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Biomedical subjects

Malcolm W Kennedy

Publications and source records attributed to Malcolm W Kennedy.

At least 19 recordsLinked to original sources
PubMed2006

Crystallization of Ranasmurfin, a blue-coloured protein from Polypedates leucomystax.

Ranasmurfin, a previously uncharacterized approximately 13 kDa blue protein found in the nests of the frog Polypedates leucomystax, has been purified and crystallized. The crystals are an intense blue colour and diffract to 1.51 A with P2(1) symmetry and unit-cell parameters a = 40.9, b = 59.9, c = 45.0 A, beta = 93.3 degrees . Self-rotation function analysis indicates the presence of a dimer in the asymmetric unit. Biochemical data suggest that the blue colour of the protein is related to dimer formation. Sequence data for the protein are incomplete, but thus far have identified no model for molecular replacement. A fluorescence scan shows a peak at 9.676 keV, indicating that the protein binds zinc and suggesting a route for structure solution.

Animals↗
PubMed2006

The perils and prospects of using phytohaemagglutinin in evolutionary ecology.

Several techniques are available for quantifying the vertebrate immune response, information that is particularly useful for understanding the contribution of immunity to the evolution of life-history strategies. The most widely used is the phytohaemagglutinin (PHA) skin-swelling technique, which is usually regarded as an index of acquired immunity. However, our understanding of the effects of PHA in skin is poor, despite the fact that it has implications for what the test can tell us about immune activity. As we discuss here, a recent study by Martin and colleagues on the response to PHA at the cellular level in wild birds has highlighted the relative extent to which PHA-induced swelling, as most commonly applied, measures innate immunity versus acquired immunity.

Animals↗
PubMed2006

A novel lipoxygenase in pea roots. Its function in wounding and biotic stress.

The genome of pea (Pisum sativum) contains genes encoding a family of distinct lipoxygenases (LOX). Among these, LOXN2 showed eight exons encoding a 93.7-kD enzyme, harboring two C-terminal deletions and an unusual arginine/threonine-tyrosine motif in the domain considered to control the substrate specificity. LOXN2, when overexpressed in yeast, exhibited normal enzyme activity with an optimum at pH 4.5, and a dual positional specificity by releasing a 3:1 ratio of C-9 and C-13 oxidized products. The predicted LOXN2 structure lacked a loop present in soybean (Glycine max) LOX1, in a position consistent with control of the degree of substrate access to the catalytic site and for LOXN2's dual positional specificity. The LOXN2 gene was tightly conserved in the Progress 9 and MG103738 genotypes, respectively, susceptible and resistant to the root cyst nematode Heterodera goettingiana. LOXN2 transcription was monitored in roots after mechanical injury and during nematode infection. The message peaked at 3 and 24 h after wounding in both genotypes and was more abundant in the resistant than in the susceptible pea. In nematode-infected roots, transcription of several LOX genes was triggered except LOXN2, which was repressed in both genotypes. In situ hybridization revealed that LOXN2 message was widespread in the cortex and endodermis of healthy roots, but specifically localized at high level in the cells bordering the nematode-induced syncytia of infected roots. However, LOXN2 transcript signal was particularly intense in collapsing syncytia of MG103738 roots, suggesting LOXN2 involvement in late mechanisms of host resistance.

Adaptation, Physiological↗
PubMed2006

Zn-alpha2-glycoprotein, an MHC class I-related glycoprotein regulator of adipose tissues: modification or abrogation of ligand binding by site-directed mutagenesis.

