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Biomedical subjects

R J Cherry

Publications and source records attributed to R J Cherry.

At least 55 records · Page 3Linked to original sources
PubMed1989

Digital fluorescence imaging of fusion of influenza virus with erythrocytes.

Fusion of influenza virus with human erythrocytes at pH 5.2 was followed by fluorescence microscopy using a cooled slow-scan CCD camera. The high sensitivity of the CCD permits repetitive digital imaging of the same cells with minimal photobleaching. The experimental conditions were such that only a small number of virus particles were adsorbed per cell. Quantitative analysis of the data indicated that for most cells only a single fusion event took place. This was, however, sufficient to cause haemolysis within 30 min at 20-22 degrees C for about 60% of cells. There was a highly variable time lag between fusion and haemolysis. The lateral diffusion coefficient of virus particles on the cell surface when bound at pH 7.4 was less than 2 x 10(-13) cm2.s-1. The technique should be of value for more detailed studies of the dynamics of viral and other membrane fusion events.

Erythrocytes↗
PubMed1989

Cytoskeletal restraints of band 3 rotational mobility in human erythrocyte membranes.

The interaction of band 3 with cytoskeletal proteins was investigated in erythrocyte membranes by measuring the rotational mobility of band 3 using the method of transient dichroism. It was found that selective proteolysis of ankyrin, a protein known to link band 3 to the spectrin-actin network, had no significant effect on band 3 rotation. Incubating ghosts to 70 degrees C, at which temperature ankyrin is expected to be denatured, also had no effect. It thus appears probable that linkage of band 3 to the cytoskeleton via ankyrin does not act as a restraint on band 3 rotational motion. It is suggested that this is a consequence of flexibility in the cytoskeletal structure. In further investigations of the effect of heat treatment, a large enhancement of band 3 rotational mobility was found to result from incubation of intact cells for 1 h at 50 degrees C. This effect was not observed if ghosts were subjected to the same treatment, nor did it occur if the incubation of cells was performed at 47 degrees C. These findings, in combination with previous studies of band 3 rotational mobility, indicate that the interactions which restrain band 3 are likely to be more complex than commonly envisaged.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1989

A comparative study of band 3 aggregation in erythrocyte membranes by melittin and other cationic agents.

The technique of laser flash-induced transient dichroism has been used to measure the rotational diffusion of eosin-labelled band 3 proteins in erythrocyte ghosts. A retardation in the mobility of band 3, measured subsequent to the addition of a variety of polyvalent cationic species, has been interpreted to reflect aggregation or 'clustering' of the protein in the plane of the membrane. A comparative study is reported between three such aggregators: melittin, polylysine and Zn2+, wherein their respective abilities to induce aggregation have been measured under varying conditions. Unlike that for melittin, band 3 aggregation by polylysine and Zn2+ is shown to be sensitive to proteolytic degradation of the membrane and to the ionic strength of the surrounding medium. Studies with fragments of melittin derived from its chymotryptic cleavage show the hydrophilic C-terminal 20-26 section to possess independent aggregating ability, but also the requirement of the 1-19 hydrophobic section to be attached in order to prevent reversibility by high ionic strength buffers. Melittin is also shown to have a unique ability to aggregate bacteriorhodopsin reconstituted into DMPC vesicles, which is partially retained by its 1-19 but not by its 20-26 fragment.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1988

Comparison of p25 presequence peptide and melittin. Red blood cell haemolysis and band 3 aggregation.

The 25 residue presequence (p25) for subunit IV of yeast cytochrome oxidase had previously been shown to possess structural and behavioural characteristics in common with the bee venom polypeptide, melittin. The present study extends the results of leakage experiments on model-membrane systems to the haemolysis of human erythrocytes, which both peptides are shown to accomplish in a manner sensitive to membrane potential. In addition, the laser flash-induced transient dichroism technique for measuring protein rotational diffusion has been used to show that both peptides aggregate band 3, the major integral membrane protein of the erythrocyte. Aggregation cannot be reversed by high ionic strength; this serves to differentiate these peptides from other positively charged species such as polylysine that aggregate band 3 at low ionic strength. These results suggest that aggregation of membrane proteins may possibly prove to be a feature of the interaction of p25 signal peptide with mitochondrial membranes.

Amino Acid Sequence↗
PubMed1986

Temperature and pH dependence of the haemolytic activity of influenza virus and of the rotational mobility of the spike glycoproteins.

Influenza virus (strain X-47) was labeled with the triplet probe, eosin 5-isothiocyanate. Most of the label was found to be associated with haemagglutinin, the major glycoprotein of the viral envelope. Rotational diffusion of the glycoprotein was investigated by measuring flash-induced transient dichroism of the eosin probe. The anisotropy decay curves showed that mobility of haemagglutinin measured at pH 7.3 increased considerably with temperature with the greatest change occurring over the range 20-30 degrees C. However, at pH 5.2 no mobility was detectable over the time range of the experiment. The activity of the virus was determined by assaying haemolysis of human erythrocytes. The haemolytic activity showed an optimum at pH 5.2 and increased markedly with temperature, being negligible below 20 degrees C. In addition, inactivation of the virus by incubation at pH 5.2 was also strongly temperature dependent. A 15 min incubation at pH 5.2 inactivated the virus above 30 degrees C but had no effect below 20 degrees C. On the basis of these results, it is proposed that mobility of haemagglutinin is significant for its functional properties. When the pH is reduced from 7.3 to 5.2, the mobility observed at higher temperatures is required for the molecular rearrangements which accompany the fusion event. In the absence of an apposing membrane, these rearrangements result in irreversible aggregation of haemagglutinin in the viral membrane, and hence loss of mobility and activity.

