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Biomedical subjects

Peter Simpson

Publications and source records attributed to Peter Simpson.

At least 19 recordsLinked to original sources
PubMed2006

The solution structure of the invasive tip complex from Afa/Dr fibrils.

Afa/Dr family of adhesins are produced by pathogenic Escherichia coli strains that are especially prevalent in chronic diarrhoeal and recurrent urinary tract infections. Most notably, they are found in up to 50% of cystitis cases in children and 30% of pyelonephritis in pregnant women. Afa/Dr adhesins are capped surface fibrils that mediate recognition of the host and subsequent bacterial internalization. Using the newly solved three-dimensional structure of the minimal invasive complex (AfaDE) combined with biochemical and cellular assays, we reveal the architecture of the fibrillar cap and identify a novel mode of synergistic integrin recognition.

Adhesins, Escherichia coli↗
PubMed2006

Embryonic growth-associated protein is one subunit of a novel N-terminal acetyltransferase complex essential for embryonic vascular development.

N-terminal protein acetylation, catalyzed by N-terminal acetyltransferases (NATs) recognizing distinct N-terminal sequences, is gaining recognition as an essential regulator of normal cell function, but little is known of its role in vertebrate development. We previously cloned a novel gene, embryonic growth-associated protein (EGAP), the expression of which is associated with rapid vascular smooth muscle cell proliferation during development. We show herein EGAP is the mammalian/zebrafish homologue of yeast Mak10p, one subunit of the yeast NatC complex, and describe the cloning of its binding partners Mak3 and Mak31. The EGAP NAT forms a functional complex in mammalian cells, is evolutionarily conserved, and developmentally regulated. It is widely but not ubiquitously expressed during early zebrafish development but undetectable in later developmental stages. We demonstrate EGAP- and Mak3-deficient zebrafish fail to develop because of, in part, decreased cell proliferation, increased apoptosis, and poor blood vessel formation contributing to embryonic lethality. We examined the role of target of rapamycin (TOR), a highly conserved protein kinase controlling cell growth, as a physiological target of EGAP NAT acetylation. Compared with controls, TOR expression and signaling is significantly reduced in EGAP morphants. Pharmacological inhibition of TOR with rapamycin phenocopied the EGAP morpholino oligonucleotide-induced growth and vessel defects. Overexpression of constitutively active TOR rescued EGAP morphants, suggesting TOR is a direct or indirect endogenous substrate of the EGAP NAT complex. These data suggest the EGAP NAT complex is an essential regulatory enzyme controlling the function of a subset of proteins required for embryonic growth control and vessel development.

Acetylation↗
PubMed2006

Face transplants.

Explore the source record for details and available documents.

Facial Injuries↗
PubMed2006

DC-SIGN and CLEC-2 mediate human immunodeficiency virus type 1 capture by platelets.

Platelets can engulf human immunodeficiency virus type 1 (HIV-1), and a significant amount of HIV-1 in the blood of infected individuals is associated with these cells. However, it is unclear how platelets capture HIV-1 and whether platelet-associated virus remains infectious. DC-SIGN and other lectins contribute to capture of HIV-1 by dendritic cells (DCs) and facilitate HIV-1 spread in DC/T-cell cocultures. Here, we show that platelets express both the C-type lectin-like receptor 2 (CLEC-2) and low levels of DC-SIGN. CLEC-2 bound to HIV-1, irrespective of the presence of the viral envelope protein, and facilitated HIV-1 capture by platelets. However, a substantial fraction of the HIV-1 binding activity of platelets was dependent on DC-SIGN. A combination of DC-SIGN and CLEC-2 inhibitors strongly reduced HIV-1 association with platelets, indicating that these lectins are required for efficient HIV-1 binding to platelets. Captured HIV-1 was maintained in an infectious state over several days, suggesting that HIV-1 can escape degradation by platelets and might use these cells to promote its spread. Our results identify CLEC-2 as a novel HIV-1 attachment factor and provide evidence that platelets capture and transfer infectious HIV-1 via DC-SIGN and CLEC-2, thereby possibly facilitating HIV-1 dissemination in infected patients.

