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Biomedical subjects

Paul A Bates

Publications and source records attributed to Paul A Bates.

At least 19 recordsLinked to original sources
PubMed2006

Probability-based model of protein-protein interactions on biological timescales.

BACKGROUND: Simulation methods can assist in describing and understanding complex networks of interacting proteins, providing fresh insights into the function and regulation of biological systems. Recent studies have investigated such processes by explicitly modelling the diffusion and interactions of individual molecules. In these approaches, two entities are considered to have interacted if they come within a set cutoff distance of each other. RESULTS: In this study, a new model of bimolecular interactions is presented that uses a simple, probability-based description of the reaction process. This description is well-suited to simulations on timescales relevant to biological systems (from seconds to hours), and provides an alternative to the previous description given by Smoluchowski. In the present approach (TFB) the diffusion process is explicitly taken into account in generating the probability that two freely diffusing chemical entities will interact within a given time interval. It is compared to the Smoluchowski method, as modified by Andrews and Bray (AB). CONCLUSION: When implemented, the AB & TFB methods give equivalent results in a variety of situations relevant to biology. Overall, the Smoluchowski method as modified by Andrews and Bray emerges as the most simple, robust and efficient method for simulating biological diffusion-reaction processes currently available.

Journal Article↗
PubMed2006

Repair of alkylated DNA: recent advances.

Cytotoxic and mutagenic methylated bases in DNA can be generated by endogenous and environmental alkylating agents. Such damaged bases are removed by three distinct strategies. The abundant toxic lesion 3-methyladenine (3-alkyladenine) is excised by a specific DNA glycosylase that initiates a base excision-repair process. The toxic lesions 1-methyladenine and 3-methylcytosine are corrected by oxidative DNA demethylation catalyzed by DNA dioxygenases. These enzymes release the methyl moiety as formaldehyde, directly reversing the base damage. The third strategy involves the mutagenic and cytotoxic lesion O(6)-methylguanine which is also repaired by direct reversal but uses a different mechanism. Here, the methyl group is transferred from the lesion to a specific cysteine residue within the methyltransferase itself. In this review, we briefly describe endogenous alkylating agents and the extensively investigated DNA repair enzymes, mammalian 3-methyladenine-DNA glycosylase and O(6)-methylguanine-DNA methyltransferase. We provide a more detailed description of the structures and biochemical properties of the recently discovered DNA dioxygenases.

Alkylating Agents↗
PubMed2006

Housekeeping by Leishmania.

The observation that differentiation of parasite life cycle stages involves structural and functional reorganisation is very familiar. A recent paper by Besteiro and colleagues provides an intriguing insight into cellular remodelling during differentiation. They show that it is essential for Leishmania to tidy up and put the house in order before it can differentiate to mammal-infective stages.

Animals↗
PubMed2006

Analysis of ESTs from Lutzomyia longipalpis sand flies and their contribution toward understanding the insect-parasite relationship.

An expressed sequence tag library has been generated from a sand fly vector of visceral leishmaniasis, Lutzomyia longipalpis. A normalized cDNA library was constructed from whole adults and 16,608 clones were sequenced from both ends and assembled into 10,203 contigs and singlets. Of these 58% showed significant similarity to known genes from other organisms, <4% were identical to described sand fly genes, and 42% had no match to any database sequence. Our analyses revealed putative proteins involved in the barrier function of the gut (peritrophins, microvillar proteins, glutamine synthase), digestive physiology (secreted and membrane-anchored hydrolytic enzymes), and the immune response (gram-negative binding proteins, thioester proteins, scavenger receptors, galectins, signaling pathway factors, caspases, serpins, and peroxidases). Sequence analysis of this transcriptome dataset has provided new insights into genes that might be associated with the response of the vector to the development of Leishmania.

Animals↗
PubMed2006

Global topological features of cancer proteins in the human interactome.

