all patients in Auckland, Green Lane and National Women's hospitals. Standard definitions for nosocomial infections were used. RESULTS: One hundred and ten (12%) of 932 patients had 129 nosocomial infections: 27 (20%) surgical site infections; 25 (19%) lower respiratory tract infections; 23 (18%) skin/ soft tissue infections; 19 (15%) urinary tract infections; 14 (11%) bloodstream infections; and 21 (17%) other infections. Predominant organisms were: Staphylococcus aureus (29%), Escherichia coli (21%), other gram negative bacilli (14%), Pseudomonas aeruginosa (6%), streptococci (6%) and Candida albicans (6%). The prevalence of nosocomial infection was lower in National Women's Hospital (5%) than either Green Lane or Auckland hospitals (15% and 14% respectively), p < 0.01. The prevalence of nosocomial infection was the same in medical and surgical patients, 53 of 394 (14%) and 42 of 297 (14%), respectively. The highest prevalence was in intensive care unit patients, 7 of 31 (23%). The prevalence of nosocomial infection increased with patient age, 17-50 yr (8%) vs > 50 yr (14%), p < 0.01, and duration of hospitalisation 2% for < 2 days, 6% for 2-7 days vs 22% for > 7 days, p < 0.01. Risk factors for nosocomial infection were present in many patients: 339 (36%) had intravenous catheters in place; 268 (29%) patients had undergone surgery during their current admission; 122 (13%) had urinary catheters in place; and 122 (13%) had other invasive devices in situ. CONCLUSION: Our results are comparable with hospitals of similar size overseas. This study provides a base line for future studies which will enable the monitoring of trends over time and the impact of focused infection control initiatives.

Adult↗
PubMed1997

Regulation of effectors by G-protein alpha- and beta gamma-subunits. Recent insights from studies of the phospholipase c-beta isoenzymes.

Both the alpha- and beta gamma-subunits of heterotrimeric guanine nucleotide-dependent regulatory proteins (G-proteins) couple members of the heptahelical class of cell-surface receptors to a diverse range of signal-generating effectors including retinal cyclic GMP phosphodiesterase, ion channels, adenylylcyclases, phosphoinositide 3-kinase, and members of the beta-class of inositol lipid-specific phospholipases C. Although the molecular details of the G-protein-regulated phospholipase C system were elucidated comparatively recently, these enzymes have become an important model for investigations of the process of G-protein effector coupling. A combination of molecular biological, biochemical, and structural studies using the phospholipase C-beta enzymes has provided some important insights into the interplay between G-proteins and their effectors and promises to reveal the mechanisms by which G-protein alpha- and beta gamma-subunits selectively associate with and activate effectors.

Animals↗
PubMed1997

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.

1-Butanol↗
PubMed1997

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.

Amino Acid Sequence↗
PubMed1997

Surveillance for antimicrobial resistance in enterococci.

AIM: To describe antimicrobial resistance patterns of Enterococcus species in Auckland. BACKGROUND: Antimicrobial resistant enterococci have emerged as major nosocomial pathogens in overseas hospitals. It is recommended that hospitals perform periodic surveys to determine local enterococcal resistance patterns. METHODS: Enterococcal isolates from four patient groups were tested: group I were recovered from routine clinical specimens; group II were stool isolates from patients at risk of having vancomycin resistant enterococci, eg, intensive care unit patients, patients receiving vancomycin, and immunocompromised patients receiving antibiotics; group III were enterococci from stool specimens sent for Clostridium difficile toxin testing; group IV were isolates from stool specimens submitted to a community laboratory for enteric pathogen testing. All enterococci isolated were tested for the presence of beta-lactamase, susceptibility to amoxycillin, teicoplanin, vancomycin, and for high level gentamicin and streptomycin resistance. RESULTS: There were 121 group I enterococcal isolates. 628 stool specimens were cultured. Enterococci were isolated from: 76/148 (51%) group II specimens; 166/279 (60%) group III specimens; and 70/201 (35%) of group IV specimens. Antimicrobial susceptibility testing was performed on 433 isolates; 74% were E faecalis, 12% E faecium, 6% E gallinarum/casseliflavus group and 8% other enterococcal species. No isolate produced beta-lactamase. All E faecalis were susceptible to amoxycillin. Two E faecium and one enterococcus species were resistant to amoxycillin (MICs all 16 mg/L). All isolates were susceptible to teicoplanin. Fourteen E gallinarum/casseliflavus group isolates had intermediate susceptibility to vancomycin (MICs of 8 mg/L). One E faecium had intermediate susceptibility to vancomycin (MIC 8 mg/L). High level gentamicin and streptomycin resistance occurred in 64 (15%) and 50 (12%) isolates respectively. CONCLUSION: Vancomycin resistance is rare and is essentially restricted to species that are rarely clinical pathogens, i.e., E casseliflavus and E gallinarum. Our results have established the local susceptibility profile for enterococcal isolates. This allows comparison with other locations and the detection of emerging trends of resistance.

Cross Infection↗
PubMed1997

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.

3T3 Cells↗
PubMed1997

Synaptojanin inhibition of phospholipase D activity by hydrolysis of phosphatidylinositol 4,5-bisphosphate.

