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Biomedical subjects

L Tilley

Publications and source records attributed to L Tilley.

At least 19 recordsLinked to original sources
PubMed2006

Sequence variation in the 5' untranslated region of the human A4GALT gene is associated with, but does not define, the P1 blood-group polymorphism.

BACKGROUND AND OBJECTIVE: The gene responsible for the P1 polymorphism of the P blood-group system remains unidentified, although the A4GALT gene, whose product is responsible for the production of P(k), has been implicated. No coding differences in A4GALT account for the P1 polymorphism, but homozygosity for two polymorphisms (-551_-550insC and -160A>G) in the 5' untranslated region of the gene has been reported to be unique to Japanese P1- individuals. This study aimed to confirm this correlation in a larger number of British individuals. MATERIALS AND METHODS: Serologically confirmed P1+ (n = 35) and P1- (n = 15) individuals were genotyped for polymorphisms in the 5' untranslated region of A4GALT. RESULTS: In addition to those previously reported, a further polymorphism, -164C>T, was identified. All P1- individuals were homozygous for -551_-550insC and -160G as compared with 10 of 35 (29%) P1+ individuals (P = 0.000003, two-tailed Fisher's exact test). Allele frequencies for all polymorphisms and estimated haplotype frequencies across the region differed significantly between P1+ and P1- groups. CONCLUSIONS: Homozygosity for the A4GALT-551_-550insC and -160G allele is significantly associated with, but not restricted to, the P1- phenotype. No single A4GALT genotype or haplotype was unique to P1- individuals. Thus, A4GALT cannot be unequivocally confirmed as the gene responsible for the P1 phenotype.

5' Untranslated Regions↗
PubMed2001

Trafficking and assembly of the cytoadherence complex in Plasmodium falciparum-infected human erythrocytes.

After invading human erythrocytes, the malarial parasite Plasmodium falciparum, initiates a remarkable process of secreting proteins into the surrounding erythrocyte cytoplasm and plasma membrane. One of these exported proteins, the knob-associated histidine-rich protein (KAHRP), is essential for microvascular sequestration, a strategy whereby infected red cells adhere via knob structures to capillary walls and thus avoid being eliminated by the spleen. This cytoadherence is an important factor in many of the deaths caused by malaria. Green fluorescent protein fusions and fluorescence recovery after photobleaching were used to follow the pathway of KAHRP deployment from the parasite endomembrane system into an intermediate depot between parasite and host, then onwards to the erythrocyte cytoplasm and eventually into knobs. Sequence elements essential to individual steps in the pathway are defined and we show that parasite-derived structures, known as Maurer's clefts, are an elaboration of the canonical secretory pathway that is transposed outside the parasite into the host cell, the first example of its kind in eukaryotic biology.

Amino Acid Sequence↗
PubMed2001

Synthesis, antimalarial activity and inhibition of haem detoxification of novel bisquinolines.

The synthesis of novel bisquinoline compounds comprising 4-(4-diethylamino-1-methylbutyl)aminoquinoline units joined through the 2-position by a (CH(2))(n) linker is described. Their ability to inhibit the growth of both chloroquine-sensitive (D10) and chloroquine-resistant (K1) strains of Plasmodium falciparum, the hydrogen peroxide-mediated pathway for decomposition of haem, and the conversion of haem to beta-haematin have been measured. The activity was affected by the length of the linker and the most active (6c, n=12) showed effects similar to chloroquine in three of the assays. However, it was even more active against the resistant strain [IC(50), 17 nM (K1); 43 nM (D10)], much superior to chloroquine (IC(50), 540 nM) and slightly better than mefloquine (IC(50), 30 nM) in this regard.

Animals↗
PubMed2001

Inhibition of heme detoxification processes underlies the antimalarial activity of terpene isonitrile compounds from marine sponges.