MOTIVATION: The study of interactomes, or networks of protein-protein interactions, is increasingly providing valuable information on biological systems. Here we report a study of cancer proteins in an extensive human protein-protein interaction network constructed by computational methods. RESULTS: We show that human proteins translated from known cancer genes exhibit a network topology that is different from that of proteins not documented as being mutated in cancer. In particular, cancer proteins show an increase in the number of proteins they interact with. They also appear to participate in central hubs rather than peripheral ones, mirroring their greater centrality and participation in networks that form the backbone of the proteome. Moreover, we show that cancer proteins contain a high ratio of highly promiscuous structural domains, i.e., domains with a high propensity for mediating protein interactions. These observations indicate an underlying evolutionary distinction between the two groups of proteins, reflecting the central roles of proteins, whose mutations lead to cancer. CONTACT: paul.bates@cancer.org.uk SUPPLEMENTARY INFORMATION: The interactome data are available though the PIP (Potential Interactions of Proteins) web server at http://bmm.cancerresearchuk.org/servers/pip. Further additional material is available at http://bmm.cancerresearchuk.org/servers/pip/bioinformatics/

Algorithms↗
PubMed2006

Synthetic glycovaccine protects against the bite of leishmania-infected sand flies.

Leishmaniasis is a vectorborne disease transmitted to human and other mammalian hosts by sand fly bite. In the present study, we show that immunization with Leishmania mexicana promastigote secretory gel (PSG) or with a chemically defined synthetic glycovaccine containing the glycans found in L. mexicana PSG can provide significant protection against challenge by the bite of infected sand flies. Only the glycan from L. mexicana was protective; those from other species did not protect against L. mexicana infection. Furthermore, neither PSG nor the glycovaccine protected against artificial needle challenge, which is traditionally used in antileishmanial vaccine development. Conversely, an antigen preparation that was effective against needle challenge offered no protection against sand fly bite. These findings provide a new target for Leishmania vaccine development and demonstrate the critical role that the vector plays in the evaluation of candidate vaccines for leishmaniasis and other vectorborne diseases.

Animals↗
PubMed2006

Developing a move-set for protein model refinement.

MOTIVATION: A wide variety of methods for the construction of an atomic model for a given amino acid sequence are known, the more accurate being those that use experimentally determined structures as templates. However, far fewer methods are aimed at refining these models. The approach presented here carefully blends models created by several different means, in an attempt to combine the good quality regions from each into a final, more refined, model. RESULTS: We describe here a number of refinement operators (collectively, 'move-set') that enable a relatively large region of conformational space to be searched. This is used within a genetic algorithm that reshuffles and repacks structural components. The utility of the move-set is demonstrated by introducing a cost function, containing both physical and other components guiding the input structures towards the target structure. We show that our move-set has the potential to improve the conformation of models and that this improvement can be beyond even the best template for some comparative modelling targets. AVAILABILITY: The populus software package and the source code are available at http://bmm.cancerresearchuk.org/~offman01/populus.html.

Algorithms↗
PubMed2006

Cluster analysis of networks generated through homology: automatic identification of important protein communities involved in cancer metastasis.

BACKGROUND: Protein-protein interactions have traditionally been studied on a small scale, using classical biochemical methods to investigate the proteins of interest. More recently large-scale methods, such as two-hybrid screens, have been utilised to survey extensive portions of genomes. Current high-throughput approaches have a relatively high rate of errors, whereas in-depth biochemical studies are too expensive and time-consuming to be practical for extensive studies. As a result, there are gaps in our knowledge of many key biological networks, for which computational approaches are particularly suitable. RESULTS: We constructed networks, or 'interactomes', of putative protein-protein interactions in the rat proteome--the rat being an organism extensively used for cancer studies. This was achieved by integrating experimental protein-protein interaction data from many species and translating this data into the reference frame of the rat. The putative rat protein interactions were given confidence scores based on their homology to proteins that have been experimentally observed to interact. The confidence score was furthermore weighted according to the extent of the experimental evidence, giving a higher weight to more frequently observed interactions. The scoring function was subsequently validated and networks constructed around key proteins, identified as being highly up- or down-regulated in rat cell lines of high metastatic potential. Using clustering methods on the networks, we have identified key protein communities involved in cancer metastasis. CONCLUSION: The protein network generation and subsequent network analysis used here, were shown to be useful for highlighting key proteins involved in metastasis. This approach, in conjunction with microarray expression data, can be extended to other species, thereby suggesting possible pathways around proteins of interest.

