Biomedical subjects

Paul A Bates

Publications and source records attributed to Paul A Bates.

At least 37 records · Page 2Linked to original sources

PubMed2004

Comparative morphometry and morphology of Anopheles aconitus Form B and C eggs under scanning electron microscope.

Comparative morphometric and morphological studies of eggs under scanning electron microscope (SEM) were undertaken in the three strains of two karyotypic forms of Anopheles aconitus, i.e., Form B (Chiang Mai and Phet Buri strains) and Form C (Chiang Mai and Mae Hong Son strains). Morphometric examination revealed the intraspecific variation with respect to the float width [36.77 +/- 2.30 microm (Form C: Chiang Mai strain) = 38.49 +/- 2.78 microm (Form B: Chiang Mai strain) = 39.06 +/- 2.37 microm (Form B: Phet Buri strain) > 32.40 +/- 3.52 microm (Form C: Mae Hong Son strain)] and number of posterior tubercles on deck [2.40 +/- 0.52 (Form B: Phet Buri strain) = 2.70 +/- 0.82 (Form B: Chiang Mai strain) < 3.10 +/- 0.32 (Form C: Chiang Mai strain) = 3.20 +/- 0.42 (Form C: Mae Hong Son strain)], whereas the surface topography of eggs among the three strains of two karyotypic forms were morphologically similar.

Animals↗

PubMed2004

Transmission of cutaneous leishmaniasis by sand flies is enhanced by regurgitation of fPPG.

Sand flies are the exclusive vectors of the protozoan parasite Leishmania, but the mechanism of transmission by fly bite has not been determined nor incorporated into experimental models of infection. In sand flies with mature Leishmania infections the anterior midgut is blocked by a gel of parasite origin, the promastigote secretory gel. Here we analyse the inocula from Leishmania mexicana-infected Lutzomyia longipalpis sand flies. Analysis revealed the size of the infectious dose, the underlying mechanism of parasite delivery by regurgitation, and the novel contribution made to infection by filamentous proteophosphoglycan (fPPG), a component of promastigote secretory gel found to accompany the parasites during transmission. Collectively these results have important implications for understanding the relationship between the parasite and its vector, the pathology of cutaneous leishmaniasis in humans and also the development of effective vaccines and drugs. These findings emphasize that to fully understand transmission of vector-borne diseases the interaction between the parasite, its vector and the mammalian host must be considered together.

Animals↗

PubMed2004

Recognition of phosphorylated-Smad2-containing complexes by a novel Smad interaction motif.

Transforming growth factor beta (TGF-beta) superfamily members signal via complexes of activated Smads, comprising phosphorylated receptor-regulated Smads, such as Smad2 and Smad3, and Smad4. These complexes are recruited to DNA by specific transcription factors. The forkhead/winged-helix transcription factors, XFast-1/XFoxH1a and XFast-3/XFoxH1b, bind an activated Smad heterotrimer comprising two Smad2s and one Smad4. Here we identify a novel Smad2 interaction motif, the Fast/FoxH1 motif (FM), present in all known Fast/FoxH1 family members, N-terminal to the common Smad interaction motif (SIM). The FM is necessary and sufficient to bind active Smad2/Smad4 complexes. The FM differs from the SIM since it discriminates between Smad2 and Smad3, and moreover only binds phosphorylated Smad2 in the context of activated Smad complexes. It is the first Smad interaction motif with this property. Site-directed mutagenesis indicates that the binding site for the FM on a Smad2/Smad4 heterotrimer is a hydrophobic pocket that incorporates the Smad/Smad interface. We demonstrate that the presence of an FM and SIM in the Fast/FoxH1 proteins allows them to compete efficiently for activated Smad2/Smad4 complexes with transcription factors such as Mixer that only contain a SIM. This establishes a hierarchy of Smad-interacting transcription factors, determined by their affinity for active Smad complexes.

Amino Acid Motifs↗

PubMed2004

Salivary gland of Toxorhynchites splendens Wiedemann (Diptera: Culicidae): ultrastructural morphology and electrophoretic protein profiles.

