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Biomedical subjects

M A Gray

Publications and source records attributed to M A Gray.

At least 73 records · Page 4Linked to original sources
PubMed1986

Increased sensitivity of virus-infected cells to inhibitors of protein synthesis does not correlate with changes in plasma membrane permeability.

Semliki Forest virus-infected BHK cells or herpes simplex virus-infected Vero cells were incubated with the protein synthesis inhibitors hygromycin B and gougerotin. Infected cells take up no more [3H]hygromycin or [3H]gougerotin than do mock-infected cells, at a time p.i. at which either compound is more inhibitory to protein synthesis in infected, than in mock-infected cells. The concentrations of hygromycin and gougerotin required to inhibit protein synthesis in intact cells (irrespective of whether they are infected or not) are several orders of magnitude higher than those required in either permeabilized cells or in cell-free systems. Infected cells take up 86Rb+ at the same rate as mock-infected cells, their intracellular content of K+ is the same, and the activity of the Na+ pump is the same. It is concluded that the increased efficacy of hygromycin and gougerotin in virus-infected cells is a consequence of altered intracellular compartmentation and that increases in permeability of the plasma membrane, if any, are so small as to be undetectable by direct methods.

Animals↗
PubMed1986

Observations on experimental inactivated vaccines for contagious bovine pleuropneumonia.

In two trials the efficacy of inactivated vaccines against contagious bovine pleuropneumonia was tested by exposing vaccinated cattle to droplet infection provided by close contact with experimentally infected 'donors'. Complete protection was given by an extreme form of vaccination in which a heavy suspension of killed Mycoplasma mycoides subsp. mycoides emulsified with Freund's complete adjuvant was given in two large doses. 'Mouse-protective antibody' (MPA) was also produced, i.e. serum transferred to mice 2-4 h before intraperitoneal challenge prevented the development of mycoplasmaemia. However, the study did not answer the question 'Is MPA protective for cattle?'. No protection was given by a milder form of vaccination in which a lighter suspension of killed mycoplasmas emulsified with Freund's incomplete adjuvant was given in a comparatively small dose on a single occasion.

Animals↗
PubMed1986

Analysis of trypanosome variable antigen types in cultures of metacyclic and mammalian forms of Trypanosoma congolense.

Cultured metacyclic forms of Trypanosoma congolense display a characteristic repertoire of metacyclic variable antigen types (M-VATs) similar to that exhibited in vitro in the tsetse fly. There appeared to be no change in expression of M-VATs in cultures of two stocks of T. congolense even after several passages, cryopreservation or long-term cultivation in vitro. Metacyclic forms transformed into mammalian forms when transferred to cultures of bovine aorta endothelial cells and whilst one stock retained expression of M-VATs without change even after 4 months, the other stock underwent antigenic variation within 14 days of transfer. Analysis of the M-VAT composition of mammalian forms of this stock using monoclonal antibodies showed that although the proportion of mammalian forms expressing certain M-VATs declined considerably, trypanosomes expressing one M-VAT increased proportionally to comprise 50% of the population. In contrast, only small changes were seen in antigen expression in cultures of metacyclic trypanosomes from which mammalian-form cultures were derived. It was possible to produce in vitro, loss and reacquisition of variable antigen surface coat, similar to the differentiation process occurring when bloodstream trypanosomes are ingested by the tsetse fly and eventually develop into metacyclic forms.

Animals↗
PubMed1985

Oxonol dyes as monitors of membrane potential: the effect of viruses and toxins on the plasma membrane potential of animal cells in monolayer culture and in suspension.