Algorithms↗
PubMed2006

Can MM-PBSA calculations predict the specificities of protein kinase inhibitors?

An application of the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) protocol to the prediction of protein kinase inhibitor selectivity is presented. Six different inhibitors are placed in equivalent orientations in each of six different receptors. Fully solvated molecular dynamics is then run for 1 ns on each of the 36 complexes, and the resulting trajectories scored, using the implicit solvent model. The results show some correlation with experimentally-determined specificities; anomalies may be attributed to a variety of causes, including difficulties in quantifying induced fit penalties and variabilities in normal modes calculations. Decomposing interaction energies on a per-residue basis yields more useful insights into the natures of the binding modes and suggests that the real value of such calculations lies in understanding interactions rather than outright prediction.

Amino Acid Sequence↗
PubMed2006

A glycine- and glutamate-rich protein is female salivary gland-specific and abundant in the malaria vector Anopheles dirus B (Diptera: Culicidae).

Before transmission, malaria parasites reside in the salivary glands of their female mosquito hosts. Saliva proteins assist in blood feeding and also may influence the ability of mosquitoes to transmit malaria. We attempted to identify and isolate cDNAs encoding proteins expressed at a high level in the salivary glands of a malaria vector, Anopheles dirus B Peyton and Harrison (= An. cracens) (Diptera: Culicidae). A major protein with an estimated molecular mass of 35 kDa and an isoelectric point (pI) of approximately 4 was detected on a two-dimensional (2D) gel. Internal peptide sequences of the protein spot showed high similarity to sequences present in the conserved C-terminal domain of glycine- and glutamate (GE)-rich proteins. A full-length cDNA encoding this protein was isolated from a salivary gland cDNA library of female An. dirus B. The cDNA encoded a 256-residue protein with a calculated molecular mass of 25.4 kDa and a pI of 3.9. BLAST analysis confirmed that it is a member of the GE-rich family. Compositional and sequence analysis of this and other family members revealed a highly acidic N-terminal region of variable length and low sequence conservation and a well conserved C-terminal domain containing 10 identical residues across the 13 known members of the gene family in mosquitoes. The An. dirus B GE-rich transcript was detected by reverse transcription-polymerase chain reaction (PCR) only in the female salivary glands, indicating that this protein is female saliva-specific. The GE-rich proteins may function as a salivary lubricant to facilitate blood feeding.

Amino Acid Sequence↗
PubMed2005

Incorporation of flexibility into rigid-body docking: applications in rounds 3-5 of CAPRI.

We have submitted models for all 9 targets in Rounds 3-5 of CAPRI and have predicted at least 30% of the correct contacts for 4 of the targets and at least 10% of the correct contacts for another 4 targets. We have employed a variety of techniques but have had the greatest success by combining established rigid-body docking with a variety of initial conformations generated by molecular dynamics.

Algorithms↗
PubMed2005

The relationship between the flexibility of proteins and their conformational states on forming protein-protein complexes with an application to protein-protein docking.

We investigate the extent to which the conformational fluctuations of proteins in solution reflect the conformational changes that they undergo when they form binary protein-protein complexes. To do this, we study a set of 41 proteins that form such complexes and whose three-dimensional structures are known, both bound in the complex and unbound. We carry out molecular dynamics simulations of each protein, starting from the unbound structure, and analyze the resulting conformational fluctuations in trajectories of 5 ns in length, comparing with the structure in the complex. It is found that fluctuations take some parts of the molecules into regions of conformational space close to the bound state (or give information about it), but at no point in the simulation does each protein as whole sample the complete bound state. Subsequent use of conformations from a clustered MD ensemble in rigid-body docking is nevertheless partially successful when compared to docking the unbound conformations, as long as the unbound conformations are themselves included with the MD conformations and the whole globally rescored. For one key example where sub-domain motion is present, a ribonuclease inhibitor, principal components analysis of the MD was applied and was also able to produce conformations for docking that gave enhanced results compared to the unbound. The most significant finding is that core interface residues show a tendency to be less mobile (by size of fluctuation or entropy) than the rest of the surface even when the other binding partner is absent, and conversely the peripheral interface residues are more mobile. This surprising result, consistent across up to 40 of the 41 proteins, suggests different roles for these regions in protein recognition and binding, and suggests ways that docking algorithms could be improved by treating these regions differently in the docking process.