A series of terpene isonitriles, isolated from marine sponges, have previously been shown to exhibit antimalarial activities. Molecular modeling studies employing 3D-QSAR with receptor modeling methodologies performed with these isonitriles showed that the modeled molecules could be used to generate a pharmacophore hypothesis consistent with the experimentally derived biological activities. It was also shown that one of the modeled compounds, diisocyanoadociane (4), as well as axisonitrile-3 (2), both of which have potent antimalarial activity, interacts with heme (FP) by forming a coordination complex with the FP iron. Furthermore, these compounds were shown to inhibit sequestration of FP into beta-hematin and to prevent both the peroxidative and glutathione-mediated destruction of FP under conditions designed to mimic the environment within the malaria parasite. By contrast, two of the modeled diterpene isonitriles, 7-isocyanoamphilecta-11(20),15-diene (12) and 7-isocyano-15-isothiocyanatoamphilecta-11(20)-ene (13), that displayed little antimalarial activity also showed little inhibitory activity in these FP detoxification assays. These studies suggest that the active isonitrile compounds, like the quinoline antimalarials, exert their antiplasmodial activity by preventing FP detoxification. Molecular dynamics simulations performed with diisocyanoadociane (4) and axisonitrile-3 (2) allowed their different binding to FP to be distinguished.

Animals↗
PubMed2001

Polymorphism in the alpha(1)-antichymotrypsin (ACT) gene promoter: effect on expression in transfected glial and liver cell lines and plasma ACT concentrations.

Alpha1-antichymotrypsin (ACT: new identification SERPINA3) is a member of the serine proteinase inhibitor (serpin) gene family and biochemically has been shown to be a constituent of the senile plaques of Alzheimer's disease. We describe a polymorphism (G-->T) in the promoter region of the ACT gene with the T allele being associated with a 22% increase in the mean plasma ACT concentrations. By reporter gene studies, the T allele is consistently associated with higher mean basal expression in both the human liver cell-line Hep G2 (32%) and in a human glial cell-line T98G (30%). Following 6-h stimulation with the cytokine oncostatin-M, there was a 30-fold increase in Hep G2 and a four-fold increase in T98G cells. The T allele in the promoter region is also in almost complete linkage disequilibrium with the T allele in the signal peptide region of the ACT gene with a standardised disequilibrium coefficient (D') of 0.97; P<0.001. This is the first description of a polymorphism in the ACT gene promoter directly associated with altered gene expression.

Aged↗
PubMed2001

Histidine-rich protein 2 of the malaria parasite, Plasmodium falciparum, is involved in detoxification of the by-products of haemoglobin degradation.

The histidine-rich protein 2 (PfHRP2) of Plasmodium falciparum has been implicated in the detoxification of ferriprotoporphyrin IX (FP) moieties that are produced as by-products of the digestion of haemoglobin. In this work, we have used a spectroscopic analysis to confirm that recombinant PfHRP2 binds FP. A monoclonal antibody that recognises both recombinant and authentic PfHRP2 was used in immunofluorescence microscopy studies. We found that PfHRP2 is mainly located in the erythrocyte cytosol of infected erythrocytes, however, dual labelling studies suggest that the location of a sub-population of the PfHRP2 molecules overlaps with that of the food vacuole-associated protein, P-glycoprotein homologue (Pgh-1). A semi-quantitative analysis of the level of PfHRP2 in infected erythrocytes suggests a concentration of a few micromolar in the food vacuole. Under conditions designed to mimic the parasite food vacuole, we found that 1.2 microM PfHRP2 is sufficient to catalyse the conversion of about 30% of a 100 microM sample of FP to beta-haematin within 24 h. Moreover, PfHRP2 is capable of promoting the H(2)O(2)-induced degradation of FP at pH 5.2. PfHRP2 also efficiently enhances the ability of FP to catalyse the H(2)O(2)-mediated oxidation of the model co-factor, ortho-phenylene diamine (OPD). These data suggest that PfHRP2 may promote the detoxification of FP and reactive oxygen species within the food vacuole. By contrast, PfHRP2 inhibits the destruction of FP by glutathione (GSH) at pH 7.4. This suggests that PfHRP2 is not a catalyst of FP degradation outside the food vacuole.

Animals↗
PubMed2001

Evidence for a role for a Plasmodium falciparum homologue of Sec31p in the export of proteins to the surface of malaria parasite-infected erythrocytes.