Optical indicators of the cationic, cyanine and anionic oxonol classes were used to evaluate the plasma membrane potential of animal cells in suspension and in monolayer culture. The optical signals were calibrated by using diffusion potentials either of K+ (in the presence of valinomycin) or of H+ (in the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone; FCCP); both classes of dye gave similar values of plasma membrane potential, in the range -40 to -90 mV for different cell types. Addition of haemolytic Sendai virus or Staphylococcus aureus alpha-toxin depolarizes cells and causes them to leak monovalent cations; these effects are antagonized by extracellular Ca2+. Cells infected with vesicular stomatitis or Semliki Forest virus become depolarized during an infectious cycle; infection with other viruses was without affect on plasma membrane potential.

Animals↗
PubMed1985

Asymmetric block of a monovalent cation-selective channel of rabbit cardiac sarcoplasmic reticulum by succinyl choline.

We have investigated the effect of the skeletal muscle relaxant succinyl choline (SC) on the conduction of potassium ions through a monovalent cation-selective channel present in the cardiac muscle sarcoplasmic reticulum membrane (CSR). This channel has been studied under voltage-clamp conditions following the fusion of purified CSR membrane vesicles with preformed planar phospholipid bilayers. The channel assumes a fixed orientation in the bilayer and displays two conducting states (B. Tomlins, A.J. Williams & R.A.P. Montgomery, 1984, J. Membrane Biol. 80:191-199). SC blocks potassium conductance through the channel in a voltage-dependent manner. Block occurs from both sides of the channel, in both conducting states and is resolved as discrete "flickering" events. Although SC is capable of blocking potassium conductance from both sides of the membrane, block is asymmetric. The zero-voltage dissociation constant for block from the cis side of the membrane is approximately threefold lower than that from the trans side. Block from the cis side displays a linear dependence on SC concentration for both open states and is competitive with potassium ions at saturating potassium activities, consistent with a single-site blocking model. The degree of SC-induced block is also influenced by membrane surface charge. SC block differs from that previously described for bis quaternary ammonium (bis Qn) compounds such as decamethonium in that SC blocks preferentially from the cis side of the channel.

Animals↗
PubMed1985

In vitro cultivation of Trypanosoma congolense: establishment of infective mammalian forms in continuous culture after isolation from the blood of infected mice.

Bloodstream form trypomastigotes of four cloned stocks of Trypanosoma congolense from West Africa were successfully adapted to continuous in vitro culture at 28 degrees C using bovine aorta endothelial cell monolayers and Eagle's minimum essential medium supplemented with 20% normal bovine serum or foetal calf serum. The trypanosomes maintained in vitro morphologically resembled bloodstream forms and remained infective for vertebrate hosts. They also induced local skin reactions in rabbits and were therefore designated "mammalian forms", possibly resembling parasites which develop extravascularly in the vertebrate host following introduction of metacyclic trypanosomes into the skin by bites of tsetse flies. Mammalian forms of two stocks were allowed to transform to procyclic trypanosomes in order to obtain cultures producing epimastigote and metacyclic stages of T. congolense. Metacyclic trypanosomes produced in this manner were shown to be neutralized by antiserum raised in rabbits against the homologous trypanosome stock transmitted by tsetse flies.

Animals↗
PubMed1985

In vitro cultivation of Trypanosoma congolense: the production of infective forms from metacyclic trypanosomes cultured on bovine endothelial cell monolayers.

After transfer to bovine endothelial cell monolayers cultured in Eagle's minimal essential medium at 28 degrees C or 37 degrees C metacyclic trypanosomes of three cloned stocks of Trypanosoma congolense became morphologically similar to parasites found in the bloodstream of the vertebrate host. The trypanosomes resumed division and grew in close association with the mammalian cells, which were essential for growth. These dividing infective forms had the ability to cause local skin reactions and systemic infections when inoculated intradermally into rabbits. Trypanosomes grown in medium supplemented with foetal calf serum (FCS) eventually differentiated into procyclic forms. No such change occurred in medium supplemented with normal bovine serum. If procyclic forms in FCS were allowed to continue their differentiation at 28 degrees C they eventually produced epimastigotes which gave rise to infective metacyclic trypanosomes once more. It was thus possible to grow and maintain several different developmental stages of T. congolense by varying culture conditions.