Animals↗
PubMed2006

Expression profiling using cDNA microarrays.

Microarray technology is revolutionizing the assessment of gene expression in human disease. Coupled with the publication of the human genome, it is becoming possible to measure the relevant levels of all 30,000-40,000 human genes in a single experiment. The applications of these methods to breast cancer research are helping us to both further our understanding of the biology of mammary tumorigenesis, as well as providing novel subclassifications of breast cancer of direct clinical relevance. Unbiased RNA amplification techniques are allow the study of small biopsy samples as well as archival pathology specimens, increasing our molecular understanding, and providing real hope for the clinical application of gene expression profiling.

Breast Neoplasms↗
PubMed2006

Gene expression analysis using filter cDNA microarrays.

The analysis of gene expression patterns by filter-based complementary (c)DNA microarray remains an important technique in the molecular biology laboratory, despite the development of large-scale cDNA microarray analysis (see Chapter 27). This chapter provides an overview of the methods necessary to carry out the production of membrane-based cDNA arrays and the subsequent synthesis and hybridization of radiolabeled cDNA probes.

Animals↗
PubMed2005

A novel galectin-like domain from Toxoplasma gondii micronemal protein 1 assists the folding, assembly, and transport of a cell adhesion complex.

Immediately prior to invasion Toxoplasma gondii tachyzoites release a large number of micronemal proteins (TgMICs) that participate in host cell attachment and penetration. The TgMIC4-MIC1-MIC6 complex was the first to be identified in T. gondii and has been recently shown to be critical in invasion. This study establishes that the N-terminal thrombospondin type I repeat-like domains (TSR1-like) from TgMIC1 function as an independent adhesin as well as promoting association with TgMIC4. Using the newly solved three-dimensional structure of the C-terminal domain of TgMIC1 we have identified a novel Galectin-like fold that does not possess carbohydrate binding properties and redefines the architecture of TgMIC1. Instead, the TgMIC1 Galectin-like domain interacts and stabilizes TgMIC6, which provides the basis for a highly specific quality control mechanism for successful exit from the early secretory compartments and for subsequent trafficking of the complex to the micronemes.

Animals↗
PubMed2005

Solution structure and interactions of the Escherichia coli cell division activator protein CedA.

CedA is a protein that is postulated to be involved in the regulation of cell division in Escherichia coli and related organisms; however, little biological data about its possible mode of action are available. Here we present a three-dimensional structure of this protein as determined by NMR spectroscopy. The protein is made up of four antiparallel beta-strands, an alpha-helix, and a large unstructured stretch of residues at the N-terminus. It shows structural similarity to a family of DNA-binding proteins which interact with dsDNA via a three-stranded beta-sheet, suggesting that CedA may be a DNA-binding protein. The putative binding surface of CedA is predominantly positively charged with a number of basic residues surrounding a groove largely dominated by aromatic residues. NMR chemical shift perturbations and gel-shift experiments performed with CedA confirm that the protein binds dsDNA, and its interaction is mediated primarily via the beta-sheet.

Amino Acid Motifs↗
PubMed2005

Zebrafish narrowminded disrupts the transcription factor prdm1 and is required for neural crest and sensory neuron specification.

Specification of both neural crest cells and Rohon-Beard (RB) sensory neurons involves a complex series of interactions between the neural and non-neural ectoderm. The molecular mechanisms directing this process are not well understood. The zebrafish narrowminded (nrd) mutation is unique, since it is one of two mutations in which defects are observed in both cell populations: it leads to a complete absence of RB neurons and a reduction in neural crest cells and their derivatives. Here, we show that nrd is a mutation in prdm1, a SET/zinc-finger domain transcription factor. A Morpholino-mediated depletion of prdm1 phenocopies the nrd mutation, and conversely overexpression of prdm1 mRNA rescues the nrd RB sensory neuron and neural crest phenotype. prdm1 is expressed at the border of the neural plate within the domain where neural crest cells and RB sensory neurons form. Analysis of prdm1 function by overexpression indicates that prdm1 functions to promote the cell fate specification of both neural crest cells and RB sensory neurons, most likely as a downstream effector of the BMP signaling pathway.