A 150-kDa protein that inhibits phospholipase D (PLD) activity stimulated by ADP-ribosylation factor and phosphatidylinositol 4, 5-bisphosphate (PI(4,5)P2) was previously purified from rat brain. The sequences of peptides derived from the purified PLD inhibitor now identify it as synaptojanin, a nerve terminal protein that has been implicated in the endocytosis of fused synaptic vesicles and shown to be a member of the inositol polyphosphate 5-phosphatase family. Further characterization of the enzymatic properties of synaptojanin now shows that it hydrolyzes only the 5-phosphate from inositol 1,4,5-trisphosphate (I(1,4,5)P3) and that it does not catalyze the dephosphorylation of either I(1,3,4)P3 or inositol 1, 4-bisphosphate. However, synaptojanin hydrolyzes both the 4- and 5-phosphates of PI(4,5)P2 and the 4-phosphate of phosphatidylinositol 4-phosphate, converting both compounds to phosphatidylinositol. Magnesium is required for the hydrolysis of I(1,4,5)P3, but not for that of phosphoinositides, by synaptojanin. The inhibition of PLD by synaptojanin is attributable to its ability to hydrolyze PI(4,5)P2. Synaptojanin did not inhibit PLD in the absence of PI(4,5)P2, and the extent of PLD inhibition was related to the extent of PI(4,5)P2 hydrolysis in substrate vesicles. It has been proposed that the biosynthesis of PI(4,5)P2 and the activation of PLD by ADP-ribosylation factor constitute a positive loop to increase rapidly the concentrations of PI(4,5)P2 and phosphatidic acid (PA) during membrane vesiculation. The PA thus produced, probably together with PI(4,5)P2, facilitates vesicle coat assembly. The hydrolysis of PI(4,5)P2, and consequent inhibition of PLD, by synaptojanin might therefore constitute a mechanism to halt the positive loop connecting PI(4,5)P2 and PA during the endocytotic cycle of synaptic vesicles and serve as a signal for uncoating.

Amino Acid Sequence↗
PubMed1997

Inhibition of phospholipase D by clathrin assembly protein 3 (AP3).

In the accompanying paper (Chung, J.-K., Sekiya, F., Kang, H.-S., Lee, C., Han, J.-S., Kim, S. R., Bae, Y. S., Morris, A. J., and Rhee, S. G. (1997) J. Biol. Chem. 272, 15980-15985), synaptojanin is identified as a protein that inhibits phospholipase D (PLD) activity stimulated by ADP-ribosylation factor and phosphatidylinositol 4, 5-bisphosphate (PI(4,5)P2). Here, the purification from rat brain cytosol of another PLD-inhibitory protein that is immunologically distinct from synaptojanin is described, and this protein is identified as clathrin assembly protein 3 (AP3) by peptide sequencing and immunoblot analysis. AP3 binds both inositol hexakisphosphate and preassembled clathrin cages with high affinity. However, neither inositol hexakisphosphate binding nor clathrin cage binding affected the ability of AP3 to inhibit PLD. AP3 also binds to PI(4,5)P2 with low affinity. But the PI(4,5)P2 binding was not responsible for PLD inhibition, because the potency and efficacy of AP3 as an inhibitor of PLD were similar in the absence and presence of PI(4,5)P2. A bacterially expressed fusion protein, glutathione S-transferase-AP3 (GST-AP3), also inhibited PLD with a potency equal to that of brain AP3. The inhibitory effect of AP3 appeared to be the result of direct interaction between AP3 and PLD because PLD bound GST-AP3 in an in vitro binding assay. Using GST fusion proteins containing various AP3 sequences, we found that the sequence extending from residues Pro-290 to Lys-320 of AP3 is critical for both inhibition of and binding to PLD. The fact that AP3 is a synapse-specific protein indicates that the AP3-dependent inhibition of PLD might play a regulatory role that is restricted to the rapid cycling of synaptic vesicles.

Adaptor Protein Complex 3↗
PubMed1997

Susceptibility patterns of bacterial isolates from intensive care and haematology/oncology patients in New Zealand.

AIMS: To determine the current susceptibility pattern of bacterial isolates from intensive care and haematology/oncology patients in New Zealand. METHOD: Over a 6 month period 417 consecutive clinically relevant bacterial isolates from intensive care and haematology/oncology patients from seven New Zealand hospitals had their susceptibility to multiple antimicrobial agents determined by the agar plate dilution method. Methicillin resistant staphylococci were not included. RESULTS: Of the 417 isolates, 224 (54%) were gram negative and 193 were gram positive. Predominant species/groups were: Escherichia coli 63 (15%), Enterobacter spp 26 (6%), other Enterobacteriacae 41 (10%), Pseudomonas aeruginosa 42 (10%), Staphylococcus aureus 111 (27%), coagulase negative staphylococci 30 (7%), Streptococcus spp 31 (7%), and Enterococcus spp 19 (5%). Isolate sources were: respiratory tract, 170 (41%); cutaneous sites, 81 (19%); blood, 64 (15%); and urine 63 (15%). Resistance was uncommon amongst staphylococci, streptococci, enterococci, and H influenzae. No vancomycin resistant or beta-lactamase-positive enterococci were encountered. For different groups of enteric gram negative bacilli: amoxycillin and amoxycillin-clavulanic acid resistance was common, 46-93% and 24-85% respectively; cefpirome was the most active cephalosporin; aminoglycoside resistance was uncommon; and no isolate possessed extended spectrum beta-lactamase. For P aeruginosa: most isolates were susceptible to cefpirome and ceftazidime, and aminoglycoside resistance was uncommon. CONCLUSION: Gram positive bacteria make up a higher proportion of isolates than in a similar European study. At present New Zealand does not have widespread resistance amongst common isolates. Several agents currently available in New Zealand provide adequate cover for commonly encountered pathogens. The choice of which agent to choose therefore rests more with their purchase and administration costs, as well as safety and efficacy data than simply susceptibility data alone.

Anti-Bacterial Agents↗
PubMed1997

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.

Amino Acid Sequence↗