Cluster Analysis↗
PubMed2005

Detailed analysis of an experimental challenge model for Leishmania infantum (JPC strain) in dogs.

In this study, disease progression after intravenous or subdermal infection of dogs with Leishmania infantum JPC strain was monitored. A challenge performed on 14 dogs via the intravenous route with 5 x 10(7) stationary phase promastigotes of the L. infantum JPC strain was 100% successful. During a follow up period of 1.5 years, several parameters were evaluated in order to find the most reliable disease markers. Parasite detection by culture and histology were found to be very sensitive (100%). Additionally, regular physical examination, serology and serum gamma-globulin levels were found to be useful parameters in the evaluation of disease severity and are recommended for inclusion in vaccination-challenge experiments. Although this intravenous challenge model has practical limitations, the data set confirms it is the best experimental model currently available for vaccine development. Two intravenously infected dogs were treated with corticosteroids for 5 months. This treatment was shown to enhance all aspects of a Leishmania infection. Five more dogs were infected by sub-dermal injection of promastigotes mixed with a proteophosphoglycan-matrix (PSG) secreted by Leishmania that assists in transmission and infection by sand fly bite. The resulting parasite burdens were low and the animals remained asymptomatic during a 2-year follow up period. However, this procedure did result in infection in 80% of the dogs and is appealing for future development as a natural challenge model in vaccine development.

Adrenal Cortex Hormones↗
PubMed2005

Production and characterization of stable amphotericin-resistant amastigotes and promastigotes of Leishmania mexicana.

The sensitivities of Leishmania mexicana amastigote and promastigote forms to amphotericin B were investigated in vitro and found to be strongly influenced by the culture media used. When differences in culture media were minimized, there was no significant difference in the 50% inhibitory concentration values between the two life cycle stages. Stable amphotericin B-resistant amastigote and promastigote lines were produced by the application of increasing drug pressure to long-term cultures. Lines capable of growth in concentrations of amphotericin B lethal to normal parasites were produced. Compared to normal parasites, these amphotericin-resistant lines showed marked differences in membrane sterol compositions, with very high levels of 4,14,dimethyl-cholesta-8,24-dienol and other methyl sterols. They also showed a consistent morphological feature, the presence of multilamellar membrane-like material in the flagellar pocket, revealed by transmission electron microscopy. Amphotericin-resistant parasites were capable of infecting BALB/c mice, but the resulting lesion growth was slower than that after infection with normal parasites. However, unlike normal parasites, the amphotericin-resistant parasites were unaffected by experimental chemotherapy with amphotericin B. These results show that amphotericin B resistance could arise as a result of increased clinical use of amphotericin B therapy.

Amphotericin B↗
PubMed2005

Sequence analyses of three nuclear ribosomal loci and a mitochondrial locus in cytologically different forms of Thai Anopheles aconitus mosquitoes.

Ribosomal DNA (rDNA) and mitochondrial DNA (mtDNA) of Anopheles aconitus mosquitoes were examined to investigate intra- and inter-species variation amongst the members of the Minimus group of Anopheles subgenus Cellia. Three rDNA loci (ITS1, ITS2 and D3 regions) and a mtDNA locus (cytochrome oxidase II) were analyzed in An. aconitus Form B and Form C collected in Chiang Mai Province, Thailand. The results show that the consensus sequences of the four loci of the two forms are consistent with those of mosquitoes in the genus Anopheles. No intraindividual variation was detected, but intrapopulation variation was present with polymorphic sequences in some forms for each gene examined. The variation rates were approximately 0.15 to 0.8%. These data indicate that An. aconitus Form B and Form C in Chiang Mai, Thailand are conspecific. In this study, the complete ITS1 sequence of An. aconitus is reported for the first time. The region showed a high variation rate (approximately 55%), compared to the closely related species An. minimus C. It is suggested that this rDNA locus may provide sequence information to differentiate the members of the Minimus group of Anopheles subgenus Cellia.

Animals↗
PubMed2005

Evidence to support two conspecific cytological races on Anopheles aconitus in Thailand.

