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A J Morgan

Publications and source records attributed to A J Morgan.

At least 37 records · Page 2Linked to original sources
PubMed1998

The identification, cloning and characterization of earthworm metallothionein.

Combining standard gel chromatographic techniques and novel molecular methodologies (Directed Differential Display and quantitative PCR), it has been possible to isolate and sequence two isoforms of the first true earthworm metallothionein. Both proteins are characteristically high in cysteine residues and possess no significant aromatic residues. Metal responsiveness was confirmed by determining metallothionein specific expression profiles in earthworms exposed to soils of differing heavy metal concentrations. Analysis of the derived amino acid sequence of isoform 2 identified two putative N-glycosylation signal sequences, suggesting that the two isoforms may have different subcellular distributions and functions. Possible implications for intracellular metal trafficking are discussed.

Amino Acid Sequence↗
PubMed1998

Identification of heavy metal induced changes in the expression patterns of the translationally controlled tumour protein (TCTP) in the earthworm Lumbricus rubellus1.

Heavy metal contaminated soils are assessed for specific human health and ecological risk by governmental regulatory agencies utilizing the abundant soil invertebrate, the earthworm, in a biomonitoring process. Fingerprinting the molecular genetic responses resulting from heavy metal exposure facilitates the identification of biomarkers for assessing the impact of such pollution on individual organisms. This paper reports the identification of a novel translationally controlled tumour protein (TCTP) in the earthworm Lumbricus rubellus. In addition to the standard molecular biological technique of differential Southern blotting, a fully quantitative approach (fluorescent microvolume PCR) was performed to assess the specific expression profiles of TCTP in earthworms exposed to different heavy metal regimes. After normalizing with actin as an invariant control, the results showed that TCTP was upregulated by at least a factor of 4 in the population originating from a Pb/Zn/Cd polluted mine, compared to an unpolluted control population. An even more pronounced increase was identified in earthworms native to a Cu polluted mine, where TCTP increased 335-fold. TCTP copies in earthworms exposed to artificial soil with a single stressor (Cd) were 14 times higher than in the appropriate control earthworms (maintained on artificial soil without Cd). The data presented are novel in two ways: first, they provide evidence for an upregulation that is induced by heavy metals (especially copper); second, they show that TCTP can also be under transcriptional control, therefore upregulation is not limited to translational modifications as TCTP's nomenclature suggests.

Amino Acid Sequence↗
PubMed1998

Indirect measurement of Epstein-Barr virus neutralising antibodies by ELISA.

A rapid and effective ELISA for measuring Epstein-Barr virus (EBV)-neutralizing antibodies in human sera was devised to replace the existing cumbersome method involving the inhibition of fetal cord blood B-cell transformation by the virus. The new method will be invaluable for assessing antibody responses in human subjects participating in EBV gp340 vaccine trials. The ELISA developed uses the human serum antibody to be tested to inhibit standardised binding of an EBV-neutralizing monoclonal antibody (mAb) to gp340 itself or its recombinant derivatives. A serum which has high EBV-neutralizing antibody titres inhibits the binding of neutralizing mAb to gp340 more than a serum with low levels. EBV neutralisation antibody titres obtained by the new inhibition ELISA correlate well with values obtained using the lengthy conventional assay where inhibition of B-cell transformation is assessed. The new assay can be carried out in a few hours compared to 4-5 weeks for the conventional test and could be automated for processing very large sample numbers in vaccine trials.

Antibodies, Blocking↗
PubMed1998

The distribution and intracellular compartmentation of metals in the endogeic earthworm Aporrectodea caliginosa sampled from an unpolluted and a metal-contaminated site.

The tissue distribution of Cd, Cu, Pb, Zn and Ca in the endogeic earthworm Aporrectodea caliginosa living in a non-polluted and a heavy metal polluted soil was investigated. The tissues of animals from the contaminated soil contained greater concentrations of Cd, Pb and Zn than the corresponding tissues of animals from the unpolluted soil. The greatest concentrations of Cd, Pb, Zn, and Ca were primarily accumulated within the posterior alimentary canal (PAC), a tissue fraction which contained the greatest proportion of the whole-worm burdens of the respective metals. Cu was distributed fairly evenly in the tissue fractions investigated. The pattern of accumulation for the 'heavy' metals is broadly similar to that for epigeic earthworms; in contrast, a different pattern of tissue accumulation was found for Ca. In animals from the uncontaminated site, the major elemental constituents of the chloragosomes were P, Ca, Zn and S. A significant positive correlation exists between P and Ca within the chloragosomal matrix. These intracellular vesicles are major foci for Pb and Zn accumulation within the PAC, with 'excess' metals associated with P ligands within the chloragosome matrix. The incorporation of Pb and Zn appears to involve the cationic displacement of Ca. Such compartmentation appears to prevent dissemination of large concentrations of these metals into other earthworm tissues, and may thus represent a detoxification strategy based on accumulative immobilization. No intracellular localization of Cd was identified in the study, although the Cd concentration in the metalliferous soils examined was not exceptionally high. The observations are discussed in the context of a contribution to enhanced understanding of metal ecotoxicology in earthworms by providing baseline data on a little investigated ecophysiological group of earthworms. Comparisons of metal distribution and mechanisms of metal sequestration are made with other ecophysiological groups of earthworms, and the significance of the findings to biomonitoring and toxicity-testing programmes is considered.