Animals↗
PubMed2005

The polypyrimidine tract binding protein is a monomer.

The polypyrimidine tract binding (PTB) protein is a potent regulator of alternative mRNA splicing. It also participates in other essential cellular functions, including translation initiation and polyadenylation. Several published reports have suggested that the protein forms a dimer in solution, a feature that has been widely incorporated into mechanistic models of protein function. However, recent studies have provided indications that full-length PTB is a monomer. Here we present new biophysical and biochemical evidence supporting the monomeric status of the protein. By use of blue-native polyacrylamide gel electrophoresis and size-exclusion chromatography, PTB was observed as a single molecular species under native reducing environments, though in oxidizing conditions, a larger protein species was also detected. Further analyses of wild-type and mutant PTB molecules with SDS-PAGE and time-of-flight electrospray ionization mass spectroscopy confirmed these observations. They also identified the single reduced species as monomeric PTB and the higher-molecular-weight nonreduced species as disulphide-linked PTB dimer mediated by Cys23. Our results indicate that the use of oxidizing environments in previous studies is likely to have contributed to the mis-assignment of PTB as a dimer. Although purified PTB may form disulphide-linked dimers under these conditions, in the reducing intracellular environment the protein will be monomeric. These findings have implications for the construction of models of PTB function in regulating mRNA metabolism.

Alternative Splicing↗
PubMed2005

Assessing the sensitivity of different life stages for sexual disruption in roach (Rutilus rutilus) exposed to effluents from wastewater treatment works.

Surveys of U.K. rivers have shown a high incidence of sexual disruption in populations of wild roach (Rutilus rutilus) living downstream from wastewater treatment works (WwTW), and the degree of intersex (gonads containing both male and female structural characteristics) has been correlated with the concentration of effluent in those rivers. In this study, we investigated feminized responses to two estrogenic WwTWs in roach exposed for periods during life stages of germ cell division (early life and the postspawning period). Roach were exposed as embryos from fertilization up to 300 days posthatch (dph; to include the period of gonadal sex differentiation) or as postspawning adult males, and including fish that had received previous estrogen exposure, for either 60 or 120 days when the annual event of germ cell proliferation occurs. Both effluents induced vitellogenin synthesis in both life stages studied, and the magnitude of the vitellogenic responses paralleled the effluent content of steroid estrogens. Feminization of the reproductive ducts occurred in male fish in a concentration-dependent manner when the exposure occurred during early life, but we found no effects on the reproductive ducts in adult males. Depuration studies (maintenance of fish in clean water after exposure to WwTW effluent) confirmed that the feminization of the reproductive duct was permanent. We found no evidence of ovotestis development in fish that had no previous estrogen exposure for any of the treatments. In wild adult roach that had previously received exposure to estrogen and were intersex, the degree of intersex increased during the study period, but this was not related to the immediate effluent exposure, suggesting a previously determined programming of ovotestis formation.

Animals↗
PubMed2004

Repression of nodal expression by maternal B1-type SOXs regulates germ layer formation in Xenopus and zebrafish.

