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Biomedical subjects

G E Jones

Publications and source records attributed to G E Jones.

At least 73 records · Page 4Linked to original sources

Recombinant ovine interferon gamma inhibits the multiplication of Chlamydia psittaci in ovine cells.

The local production of interferon-gamma (IFN-gamma) in sheep in response to Chlamydia psittaci was measured by cannulation of the efferent lymph duct draining the site of challenge inoculation. Peak production of IFN-gamma (256 U/ml) was detected 24 h after challenge. Based on these physiological data, functional studies were carried out in vitro to determine the effect of recombinant ovine (rOv) IFN-gamma on the multiplication of C. psittaci in ovine fibroblasts. IFN-gamma inhibited the multiplication of C. psittaci in ovine cells over a range of concentrations (250 U/ml to 2.5 U/ml) in a dose-dependent manner. The inhibition of chlamydial multiplication was most pronounced when cells were treated with rOvIFN-gamma for 24 h before infection. The addition of exogenous L-tryptophan (500 micrograms/ml) to cultures within 48th of infection abrogated the anti-chlamydial effect of rOvIFN-gamma thus suggesting that tryptophan deprivation is an anti-chlamydial mechanism induced by rOvIFN-gamma in these ovine cells.

Animals↗

Electrophoretic analysis of the major outer membrane protein of Chlamydia psittaci reveals multimers which are recognized by protective monoclonal antibodies.

Purified major outer membrane protein, detergent solubilized and reduced with dithiothreitol but not heated, gave an apparent molecular weight in sodium dodecyl sulfate (SDS)-polyacrylamide gels almost three times that observed for the heat-denatured SDS-treated peptide. This is similar to the behavior of porin trimers from gram-negative bacteria. Two protective monoclonal antibodies showed strong binding to the proposed trimer but not to denatured, monomeric major outer membrane protein.

Animals↗

Perceived discomfort associated with working with persons with varying disabilities.

Perceptions of discomfort by nondisabled coworkers are a major barrier to the acceptance of disabled persons into work groups. This research examined whether reported discomfort varied by the type or nature of the disability. 151 subjects rated 20 types of disabilities in terms of how uncomfortable or comfortable they would be working closely at a nonspecified task with a person with the particular disability. A stable hierarchy of the 20 disabilities was found. Patterns and implications are discussed. Gender of the rater influenced the ratings, specifically females exhibited less discomfort with disabilities over-all than did males. Prior contact with a disabled person, either personally or at unspecified work, did not affect the ratings of discomfort.

Adult↗

Masseter muscle hyperactivity and myofascial pain dysfunction syndrome: a relationship under stress.

Myofascial pain dysfunction syndrome (MPD) of the temporomandibular joint (TMJ) is a psychophysiological disorder that develops because of hyperactive muscles of mastication. Ten women meeting criteria for MPD and 12 symptom-free women participated in the study. The rationale for this study was to observe cardiovascular and masseter muscle changes during four contiguous experimental periods: baseline/adaptation, reaction time, recovery, and relaxation. MPD patients showed less masseter muscle activity and higher heart rates at baseline than controls. Controls had significantly higher masseter EMG activity during reaction time. Both groups showed significant elevation in masseter muscle activity and heart rate over the 14-min reaction period. MPD patients' recovery from stress was equivalent to controls' for both heart rate and masseter muscle activity. MPD patients exhibited significantly slower reaction times than controls. The results suggest that masseter muscle hyperactivity may not account for the development and maintenance of MPD.

Adult↗

Direct observation of hexokinase translocation in stimulated macrophages.

1. Fluorescence imaging of antibodies was used to show that phorbol 12-myristate 13-acetate (PMA) induces a 4-fold increase in the amount of hexokinase relative to the control in the cortical shell of rat peritoneal macrophage cytosol adjacent to the plasma membrane, and a corresponding depletion in the amount of hexokinase in the central core of the cytosol. However, there was no significant PMA-dependent change in the distribution of glucose-6-phosphate dehydrogenase. 2. Cytochalasin D, an inhibitor of actin microfilament polymerization, prevented the PMA-induced hexokinase translocation and also reduced the PMA-dependent increases in 2-deoxy-D-glucose transport and glucose-dependent PMA-stimulated superoxide production. 3. PMA caused a contraction of the width of the cortical F-actin zone. Cytochalasin D caused some dispersal of F-actin within the cell, increasing the density of F-actin within the central cytosolic core and causing aggregation of the F-actin within the cortex. These data are consistent with the view that PMA induces attachment of hexokinase to microfilaments within the cortical zone adjacent to the cell membrane of macrophages, and cytochalasin D prevents this attachment. This is the first direct demonstration of the translocation of hexokinase to the plasma membrane in activated cells, and supports the view that enhanced hexokinase activity in the cortical region of the cytosol is an important early component of the macrophage activation process.