Journal Article↗
PubMed1998

Cu-Cd interactions in earthworms maintained in laboratory microcosms: the examination of a putative copper paradox.

Earthworms (Lumbricus rubellus) from Ecton (predominantly Cu-contaminated), Shipham (Cd-contaminated) and Dinas Powys (uncontaminated, reference) were maintained in the laboratory on soil from the sampling sites. Two principle exposure protocols were used: (1) a 4-week 'no pre-exposure experiment', where batches of earthworms were maintained on soils from each habitat and (2) a 'pre-exposure experiment' where uncontaminated control worms were maintained on Shipham soil for 4 weeks (the pre-exposure period), and then transferred to Ecton soil for a further 4 weeks, and vice versa. In summary, there were two primary findings. Firstly, exposure of worms to Cd-rich soil (Shipham) before exposure to Cu-rich soil (Ecton) increased the Cu burden and secondly, biochemical analysis of metal binding proteins in analogous earthworm populations indicated that Cu and Cd, but not Zn, are bound to a metallothionein homologue. These observations are consistent with the hypothesis that Cu does not induce thionein synthesis efficiently in earthworm tissues, but may bind avidly to Cd-induced thionein by substituting for Cd in the molecule because Cu-thionein is a more stable complex.

Animals↗
PubMed1998

CD4+ T cells inhibit growth of Epstein-Barr virus-transformed B cells through CD95-CD95 ligand-mediated apoptosis.

Greater than 90% of the human population acquire Epstein-Barr virus (EBV) in infancy and retain a lifelong latent infection without any clinical consequences. Nevertheless EBV has been identified as the causal agent of infectious mononucleosis, and is associated with several tumours including endemic Burkitt's lymphoma and B cell lymphomas in immunosupressed patients. B cells infected with EBV are transformed in vitro and grow continuously as lymphoblastoid cell lines. The growth of EBV-transformed B cells in vivo is controlled by the immune system. Studies on immunity to EBV have mainly focused on MHC class I-restricted CD8+ cytotoxic T cells specific for viral latent antigens. Here it is reported that in vitro stimulation of peripheral blood lymphocytes by autologous EBV-infected B cells, which have been induced to express lytic cycle antigens, gives rise to a predominantly CD4+ T cell response. Furthermore, the growth of EBV-infected B cells can also be regulated by these activated CD4+ T cells through apoptosis mediated by CD95-CD95 ligand (CD95L). CD95-CD95L-mediated apoptosis is an important mechanism of normal B cell growth regulation. As EBV-transformed B cells remain susceptible to this mechanism, the control of EBV in vivo may be not only by virus-specific CD8+ cytotoxic T cell immunity but also by normal mechanisms of immune regulation of B cell growth.

Acyclovir↗
PubMed1997

Immunomodulation by Quillaja saponaria adjuvant formulations: in vivo stimulation of interleukin 12 and its effects on the antibody response.

The capacity of adjuvants to activate Ag-presenting cells during the induction of the primary immune response is of critical importance for the development of protective immunity to a number of pathogens. In this context, interleukin 12 (IL-12) has a key role by controlling the differentiation of T helper cells and favouring the expansion of Th1 cells. The capacity of iscoms with influenza virus Ag (flu-iscoms) and iscom matrix with EBV gp340 Ag to induce IL-12 was analysed in mice. The flu-iscom drives the immune response towards a Th1 type subsequent to IL-12 induction as measured in the serum of H2b, H2d and H2k mice. The iscom presenting the Ag and adjuvant in the same particle was considerably more efficient than the formulation of matrix and Ag in separate particles. Inhibition experiments with mAb neutralizing IL-12, interferon gamma (IFN-gamma) or IL-4, the latter two cytokines representing the Th1 and Th2 type of responses, showed that iscoms induce a broader immune response than that involving IL-12. This was shown by the additional effect that IL-4 neutralization had on the immune response to iscoms. Anti-IL 12 reduced the specific total Ab as well as IgG1, IgG2a and IgG2b while anti-IL 4 influenced the response to iscom by decreasing IgG2a and increasing IgG1. Further, the neutralization experiments indicate that IL-12 has a broader effect than IFN-gamma on the Ab response by influencing the production of IgG1, IgG2a and IgG2b.