The salivary glands of male and female Toxorhynchites splendens have the same morphology, and they are paired organs lying on either side of the esophagus. Each gland is composed of two identical tubular lobes, joined together at the end of the proximal region. In the gland, a salivary duct extends through the length of each lobe. The general cellular architecture of the salivary gland of this mosquito is unique. No secretory cavity was found in any cell, and the salivary materials are secreted from long microvilli and collect in a periductal space surrounding the duct. In addition, a number of mitochondria, rough endoplasmic reticulum, and a very large nucleus were observed, suggesting a high energy requirement for producing the salivary proteins involved in sugar feeding. The size of the gland is approximately 50 microm in diameter and 1.5 mm in length. These dimensions correlate with high protein content of these salivary glands (2.88+/-0.14 microg/gland pair). Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that the electrophoretic protein profiles of the male and female salivary glands were identical. No dominant major proteins were found. Compared with Aedes and Anopheles mosquitoes, the protein profile of T. splendens was similar to that observed in the males of these other species but different to that shown by the females, thus making T. splendens an excellent organism for studying the biochemistry of sugar feeding in mosquitoes.

Animals↗

PubMed2003

Structural context of exons in protein domains: implications for protein modelling and design.

Intron boundaries were extracted from genomic data and mapped onto single-domain human and murine protein structures taken from the Protein Data Bank. A first analysis of this set of proteins shows that intron boundaries prefer to be in non-regular secondary structure elements, while avoiding alpha-helices and beta-strands. This fact alone suggests an evolutionary model in which introns are constrained by protein structure, particularly by tertiary structure contacts. In addition, in silico recombination experiments of a subset of these proteins together with their homologues, including those in different species, show that introns have a tendency to occur away from artificial crossover hot spots. Altogether, these findings support a model in which genes can preferentially harbour introns in less constrained regions of the protein fold they code for. In the light of these findings, we discuss some implications for protein modelling and design.

Animals↗

PubMed2003

Functional recycling of C2 domains throughout evolution: a comparative study of synaptotagmin, protein kinase C and phospholipase C by sequence, structural and modelling approaches.

The C2 domain is one of the most frequent and widely distributed calcium-binding motifs. Its structure comprises an eight-stranded beta-sandwich with two structural types as if the result of a circular permutation. Combining sequence, structural and modelling information, we have explored, at different levels of granularity, the functional characteristics of several families of C2 domains. At the coarsest level, the similarity correlates with key structural determinants of the C2 domain fold and, at the finest level, with the domain architecture of the proteins containing them, highlighting the functional diversity between the various sub-families. The functional diversity appears as different conserved surface patches throughout this common fold. In some cases, these patches are related to substrate-binding sites whereas in others they correspond to interfaces of presumably permanent interaction between other domains within the same polypeptide chain. For those related to substrate-binding sites, the predictions overlap with biochemical data in addition to providing some novel observations. For those acting as protein-protein interfaces, our modelling analysis suggests that slight variations between families are a result of not only complementary adaptations in the interfaces involved but also different domain architecture. In the light of the sequence and structural genomic projects, the work presented here shows that modelling approaches along with careful sub-typing of protein families will be a powerful combination for a broader coverage in proteomics.

Amino Acid Sequence↗

PubMed2003

Two separate growth phases during the development of Leishmania in sand flies: implications for understanding the life cycle.

