Graham L. Jones

Decreased rotational diffusion of band 3 in melanesian ovalocytes from Papua, New Guinea

SummaryMelanesian ovalocytes from Papua New Guinea have an N-terminal extension of the band 3 polypeptide (Jones, G.L., Edmunson. H.M., Wesche, D., Saul, A. 1990.Biochim. Biophys. Acta1096:33–40). The ovalocytes showed a threefold increase in shear elastic modulus as determined by micropipette aspiration measurements of membrane rigidity. Time-resolved phosphorescence anisotropy has been used to study the rotational freedom of band 3 in membranes prepared from ovalocytes. The ovalocytic polymorphism was found to be associated with a marked decrease in the rotational mobility of band 3. This may indicate participation of band 3 in large homoaggregates or in complexes with other proteins at the cytoplasmic surface. There was no morphological clustering of band 3 detectable by immunofluorescence microscopy.

Age-related attenuation in the expression of the major heat shock proteins in human peripheral lymphocytes

A defining characteristic of human ageing is the reduced ability to maintain homeostasis in the face of adverse environmental stresses. This progressive impairment may be a major cause for the increased incidence of infections, and general morbidity and mortality in the elderly. Heat shock proteins (hsps) or stress proteins, induced in response to hyperthermia and to various other physical, chemical and biological stressors, are often also expressed constitutively at a lower level and perform many essential functions in the cell. Here we investigate age-related changes in the heat induced expression of a comprehensive range of hsps at the translational level using primary human peripheral lymphocytes in short term culture. Our study reveals age-related attenuation in the response of the well characterised up-regulated molecular chaperone system hsp 70, the steroid-receptor binding hsp 90 and the chaperonin hsp 60. A diminution with age is also demonstrated in the heat induced response of hsps 105, 56, 47, 40, 27, and 16. Differentially down-regulated proteins at 100, 38, and 18 kDa were also noted.

Influence of ageing, heat shock treatment and in vivo total antioxidant status on gene-expression profile and protein synthesis in human peripheral lymphocytes.

Ageing results in a progressive, intrinsic and generalised imbalance of the control of regulatory systems. A key manifestation of this complex biological process includes the attenuation of the universal stress response. Here we provide the first global assessment of the ageing process as it affects the heat shock response, utilising human peripheral lymphocytes and cDNA microarray analysis. The genomic approach employed in our preliminary study was supplemented with a proteomic approach. In addition, the current study correlates the in vivo total antioxidant status with the age-related differential gene expression as well as the translational kinetics of heat shock proteins (hsps). Most of the genes encoding stress response proteins on the 4224 element microarray used in this study were significantly elevated after heat shock treatment of lymphocytes obtained from both young and old individuals albeit to a greater extent in the young. Cell signaling and signal transduction genes as well as some oxidoreductases showed varied response. Results from translational kinetics of induction of major hsps, from 0 to 24 h recovery period were broadly consistent with the differential expression of HSC 70 and HSP 40 genes. Total antioxidant levels in plasma from old individuals were found to be significantly lower by comparison with young, in agreement with the widely acknowledged role of oxidant homeostasis in the ageing process.

Cross–reactivity of antibody against an epitope of the Plasmodium falciparum second merozoite surface antigen

Summary Monoclonal antibodies directed against the 51 kD merozoite surface antigen of Plasmodium falciparum also bind to other antigens within the infected cell. The sizes of these cross–reacting antigens have been characterized. Immunofluorescence due to the reaction of one of the monoclonal antibodies with these cross–reacting antigens was localized in the intra–erythrocytic parasite and in granules in the infected red cell cytoplasm. This immunofluorescence could be distinguished from the merozoite surface antigen in parasite lines with a variant serotype of the merozoite surface antigen which fails to react with the monoclonal antibodies. It was found that the in–vitro growth inhibition caused by the presence of one of the monoclonal antibodies, 8G10/48, was dependent on the expression of the corresponding serotype of merozoite surface antigen, a finding consistent with the inhibitory effect of this antibody being primarily directed against the merozoite surface antigen and not the cross–reacting antigens. Analysis of the frequency at which epitopes occur suggests that such cross–reacting proteins will be commonly seen in malaria, without the need to postulate a selective advantage for such cross–reacting specificities.

Characterisation of an inhibitory monoclonal antibody-defined epitope on a malaria vaccine candidate antigen

A monoclonal antibody that recognizes a recently characterised 45-kDa merozoite surface antigen of the human malaria parasite Plasmodium falciparum inhibits the growth of the asexual blood stages of the parasite in vitro. The corresponding epitope has been determined by testing the reactivity of the antibody with sequentially overlapping octapeptides. A synthetic peptide containing the epitope elicits antibodies that react with the native antigen. Epitope mapping in this manner is useful in the design of synthetic vaccines against malaria.

Rotational dynamics of the integral membrane protein, band 3, as a probe of the membrane events associated with Plasmodium falciparum infections of human erythrocytes.