Adjuvants, Immunologic↗
PubMed1997

Immunocytochemical identification of metallothionein-positive cells in rheumatoid synovium and analysis of their cell lineage.

Metallothionein is a ubiquitous low molecular weight metalloprotein with powerful protective properties against oxygen radical-mediated cytotoxicity associated with inflammatory processes. In rheumatoid arthritis, the inflammatory damage to the synovium appears to be mediated by free radicals released by the high concentration of neutrophils found in the synovial fluid of the inflamed joint. Synovial tissue obtained during routine surgery on rheumatoid and non-rheumatoid joints was subjected to an indirect immunoperoxidase protocol for the immunolocalization of metallothionein using mouse monoclonal anti-metallothionein antibody E9, reactive against the two major isoforms of mammalian metallothionein. A layer of large dendritic-like cells situated subsynovially in the rheumatoid synovium stained very positively for the metalloprotein, both cytoplasmically and in their nuclei. These cells were not found in non-rheumatoid osteoarthritic or in undamaged synovial tissue associated with traumatic joint injury. An attempt was made to investigate their lineage using a series of antibody markers against epithelial cells, endothelial cells, smooth muscle, mesothelial cells, fibroblasts, neutrophils, dermal dendrocytes, macrophages, low and high molecular weight cytokeratin as well as a cell proliferation marker. From our results, it is suggested that these metallothionein-positive cells are probably myofibroblasts similar to the highly motile cells present in granulation tissue. They may originate from perivascular areas of synovium and their movement into the inflamed synovium may reflect the cytoprotective role of metallothionein acting as a free radical scavenger against oxidative damage.

Antigens, CD↗
PubMed1997

Lack of susceptibility of the cottontop tamarin to hepatitis C infection.

The only species apart from man that is known to be susceptible to HCV infection is the chimpanzee but the availability of this primate for research is strictly limited. In an attempt to find an alternative and more practical model for HCV studies three cottontop tamarins were inoculated intravenously with HCV-containing serum from patients with chronic HCV infection. The tamarins were monitored regularly for biochemical indications of hepatic inflammation and serum samples were assayed at weekly intervals for the presence of HCV-RNA and HCV antibodies. HCV-RNA was detectable at 10 minutes postinoculation in all three animals but not at any later time point over a 6 month period. No evidence of an active humoral immune response to the inoculated HCV was obtained although passively transferred anti-HCV was detectable in one animal until 1 week postinoculation. Biochemical findings did not indicate hepatic inflammation and liver histology remained normal. It is concluded on the basis of these negative findings that the cottontop tamarin is not susceptible to HCV infection.

Animals↗
PubMed1996

Ca2+ influx does more than provide releasable Ca2+ to maintain repetitive spiking in human umbilical vein endothelial cells.

We investigated why oscillations of intracellular Ca2+ concentrations ([Ca2+]i) in endothelial cells challenged by sub-maximal histamine run down in Ca(2+)-free medium despite stores retaining most of their Ca2+. One explantation is that only a small subpopulation of the Ca2+ stores oscillate and are completely emptied of Ca2+. To investigate if influx refills an empty store subpopulation, we differentiated between cations entering the cell and those released from internal stores by using extracellular Sr2+ as a Ca2+ surrogate; we distinguished between [Sr2+]i and [Ca2+]i by using the larger effect of Sr2+ on fura 2 fluorescence at 360 nm (F360). Ca2+ was still available for release when oscillations had run down since oscillations promptly reappeared on addition of Sr2+o and these were predominantly of Ca2+ (indicated by F360 changes). Also, totally depleting Ca2+ stores inhibited Sr(2+)-induced oscillations, suggesting that Sr2+ entry leads to Ca2+ release. In contrast, Ba2+o was unable to stimulate oscillations. Finally, oscillations generated by photolytic release of inositol trisphosphate (IP3) analogues were similarly sensitive to extracellular Ca2+ and Sr2+. We conclude that stores (or a sub-population) are not completely depleted of Ca2+ when oscillations run down in Ca(2+)-free medium. Bivalent cation entry therefore maintains sensitivity to IP3, possibly by maintaining luminal bivalent cation levels.