The life cycle of Leishmania alternates between two main morphological forms: intracellular amastigotes in the mammalian host and motile promastigotes in the sand fly vector. Several different forms of promastigote have been described in sandfly infections, the best known of these being metacyclic promastigotes, the mammal-infective stages. Here we provide evidence that for Leishmania (Leishmania) mexicana and Leishmania (Leishmania) infantum (syn. chagasi) there are two separate, consecutive growth cycles during development in Lutzomyia longipalpis sand flies involving four distinct life cycle stages. The first growth cycle is initiated by procyclic promastigotes, which divide in the bloodmeal in the abdominal midgut and subsequently give rise to non-dividing nectomonad promastigotes. Nectomonad forms are responsible for anterior migration of the infection and in turn transform into leptomonad promastigotes that initiate a second growth cycle in the anterior midgut. Subsequently, leptomonad promastigotes differentiate into non-dividing metacyclic promastigotes in preparation for transmission to a mammalian host. Differences in timing, prevalence and persistence of the four promastigote stages were observed between L. mexicana and L. infantum in vivo, which were reproduced in cultures initiated with lesion amastigotes, indicating that development is to some extent governed by a programmed series of events. A new scheme for the life cycle in the subgenus Leishmania (Leishmania) is proposed that incorporates these findings.

Animals↗

PubMed2003

Guided docking: first step to locate potential binding sites.

The long-range electrostatic forces of the targets in round 2 of the Critical Assessment of PRediction of Interactions (CAPRI) experiment were examined and a simple guided docking method, based on these forces, was applied. The method described consists of calculating an initial rigid body trajectory and an optional final, fully flexible refinement stage. Although only limited success was found in predicting the final complexes, some interesting information was discovered. In particular, the long-range forces seem to give some insight into the unusual binding mode of target 4 while raising some questions about target 7, which warrant further investigation.

Algorithms↗

PubMed2003

In silico protein recombination: enhancing template and sequence alignment selection for comparative protein modelling.

Comparative modelling of proteins is a predictive technique to build an atomic model for a given amino acid sequence, on the basis of the structures of other proteins (templates) that have been determined experimentally. Critical problems arise in this procedure: selecting the correct templates, aligning the query sequence with them and building the non-conserved surface loops. In this work, we apply a genetic algorithm, with crossover and mutation, as a new tool to overcome the first two. In silico protein recombination proves to be an effective way to exploit the variability of templates and sequence alignments to produce populations of optimized models by artificial selection. Despite some limitations, the procedure is shown to be robust to alignment errors, while simplifying the task of selecting templates, making it a good candidate for automatic building of reliable protein models.

Algorithms↗

PubMed2003

Novel use of a genetic algorithm for protein structure prediction: searching template and sequence alignment space.

A novel genetic algorithm was applied to all CASP5 targets. The algorithm simultaneously searches template and alignment space. Results show that the current implementation of the method is perhaps most useful in recognizing and refining remote homology targets. This new method is briefly described and results are analyzed. Strengths and weaknesses of the current implementation of the algorithm are discussed.

Algorithms↗

PubMed2003

OPCML at 11q25 is epigenetically inactivated and has tumor-suppressor function in epithelial ovarian cancer.

Epithelial ovarian cancer (EOC), the leading cause of death from gynecological malignancy, is a poorly understood disease. The typically advanced presentation of EOC with loco-regional dissemination in the peritoneal cavity and the rare incidence of visceral metastases are hallmarks of the disease. These features relate to the biology of the disease, which is a principal determinant of outcome. EOC arises as a result of genetic alterations sustained by the ovarian surface epithelium (OSE; ref. 3). The causes of these changes are unknown but are manifest by activation of oncogenes and inactivation of tumor-suppressor genes (TSGs). Our analysis of loss of heterozygosity at 11q25 identified OPCML (also called OBCAM), a member of the IgLON family of immunoglobulin (Ig) domain-containing glycosylphosphatidylinositol (GPI)-anchored cell adhesion molecules, as a candidate TSG in EOC. OPCML is frequently somatically inactivated in EOC by allele loss and by CpG island methylation. OPCML has functional characteristics consistent with TSG properties both in vitro and in vivo. A somatic missense mutation from an individual with EOC shows clear evidence of loss of function. These findings suggest that OPCML is an excellent candidate for the 11q25 ovarian cancer TSG. This is the first description to our knowledge of the involvement of the IgLON family in cancer.

Animals↗

PubMed2002

Reversal of DNA alkylation damage by two human dioxygenases.