Circular Dichroism↗
PubMed1986

The value of a next day hand injury review clinic.

A daily hand injury review clinic takes place at East Birmingham Hospital. The purpose of this study is to determine whether this clinic is beneficial in reducing the morbidity of neglected hand injuries, and to compare the running of the clinic with the original criteria laid down at its inception as a "once only' screening clinic. We conclude that such a clinic is highly beneficial, and recommended that it should be standard practice in other hospitals.

England↗
PubMed1985

Lipid peroxidation decreases the rotational mobility of cytochrome P-450 in rat liver microsomes.

Phenobarbital-induced rat liver microsomes were subjected to NADPH- and Fe2+-catalyzed lipid peroxidation. The formation of approx. 95 nmol malondialdehyde/mg protein during 18 min peroxidation at 37 degrees C was observed. Membrane rigidity measured by means of the steady-state fluorescence anisotropy rs of diphenylhexatriene increased in parallel with the malondialdehyde formation. Both the amount of malondialdehyde and rs remained constant thereafter during incubation of the peroxidized membranes for 2 h. The aminopyrine demethylase activity decreased by about 60% upon lipid peroxidation for 18 min, whereas no significant loss of benzphetamine demethylase activity within the same time range was observed. A time-dependent formation of protein complexes of high molecular weight, comprising most of the microsomal polypeptides, upon lipid peroxidation was observed in SDS-polyacrylamide gel electrophoresis. The effect of microsomal lipid peroxidation on protein-protein interactions was examined by measuring the rotational mobility of intact cytochrome P-450. Rotational diffusion was measured by observing the decay of flash-induced absorption anisotropy r(t) of the P-450 X CO complex. Analysis was based on a 'rotation-about-membrane normal' model with the equation r(t) = r1exp(-t/phi 1) + r2exp(-t/phi 2). In control microsomes, two classes (rapid and slow) of rotating populations of cytochrome P-450 were observed with phi 1 approximately equal to 150 microseconds, fraction r1/(r1 + r2) approximately equal to 40% and phi 2 approximately equal to 2 ms, fraction r2/(r1 + r2) approximately equal to 60%. A relatively small decrease in the rotational mobility of P-450 was observed by a 18-min lipid peroxidation, while a subsequent incubation of peroxidized microsomes for 2 h at 37 degrees C resulted in a dramatic immobilization of P-450 by the increase of both r2/(r1 + r2) approximately equal to 75% and phi 2 approximately equal to 10-25 ms. The decrease in the P-450 mobility during 18-min lipid peroxidation would be due to the rigidification of the lipid bilayer. However, because the lipid fluidity remained unchanged thereafter, the significant immobilization of P-450 by the subsequent 2-h incubation is deduced to be due to formation of protein aggregates.

Animals↗
PubMed1985

Protein rotational diffusion measurements on the interaction of bee venom melittin with bacteriorhodopsin in lipid vesicles.

The rotational diffusion of bacteriorhodopsin reconstituted into dimyristoylphosphatidylcholine vesicles was measured by the technique of flash-induced transient dichroism. In the presence of melittin, a cell lysing peptide from honey bee (Apis mellifera) venom, dose-dependent loss of rotational mobility was observed. Chemically modified melittin derivatives, in which free amine groups were either acetylated or succinylated, were impaired in their ability to induce immobilisation of bacteriorhodopsin. Bacteriorhodopsin reconstitutions of differing lipid/protein ratio were tested and it was found that the bacteriorhodopsin immobilisation phenomena depended on the melittin/protein ratio, not the melittin/lipid ratio. This suggests that melittin produces its effect via direct interaction with bacteriorhodopsin. A mechanism is proposed in which the aggregation of bacteriorhodopsin is induced by electrostatic attraction between its anionic surface moieties and the highly cationic C-terminal segment of melittin.

Acetylation↗
PubMed1985

Rotational diffusion and self-association of band 3 in reconstituted lipid vesicles.

Band 3, the anion transporter of the human erythrocyte membrane, has been purified and reconstituted into phospholipid vesicles of varying composition. Rotational diffusion of band 3 in these vesicles was measured by observing flash-induced transient dichroism of an eosin triplet probe covalently bound to the protein. In egg phosphatidylcholine vesicles of high lipid/protein ratio at temperatures well above the gel to liquid-crystalline phase transition, the absorption anisotropy decays to a constant value of 12 +/- 1% (expressed as a percentage of the initial anisotropy). However, higher values of the residual anisotropy and a slower decay are observed upon decreasing the temperature, even though the lipids remain in a liquid-crystalline phase. A similar effect is observed upon decreasing the lipid/protein ratio at constant temperature. It is concluded that self-association of band 3 occurs which is dependent on temperature and protein concentration in the bilayer. It is very probable that similar effects occur in the erythrocyte membrane, where a strong temperature dependence of band 3 rotational mobility is also observed. Reconstitution of band 3 into dioleoylphosphatidylcholine vesicles yields results similar to those obtained with egg phosphatidylcholine. When dimyristoylphosphatidylcholine is used for reconstitution, band 3 is immobilized below the lipid phase transition and remains partially associated above the transition. A precise analysis of the anisotropy decay curves is hampered by the presence of multiple rotating species.(ABSTRACT TRUNCATED AT 250 WORDS)

Anion Exchange Protein 1, Erythrocyte↗