Calcium↗
PubMed1996

Protein tyrosine kinases regulate agonist-stimulated prostacyclin release but not von Willebrand factor secretion from human umbilical vein endothelial cells.

The rapid synthesis and release of prostacyclin (PGI2) and the exocytotic secretion of von Willebrand Factor (vWF) elicited by activation of G-protein-coupled receptors on endothelium occur via signaling mechanisms which are incompletely defined. Activation of protein tyrosine kinases (PTKs) and modulation of the tyrosine-phosphorylation state of endogenous proteins have been implicated in several cellular processes including arachidonate release and exocytosis. In the present study we have examined the regulatory role of PTKs in agonist-stimulated release of PGI2 and vWF from human umbilical vein endothelial cells (HUVECs) using two chemically and mechanistically dissimilar PTK inhibitors (genistein and ST271). Genistein, but not the less active analogue daidzein, dose-dependently attenuated PGI2 release in response to thrombin and histamine (IC50 approx. 20 microM), and to the thrombin-receptor-activating peptide. A more potent inhibition of thrombin- and histamine-induced PGI2 synthesis was observed in cells exposed to ST271. In contrast, neither genistein nor ST271 modulated agonist-drive vWF secretion. At concentrations that abolished PGI2 release, genistein blocked thrombin- or histamine-evoked tyrosine phosphorylation of a 42 kDa protein. Ca2+ ionophore-induced PGI2 generation, but not vWF secretion, was also inhibited by both genistein and ST271, suggesting that these agents modulate PGI2 synthesis by acting at, or distal to, agonist-induced changes in intracellular CA2+ ([Ca2+]i). In fura-2-loaded HUVECs genistein partially reduced the histamine-induced peak [Ca2+]i but had no effect on the thrombin response. Ca(2+)-induced PGI2 release from electrically permeabilized HUVECs was abolished in the presence of ST271 or genistein, but not diadzein. The generation of PGI2 in response to exogenous arachidonic acid was not modulated by genistein or ST271, suggesting that PTK inhibitors do not directly inhibit cyclo-oxygenase activity. Taken together, these results suggest that PTKs regulate PGI2 synthesis and release in HUVECs by modulating, directly or indirectly, a CA(2+)-sensitive step upstream of cyclo-oxygenase.

Calcium↗
PubMed1996

Virus-specific cytotoxic T cell responses are associated with immunity of the cottontop tamarin to Epstein-Barr virus (EBV).

The cytotoxic responses of peripheral blood lymphocytes from cottontop tamarins to in vitro restimulation with autologous lymphoblastoid cell lines (LCL) were assayed. Lymphocytes from immune tamarins that had recovered from EBV challenge developed potent cytotoxicity for natural killer (NK) cell targets and for autologous LCL. The cytotoxicity for LCL targets was EBV-specific, as B cell blasts uninfected with EBV were not killed. The cell lines could be maintained by repeated stimulation with LCL and the addition of IL-2. Flow cytometry showed that they were T cell lines expressing CD2, CD3, CD4, CD8 and CD25. Dual-colour flow cytometry revealed two subpopulations, one CD4+ CD8+ population and the other CD4- CD8+. After separation by magnetic cell sorting both subpopulations were shown to be cytotoxic and the CD4+ CD8+ fraction was also shown to be MHC class II-restricted; the MHC restriction of the CD8+ subpopulation could not be determined. The unseparated T cells and both the subpopulations were able to inhibit LCL outgrowth in vitro. In contrast, PBL from naive tamarins stimulated by autologous LCL developed less NK cell cytotoxicity and little cytotoxicity for LCL. The cytotoxic response was enhanced at higher levels of LCL stimulation, but the cells were unable to inhibit LCL outgrowth in vitro. We conclude that cytotoxic responses capable of inhibiting LCL growth in vitro correlate with in vivo immunity in the tamarin model and provide a basis for understanding the mechanism of vaccine-induced immune protection.

Animals↗
PubMed1996

Increased small intestinal fermentation is partly responsible for the anti-nutritive activity of non-starch polysaccharides in chickens.