The Escherichia coli AlkB protein protects against the cytotoxicity of methylating agents by repair of the DNA lesions 1-methyladenine and 3-methylcytosine, which are generated in single-stranded stretches of DNA. AlkB is an alpha-ketoglutarate- and Fe(II)-dependent dioxygenase that oxidizes the relevant methyl groups and releases them as formaldehyde. Here, we identify two human AlkB homologs, ABH2 and ABH3, by sequence and fold similarity, functional assays, and complementation of the E. coli alkB mutant phenotype. The levels of their mRNAs do not appear to correlate with cell proliferation but tissue distributions are different. Both enzymes remove 1-methyladenine and 3-methylcytosine from methylated polynucleotides in an alpha-ketoglutarate-dependent reaction, and act by direct damage reversal with the regeneration of the unsubstituted bases. AlkB, ABH2, and ABH3 can also repair 1-ethyladenine residues in DNA with the release of acetaldehyde.

Adenine↗

PubMed2002

Different Smad2 partners bind a common hydrophobic pocket in Smad2 via a defined proline-rich motif.

Transforming growth factor-beta (TGF-beta)/activin-induced Smad2/Smad4 complexes are recruited to different promoter elements by transcription factors, such as Fast-1 or the Mix family proteins Mixer and Milk, through a direct interaction between Smad2 and a common Smad interaction motif (SIM) in the transcription factors. Here we identify residues in the SIM critical for Mixer-Smad2 interaction and confirm their functional importance by demonstrating that only Xenopus and zebrafish Mix family members containing a SIM with all the correct critical residues can bind Smad2 and mediate TGF-beta-induced transcriptional activation in vivo. We identify significant sequence similarity between the SIM and the Smad-binding domain (SBD) of the membrane-associated protein SARA (Smad anchor for receptor activation). Molecular modelling, supported by mutational analyses of Smad2 and the SIM and the demonstration that the SARA SBD competes directly with the SIM for binding to Smad2, indicates that the SIM binds Smad2 in the same hydrophobic pocket as does the proline-rich rigid coil region of the SARA SBD. Thus, different Smad2 partners, whether cytoplasmic or nuclear, interact with the same binding pocket in Smad2 through a common proline-rich motif.

3T3 Cells↗

PubMed2002

Developmentally regulated expression of a cell surface class I nuclease in Leishmania mexicana.

Leishmania mexicana, like other trypanosomatid parasites, is a purine auxotroph and must obtain these essential nutrients from its sandfly and mammalian hosts. A single copy gene encoding its unique externally oriented, surface membrane, purine salvage enzyme 3'-nucleotidase/nuclease, was isolated. Structural features of the deduced protein included: an endoplasmic reticulum-directed signal peptide, several conserved class I catalytic and metal co-factor (Zn(2+)) binding domains, transmembrane anchor sequence and a C-terminal cytoplasmic tail. 3'-Nucleotidase/nuclease gene (mRNA) and protein (enzyme activity) expression were examined in three different L. mexicana developmental forms: procyclic promastigotes, metacyclic promastigotes and amastigotes. Results of both approaches demonstrated that the 3'-nucleotidase/nuclease was a stage-specific enzyme, being expressed by promastigote forms (stages restricted to the insect vector), but not by amastigotes (which produce disease in mammalian hosts). Starvation of these parasites for purines resulted in the significant up-regulation of both 3'-nucleotidase/nuclease mRNA and enzyme activity in promastigotes, but not in amastigotes. These results underscore the critical role that the 3'-nucleotidase/nuclease must play in purine salvage during the rapid multiplicative expansion of the parasite population within its insect vector. To our knowledge, the L. mexicana 3'-nucleotidase/nuclease is the first example of a nutrient-induced and developmentally regulated enzyme in any parasitic protozoan.

Amino Acid Sequence↗

PubMed2002

Comparative modelling: an essential methodology for protein structure prediction in the post-genomic era.