Iso-female lines (isolines) of Anopheles aconitus collected from Mae Hong Son, Phet Buri, and Chiang Mai Provinces were successfully identified to karyotypic forms. The results of identification revealed that An. aconitus Form B (X1, X2, Y2) was obtained from four and 48 isolines in Phet Buri and Chiang Mai Provinces, respectively, and Form C (X1, X2, Y3) was recovered from three and 41 isolines in Mae Hong Son and Chiang Mai Provinces, respectively. When comparing band to band on the same arm of ovarian nurse cell polytene chromosomes of An. aconitus Form B (Phet Buri: four isolines) and C (Mae Hong Son: three isolines, Chiang Mai: 20 isolines) to the standard chromosome mapping of An. aconitus Form B (Chiang Mai: 20 isolines), no major chromosomal rearrangements that related to the karyotype variations were demonstrated. The investigations on allelic frequencies of 4th stage larvae and adult females of three (Form C: Mae Hong Son), four (Form B: Phet Buri), 41 (Form C: Chiang Mai) and 48 (Form B: Chiang Mai) isolines suggested that An. aconitus Form B and C of all strains have similar allelic frequencies. This was observed at 10 isoenzymes 16 loci in 4th stage larvae, and 11 isoenzymes 13 loci in adult females. Hybridization tests among the four laboratory-raised isolines of An. aconitus Form B (Chiang Mai and Phet Buri) and C (Chiang Mai and Mae Hong Son) were employed by induced copulation. The results of crosses indicated that they were genetically compatible, providing viable progeny and completely synaptic salivary gland polytene chromosomes. The complete sequences ofrDNA internal-transcribed spacer two (ITS2) and partial sequences of mitochondrial cytochrome c oxidase subunit I and II (COI and COII) from genomic DNA of 12 isolines of An. aconitus Form B and C were identified. Total sequence lengths (ITS2+COI+COII) of An. aconitus isolines varied from 1550bp to 1556bp. Conspecific relationships between the two An. aconitus forms were well supported by low values of intraspecific distances (ranged from 0.1% to 1.0%) and genetic differentiation (d(xy): 0.01322) between the two forms. Based on evidence of no pre- and post-mating isolations, and nearly identical of DNA sequences of ITS2, COI and COII regions between An. aconitus Form B and C, we conclude that they are conspecific cytological races in the Thai population.

Animals↗
PubMed2004

Molecular characterization, expression, and in vivo analysis of LmexCht1: the chitinase of the human pathogen, Leishmania mexicana.

Chitinases have been implicated to be of importance in the life cycle development and transmission of a variety of parasitic organisms. Using a molecular approach, we identified and characterized the structure of a single copy LmexCht1-chitinase gene from the primitive trypanosomatid pathogen of humans, Leishmania mexicana. The LmexCht1 encodes an approximately 50 kDa protein, with well conserved substrate binding and catalytic domains characteristic of members of the chitinase-18 protein family. Further, we showed that LmexCht1 mRNA is constitutively expressed by both the insect vector (i.e. promastigote) and mammalian (i.e. amastigote) life cycle developmental forms of this protozoan parasite. Interestingly, however, amastigotes were found to secrete/release approximately >2-4-fold higher levels of chitinase activity during their growth in vitro than promastigotes. Moreover, a homologous episomal expression system was devised and used to express an epitope-tagged LmexCht1 chimeric construct in these parasites. Expression of the LmexCht1 chimera was verified in these transfectants by reverse transcription-PCR, Western blots, and indirect immunofluorescence analyses. Further, results of coupled immunoprecipitation/enzyme activity experiments demonstrated that the LmexCht1 chimeric protein was secreted/released by these transfected L. mexicana parasites and that it possessed functional chitinase enzyme activity. Such transfectants were also evaluated for their infectivity both in human macrophages in vitro and in two different strains of mice. Results of those experiments demonstrated that the LmexCht1 transfectants survived significantly better in human macrophages and also produced significantly larger lesions in mice than control parasites. Taken together, our results indicate that the LmexCht1-chimera afforded a definitive survival advantage to the parasite within these mammalian hosts. Thus, the LmexCht1 could potentially represent a new virulence determinant in the mammalian phase of this important human pathogen.

Amino Acid Sequence↗