B1-type SOXs (SOXs 1, 2, and 3) are the most evolutionarily conserved subgroup of the SOX transcription factor family. To study their maternal functions, we used the affinity-purified antibody antiSOX3c, which inhibits the binding of Xenopus SOX3 to target DNA sequences [Development. 130(2003)5609]. The antibody also cross-reacts with zebrafish embryos. When injected into fertilized Xenopus or zebrafish eggs, antiSOX3c caused a profound gastrulation defect; this defect could be rescued by the injection of RNA encoding SOX3DeltaC-EnR, a SOX3-engrailed repression domain chimera. In antiSOX3c-injected Xenopus embryos, normal animal-vegetal patterning of mesodermal and endodermal markers was disrupted, expression domains were shifted toward the animal pole, and the levels of the endodermal markers SOX17 and endodermin increased. In Xenopus, SOX3 acts as a negative regulator of Xnr5, which encodes a nodal-related TGFbeta-family protein. Two nodal-related proteins are expressed in the early zebrafish embryo, squint and cyclops; antiSOX3c-injection leads to an increase in the level of cyclops expression. In both Xenopus and zebrafish, the antiSOX3c phenotype was rescued by the injection of RNA encoding the nodal inhibitor Cerberus-short (CerS). In Xenopus, antiSOX3c's effects on endodermin expression were suppressed by injection of RNA encoding a dominant negative version of Mixer or a morpholino against SOX17alpha2, both of which act downstream of nodal signaling in the endoderm specification pathway. Based on these data, it appears that maternal B1-type SOX functions together with the VegT/beta-catenin system to regulate nodal expression and to establish the normal pattern of germ layer formation in Xenopus. A mechanistically conserved system appears to act in a similar manner in the zebrafish.

Amino Acid Sequence↗
PubMed2004

An atomic resolution model for assembly, architecture, and function of the Dr adhesins.

Pathogenic bacteria possess adhesion protein complexes that play essential roles in targeting host cells and in propagating infection. Although each family of adhesion proteins is generally associated with a specific human disease, the Dr family from Escherichia coli is a notable exception, as its members are associated with both diarrheal and urinary tract infections. These proteins are reported to form both fimbrial and afimbrial structures at the bacterial cell surface and target a common host cell receptor, the decay-accelerating factor (DAF or CD55). Using the newly solved three-dimensional structure of AfaE, we have constructed a robust atomic resolution model that reveals the structural basis for assembly by donor strand complementation and for the architecture of capped surface fibers.

Adhesins, Escherichia coli↗
PubMed2004

High resolution studies of the Afa/Dr adhesin DraE and its interaction with chloramphenicol.

Pathogenic Escherichia coli expressing Afa/Dr adhesins are able to cause both urinary tract and diarrheal infections. The Afa/Dr adhesins confer adherence to epithelial cells via interactions with the human complement regulating protein, decay accelerating factor (DAF or CD55). Two of the Afa/Dr adhesions, AfaE-III and DraE, differ from each other by only three residues but are reported to have several different properties. One such difference is disruption of the interaction between DraE and CD55 by chloramphenicol, whereas binding of AfaE-III to CD55 is unaffected. Here we present a crystal structure of a strand-swapped trimer of wild type DraE. We also present a crystal structure of this trimer in complex with chloramphenicol, as well as NMR data supporting the binding position of chloramphenicol within the crystal. The crystal structure reveals the precise atomic basis for the sensitivity of DraE-CD55 binding to chloramphenicol and demonstrates that in contrast to other chloramphenicol-protein complexes, drug binding is mediated via recognition of the chlorine "tail" rather than via intercalation of the benzene rings into a hydrophobic pocket.

Adhesins, Bacterial↗
PubMed2004

Structure, dynamics and interactions of p47, a major adaptor of the AAA ATPase, p97.

p47 is a major adaptor molecule of the cytosolic AAA ATPase p97. The principal role of the p97-p47 complex is in regulation of membrane fusion events. Mono-ubiquitin recognition by p47 has also been shown to be crucial in the p97-p47-mediated Golgi membrane fusion events. Here, we describe the high-resolution solution structures of the N-terminal UBA domain and the central domain (SEP) from p47. The p47 UBA domain has the characteristic three-helix bundle fold and forms a highly stable complex with ubiquitin. We report the interaction surfaces of the two proteins and present a structure for the p47 UBA-ubiquitin complex. The p47 SEP domain adopts a novel fold with a betabetabetaalphaalphabeta secondary structure arrangement, where beta4 pairs in a parallel fashion to beta1. Based on biophysical studies, we demonstrate a clear propensity for the self-association of p47. Furthermore, p97 N binding abolishes p47 self-association, revealing the potential interaction surfaces for recognition of other domains within p97 or the substrate.

Adaptor Proteins, Signal Transducing↗