Zn-alpha(2)-glycoprotein (ZAG) is a soluble lipid-mobilizing factor associated with cancer cachexia and is a novel adipokine. Its X-ray crystal structure reveals a poly(ethylene glycol) molecule, presumably substituting for a higher affinity natural ligand, occupying an apolar groove between its alpha(1) and alpha(2) domain helices that corresponds to the peptide binding groove in class I MHC proteins. We previously provided evidence that the groove is a binding site for hydrophobic ligands that may relate to the protein's signaling function and that the natural ligands are probably (polyunsaturated) fatty acid-like. Using fluorescence-based binding assays and site-directed mutagenesis, we now demonstrate formally that the groove is indeed the binding site for hydrophobic ligands. We also identify amino acid positions that are involved in ligand binding and those that control the shape and exposure to solvent of the binding site itself. Some of the mutants showed minimal effects on their binding potential, one showed enhanced binding, and several were completely nonbinding. Particularly notable is Arg-73, which projects into one end of the binding groove and is the sole charged amino acid adjacent to the ligand. Replacing this amino acid with alanine abolished ligand binding and closed the groove to solvent. Arg-73 may therefore have an unexpected dual role in binding site access and anchor for an amphiphilic ligand. These data add weight to the distinctiveness of ZAG among MHC class I-like proteins in addition to providing defined binding-altered mutants for cellular signaling studies and potential medical applications.

Amino Acid Sequence↗
PubMed2005

Allergen-specific IgE and IgG4 are markers of resistance and susceptibility in a human intestinal nematode infection.

IgG4 has been proposed to act as a 'blocking antibody' due to its ability to compete for the same epitopes as IgE thus preventing IgE-dependent allergic responses. IgG4 and IgE are both elevated in helminth infections and strong anti-parasite IgE responses are associated with resistance to infection. We wished to determine the relationship between anti-parasite IgG4 and IgE and Ascaris lumbricoides infection status. We examined anti-parasite responses, including antibody levels to recombinant Ascaris allergen-1A (rABA-1A), a target of serum IgE in endemic populations. Worm burden was indirectly estimated by measuring parasite egg output in a cross-sectional human population (N = 105). Levels of anti-parasite IgG4 and IgE in patients' plasma were quantified by immunoassay. Global anti-parasite antibody responses did not bear any significant relationships with intensity of Ascaris infection. Individuals who had detectable levels of IgE but not IgG4 to rABA-1A (11%) had lower average levels of infection compared with individuals who produced anti-rABA-1A IgG4 (40%) and sero-negative individuals (49%) (P = 0.008). The ratio of IgG4/IgE in rABA-1A responders positively correlated with intensity of infection (P < 0.025). IgG4 levels positively correlated with infection level in younger children (age 4-11) where average levels of infection were increasing (P = 0.038), whereas allergen specific IgE emerged as a correlate of immunity in older children and adults (age 12-36) where infection levels were decreasing (P = 0.048). Therefore, in a gastrointestinal helminth infection, differential regulation of anti-allergen antibody isotypes relate to infection level. Our results are consistent with the concept that IgG4 antibody can block IgE-mediated immunity and therefore allergic processes in humans.

Adolescent↗
PubMed2004

Adsorption of frog foam nest proteins at the air-water interface.

The surfactant properties of aqueous protein mixtures (ranaspumins) from the foam nests of the tropical frog Physalaemus pustulosus have been investigated by surface tension, two-photon excitation fluorescence microscopy, specular neutron reflection, and related biophysical techniques. Ranaspumins lower the surface tension of water more rapidly and more effectively than standard globular proteins under similar conditions. Two-photon excitation fluorescence microscopy of nest foams treated with fluorescent marker (anilinonaphthalene sulfonic acid) shows partitioning of hydrophobic proteins into the air-water interface and allows imaging of the foam structure. The surface excess of the adsorbed protein layers, determined from measurements of neutron reflection from the surface of water utilizing H(2)O/D(2)O mixtures, shows a persistent increase of surface excess and layer thickness with bulk concentration. At the highest concentration studied (0.5 mg ml(-1)), the adsorbed layer is characterized by three distinct regions: a protruding top layer of approximately 20 angstroms, a middle layer of approximately 30 angstroms, and a more diffuse submerged layer projecting some 25 angstroms into bulk solution. This suggests a model involving self-assembly of protein aggregates at the air-water interface in which initial foam formation is facilitated by specific surfactant proteins in the mixture, further stabilized by subsequent aggregation and cross-linking into a multilayer surface complex.

Adsorption↗
PubMed2004

Crystallographic studies of ligand binding by Zn-alpha2-glycoprotein.