1. The mechanism of the anti-nutritive activities of soluble non-starch polysaccharides (NSPs) in broiler diets was investigated with emphasis on the inter-relationship between viscosity and fermentation along the gut. Isolated soluble NSP were added to a control diet to effect high gut viscosity, and in vivo depolymerisation of the NSP was achieved using a commercial glycanase. 2. Addition of soluble NSPs significantly (P < 0.01) increased gut viscosity, reduced the AME of the diet and depressed the growth and FCE of the birds. Enzyme supplementation of the NSP-enriched diet reversed the adverse effects, increasing (P < 0.01) weight gain, FCE and AME. Comparisons of the viscosities (mPa) in birds fed on the NSP-enriched diet and the same diet supplemented with enzyme were respectively: 11.9 v. 2.3 in the duodenum; 78.3 v. 4.4 in the jejunum and 409.3 v. 10.8 in the ileum. 3. Caecal volatile fatty acid concentration was markedly (P < 0.01) elevated by enzyme supplementation, whereas ileal fermentation was inhibited. 4. Microscopic examination revealed that, among birds fed on the NSP-enriched diet, there had been extensive small intestinal fermentation, which was eliminated by the enzyme supplementation. 5. Addition of a synthetic antibiotic (Amoxil) had no beneficial effects. 6. The current study demonstrated that increased fermentation occurs in the small intestine when a large amount of viscous NSPs is present in the diet and this is detrimental to the performance and well-being of poultry.

Animal Feed↗
PubMed1995

Selection of monoclonal antibodies for the identification of lymphocyte surface antigens in the New World primate Saguinus oedipus oedipus (cotton top tamarin).

32 monoclonal antibodies reactive with human CD antigens were tested against tamarin peripheral blood lymphocytes, ConA blasts and lymphoblastoid B cell lines derived from tamarin cells. Reagents that cross-react with MHC class I and II, B cells (CD20, -21 and -23), monocytes (CD14) and NK cells (CD16, -56) have been identified. In addition monoclonals that cross-react with T cells (CD2, CD3), the CD4/CD8 subsets of T cells and the IL-2 receptor (CD25) are reported. A monoclonal against the beta chain of LFA-1 (CD18) cross-reacted strongly, but there was only a very poor cross-reaction with a monoclonal against the alpha chain of CD11a. Two monoclonals tested against ICAM-1(CD54) were negative.

Animals↗
PubMed1995

Co-expression of the Epstein-Barr virus BXLF2 and BKRF2 genes with a recombinant baculovirus produces gp85 on the cell surface with antigenic similarity to the native protein.

Glycoprotein H (gH) is a conserved herpesvirus gene product functionally implicated in the penetration of the virus into the host cell. Other human herpesviruses require an accessory glycoprotein named gL for the synthesis of mature gH. We constructed a series of recombinant baculoviruses to determine whether gL expression can overcome the constraints upon Epstein-Barr virus (EBV) gH (gp85) folding and transport in this expression system. When gH and gL were co-expressed some EBV gH was transported to the insect cell surface. Deletion of 27 amino acids from the gH carboxy terminus resulted in the secretion of an 80 kDa protein (gHt) into the culture medium when it was expressed either in the presence or absence of gL and this protein could also be immunoprecipitated with E1D1. In contrast, gL was not secreted into the culture medium. Our results suggests that either co-expression of gH with gL or removal of the predicted transmembrane anchor sequence can overcome some of the constraints upon EBV gH expression in the baculovirus system.

Animals↗
PubMed1995

The Epstein-Barr virus open reading frame BDLF3 codes for a 100-150 kDa glycoprotein.

The Epstein-Barr virus (EBV) open reading frame BDLF3 is predicted to code for a glycoprotein on the basis that it contains sequences with signal peptide and transdomain characteristics and nine potential N-linked glycosylation sites. No sequential or positional homologues of BDLF3 have been located in other herpesviruses. A bacterial glutathione S-transferase (GST)-BDLF3 fusion protein was used to demonstrate that over one-third of EBV-immune human sera tested recognized the fusion protein but not GST alone on Western blots. The fusion protein was used to raise polyclonal sera in rabbits. A BDLF3 recombinant baculovirus was constructed using the full-length BDLF3 sequence (AcBDLF3). Rabbit anti-fusion protein sera and some human EBV-immune sera recognized products of approximately 30 and 55 kDa from AcBDLF3-infected insect cells by Western blotting. A peptide representing the carboxy-terminal amino acids 215-234 of the BDLF3 sequence was used to raise anti-peptide sera in rabbits. Anti-peptide serum detected a product by indirect immunofluorescence in acetone-fixed EBV-infected B cells from all cell lines tested. A diffuse band with a molecular mass of 100-150 kDa was detected by Western blot in B95-8 cell lysates, partially purified B95-8 virus and B95-8-infected cell membranes after probing with anti-BDLF3 peptide serum. This product was shown to be glycosylated after enzymatic deglycosylation of a B95-8 virus preparation using neuraminidase, O-glycosidase or N-glycosidase F. The BDLF3 protein products have no known function.

Animals↗