Animals↗
PubMed1984

'Impermeant' inhibitors of protein synthesis do not affect virally-infected cells as a result of a permeability change.

The effect of certain antiviral compounds said to act because of an increased permeability of virally infected cells has been tested in SFV-infected BHK cells. The time at which SFV-infected BHK cells become sensitive to the action of GppCH2p is more than an hour later than the time at which protein synthesis in such cells becomes depressed. The uptake of [3H]GppCH2p is the same in infected and uninfected cells, whether measured at 19 or 37 degrees C. We conclude that GppCH2p, and probably other 'impermeant' inhibitors of protein synthesis also, affect virally-infected cells selectively not because of an increased permeability, but because of a general impairment of protein synthesis in such cells.

Animals↗
PubMed1984

In vitro cultivation of Trypanosoma congolense: the production of infective metacyclic trypanosomes in cultures initiated from cloned stocks.

Glossina morsitans were infected with two cloned stocks of T. congolense. The proboscides, foreguts and midguts of infected flies were then used as sources of trypanosomes in vitro at 28 degrees C in the presence of bovine dermal collagen explants. Cultures were established in which trypanosomes differentiated into adhering colonies of epimastigote forms which could then be maintained and subcultured in Eagle's Minimum Essential Medium supplemented with foetal calf serum for over 40 weeks. Within 2-4 weeks of establishment of each culture or subculture the epimastigote trypanosomes differentiated into metacyclic trypanosomes which could be harvested from supernatant medium at concentrations of 1 X 10(5)-3 X 10(6) parasites/ml. These organisms were used to induce the formation of local skin reactions in rabbits. Successful cultivation of infective trypanosomes appeared to depend on the initial adhesion of the parasites to the surface of the flask where they subsequently differentiated first into epimastigote and then to metacyclic forms.

Animals↗
PubMed1983

Protein synthesis in cells infected with Semliki Forest virus is not controlled by intracellular cation changes.

Treatment of BHK cells with 1 microM nigericin results in a 55% decrease in K+ and a 3.3-fold increase in intracellular Na+; protein synthesis under these conditions is depressed by 35%. In BHK cells infected with Semliki Forest virus (SFV), protein synthesis is depressed by 76% 6.5 h after infection; intracellular K+ is unchanged, and intracellular Na+ is increased 1.8-fold at this time. These results suggest that the increase in intracellular Na+ in SFV-infected BHK cells does not adequately account for the decrease in protein synthesis, and makes it likely that an increased Na+ concentration is a consequence, not a cause, of alterations in protein synthesis in virally-infected cells. No evidence was obtained for the purported [Alonso, M. A. and Carrasco, L. (1980) Eur. J. Biochem. 109, 535-540; (1981) Eur. J. Biochem. 118, 289-294; (1981) FEBS Lett. 127, 112-114] ability of 1 microM nigericin to permeabilize' cells.

Animals↗
PubMed1983

Protein synthesis in Semliki Forest virus-infected cells is not controlled by permeability changes.

The uptake of the GTP analogue guanylyl(beta,gamma-methylene)diphosphonate (GppCH2p) is the same in Semliki Forest virus (SFV)-infected BHK cells as in mock-infected cells, in spite of the fact that protein synthesis is inhibited by GppCH2p more markedly in SFV-infected cells than in control cells. A possible explanation for this difference is that infected cells have a lower concentration of GTP and a lower ratio of GTP:GDP than uninfected cells, and the analogue may thus be a more effective competitive inhibitor of translation. We conclude that in this system, the difference between infected and uninfected cells lies not at the plasma membrane but within the cytoplasm.

Animals↗
PubMed1983

Effect of vesicular stomatitis virus and Semliki Forest Virus on uptake of nutrients and intracellular cation concentration.

