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Biomedical subjects

A J Morgan

Publications and source records attributed to A J Morgan.

At least 55 records · Page 3Linked to original sources
PubMed1995

In vivo metal substitutions in metal sequestering subcellular compartments: X-ray mapping in cryosections.

Qualitative digital X-ray mapping techniques were employed to determine the distributions of essential and non-essential elements in three invertebrate "models": (1) Pb, Zn, Cd, Cu, Fe in thin cryosections of the hepatopancreas of the terrestrial isopod, Oniscus asellus; (2) Pb, Zn, Cd, Ca in thin cryosections of the chloragogenous tissue of the earthworm, Lumbricus rubellus; and (3) As in air-dried smears and thin cryosections of chloragogen in L. rubellus. Four general conclusions were drawn from the results of these studies: (a) non-essential elements can accumulate, distribute and be compartmentalized because they, or the organo-complexes that they form, act as "mimics" of essential elements with which they share to a greater or lesser extent certain chemical affinities; (b) thermodynamic considerations notwithstanding, the influence of biological factors on the sequestration and fates of certain elements (e.g., arsenic) is profound through modifications of redox states and organo-compound formation; (c) X-ray mapping, combined with anhydrous preparative procedures, yields unbiased information concerning the relative spatial distributions of several elements in structurally heterogeneous sampling "fields", although the morphological characterization of (occasionally unsuspected) subcellular compartments may be constrained by the intrinsic quality of the preparation; and (d) X-ray microanalysis yields codistribution data, when integrated with biochemical information from other sources, which give strong pointers to the identity of binding ligands and of the valence state of sequestered cations.

Animals↗
PubMed1994

Ionomycin enhances Ca2+ influx by stimulating store-regulated cation entry and not by a direct action at the plasma membrane.

In fura-2-loaded ECV304 cells ionomycin elicited a saturable biphasic change in intracellular Ca2+ concentration ([Ca2+]i), where the initial phase represented mobilization of intracellular stores and the sustained component represented Ca2+ influx. To examine whether ionomycin could stimulate influx via a store-dependent mechanism. Mn2+ entry was monitored by the quenching of fura-2 fluorescence: influx was enhanced even after ionomycin wash-out, provided that internal stores were not refilled with Ca2+. Moreover, the maximal rate of histamine-stimulated Mn2+ entry was unaffected by ionomycin, suggesting a common route of entry. The Ca(2+)-entry blocker SK&F 96365 inhibited both the ionomycin-induced Mn2+ entry and the sustained [Ca2+]i response to the ionophore (leaving the initial peak [Ca2+]i response unaffected). In other experiments, although addition of ionomycin further increased the plateau phase induced by 100 microM histamine, the increase was completely abolished by pretreatment with the store Ca(2+)-ATPase inhibitor cyclopiazonic acid (CPA). Furthermore, in store-depleted cells, re-addition of 1 mM extracellular Ca2+ (in the presence of CPA plus histamine) led to a rapid rise in [Ca2+]i, dependent on Ca2+ influx, with kinetics that were not enhanced by ionomycin. These data suggest that ionomycin acts primarily at the level of the internal Ca2+ stores, so that, at the concentrations used here (< or = 1 microM), it increases Ca2+ (and Mn2+) influx via activation of endogenous entry pathways and not by plasmalemmal translocation.

Biological Transport↗
PubMed1994

The localization of mercury and metallothionein in the cerebellum of rats experimentally exposed to methylmercury.

Rats were dosed with methylmercuric chloride, either by gastric gavage (5 x 10 mg kg-1 body weight over a 15-day period), or in their drinking water (20 mg methylmercuric chloride l-1 for 14 or 42 days). Localization of mercury within the cerebellum was performed with a silver physical development technique, and metallothionein with dinitrophenyl hapten-sandwich immunohistochemistry. Mercury was detected in structurally undamaged Purkinje neurones and adjacent Bergmann glial cells; no mercury was detected in granule cells even though these small cells nearest the Purkinje layer had a high incidence of pyknotic nuclei. In general, metallothionein was detected mainly in Bergmann glial cells, Purkinje cells, astrocytes and glial cells of white matter; no metallothionein was detected in granule cells. We hypothesized that the resistance of Purkinje cells to methylmercuric chloride reflects their ability to transform organic mercurials to inorganic mercury that, in turn, induces the synthesis of radical-scavenging metallothionein molecules.