Actins↗

Low level of genetic drift in foreign sequences replicating in an RNA virus in plants.

The accumulation of mutations was measured in foreign sequences constituting a portion of a hybrid virus derived from the 6.4-kb (+) RNA virus, tobacco mosaic tobamovirus (TMV). Neither of the two foreign sequences tested (dihydrofolate reductase and neomycin phosphotransferase II) are functionally required by the virus, so they should be free of selective pressures and should be a true measure of viral sequence drift in whole plants. Four hybrid virus populations, two of each foreign sequence, were taken through 9-10 passages in whole plants of Nicotiana benthamiana. Sequences were sampled from these populations by conversion to cDNA, amplification by the polymerase chain reaction, and sequencing resulting bacterial clones. The background mutation rate contributed by the enzymes of this assay system allowed viral mutation rates greater than 10(-4) mutations per base per passage to be measured. Surprisingly, all native and foreign genes accumulated mutations at a very low rate, lower than could be detected by the assay procedure. This low mutation accumulation rate of < or = 10(-4) mutations per base per passage may be due to replicase fidelity or populational "bottlenecking." Sequence drift should not be a practical limitation to most uses of TMV as a vector, although deletion phenomena observed in this study may present difficulties.

Base Sequence↗

An evaluation of cell separation techniques in a model mixed cell population.

Muscle precursor cells may act not only as a means of inserting normal genes into diseased muscle fibres, in order to correct or alleviate a genetically inherited myopathy, but recent demonstrations have shown they may prove an invaluable tool for the expression of, and systemic dissemination of, non-muscle gene products. If muscle precursor cells are proved to act as such widespread vectors in terms of gene therapy, then it is imperative that methods are properly elucidated to produce large populations of pure viable myogenic cells for such purposes. In the past, many methods of cell separation have been investigated but carry with them the problems of either a lack of myogenic purity of the population or poor percentage recovery of the original cell population. In the present work we have investigated two methods for segregating myogenic from non-myogenic cells and have critically reviewed the efficiency of separation of the two techniques used. To obtain a quantitative measure of separation efficiency, segregation was carried out on a 1:1 mixture of murine C2 myogenic and murine 3T3 fibroblastic cells. To distinguish between C2 and 3T3 cells, the latter were prelabelled with the fluorescent strain carboxyfluorescein diacetate succinimyl ester (CFSE). Once incorporated into the cell, CFSE remains there, thus preventing transfer of the label to C2 cells. Both methods of separation used depend on the affinity of myogenic cells for the monoclonal antibody Mab H28, which specifically binds to the mouse neuronal cell adhesion molecule N-CAM, but differ in that one method, "panning", completes segregation by adherence of N-CAM positive cells to a dish precoated with secondary IgG antibody whereas in the other separation proceeds by the use of commercially available IgG-coated magnetic beads. Results indicate magnetic bead separation to be more efficient than panning if the beads are precoated with 0.1% gelatin.

3T3 Cells↗

Effects of macrophage colony-stimulating factor and phorbol myristate acetate on 2-D-deoxyglucose transport and superoxide production in rat peritoneal macrophages.