The gap between the number of protein sequences and protein structures is increasing rapidly, exacerbated by the completion of numerous genome projects now flooding into public databases. To fill this gap, comparative protein modelling is widely considered the most accurate technique for predicting the three-dimensional shape of proteins. High-throughput, automatic protein modelling should considerably increase our access to protein structures other than those determined by experimental techniques such as X-ray crystallography and NMR (nuclear magnetic resonance) spectroscopy. The uses for these complete three-dimensional models are growing rapidly, ranging from guiding site-directed mutagenesis experiments to protein-protein interaction predictions. In recognition of this, a number of very useful comparative modelling servers have begun to emerge on the Web. Molecular biologists now have a powerful web-based toolkit to construct models, assess their accuracy, and use them to explain and predict experiments. There is, however, still much to do by those engaged in algorithmic development if comparative modelling is to compete on an equal footing with experimental protein structure determination techniques.

Computational Biology↗

PubMedDate not supplied

Flagellar attachment of Leishmania promastigotes to plastic film in vitro.

Trypanosomatid parasites are able to use their flagella for attachment to cuticular surfaces within their arthropod hosts. In this study the attachment mechanism of Leishmania promastigotes was investigated using a new and quantifiable in vitro assay system. The results showed that hemidesmosomal flagellar attachment to three different plastic substrates occurred (Melinex, Polyvinyl, Thermanox). Attachment density was increased by scratching the surface of the substrate or by coating with the hydrocarbons n-octacosane and paraffin. Variation in attachment density was observed, depending on the culture medium and the parasite isolate used. All four species examined, L. braziliensis, L. donovani, L. major and L. mexicana, were capable of flagellar attachment in vitro. Collectively, these data indicate that flagellar attachment is mediated by a non-specific hydrophobic interaction in Leishmania species.

Animals↗

PubMedDate not supplied

A link between sequence conservation and domain motion within the AAA+ family.

The AAA+ family of proteins play fundamental roles in all three kingdoms of life. It is thought that they act as molecular chaperones in aiding the assembly or disassembly of proteins or protein complexes. Recent structural studies on a number of AAA+ family proteins have revealed that they share similar structural elements. These structures provide a possible link between nucleotide binding/hydrolysis and the conformational changes which are then amplified to generate mechanical forces for their specific functions. However, from these individual studies it is far from clear whether AAA+ proteins in general share properties in terms of nucleotide induced conformational changes. In this study, we analyze sequence conservation within the AAA+ family and identify two subfamilies, each with a distinct conserved linker sequence that may transfer conformational changes upon ATP binding/release to movements between subdomains and attached domains. To investigate the relation of these linker sequences to conformational changes, molecular dynamics (MD) simulations on X-ray structures of AAA+ proteins from each subfamily have been performed. These simulations show differences in both the N-linker peptide, subdomain motion, and cooperativity between elements of quaternary structure. Extrapolation of subdomain movements from one MD simulation enables us to produce a structure in close agreement with cryo-EM experiments.

Adenosine Triphosphatases↗

PubMedDate not supplied

C-reactive protein-mediated phagocytosis of Leishmania donovani promastigotes does not alter parasite survival or macrophage responses.

C-reactive protein (CRP) is an acute phase protein that binds to surface structures of a number of different organisms. Leishmania donovani express CRP ligand when first entering the mammalian host and CRP has been shown to alter macrophage function. The aim of this study was to investigate the functional significance of CRP-mediated uptake of L. donovani on survival of the parasite within human macrophages and macrophage cell responses to the infection. CRP opsonized L. donovani uptake was inhibitable by including excess CRP in the fluid phase, suggesting Fc receptor usage rather than indirect complement-mediated uptake. Comparing equivalent initial infection loads, parasite survival over 72 h within peripheral blood derived macrophages (PBMs) and differentiated U937 cells was unaltered by CRP. Whereas CRP increased macrophage responses to phosphorylcholine coated erythrocytes, no significant alteration in tumour necrosis factor-alpha, interleukin (IL)-10 or IL-12 production from PBMs was observed between CRP opsonized or unopsonized L. donovani promastigotes. Thus, in contrast to other systems, where CRP opsonization results in macrophage activation, Leishmania can use CRP to improve infection without inducing detrimental macrophage activation.

Animals↗