Administration, Oral↗
PubMed1994

Immunization of cottontop tamarins and rabbits with a candidate vaccine against the Epstein-Barr virus based on the major viral envelope glycoprotein gp340 and alum.

The Epstein-Barr virus (EBV) is associated with a range of life-threatening diseases in humans. Development of an effective vaccine has therefore been an important objective. One problem in the development of a subunit vaccine for human administration is the selection of a satisfactory adjuvant since the only one currently licensed for human use is alum, although this is not considered to be very effective. The present study demonstrated that a subunit vaccine composed of the EBV envelope glycoprotein gp340 with alum as the adjuvant did elicit protective immunity against EBV-induced lymphoma in three out of five cottontop tamarins. Furthermore, rabbits immunized with gp340/alum developed the same range of antibody responses as rabbits immunized with gp340/SAF-1, an experimental adjuvant claimed to be more effective than alum. Therefore, these results indicate that alum should be evaluated as an adjuvant as part of a human trial of a gp340-based subunit vaccine.

Adjuvants, Immunologic↗
PubMed1994

Expression of the Epstein-Barr virus envelope fusion glycoprotein gp85 gene by a recombinant baculovirus.

The gp85 envelope glycoprotein of Epstein-Barr virus (EBV) has a role in the molecular mechanism of infection, enabling fusion between the viral and host cell envelopes, a role in common with the homologous gH glycoproteins in other herpesviruses. A glutathione S-transferase bacterial fusion protein (GST85N-S) was generated, containing 178 amino acids from the C terminus of gp85 and including a known gp85 linear epitope. A panel of EBV-positive human antisera contained no antibodies to linear epitopes presented on the purified GST85N-S protein, indicating that primary protein structure in this region of gp85 is not a B cell target. This bacterial fusion protein was used to raise a rabbit monospecific polyclonal antiserum capable of detecting gp85 in a Western blot. The majority of recombinant baculovirus-expressed gp85 obtained from cell extracts prepared with SDS appeared on Western blots as heterogeneous high M(r) protein aggregates and consistently included 84K, 81K and 70K bands. Recombinant gp85 aggregation was increased by boiling the sample prior to gel electrophoresis. The 84K and 81K proteins were completely sensitive to endoglycosidase H treatment, indicating that these glycosylated species did not undergo further post-translational processing. Immunofluorescence studies revealed that recombinant gp85 was not transported to the insect cell surface. It reacted only with antibodies recognizing denatured gp85 and not with antibody to native gp85. Therefore expression of the gene encoding gp85, BXLF2, alone in the baculovirus expression system is insufficient for the synthesis of a correctly transported, processed, folded and antigenically native form of recombinant gp85.

Animals↗
PubMed1993

Comparison of the effect of isobutylmethylxanthine and phosphodiesterase-selective inhibitors on cAMP levels in SH-SY5Y neuroblastoma cells.

A comparison of the effects of various phosphodiesterase (PDE) inhibitors upon cellular cAMP levels was undertaken in human neuroblastoma SH-SY5Y cells. When inhibitors such as rolipram and Ro 20 1724 (selective for the low Km cAMP-specific PDE) were used, cAMP levels were seen to rise dramatically under basal (< or = 60 fold) or forskolin-stimulated (< or = 200 fold) conditions. However, the non-selective PDE inhibitor isobutylmethylxanthine (IBMX) was 7-18% as effective as these other agents even at 1 mM. The poor efficacy of IBMX was not attributable to concomitant increases in cGMP, to alterations in cAMP egress or to a lack of sensitivity of the cellular PDEs to IBMX inhibition. In additivity experiments, IBMX potently and rapidly reduced cAMP that had accumulated after rolipram treatment. The fact that the agonist 2-chloroadenosine can enhance cAMP accumulation in these cells, and that cAMP elevated by rolipram or forskolin can be reduced by adenosine deaminase and theophylline suggest that cell-derived adenosine enhances cAMP in these cells in an autocrine fashion. Since IBMX is an adenosine receptor antagonist, it is suggested that its blockade of endogenous adenosine effects is at least partly responsible for its poor response when compared to other PDE inhibitors which are weaker adenosine receptor antagonists. These results forewarn against assuming that similar levels of cAMP accumulate after application of PDE inhibitors in these cells.