2-D-Deoxyglucose (2-dGlc) uptake and accumulation into rat peritoneal macrophages was increased by colony-stimulating factor (mCSF) by stimulating the coupling between endofacial hexokinase activity and the sugar transporter. The evidence for this is as follows: (1) mCSF significantly decreased the Km for zero-trans uptake (P less than 0.05), without altering Vmax.; (2) the accumulation of free 2-dGlc was increased by mCSF (P less than 0.05); (3) mCSF retarded the rate of exit of accumulated free 2-dGlc. The mCSF-dependent increase in 2-dGlc uptake by macrophages was enhanced by preincubation of the cells in mCSF-free solution. The activity of the hexose monophosphate shunt (HMPS) measured by the differential uptake of 2-d[1-3H]Glc and 2-d[2,6-3H]Glc was not stimulated by mCSF. Also, in quiescent cells, superoxide production, as determined by cytochrome c reduction, was unaffected by mCSF. Phorbol myristate acetate (PMA; 40 nM) stimulated both the HMPS activity and superoxide production. Both these effects were dependent on the uptake of external sugar (2-dGlc). Incubation of the macrophages with mCSF enhanced the sugar transport and PMA-dependent stimulation of HMPS activity and superoxide production, indicating a role for mCSF in the 'priming' of macrophage functions. Both HMPS activity and superoxide production are entirely dependent on uptake of exogenous sugar, since the potent sugar-transport inhibitor cytochalasin B competitively inhibited 2-dGlc uptake, HMPS activity and superoxide generation in PMA-activated cells (Ki approximately 0.3 microM for all three processes). Over a wide range of 2-dGlc concentrations, 4 mol of superoxide were generated/mol of 2-dGlc metabolized in the HMPS pathway, indicating coupling between these processes. The Km of 2-d[2,6-3H]Glc uptake in PMA-treated cells was 0.45 +/- 0.07 mM, and Vmax. was 1.32 +/- 0.05 mumol.min-1.ml of cell water-1. It is evident that there is a large degree of slippage between HMPS activity and membrane-associated hexokinase activity, since the Km for HMPS activity was 0.06 +/- 0.02 mM and the Vmax. was 0.10 +/- 0.03 mumol.min-1.ml of cell water-1.

Animals↗

Cross-reacting antigens between Mycoplasma ovipneumoniae and other species of mycoplasma of animal origin, shown by ELISA and immunoblotting with reference antisera.

Using enzyme-linked immunosorbent assays with Mycoplasma ovipneumoniae as antigen, the cross-reactivity of antigens between this species and 22 other mycoplasma species was examined using reference polyclonal antisera. Significant cross-reactivity with M. ovipneumoniae was demonstrated by five species, only, viz. M. bovoculi, M. dispar, M. flocculare, M. hyopneumoniae and M. hyorhinis. Using one-dimensional SDS-PAGE and immunoblotting techniques with homologous and heterologous antisera, cross-reacting antigens of M. dispar, M. flocculare, M. hyopneumoniae and M. ovipneumoniae were further investigated. Cross-reacting antigens with apparent molecular weights of 64, 44 and 32 kDa were common to all and a 184 kDa cross-reacting antigen occurred in all except M. ovipneumoniae. Further cross-reacting antigens (one-way and two-way) between two of the four species are reported. Four monoclonal antibodies against different antigens of M. ovipneumoniae did not recognise any antigen in the other three species examined.

Animals↗

Vaccine containing iron-regulated proteins of Pasteurella haemolytica A2 enhances protection against experimental pasteurellosis in lambs.

A vaccine containing sodium salicylate extract (SSE) of Pasteurella haemolytica A2 cells grown in a medium chemically depleted of available iron by the addition of alpha alpha dipyridyl to induce iron-regulated proteins (IRPs) conferred protection to specific pathogen-free (SPF) lambs exposed to an aerosol of P. haemolytica A2. The disease score in these lambs was significantly lower (p less than 0.005) than those in unvaccinated lambs or in lambs immunized with SSE prepared from cells grown in iron-replete medium. Immunoblotting of sera from these SPF lambs against whole cell antigens of P. haemolytica A2 grown under iron-restricted conditions demonstrated that antibodies to IRPs were present only in the sera of animals immunized with SSE-IRP. The antibody profile of sera from the SSE-IRP group was similar to that obtained with serum from a lamb which had recovered from P. haemolytica A2 disease produced experimentally. Negligible levels of cytotoxin-neutralizing and bactericidal antibodies were detectable in the SSE-IRP group and therefore appear not to be involved in the protection observed in this experiment.

Animals↗

Synergistic activation of 2-deoxy-D-glucose uptake in rat and murine peritoneal macrophages by human macrophage colony-stimulating factor-stimulated coupling between transport and hexokinase activity and phorbol-dependent stimulation of pentose phosphate-shunt activity.

