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Biomedical subjects

B D Robertson

Publications and source records attributed to B D Robertson.

29 records · Page 2Linked to original sources
PubMed1993

The role of galE in the biosynthesis and function of gonococcal lipopolysaccharide.

Lipopolysaccharide is an essential component of the outer membrane of Gram-negative bacteria and an important virulence factor of many pathogens, such as Neisseria gonorrhoeae. We have cloned the gonococcal galE gene which was found to be located in the gonococcal homologue of the meningococcal capsule gene complex region D. Sequence alignment indicated extensive homology with the Escherichia coli and Salmonella GalE proteins. Mutants with insertions in the galE gene were used as a tool to characterize the structure and function of gonococcal lipopolysaccharide. They displayed deep rough phenotypes, and chemical analysis confirmed the loss of galactose from the mutant lipopolysaccharide. Functional analysis indicated that the terminal oligosaccharides contain galactose and that these are lost in galE mutants. The importance of these oligosaccharides in gonococcal biology is clear from the fact that they contain the epitopes that are the targets for killing by normal human serum, and the acceptor site for sialic acid, which acts to protect the gonococcus from this killing. Furthermore, infection experiments in vitro indicate that the galE mutants exhibit unaltered intergonococcal adhesion as well as adhesion to, and invasion of, epithelial cells.

Amino Acid Sequence↗
PubMed1993

Diagnosis of human toxocariasis by antigen capture enzyme linked immunosorbent assay.

AIMS: To evaluate an antigen capture enzyme linked immunosorbent assay (ELISA) which detects a carbohydrate epitope on the excretory-secretory (ES) antigens of Toxocara canis in clinical practice. METHODS: Serum specimens from healthy adults, patients with acute visceral larva migrans, ocular and inactive toxocariasis, and with other helminth infections were examined by two site antigen capture ELISA. RESULTS: Over half of the patients (19/28) with acute toxocariasis had a positive result in contrast to a small proportion of those with inactive disease (1/10) or ocular infection (2/7). False positive reactions, however, were found in 25% of the patients with serologically confirmed schistosomiasis and filariasis. CONCLUSIONS: This assay is useful in confirming the diagnosis of acute visceral larva migrans but could not be used alone in diagnosis because of false positive reactions in patients with other helminth infections.

Adult↗
PubMed1992

Genetic variation in pathogenic bacteria.

In contrast to textbook ideas of pure cultures and defined strains, genetic variation is a fact of life in the microbial world. It not only allows pathogens to establish themselves in their chosen host, but also allows them to resist that host's subsequent attempts to evict them. Here we review some of the mechanisms that bring about this variation, and some of the functional consequences that result from it.

Adaptation, Biological↗
PubMed1990

Developmentally regulated expression and secretion of a polymorphic antigen by Onchocerca infective-stage larvae.

In order to analyse the developmental biology of Onchocerca spp. with a view to identifying molecules with specialised functions, we have devised a novel method for labelling proteins synthesised by larvae during growth in the vectors. Pulse labelling of Onchocerca lienalis by micro-injections of [35S]methionine into blackflies have revealed a major acidic protein of 23 kDa which is developmentally expressed almost exclusively by infective, third-stage larvae. The protein appears to be antigenically conserved between O. lienalis and Onchocerca volvulus, but exhibits size polymorphisms both among species and among individual organisms. It continues to be elaborated after terminal differentiation of the parasite in flies, but not by post-infective larvae entering the phase of development in the vertebrate host. A shift in temperature from 26 degrees C to 37 degrees C triggers secretion of the 23-kDa molecule as a discrete event 24-72 h after transmission. The labelling technique has been successfully employed with filarial species that develop in mosquitoes, and in principle should be widely applicable to the study of endoparasite gene expression within arthropods.

Animals↗
PubMed1989

Toxocara canis: proteolytic enzymes secreted by the infective larvae in vitro.

Second-stage larvae of the dog nematode Toxocara canis are infective to man and cause the syndromes of visceral larva migrans and ocular toxocariasis. Larvae cultured in vitro secrete proteases which degrade components of a model of extracellular matrix and basement membranes. These enzymes have been characterized using a variety of techniques. Multiple enzyme activities were demonstrated by substrate gel electrophoresis, associated with proteins of molecular weights of 120 and 32 kDa. The enzyme activity was inhibited both in substrate gels and in a radiogelatin microplate assay by phenylmethylsulfonyl fluoride. Optimal activity occurred at pH 9, with minor activities apparent at pH 5 and 7; the relationship between these proteolytic activities is currently under investigation.

Animals↗
PubMed1988

A sensitive microplate assay for the detection of proteolytic enzymes using radiolabeled gelatin.

A sensitive, microplate assay is described for the detection of a wide range of proteolytic enzymes, using radio-iodine-labeled gelatin as substrate. The technique uses the Bolton-Hunter reagent to label the substrate, which is then coated onto the wells of polyvinyl chloride microtiter plates. By measuring the radioactivity released the assay is able to detect elastase, trypsin, and collagenase in concentrations of 1 ng/ml or less, while the microtiter format permits multiple sample handling and minimizes sample volumes required for analysis.