1-Methyl-3-isobutylxanthine↗
PubMed1993

Seasonal changes in the tissue-metal (Cd, Zn and Pb) concentrations in two ecophysiologically dissimilar earthworm species: pollution-monitoring implications.

Metal (Cd, Pb, Zn) concentrations and contents were measured each month for thirteen consecutive months in two ecophysiologically distinct earthworm species, sampled from the vicinity of a disused Pb/Zn mine. Aporrectodea caliginosa, an endogeic species, enters diapause during unfavourable conditions; Lumbricus rubellus, an epigeic species, does not enter diapause. Cd concentrations were higher in A. caliginosa than in L. rubellus throughout the year: Pb concentrations were also usually higher in the endogenic species. Zn concentrations were fairly similar in both species, except during Winter and early Spring, when L. rubellus has significantly higher concentrations. Analysis of covariance indicated that the concentrations of Cd, Pb and Zn varied significantly over the thirteen-month period in L. rubellus, but only the non-essential metals (Pb, Cd) varied significantly in A. caliginosa. During diapause (observed only in the July sample), tissue concentrations of Cd and Zn in A. caliginosa were significantly lower than in 'active' worms; Pb concentrations were significantly higher during diapause. Since diapause was accompanied by a significant decrease in dry body mass, it was concluded that Pb is retained by earthworm tissues during the resting phase; but Cd and Zn are actively eliminated from the tissues before or during diapause. The implications of these findings on the use of earthworms for monitoring the relative degrees of metal contamination at different sites are discussed.

Journal Article↗
PubMed1993

Short-term Pb accumulation by slugs sampled from clean and contaminated sites when fed artificial Pb-spiked diets.

Slugs (Deroceras reticulatum and Arion subfuscus) were sampled from two distinct sites, an uncontaminated site at Dinas Powys and a Pb-contaminated, disusedmine site at Llantrisant. Batches of the slugs were exposed for ten days under laboratory conditions to an artificial diet consisting of a 5% (w/v) agar + 5% (w/v) sucrose matrix contaminated with different concentrations of Pb, as Pb (NO(3))(2). The food 'cubes' contained approximately 0, 10, 100, and 1000 microg Pb ml(-1). Tissue-Pb concentrations increased with increasing dietary-Pb concentration in both species. However, at the highest Pb exposure (1000 microg ml(-1)) the tissue-Pb concentrations in D. reticulatum and A. subfuscus sampled originally from the clean site were higher than those in their counterparts from the contaminated site, even though the Llantrisant slugs had significantly higher baseline Pb concentrations than the Dinas Powys slugs. It is suggested that these observations indicate metal tolerance phenotypically expressed as reduced accumulation in the populations with a history of Pb-exposure in their natural habitat. We discuss how biomonitoring may be affected by the way in which different slug populations deal with metals.

Journal Article↗
PubMed1993

Replication-defective recombinant adenovirus expressing the Epstein-Barr virus (EBV) envelope glycoprotein gp340/220 induces protective immunity against EBV-induced lymphomas in the cottontop tamarin.

A replication-defective recombinant adenovirus (Ad) expressing the full length Epstein-Barr virus (EBV) major envelope glycoprotein gp340/220 was tested for its ability to protect against EBV-induced lymphoma in the cottontop tamarin. Antibody responses against Ad capsid proteins and EBV gp340/220 were observed but these antibodies did not neutralize EBV in vitro. However, all immunized animals were protected against challenge following three intramuscular doses of the recombinant Ad. These data indicate that the recombinant Ad is potentially a useful vector for vaccination.

Adenoviridae↗
PubMed1993

Analysis of Epstein-Barr virus gene transcription in lymphoma induced by the virus in the cottontop tamarin by construction of a cDNA library with RNA extracted from a tumour biopsy.

Inoculation of the cottontop tamarin with Epstein-Barr virus (EBV) gives rise to the development of mono- and/or oligoclonal large cell malignant lymphoma. A cDNA library was generated with the RNA extracted from an EBV-induced tamarin lymphoma biopsy in order to study the transcripts expressed in the tumour tissue. Fifteen EBV-specific cDNA clones were localized in the corresponding viral genomic fragments. Among them, two correspond to the EBNA-2 gene, and two others to the latent membrane protein gene. The majority of the cDNA clones were localized in the BamHI A fragment which has not been associated with latent expression. Furthermore, cDNAs were also found from the BamHI D and I fragments. Sequence analysis of the cDNAs localized in BamHI A showed that they correspond to a rightward transcript in the BALF-3 region, with the one clone that was sequenced containing four exons and three introns. The above results were confirmed by testing three different biopsies with the rapid amplification of cDNA ends-PCR method.

