Biomedical subjects
Publications and source records attributed to A J Morgan.
[reaction: see text]An efficient formal synthesis of the potent protein kinase C inhibitor (-)-balanol that relies on a modified asymmetric aminohydroxylation of the alpha,beta-unsaturated aryl ester (1) is reported. The aryl ester functionality and the dihydroquinyl alkaloid ligand system (DHQ)2-AQN are used to control the regio- and enantioselectivity of the process.
We report the identification of two earthworm cyclophilin genes, which resemble the cytosolic cyclophilin-A and the signal sequence containing cyclophilin-B. Using fully quantitative PCR we were able to assess the transcript regulation of both cyclophilin isoforms, as well as a further independent control gene (actin), during exposure to heavy metals. Whilst the expression of cyclophilin-B and actin remained exceptionally constant, cyclophilin-A was up-regulated 38-fold. This intriguing observation has profound implications regarding cyclophilin's use as an invariant control and highlights the fact that it is essential to treat cyclophilin isoforms as separate entities, rather than one functional unit.
[formula: see text] The asymmetric synthesis of beta-hydroxy-alpha-amino acids is reported which relies on the use of alpha,beta-unsaturated aryl ester substrates and the dihydroquinyl alkaloid ligand system (DHQ)2-AQN to control the regio- and enantioselectivity of the asymmetric aminohydroxylation (AA) process. alpha,beta-Unsaturated ester substrates of type 1 have a significant effect on the substrate-ligand recognition event which results in a reversal of regioselectivity in the AA reaction.
A recombinant form of the EBV envelope glycoprotein and vaccine candidate gp340, lacking its hydrophobic transmembrane region, was incorporated into Iscoms after coupling to phosphatidyl ethanolamine via carbohydrate residues. Coupling by partial oxidation of gp340 carbohydrate with sodium periodate partly denatured the incorporated gp340 as indicated by its reduced reactivity with monoclonal antibodies that recognise the major neutralising epitope. Immunisation of cottontop tamarins with these Iscoms elicited antibody responses to gp340, but these antibodies only poorly recognised the major neutralising epitope in a competition ELISA and were unable to neutralise EBV in vitro. Despite the lack of neutralising antibody, immunisation with these Iscoms primed significant in vitro proliferative responses to soluble gp340 in lymphocytes from the draining lymph nodes and spleen. T-cell lines were raised from both immunised and control animals by in vitro stimulation of peripheral blood lymphocytes or spleen cells with autologous EBV-transformed lymphoblastoid cell lines. The T-cell lines from control animals had higher numbers of CD4+ T-cells than CD8+ T-cells and were not cytotoxic for autologous lymphoblastoid cell lines (LCL). In contrast the lines from immunised animals contained more CD8+ T-cells than CD4+ T-cells and had marked cytotoxicity for autologous LCL.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
1. Histamine-stimulated cytosolic free Ca2+ ([Ca2+]i) oscillations in human umbilical vein endothelial cells (HUVECs) comprise repetitive spikes generated by pulsatile release from stores. We have investigated the roles of the store Ca2+-ATPases in regulating both the upstroke and downstroke of a Ca2+ spike. 2. The sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor cyclopiazonic acid (CPA) dramatically affected oscillations whereas inhibition of the plasma membrane Ca2+-ATPase (PMCA) with La3+ had little effect. This and other evidence suggested that the downstroke of a spike is predominantly mediated by SERCA. 3. Artificial [Ca2+]i spiking generated by repetitive pulsatile application of 0.3 microM histamine in Ca2+-free medium did not cause net loss of Ca2+ from the cell whereas repetitive pulsatile application of 1 and 10 microM histamine did, with the higher concentration being more effective. We conclude that there is an inverse relationship between stimulus intensity and relative SERCA activity. 4. For a Ca2+ transient, the initiation of release was suppressed by SERCA during either the lag phase or the interspike period (ISP) since: (i) the ISP was shortened by low CPA concentrations, (ii) higher concentrations of CPA stimulated an explosive Ca2+ release when applied during the ISP but not when applied in the absence of agonist, and (iii) CPA synchronized the initial Ca2+ response to a low histamine dose (even recruiting silent, histamine-unresponsive cells). 5. Two aspects of the regenerative upstroke of a spike were differently affected by SERCA inhibition: Ca2+ wave velocity was entirely unaffected by CPA whereas the local rate of rise was increased. 6. The [Ca2+]i at which a Ca2+ spike terminated depended on SERCA since CPA dose dependently enhanced the peak [Ca2+]i. 7. We conclude that SERCA plays a powerful and dynamic role in regulating [Ca2+]i oscillations in HUVECs. SERCA differentially modulates the phases of Ca2+ release in addition to bringing about the falling phase of a Ca2+ spike.
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.
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.
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.
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.
Explore the source record for details and available documents.
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.
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.
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.
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.
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.
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.