BHK-21 cells showed an increased ability to concentrate 2-deoxy-D-glucose (dGlc) 2 to 3 h after infection with vesicular stomatitis virus (VSV) or Semliki Forest virus (SFV), which began to be released at 2 and 3 h post-infection respectively; uptake of other nutrients was not affected in this way. Intracellular Na+ was either unchanged (VSV-infected cells) or increased (SFV-infected cells); K+ content was unchanged. These results do not support the current hypothesis that a non-specific increase in membrane permeability occurs in cells infected with rhabdoviruses or togaviruses.

Animals↗
PubMed1982

Membrane changes during viral infection.

The effect of viruses on the surface membrane of susceptible cells during the entry and exit process has been studied. Haemolytic paramyxoviruses induce a non-specific leakage to low-molecular-weight compounds during entry; other viruses do not show this effect. During exit, no such changes occur with any virus so far studied: some viruses are released without any obvious change at all in surface membrane function; in other cases, uptake of some nutrients is altered and there is a fall in membrane potential. These results do not support the hypothesis that a generalized membrane leakiness is a prerequisite for the synthesis and release of virus particles.

Animals↗
PubMed1981

Cultivation of infective forms of Trypanosoma congolense from trypanosomes in the proboscis of Glossina morsitans.

Two stocks of Trypanosoma congolense were established in culture at 28 degrees C using trypanosomes from the proboscides of infective Glossina morsitans. Successful primary cultures were initiated by placing an infected tsetse proboscis beside a bovine dermal collagen explant in Eagle's minimum essential medium supplemented with foetal calf serum. The trypanosomes multiplied rapidly in the medium and also gradually formed an adherent layer o the plastic surface of the culture vessel. Three primary cultures produced organisms infective for mice from 14, 20 and 35 days after initiation and thereafter continuously until days 76, 76 and 52 when they were discarded. Four attempts to initiate infective cultures using infected tsetse proboscides in medium without dermal explants were unsuccessful. When trypanosomes from primary cultures were placed in culture medium with proboscides from uninfected tsetse flies, the parasites multiplied, formed an adherent layer in the culture flasks and were seen in the proboscides within 24 h. A line of 1 stock was serially sub-passaged in this way 4 times during a period of 215 days. Infectivity titrations in mice indicated that primary and sub-passaged cultures each contained similar numbers of infective organisms. Another line of the same stock was also sub-passaged 4 times in medium alone over a period of 186 days. These sub-cultures again retained infectivity for mice, but titrations showed a decrease in infective organism production in the 4th sub-culture. Primary and sub-passaged cultures all included a variety of morphologically different developmental forms of T. congolense, closely resembling those described in the labrum and hypopharynx of Glossina by previous workers. Short metacyclic-like trypanosomes and organisms with proteinaceous surface coats were present in infective cultures. Cultures were successfully re-established after cryopreservation at -196 degrees C and retained the ability to produce infective organisms.

Animals↗
PubMed1980

Evaluation of an enzyme immunoassay for serodiagnosis of infections with Theileria parva and T annulata.

An enzyme linked immunosorbent assay (ELISA) was used to determine antibody levels in cattle infected with Theileria parva and T annulata, using antigens prepared from the intra-erythrocytic piroplasm stage of the parasites. Antibody levels in calves infected with T parva increased from the 16th day after infection to reach peak values at days 28 to 35 and then declined rapidly, but in calves infected with T annulata antibody levels rose steadily up to day 40. Similar patterns of antibody production were shown by indirect fluorescent antibody tests. Sera from animals infected with T parva gave higher ELISA values with the antigen prepared from the homologous parasite species than with the antigen prepared from T annulata, but sera from cattle infected with T annulata gave similar high ELISA values with antigens prepared from both T parva and T annulata. Sera from animals infected with T mutans, T sergenti, T velifera, Babesia divergens, B major and B bovis gave only slight or no cross reactions with the piroplasm antigens, but serum from a calf infected with B bigemina cross reacted at a significant level with both piroplasm antigens.

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