Animals↗
PubMed1993

The effect of zinc in the form of erythromycin-zinc complex (Zineryt lotion) and zinc acetate on metallothionein expression and distribution in hamster skin.

The occurrence of zinc-induced synthesis of metallothionein in skin after topical application of the anti-acne drug Zineryt lotion was investigated in hamster ears. The dinitrophenyl hapten-sandwich immunohistochemical method involving a monoclonal anti-metallothionein (MT) antibody (E9) was used to detect and localize zinc-binding MT in the 'treated' and untreated hamster skin. Atomic absorption spectrophotometry and dithizone histochemistry indicated that zinc penetrated the skin more readily, and accumulated more efficiently within the sebaceous glands, when applied to the skin surface as the organo-zinc complex, rather than as the inorganic zinc salt. MT and zinc had similar distributions in hamster skin exposed to the metal. Thus, MT immunoreactivity was especially intense in the sebaceous glands of Zineryt lotion-treated skin, with evidence of nuclear distribution in some cells. Zinc delivered to the sebaceous glands, and released from the organo-complex under the prevailing aqueous conditions, certainly induced MT synthesis; the cysteine-rich protein may protect the pilosebaceous units during the inflammatory phase of acne by scavenging generated oxyradical species.

Acetates↗
PubMed1992

Epstein-Barr virus vaccines.

Epstein-Barr virus (EBV) is a human viral pathogen of considerable importance. More than 95% of the human population world-wide becomes infected with the virus during childhood, although in the West infection may be delayed until adolescence. The infection only has an undesirable significant clinical outcome in a tiny minority of cases, but because the virus is so ubiquitous the minority is numerically very significant. The virus is associated with two important human cancers, endemic Burkitt's lymphoma (BL) and undifferentiated nasopharyngeal carcinoma (NPC). These diseases have a very clearly defined geographical distribution in the Third World indicating a strong co-factor dependence. In the West, Epstein-Barr virus infection, when delayed to adolescence, is associated with infectious mononucleosis. The virus is also associated in the West with tumours arising in individuals undergoing immunosuppressive treatment or who are immunosuppressed as a result of HIV infection. More recently evidence has been obtained of an association with Hodgkin's disease which is very common in the West. A number of vaccines have been developed based on the EBV envelope glycoprotein gp340. Vaccination of those populations at risk from developing NPC or BL should lead to a reduction or elimination of these diseases. A safe and effective vaccine may also have a role in the prevention of EBV-related diseases in the West. Recombinant vaccinia, varicella and adenovirus vaccine vectors expressing gp340 are being developed and a recombinant-derived subunit vaccine based on the gp340 molecule is shortly to enter phase I human trials.

Animals↗
PubMed1992

Purification and characterization of Epstein-Barr virus gp340/220 produced by a bovine papillomavirus virus expression vector system.

Our initial results with a bovine papilloma virus (BPV) vector expression system indicated that we could produce significant amounts of Epstein-Barr virus (EBV) gp340/220 in the supernatant of a mouse fibroblast cell line. We have now extended these findings to show that the truncated version of gp340/220, where the membrane anchor sequence is deleted, is produced even after extended passage of the cells, at a level of approximately 1 mg/4 x 10(8) cells. A simple purification protocol using Sephacryl S300HR and gelatin agarose gives a product which is greater than 90% pure. This product is recognized by anti-gp340 monoclonal antibodies from five different epitope groups and induces antibody that recognizes the authentic gp340/220 and neutralizes EBV in vitro. The purified gp340/220 can be used in ELISA and stimulates the proliferation of T-cell clones specific for gp340/220. These characteristics, together with the fact that BPV-transformed lines have been utilized for the production of pharmaceuticals for use in humans, suggest that this gp340/220 is suitable as a source of antigen for vaccination to prevent EBV infection and related diseases.