Gelatin↗
PubMed1988

Detection of circulating parasite antigen and specific antibody in Toxocara canis infections.

Serological surveys, measuring humoral antibody responses, have indicated significant levels of human infection with the zoonotic nematode Toxocara canis, and raised concern about the resultant risk of ocular and neurological damage. Such measurements do not distinguish with certainty current infection from past exposure. Thus, we have developed a test for circulating Toxocara antigen released by parasites in the host. This monoclonal antibody-based two-site 'sandwich' assay discriminates between T. canis and the related feline ascarid T. cati, and has been used, in tandem with the standard ELISA, to examine experimental and human infections. In experimental animals, antigen is transiently detectable, disappearing when immunocomplexed with host antibody. Antigen was also found in sera from UK patients diagnosed with visceral or ocular toxocariasis, and in four asymptomatic Papua New Guinean children. In the latter population, individuals positive for parasite antigens were not necessarily positive for antibody, implying that some infected cases may be negative in the current diagnostic ELISA. The antibody test was also adapted to measure host antibody directed to single monoclonal antibody-defined epitopes, revealing evidence of differential temporal regulation of distinct antibody specificities.

Animals↗
PubMed1987

Shared carbohydrate epitopes on distinct surface and secreted antigens of the parasitic nematode Toxocara canis.

The nematode parasite Toxocara canis is found in all dog populations and poses a poorly defined health hazard to humans. We have studied excretory-secretory antigen (ES) and surface antigens of the infective larval stage which is tissue-invasive in mammalian hosts. Antigens were probed with a panel of eight monoclonal antibodies raised in mice to whole ES. Six of eight antibodies reacted with periodate-sensitive carbohydrate epitopes on ES molecules, and the remaining two (Tcn-3 and Tcn-6) recognized either peptide or periodate-resistant sugar determinants. By immunoprecipitation and immunoblotting, the anti-carbohydrate monoclonals each reacted with several distinct ES molecules, known from previously published work to possess contrasting biochemical properties. Tcn-3 and -6 were directed predominantly against 32,000 and 120,000 m.w. molecules, respectively. Iodinated surface antigens of similar m.w. were precipitated by each antibody after detergent solubilization, but only two clones (Tcn-2 and -8) were able to bind exposed sites on the epicuticle of intact Toxocara larvae. Significantly, these antibodies do not bind to newly hatched larvae, and their target antigens are poorly expressed until the second day of in vitro cultivation. The specificities of the monoclonals were further studied by cold antibody inhibition of radiolabeled monoclonal binding, and by a matrix of two-site binding assays. These data show that Tcn-2, -4, -5, and -8 recognize a related group of repetitive carbohydrate epitopes, whereas Tcn-1, -6, and -7 bind discrete determinants on the same molecules. These studies are being continued to define further the structure of antigenic Toxocara carbohydrates and to compare the diagnostic utility of carbohydrate and peptide antigens.

Animals↗
PubMed1987

Secretory acetylcholinesterases from Brugia malayi adult and microfilarial parasites.

Brugia malayi, a lymphatic filarial parasite, secretes acetylcholinesterase during in vitro cultivation. A significant amount of enzyme activity was detected both in culture media and somatic extracts of adult and microfilarial stages of the parasite. The microfilarial stage produces three times more enzyme than adult parasites as a proportion of total protein. The enzyme has true acetylcholinesterase (AChE) activity as hydrolysis of acetylthiocholine is three times faster than butyrylthiocholine and is inhibited by eserine, a specific inhibitor of AChE. Secretory enzyme from adult female parasite excretory-secretory material (ES) was enriched 23 fold using copper chelating and concanavalin A (Con A) affinity chromatography. The Con A eluate showed a major protein band of 100 kDa and a minor 200 kDa component. The ES enzyme is antigenic and cross reacts with antibodies raised in mice against AChE from electric eel by enzyme-linked immunosorbent assay and immunoprecipitation. Immunoprecipitation of 125I-labelled microfilarial ES and adult ES with anti-electric eel AChE antibodies revealed three proteins of 30, 40 and 200 kDa in microfilariae and two proteins of 100 and 200 kDa in adult female ES. It appears that filarial secretory AChE exists in multiple molecular forms.

Acetylcholinesterase↗
PubMed1987

Age-relationships of Toxocara canis seropositivity and geohelminth infection prevalence in two communities in St. Lucia, West Indies.

Sera were examined from an age-stratified sample of two Caribbean communities using the Toxocara-ELISA with larval ES antigen. Seropositivity was markedly age dependent, attaining maximal values (40 and 60%) in 5-15 year olds and declining in adults. The rate of acquisition of infection with Toxocara canis and the age-prevalence profile are similar to those of Ascaris lumbricoides and Trichuris trichiura. It is suggested that toxocariasis is likely to be prevalent in tropical areas with endemic geohelminthiasis.

Adolescent↗