Zn-alpha2-glycoprotein (ZAG) is a 41 kDa soluble protein that is present in most bodily fluids. The previously reported 2.8 A crystal structure of ZAG isolated from human serum demonstrated the structural similarity between ZAG and class I major histocompatibility complex (MHC) molecules and revealed a non-peptidic ligand in the ZAG counterpart of the MHC peptide-binding groove. Here we present crystallographic studies to explore further the nature of the non-peptidic ligand in the ZAG groove. Comparison of the structures of several forms of recombinant ZAG, including a 1.95 A structure derived from ZAG expressed in insect cells, suggests that the non-peptidic ligand in the current structures and in the structure of serum ZAG is a polyethylene glycol (PEG), which is present in the crystallization conditions used. Further support for PEG binding in the ZAG groove is provided by the finding that PEG displaces a fluorophore-tagged fatty acid from the ZAG binding site. From these results we hypothesize that our purified forms of ZAG do not contain a bound endogenous ligand, but that the ZAG groove is capable of binding hydrophobic molecules, which may relate to its function.

Adipokines↗
PubMed2003

Direct measurement of antigen binding properties of CD1 proteins using fluorescent lipid probes.

CD1 proteins are antigen-presenting molecules that bind foreign and self-lipids and stimulate specific T cell responses. In the current study, we investigated ligand binding by CD1 proteins by developing a fluorescent probe binding approach using soluble recombinant human CD1 proteins. To increase stability and yield, soluble group 1 CD1 (CD1b and CD1c) and group 2 CD1 (CD1d) proteins were produced as single chain secreted CD1 proteins in which beta2-microglobulin was fused to the N termini of the CD1 heavy chains by a flexible peptide linker sequence. Analysis of ligand binding properties of single chain secreted CD1 proteins by using fluorescent lipid probes indicated significant differences in ligand preference and in pH dependence of binding by group 1 versus group 2 CD1 proteins. Whereas group 1 CD1 isoforms (CD1b and CD1c) show stronger binding of nitrobenzoxadiazole (NBD)-labeled dialkyl-based ligands (phosphatidylcholine, sphingomyelin, and ceramide), group 2 CD1 (CD1d) proteins were stronger binders of small hydrophobic probes such as 1-anilinonaphthalene-8-sulfonic acid and 4,4'-dianilino-1,1'-naphthyl-5,5'-disulfonic acid. Competition studies indicated that binding of fluorescent lipid probes involved association of the probe with the hydrophobic ligand binding groove of CD1 proteins. Analysis of selected alanine substitution mutants of human CD1b known to inhibit antigen presentation showed that NBD-labeled lipid probe binding could be used to distinguish mutations that interfere with ligand binding from those that affect T cell receptor docking. Our findings provide further evidence for the functional specialization of different CD1 isoforms and demonstrate the value of the fluorescent lipid probe binding method for assisting structure-based studies of CD1 function.

Amino Acid Sequence↗
PubMed2003

Investigating the impact of helminth products on immune responsiveness using a TCR transgenic adoptive transfer system.

Helminth infections and their products have a potent immunomodulatory effect on the host immune system and can impair immune responses against unrelated Ags. In vitro studies have suggested that the immunomodulation by helminth extracts may be the result of bystander response bias toward a Th2 phenotype and/or an Ag-specific T lymphocyte proliferative hyporesponsiveness. The aim of this study was to determine the role of these potential mechanisms of immunosuppression in vivo. Therefore, using a sensitive model of CFSE-labeled OVA-specific TCR transgenic T lymphocyte adoptive transfer, we analyzed the effect of Ascaris suum body fluid (ABF) on the kinetics and amplitude of a primary OVA-specific T cell response as well as the Th1/Th2 profile of the response in wild-type and IL-4 knockout (KO) mice. We find that inhibition of delayed-type hypersensitivity by ABF was associated with a Th1/Th2 shift in wild-type animals, but not in IL-4 KO mice. The use of this model has allowed us to demonstrate that although the kinetics of the OVA-specific primary response was not affected by ABF, the expansion of the OVA-specific T lymphocytes was significantly inhibited in both wild-type and IL-4 KO mice. This inhibition was associated with a reduced proliferative capacity of these cells in vivo, distinct from anergy.