Time-resolved phosphorescence anisotropy was used to study the molecular organisation of band 3 in the erythrocyte membrane. Three different rotational relaxation regimes of mobile band 3 were resolved. These populations may represent different aggregation states of band 3 within the membrane, or they may result from association of band 3 with other proteins at the cytoplasmic surface. The polycation spermine decreases the apparent mobility of band 3 by a mechanism that does not involve the underlying cytoskeleton. A monoclonal antibody directed against the cytoplasmic portion of band 3 can also cause an increase in the immobile fraction of band 3 molecules. This monoclonal antibody will inhibit invasion of erythrocytes by malaria parasites. Membranes prepared from erythrocytes infected with mature stages of the malaria parasite, Plasmodium falciparum, show altered dynamic properties corresponding to a marked restriction of band 3 mobility.

Human erythrocyte Band-3 has an altered N terminus in malaria-resistant Melanesion ovalocytosis

There is a high prevalence of the erythrocyte polymorphism ovalocytosis associated with reduced susceptibility to malaria in Papua New Guinea. The major erythrocyte integral membrane protein, Band-3, showed markedly increased phosphorylation in whole cells or isolated ghosts from ovalocytic individuals. The cytoplasmic domain of the ovalocyte Band-3 was found to be approx. 3 kDa larger than the normocytic protein. The N-terminal sequence of the ovalocytic Band-3 was different from the reported sequence for human Band-3, suggesting that the increased size results from an N-terminal extension. Since this is the region of Band-3 which is phosphorylated and interacts with the red cell cytoskeleton, it is likely that this alteration in ovalocytic Band-3 is the underlying cause of the diverse alterations in ovalocytic cells including increased phosphorylation, increased membrane rigidity, decreased agglutinability by blood group antibodies and refractoriness to invasion by malarial parasites.

Peptide vaccines derived from a malarial surface antigen: effects of dose and adjuvants on immunogenicity

Peptides P2122 (CKNNNSTNSGI) and P513 (CSQRSTNSAST) containing an epitope of a malarial surface antigen (MSA2) recognised by inhibitory monoclonal antibodies were conjugated to diphtheria toxoid (DT) protein and formulated with various gel-based and water in oil emulsion adjuvants in vaccine trials in mice and rabbits. The P2122-DT construct was effective in raising antibodies reactive with both the immunising peptide and the native antigen. Effective adjuvanticity as measured by the titre of the anti-peptide or anti-protein response in mice varied in the order: Algammulin, Montanide ISA 50 greater than or equal to Freunds adjuvant, Montanide ISA 708, 721, 70 much greater than alum, Squalene Arlacel greater than SAF-1. A similar order of adjuvant efficacy: Freunds greater than alum greater than Squalene Arlacel greater than SAF-1, was observed in rabbits.

Changes in lysosomal cathepsins during liver regeneration

The effect of 2/3 hepatectomy in the adult rat on the specific and total activities of two lysosomal endopeptidases, cathepsins B1 and D, has been examined. The specific activity of both enzymes fell rapidly following hepatectomy when compared to paired, sham-operated controls. When changes in total protein were compared to changes in cathepsins B1 and D, all three decreased in a parallel fashion for the first 18 h. At 24 h, total liver protein increased rapidly while cathepsins B1 and D continued to decrease. Cathepsin B1 fell to a level of 12% of non-operated control levels at 36 h, while total protein was already back to 40% of control levels. In contrast to the decreases in the activities of the cathepsins, there was an increase in the activities of lactate dehydrogenase and malate dehydrogenase during the first two days of regeneration. The clear lag in replacement of the cathepsins relative to other liver proteins following partial hepatectomy suggests that cathepsin activity is selectively controlled and that lowering the levels of cathepsins B1 and D may play an important role in the decreased degradation of protein seen during the early phases of liver regeneration.

Immunological fine structure of the variable and constant regions of a polymorphic malarial surface antigen from Plasmodium falciparum

The 51-kDa merozoite surface antigen MSA2 of Plasmodium falciparum shows considerable strain-dependent polymorphism. Although marked sequence variation occurs in the central region of the molecule, the N and C-terminal sequences are highly conserved. A number of monoclonal antibodies directed against MSA2 have been described which inhibit parasite growth in vitro, but these are all directed against variable regions. In an attempt to raise strain independent antibodies we have prepared peptide-diphtheria toxoid (DT) constructs from 36 N-terminal octapeptides spanning the constant region and extending into the variable region of the FCQ/27 PNG variant staggered by one amino acid at either end. Similarly, we prepared 26 C-terminal octapeptides spanning the C-terminal constant region as well as 10 octapeptides from the variable region of the Indochina I variant MSA2. Most of the peptides elicited antipeptide titres in excess of 1/10(4) when administered to mice as peptide-DT adducts emulsified with Freunds complete adjuvant. Only 3 of the 43 N- and C-terminal constant region peptides elicited antibodies which reacted appropriately on immunofluorescence (IFA) or immunoblotting analysis with the intact MSA2 of both strains studied (FCQ/27 and Indochina I), whereas 3 other peptides from the variable region elicited antibodies reactive with the parent MSA2 only. Peptide constructs eliciting antibodies recognising the intact protein corresponded to elements in the cognate sequence of high antigenicity as predicted by the Jameson and Wolf algorithm.