The malaria parasite, Plasmodium falciparum, spends part of its life cycle inside the enucleated erythrocytes of its human host. The parasite modifies the cytoplasm and plasma membrane of its host cell by exporting proteins beyond the confines of its own plasma membrane. We have previously provided evidence that a plasmodial homologue of the COPII protein, Sar1p, is involved in the trafficking of proteins across the erythrocyte cytoplasm. We have now characterised an additional plasmodial COPII protein homologue, namely Sec31p. Recombinant proteins corresponding to the WD-40 and the intervening domains of the PfSec31p sequence were used to raise antibodies. The affinity-purified antisera recognised a protein with an apparent relative molecular mass of 1.6 x 10(5) on western blots of malaria parasite-infected erythrocytes but not on blots of uninfected erythrocytes. PfSec31p was shown to be largely insoluble in nonionic detergent, suggesting cytoskeletal attachment. Confocal immunofluorescence microscopy of malaria parasite-infected erythrocytes was used to show that PfSec31p is partly located within the parasite and partly exported to structures outside the parasite in the erythrocyte cytoplasm. We have also shown that PfSec31p and PfSar1p occupy overlapping locations. Furthermore, the location of PfSec31p overlaps that of the cytoadherence-mediating protein PfEMP1. These data support the suggestion that the malaria parasite establishes a vesicle-mediated trafficking pathway outside the boundaries of its own plasma membrane - a novel paradigm in eukaryotic biology.

Animals↗
PubMed2000

The multidrug resistance protein is photoaffinity labeled by a quinoline-based drug at multiple sites.

Tumor cells overcome cytotoxic drug pressure by the overexpression of either or both transmembrane proteins, the P-glycoprotein (P-gp) and the multidrug resistance protein (MRP). The MRP has been shown to mediate the transport of cytotoxic natural products, in addition to glutathione-, glucuronidate-, and sulfate-conjugated cell metabolites. However, the mechanism of MRP drug binding and transport is at present not clear. In this study, we have used a photoreactive quinoline-based drug, N-(hydrocinchonidin-8'-yl)-4-azido-2-hydroxybenzamide (IACI), to show the photoaffinity labeling of the 190 kDa protein in membranes from the drug resistant SCLC H69/AR cells. The photoaffinity labeling of the 190 kDa protein by IACI was saturable and specific. The identity of the IACI-photolabeled protein as the MRP was confirmed by immunoprecipitation with the monoclonal antibody QCRL-1. Furthermore, a molar excess of leukotriene C(4), doxorubicin, colchicine, and other quinoline-based drugs, including MK571, inhibited the photoaffinity labeling of the MRP. Drug transport studies showed lower IACI accumulation in MRP-expressing cells which was reversed by depleting ATP levels in H69/AR cells. Mild digestion of the purified IACI-photolabeled MRP with trypsin showed two large polypeptides ( approximately 111 and approximately 85 kDa). The 85 kDa polypeptide which contains the QCRL-1 and MRPm6 monoclonal antibody epitopes corresponds to the C-terminal half of the MRP (amino acids approximately 900-1531) containing the third multiple spanning domain (MSD3) and the second nucleotide binding site. The 111 kDa polypeptide which contains the epitope sequence of the MRPr1 monoclonal antibody encodes the remainder of the MRP sequence (amino acids 1-900) containing the MSD1 and MSD2 plus the first nucleotide binding domain. Cleveland maps of purified IACI-labeled 85 and 111 kDa polypeptides revealed 6 kDa and approximately 6 plus 4 kDa photolabeled peptides, respectively. In addition, resolution of the exhaustively digested IACI-photolabeled MRP by HPLC showed two major and one minor radiolabeled peaks that eluted late in the gradient (60 to 72% acetonitrile). Taken together, the results of this study show direct binding of IACI to the MRP at physiologically relevant sites. Moreover, IACI photolabels three small peptides which localize to the N- and C-halves of the MRP. Finally, IACI provides a sensitive and specific probe for studying MRP-drug interactions.

ATP-Binding Cassette Transporters↗
PubMed2000

Antimalarial drug development and new targets.