1. Transport and accumulation of 2-deoxy-D-glucose (2dGlc) in rat and murine peritoneal macrophages were investigated by using C-1-3H-labelled and C-2,6-3H-labelled 2dGlc. 2. There was active accumulation of both C-1- and C-2,6-labelled 2dGlc by quiescent rat and murine macrophages via a phloretin-inhibitable transport system. 3. The rate of uptake and accumulation of 2dGlc (C-1 label) was increased by exposure to human macrophage colony-stimulating factor (mCSF-1) (1000 units/ml) in both murine and rat macrophages. This indicates that mCSF-1 enhances coupling between hexokinase activity and glucose transport at the endofacial surface of the transporter. 4. Phorbol 12-myristate 13-acetate ('phorbol') at 40 nM stimulated 2dGlc in rat macrophages entirely by increasing the C-2,6 label uptake. This indicates that phorbol stimulates 2dGlc uptake mainly by increasing the activity of the pentose phosphate pathway. 5. Simultaneous exposure to phorbol and mCSF-1 stimulates 2dGlc uptake to a greater extent than found with either phorbol or mCSF-1 alone. This result is explained by a simultaneous enhancement of pentose phosphate-pathway activity and of hexokinase activity acting at the endofacial surface of the cell membrane. The dual activation of these serial processes coupled to the loss of the reaction products of the pentose phosphate-shunt pathway from the cells in the form of reactive oxygen intermediates, protons and CO2 could explain the synergistic action of phorbol and mCSF-1 in activation of sugar transport in macrophages.

Animals↗

Polypeptide and antigenic variability among strains of Mycoplasma ovipneumoniae demonstrated by SDS-PAGE and immunoblotting.

Comparison of the polypeptide patterns of 22 isolates of M. ovipneumoniae by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a marked degree of heterogeneity with only limited groupings identifiable. Of the 50 major polypeptides identified in one strain (956/2), 35 were shown to be antigenic using immunoblotting with a homologous polyclonal serum. Radioimmune precipitation of 125I-surface-labelled proteins and phase partition using Triton X-114 detergent indicated that these were membrane associated. Cross-reactivity between the isolates was examined by immunoblotting using one polyclonal serum and four monoclonal antibodies (MAbs), all raised against strain 956/2. The polyclonal serum revealed considerable antigenic heterogeneity, but at least nine major antigens were conserved across all isolates. Two MAbs cross-reacted with all 22 strains, but the other two MAbs allowed some differentiation of the strains. One (MO/3) divided the isolates into groups of 16 and 6 based on the presence of absence of a 26-kDa antigen. All strains isolated from sheep with pulmonary adenomatosis fell into the smaller group and did not possess the 26-kDa antigen.

Animals↗

Efficacy against ovine enzootic abortion of an experimental vaccine containing purified elementary bodies of Chlamydia psittaci.

A vaccine prepared from purified, inactivated elementary bodies of Chlamydia psittaci protected sheep against abortion after subcutaneous challenge with live chlamydiae. Immunoblot analysis of serum samples revealed a consistently dominant antibody response against the chlamydial major outer membrane protein in all vaccinated sheep. Reactions to other chlamydial antigens were also detected but were less pronounced or inconsistent. Serological responses detected by complement fixation were variable and did not correlate with immunity.

Abortion, Veterinary↗

The humoral immune response of lambs experimentally infected with Mycoplasma ovipneumoniae.

Using sera from lambs experimentally infected with Mycoplasma ovipneumoniae and Pasteurella haemolytica, the development of a good humoral immune response to M. ovipneumoniae was detected by ELISA. The antibody titres peaked 41 days post-infection and good antibody titres were maintained over the 16-week experimental period. Immunoblotting revealed that antibodies to specific antigens appeared in the sera in a sequential manner, some being seen shortly after infection and others developing only after a substantial time lag. Antibodies were raised against almost all the major antigens detected in one laboratory strain (956/2) and against all antigens previously shown to be conserved in 22 Scottish field isolates of M. ovipneumoniae.

Animals↗

Synthesis and evaluation of a series of aryl[e]fused pyrazolo[4,3-c]pyridines with potential anxiolytic activity.

A series of pyrazolo[4,3-c]pyridines has been synthesized and evaluated as potential anxiolytic agents. Selected compounds from this series show a pharmacological profile of action different from that of diazepam. A number of the compounds possess higher affinity for central benzodiazepine receptors than diazepam, yet show less anticonvulsant activity and are less sedative. The structure-activity relationships of these potential anxiolytic agents are discussed.

Animals↗

The susceptibility of in vivo-grown Pasteurella haemolytica to ovine defence mechanisms in vitro.

Pasteurella haemolytica organisms grown in vivo were examined for their susceptibility to ovine immune mechanisms in vitro. Compared with in vitro grown organisms they were less susceptible to opsonophagocytosis and, in contrast, susceptible to complement-dependent killing in the absence of exogenous antibody. These differences were not associated with phenotypic changes in the surface of the bacterial cell. However, overproduction and de novo synthesis of proteins was observed in in vivo grown organisms. Also, bound host-immunoglobulin was observed on in vivo grown organisms and a role for this in modifying the interaction with immune mechanisms is discussed.

Animals↗