Adoptive Transfer↗
PubMed2003

The polyprotein and FAR lipid binding proteins of nematodes: shape and monomer/dimer states in ligand-free and bound forms.

Nematodes produce two classes of small, helix-rich fatty acid- and retinol-binding proteins whose structures and in vivo functions remain to be elucidated. These are the polyprotein allergens (NPA) and the FAR proteins. The solution properties of recombinant forms of these proteins from parasitic [Ascaris suum (As) and Onchocerca volvulus (Ov)] and free-living [Caenorhabditis elegans (Ce)] nematodes have been examined. Analytical ultracentrifugation (AUC) showed that, contrary to previous findings, the rAs-NPA-1A polyprotein unit (approximately 15 kDa) is a monomer, and this stoichiometry is unaltered by ligand (oleic acid) binding. The rOv-FAR-1 and rCe-FAR-5 proteins differ in that the former forms a tight dimer and the latter a monomer, and these oligomeric states are also unaffected by ligand binding or protein concentration. Sedimentation equilibrium experiments showed that the partial specific volume v of the unliganded proteins agree well with the value calculated from amino acid composition extrapolated to experimental temperature, and was unaffected upon ligand binding. Data from small-angle X-ray scattering (SAXS) indicated that both of the monomeric proteins rAs-NPA-1A and rCe-FAR-5 are globular, although slightly elongated and flattened. These data are in good agreement with shapes predicted from sedimentation velocity experiments and hydrodynamic bead modelling. On the basis of functional and secondary structural homology with the ligand-binding domain of the retinoic acid receptor RXRalpha, de novo atomic resolution structures for rAs-NPA-1A and rCe-FAR-5 have been constructed which are consistent with the SAXS and hydrodynamic data.

Allergens↗
PubMed2003

Ac-FAR-1, a 20 kDa fatty acid- and retinol-binding protein secreted by adult Ancylostoma caninum hookworms: gene transcription pattern, ligand binding properties and structural characterisation.

Antibody against adult Ancylostoma caninum excretory-secretory (ES) products was used to immunoscreen a cDNA expression library leading to the isolation of cDNAs encoding putative hookworm fatty-acid and retinol-binding proteins. Ac-far-1 and Ac-far-2 cDNAs encode open reading frames corresponding to approximately 20kDa proteins with 91 percent amino acid identity. Ac-FAR-1 and Ac-FAR-2 exhibit clear similarities to other FARs of parasitic nematodes, most closely to two of the FAR proteins of Caenorhabditis elegans (Ce-FAR-1 and Ce-FAR-2). By reverse transcriptase polymerase chain reaction (RT-PCR) assay, Ac-far-1 mRNA was detected in both adult and third-stage larvae of A. caninum. However, the respective proteins were detectable by immunoblot only in adult hookworm ES products and adult extracts. Using fluorescence-based binding assays, bacterial recombinant Ac-FAR-1 was found to bind fatty acids and retinol (Vitamin A) with dissociation constants in the micromolar region. Circular dichroism spectra indicated that Ac-FAR-1 possesses a high level of alpha-helix, similar to Ov-FAR-1 from Onchocerca volvulus. This is the first demonstration of a functional FAR secreted by adult hookworms and provides further evidence that FAR proteins secreted by parasitic nematodes are crucial to parasitism.

Amino Acid Sequence↗
PubMed2002

The FAR protein family of the nematode Caenorhabditis elegans. Differential lipid binding properties, structural characteristics, and developmental regulation.