The Molecular Approaches to Malaria (MAM2000) conference, Lorne, Australia, 2-5 February 2000, brought together world-class malaria research scientists. The development of new tools and technologies - transfection, DNA microarrays and proteomic analysis - and the availability of DNA sequences generated by the Malaria Genome Project, along with more classic approaches, have facilitated the identification of novel drug targets, the development of new antimalarials and the generation of a deeper understanding of the molecular mechanism(s) of drug resistance in malaria. It is hoped that combinations of these technologies could lead to strategies that enable the development of effective, efficient and affordable new drugs to overcome drug-resistant malaria, as discussed at MAM2000 and outlined here by Ian Macreadie and colleagues.

Animals↗
PubMed2000

Animal models of cardiac disease: potential usefulness for studying health effects of inhaled particles.

Cardiac disease is one of the major causes of morbidity and mortality worldwide and is the leading cause of death in the United States. Epidemiologic studies have shown a close association between morbidity and mortality from cardiac disease, primarily in persons already affected, and with modest increases in levels of air pollution. At present, no mechanisms are known by which inhaled air pollutants interact with the heart to cause death. Thus, animal models of cardiac disease are needed to study possible interactions between inhaled pollutants and the heart and the resultant morbidity and mortality. Very little research in animals has been conducted in this area, and appropriate animal models must be carefully selected. The purpose of this review is to examine several potential animal models and to discuss their advantages and disadvantages in the study of cardiac disease and air pollution.

Air Pollutants↗
PubMed1999

Inhibition of the peroxidative degradation of haem as the basis of action of chloroquine and other quinoline antimalarials.

The malaria parasite feeds by degrading haemoglobin in an acidic food vacuole, producing free haem moieties as a by-product. The haem in oxyhaemoglobin is oxidized from the Fe(II) state to the Fe(III) state with the consequent production of an equimolar concentration of H2O2. We have analysed the fate of haem molecules in Plasmodium falciparum-infected erythrocytes and have found that only about one third of the haem is polymerized to form haemozoin. The remainder appears to be degraded by a non-enzymic process which leads to an accumulation of iron in the parasite. A possible route for degradation of the haem is by reacting with H2O2, and we show that, under conditions designed to resemble those found in the food vacuole, i.e., at pH5.2 in the presence of protein, free haem undergoes rapid peroxidative decomposition. Chloroquine and quinacrine are shown to be efficient inhibitors of the peroxidative destruction of haem, while epiquinine, a quinoline compound with very low antimalarial activity, has little inhibitory effect. We also show that chloroquine enhances the association of haem with membranes, while epiquinine inhibits this association, and that treatment of parasitized erythrocytes with chloroquine leads to a build-up of membrane-associated haem in the parasite. We suggest that chloroquine exerts its antimalarial activity by causing a build-up of toxic membrane-associated haem molecules that eventually destroy the integrity of the malaria parasite. We have further shown that resistance-modulating compounds, such as chlorpromazine, interact with haem and efficiently inhibit its degradation. This may explain the weak antimalarial activities of these compounds.

Animals↗
PubMed1999

Export of parasite proteins to the erythrocyte cytoplasm: secretory machinery and traffic signals.

To the malaria parasite, the prospect of setting up residence within a human erythrocyte represents a formidable challenge. The mature human erythrocyte is essentially a bag of haemoglobin with no internal organelles and no protein synthesis machinery. The parasite needs, therefore, to assemble all the essential amenities--foundations, plumbing and furnishings--from scratch. The parasite remodels its adopted home by exporting proteins to the erythrocyte membrane. To reach their final destinations, the exported proteins must cross the parasite plasma membrane, the parasitophorous vacuole membrane and the erythrocyte cytosol. To further understand this unusual and complex trafficking pathway, we have searched for proteins that may form part of the trafficking machinery of the infected erythrocyte. We have identified an ER-located, calcium-binding homologue of reticulocalbin (PfERC) that co-localizes with the ER molecular chaperone, PfGRP. We have also identified a homologue of the GTP-binding protein, Sar1p, a small GTPase that, in other eukaryotic cells, is thought to play a crucial role in trafficking proteins between the ER and the Golgi. PfSar1p is located in discrete structures near the periphery of the parasite cytoplasm that may represent specialized export compartments. PfSar1p is exported to structures outside the parasite in the erythrocyte cytoplasm. The malaria parasite appears to be capable of elaborating components of the 'classical' vesicle mediated trafficking machinery outside the boundaries of its own plasma membrane.