Animals↗
PubMed1992

Protective immunization against Epstein-Barr virus-induced disease in cottontop tamarins using the virus envelope glycoprotein gp340 produced from a bovine papillomavirus expression vector.

Inoculation with Epstein-Barr virus (EBV) induces malignant lymphomas in the cottontop tamarin (Saguinus oedipus oedipus). This provides an experimental animal model for assessing the efficacy of candidate EBV vaccines which are intended to reduce the incidence of human tumours associated with EBV infection. Previous work has shown that experimental vaccines based on the major virus envelope glycoprotein gp340 prepared from the membranes of EBV-infected cells are effective in protecting cottontop tamarins against EBV-induced disease. However, not all purified gp340 preparations induce protective immunity against EBV lymphoma in the tamarin. In this work, cottontop tamarins were immunized with recombinant gp340, produced using a bovine papillomavirus (BPV) expression vector, and a threonyl muramyl dipeptide adjuvant formulation. Although the recombinant-derived gp340 lacked the membrane anchor sequence of authentic gp340 and was expressed in mouse cells, it was immunogenic and induced virus-neutralizing antibodies. Healthy vaccinated tamarins were protected against EBV-induced disease. The demonstration that a recombinant gp340 product is able to elicit protective immunity in the cottontop tamarin is a significant step in the development of an EBV vaccine because previously it had not been clear whether a recombinant product would have the exact tertiary structure, including the necessary carbohydrate components, to induce protective immunity. A recombinant gp340 vaccine offers various advantages over production of the authentic molecule by laborious biochemical separation, including lower cost and the absence of potentially oncogenic EBV DNA. Therefore, recombinant gp340 produced using the BPV expression vector is suitable for development as a candidate EBV vaccine for a human Phase I trial and beyond.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1992

Distribution of epitopes within the amino acid sequence of the Epstein-Barr virus major envelope glycoprotein, gp340, recognized by hyperimmune rabbit sera.

Epstein-Barr virus (EBV) is a major human pathogen for which the development of an effective vaccine remains an important goal. Rabbits were immunized with one of a set of 10 fusion proteins representing protein fragments from the EBV receptor-binding ligand and candidate subunit vaccine gp340. Sera from recipients of fragments from the amino-terminal half of the polypeptide chain bound gp340 in Western blot assays and ELISA but were not virus-neutralizing. The fine epitope specificity of these sera, and of EBV-neutralizing rabbit sera raised against whole EBV and gp340-containing immune-stimulating complexes, were assessed in a peptide ELISA. All but two of these sera bound peptides located between positions 236 and 327 in the 907 amino acids of the gp340 polypeptide chain. Among these it was possible to identify regions containing candidate virus-neutralizing B cell epitopes. The use of a gp340 fusion protein affinity column to isolate antibodies from EBV-neutralizing rabbit sera specific for this region suggests the presence of both continuous and discontinuous B cell epitopes with potential roles in EBV neutralization.

Amino Acid Sequence↗
PubMed1992

Mapping of B-cell epitopes on the polypeptide chain of the Epstein-Barr virus major envelope glycoprotein and candidate vaccine molecule gp340.

The Epstein-Barr virus (EBV) major envelope glycoprotein gp340 is the subject of current efforts to develop an EBV subunit vaccine. The importance of gp340-specific humoral immunity has been highlighted by studies of natural infection in humans and gp340 immunization of experimental animals. The former studies have demonstrated the presence of gp340-specific serum antibodies which mediate EBV neutralization, complement fixation, and antibody-dependent cellular cytotoxicity. The latter studies have often shown a correlation between the induction of gp340-specific EBV-neutralizing antibodies and protection from virus challenge. We have used a series of bacterial beta-galactosidase-gp340 fusion proteins and overlapping synthetic peptides from the gp340 open reading frame to map the positions of B-cell epitopes within the gp340 primary amino acid sequence. The data reported here indicate the presence of B-cell epitopes within the carboxy-terminal third of the gp340 polypeptide chain. These epitopes could not be detected with a peptide enzyme-linked immunosorbent assay, thereby suggesting that they are discontinuous. Affinity purification of antibodies with a gp340 fusion protein from the carboxy terminus of the gp340 polypeptide chain has been used to show that these antibodies are not EBV neutralizing in vitro. The consequences of these findings for future EBV vaccine development are considered.

Amino Acid Sequence↗