Parasitic nematodes of humans and plants secrete a structurally novel type of fatty acid- and retinol-binding protein, FAR, into the tissues they occupy. These proteins may interfere with intercellular lipid signaling to manipulate the defense reactions of the host or acquire essential lipids for the parasites. The genome of the nematode Caenorhabditis elegans encodes eight FAR-like proteins (Ce-FAR-1 to -8). These fall into three discrete groups as indicated by phylogenetic sequence comparisons and intron positions, the proteins from parasitic nematodes falling into group A. Recombinant Ce-FAR-1 to -7 were produced in Escherichia coli and tested for lipid binding in fluorescence-based assays. Ce-FAR-1 to -6 bound DAUDA (11-((5-dimethylaminonaphthalene-1-sulfonyl)amino)undecanoic acid), cis-parinaric acid, and retinol with dissociation constants in the micromolar range, whereas Ce-FAR-7 bound the latter two lipids relatively poorly. Each protein produced a characteristic shift in peak fluorescence emission of DAUDA, and one (Ce-FAR-5) produced a shift greater than has been observed previously for any lipid-binding protein. Selected Ce-FAR proteins were analyzed by circular dichroism (CD) and differential scanning calorimetry, were found to be helix-rich, and exhibited high thermal stability (transition midpoint, 82.7 degrees C). CD and secondary structure predictions, however, both indicated that Ce-FAR-7 possesses substantially less helix than the other FAR proteins. The genes encoding the Ce-FAR proteins were found to be transcribed differentially through the life cycle of C. elegans, such that Ce-far-4 was transcribed at highest levels in the fourth larval stage, and Ce-far-3 and -7 predominated in males.

Amino Acid Sequence↗
PubMed2002

The FAR proteins of parasitic nematodes: their possible involvement in the pathogenesis of infection and the use of Caenorhabditis elegans as a model system to evaluate their function.

Parasitic nematodes secrete a structurally novel class of fatty acid and retinol-binding (FAR) protein into the surrounding tissues of the host. These proteins are of interest because they may play an important role in scavenging fatty acids and retinoids from the host that are essential for the survival of the parasite and also because the localised depletion of such lipids may have immunomodulatory effects that compromise the host immune response. Research into the biological function(s) of the FAR proteins has been severely hampered by the difficulties associated with the life-cycle propagation of parasitic nematodes and the current intractability of the parasites to reverse-genetic studies. The genome of the free-living nematode Caenorhabditis elegans, however, encodes eight FAR-like proteins, and in this review we compare the FAR proteins of C. elegans and parasitic nematodes, and we discuss the suitability of C. elegans as a model system to investigate the biological function(s) of the FAR proteins of parasitic nematodes.

Amino Acid Sequence↗
PubMed2002

How helminth lipid-binding proteins offload their ligands to membranes: differential mechanisms of fatty acid transfer by the ABA-1 polyprotein allergen and Ov-FAR-1 proteins of nematodes and Sj-FABPc of schistosomes.

Three different classes of small lipid-binding protein (LBP) are found in helminth parasites. Although of similar size, the ABA-1A1 (also designated As-NPA-A1) and Ov-FAR-1 (formerly known as Ov20) proteins of nematodes are mainly alpha-helical and have no known structural counterparts in mammals, whereas Sj-FABPc of schistosomes is predicted to form a beta-barrel structure similar to the mammalian family of intracellular fatty acid binding proteins. The parasites that produce these proteins are unable to synthesize their own complex lipids and, instead, rely entirely upon their hosts for supply. As a first step in elucidating whether these helminth proteins are involved in the acquisition of host lipid, the process by which these LBPs deliver their ligands to acceptor membranes was examined, by comparing the rates and mechanisms of ligand transfer from the proteins to artificial phospholipid vesicles using a fluorescence resonance energy transfer assay. All three proteins bound the fluorescent fatty acid 2-(9-anthroyloxy)palmitic acid (2AP) similarly, but there were clear differences in the rates and mechanisms of fatty acid transfer. Sj-FABPc displayed a collisional mechanism; 2AP transfer rates increased with acceptor membrane concentration, were modulated by acceptor membrane charge, and were not diminished in the presence of increasing salt concentrations. In contrast, transfer of ligand from Ov-FAR-1 and ABA-1A1 involved an aqueous diffusion step; transfer rates from these proteins were not modulated by acceptor membrane concentration or charge but did decrease with the ionic strength of the buffer. Despite these differences, all of the proteins interacted directly with membranes, as determined using a cytochrome c competition assay, although Sj-FABPc interacted to a greater extent than did Ov-FAR-1 or rABA-1A1. Together, these results suggest that Sj-FABPc is most likely to be involved in the intracellular targeted transport and metabolism of fatty acids, whereas Ov-FAR-1 and ABA-1A1 may behave in a manner analogous to that of extracellular LBPs such as serum albumin and plasma retinol binding protein.

Allergens↗