Animals↗
PubMed1999

A homologue of Sar1p localises to a novel trafficking pathway in malaria-infected erythrocytes.

We have identified a homologue of the GTP-binding protein, Sar1p, in Plasmodium falciparum. Sar1p is a small GTPase that is thought to play a crucial role in trafficking of proteins between the endoplasmic reticulum and the Golgi. The P.falciparum SAR1 gene is located on chromosome 4 and comprises two exons separated by a 508 bp intron. The deduced amino acid sequence of PfSar1p (GenBank accession number AF104306) shows 71% similarity (58% identity) to Sar1p from Saccharomyces cerevisiae. Expression of PfSar1p in erythrocytic stages of P. falciparum was confirmed by sequencing of a tryptic peptide derived from a polypeptide excised from an SDS-polyacrylamide gel. A recombinant protein corresponding to approximately 70% of the PfSar1p sequence was used to raise antibodies. The affinity-purified antiserum recognised a protein with an apparent molecular weight of 23 K in Western blots of malaria-infected erythrocytes but not in uninfected erythrocytes. PfSar1p was shown to be largely insoluble in non-ionic detergent and a low ionic strength buffer. Confocal immunofluorescence microscopy of malaria-infected erythrocytes was used to show that PfSar1p is located near the periphery of the parasite in discrete compartments, which appear to be distinct from the parasite endoplasmic reticulum. In addition, PfSar1p appears to be exported to structures outside the parasite in the erythrocyte cytoplasm. The export of PfSar1p to the erythrocyte cytosol is inhibited by treatment with brefeldin A. This provides the first evidence that the malaria parasite is capable of elaborating components of the classical vesicle-mediated trafficking machinery outside the boundaries of its own plasma membrane.

Amino Acid Sequence↗
PubMed1999

Evaluation of polymorphisms in the presenilin-1 gene and the butyrylcholinesterase gene as risk factors in sporadic Alzheimer's disease.

The E4 allele of the apolipoprotein E gene (APOE) is a major risk factor for late-onset Alzheimer's disease (LOAD) but is neither necessary nor sufficient to cause the disease. In this study, we investigated polymorphisms in the presenilin-1 (PS-1), and butyrylcholinesterase (BChE) genes, which have been implicated as risk factors for LOAD. Our data-set comprised 177 AD and 118 control patients, all of whom had been histopathologically confirmed following autopsy. We have tested homozygosity for the PS-1 allele 1 and possession of the BChE-K variant in association with APOE epsilon4 as risk factors in LOAD. Our findings support an association between the PS-1 polymorphism and LOAD, finding homozygosity for allele 1 associated with an approximately two-fold increased risk. Our data also show that in subjects greater than 75 years of age possession of both BChE-K and APOE-epsilon4 alleles is associated with an increased risk of LOAD, whilst the risk associated with APOE-epsilon4 allele alone is not significant.

Aged↗
PubMed1999

Specific inhibition of the eubacterial DNA ligase by arylamino compounds.

All known DNA ligases catalyze the formation of a phosphodiester linkage between adjacent termini in double-stranded DNA via very similar mechanisms. The ligase family can, however, be divided into two classes: eubacterial ligases, which require NAD(+) as a cofactor, and other ligases, from viruses, archaea, and eukaryotes, which use ATP. Drugs that discriminate between DNA ligases from different sources may have antieubacterial activity. We now report that a group of arylamino compounds, including some commonly used antimalarial and anti-inflammatory drugs and a novel series of bisquinoline compounds, are specific inhibitors of eubacterial DNA ligases. Members of this group of inhibitors have different heterocyclic ring systems with a common amino side chain in which the two nitrogens are separated by four carbon atoms. The potency, but not the specificity of action, is influenced by the DNA-binding characteristics of the inhibitor, and the inhibition is noncompetitive with respect to NAD(+). The arylamino compounds appear to target eubacterial DNA ligase in vivo, since a Salmonella Lig(-) strain that has been rescued with the ATP-dependent T4 DNA ligase is less sensitive than the parental Salmonella strain.

Adenosine Triphosphate↗