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Biomedical subjects

A H Maddy

Publications and source records attributed to A H Maddy.

At least 19 recordsLinked to original sources
PubMed1993

Alternative pathways of apoptosis induced by methylprednisolone and valinomycin analyzed by flow cytometry.

Apoptosis of murine thymocytes induced by either methylprednisolone or valinomycin was studied by flow cytometry. The apoptosis induced by methylprednisolone followed three stages: an initial decrease in cell volume, indicated by a fall in forward scatter accompanied by faint ethidium bromide staining, a second stage in which the cells became brightly stained by ethidium bromide, and a final stage when the cells were apparently less fluorescent as the nuclei disintegrated into apoptotic bodies. As the forward scatter of cells decreased there was a simultaneous depolarization of the cells and an elevation of intracellular calcium. These early changes preceded the fragmentation of the DNA which also preceded the intense staining of the cells by ethidium bromide. Methylprednisolone-induced apoptosis was inhibited by low concentrations (1 x 10(-7) M) of valinomycin and nonactin, neither of which could themselves induce apoptosis at these low concentrations. Cadmidazolium and cycloheximide arrested the program at an early stage. Okadaic acid allowed volume loss and ethidium bromide staining to proceed in the absence of DNA fragmentation. At high concentrations (1 x 10(-5) M) valinomycin induced a form of apoptosis, but nonactin only caused the cells to fragment. The valinomycin-induced apoptosis, although it involved the degradation of DNA and the disintegration of the nuclei into apoptotic bodies, differed from the methylprednisolone apoptosis as it did not involve a decrease of cell volume and was not inhibited by cycloheximide or affected by okadaic acid.

Animals↗
PubMed1992

Simultaneous analysis of immunophenotype and apoptosis of murine thymocytes by single laser flow cytometry.

The study of the role of apoptosis in thymocyte development has been hampered by the lack of a means of directly immunophenotyping cells undergoing the early phase of apoptosis. This restriction has been overcome by single laser flow cytometry in which apoptosis is detected by Ethidium Bromide (EBr) staining and cell phenotype by binding of FITC-labelled antibody. The initial phase of apoptosis is observed as a cell population that stains faintly with EBr preceding the characteristically bright EBr-staining normally associated with cell death. Here we directly demonstrate using single laser flow cytometry that CD4+ CD8+ CD3low/CD3intermediate thymocytes undergo apoptosis in vitro in response to glucocorticoid treatment.

Animals↗
PubMed1990

The analysis of the variation of the surface proteins of leukemic lymphocytes of B-chronic lymphocytic leukemia patients by high performance liquid chromatography.

Surface phenotypes of leukemic lymphocytes are analyzed by high performance liquid chromatography of the vectorially iodinated surface proteins and the variation of the phenotypes of B-chronic lymphocytic leukemia (B-CLL) lymphocytes from different individuals is studied. A number of surface molecules show a coordinated variation between patients in their level of expression. Some of these molecules belong to recognized clusters of differentiation, e.g., CD45 and CD21 and their variation can be confirmed by flow cytometry. But the HPLC also reveals other components that have not been assigned to known clusters, e.g., a component of Mr of around 300 kD. Two types of B CLL lymphocytes can be recognized by this set of molecules and the patients ranked according to the level of expression of these markers on their leukemic cells. The effects of TPA treatment on expression suggests that these molecules represent a maturational sequence and that the leukemias are derived from progressive stages along this process.

Antigens, Differentiation↗
PubMed1989

The identification of antigens in antibody/antigen complexes using high performance liquid chromatography.

A method for the identification of antigens in a complex mixture of proteins is described. It exploits the effect which addition of a monoclonal antibody and resultant formation of antibody/antigen complexes has on the apparent retention time of the antigen on a size exclusion column. An antigen in the mixture can be identified as that component whose retention time is decreased by addition of its antibody. The principle of the method is demonstrated using an HPLC size exclusion column (DuPont GF450) and three MoAbs, PD7/26, DA6 147 and W6/32 which are directed against three antigens of the lymphocyte surface, the leucocyte-common antigen, class II MHC and class I MHC respectively.

Antibodies, Monoclonal↗
PubMed1989

The role of cell maturation in the generation of phenotypic heterogeneity in B-cell chronic lymphocytic leukaemia.

B-chronic lymphocytic leukaemia (B-CLL) patients can be ranked along a progression of phenotypes characterized by a decreasing surface expression of CD20, CD21, CD22 and membrane immunoglobulin and a gradual replacement of the high molecular weight (MW) glycoproteins of the leucocyte-common antigen (LC) CD45RA by the lower MW components, including the CD45RO determinant. As CD20, CD21, CD22 and membrane immunoglobulin change during or after B-cell activation, and the CD45RA/CD45RO inversion is implicated in T-cell maturation, the possibility that the phenotypic differences are generated by a maturational diversity of the CLL clones has been investigated by testing the effects of TPA treatment of the leukaemic cells. TPA reduces the level of expression of CD20, CD21, mIg and CD45RA and increases CD45RO binding, thereby minimizing the phenotypic heterogeneity of the CLL clones and causing them to converge towards one end of the natural range. We propose that the phenotypic diversity in CLL is, at least in part, a consequence of maturational diversity where lymphocyte development is disrupted at different stages in different patients.

Antigens, CD↗
PubMed1985

Abnormalities in the expression of the leucocyte-common antigen in chronic lymphocytic leukaemia.

The surface glycoproteins of lymphocytes isolated from patients suffering from B cell chronic lymphocytic leukaemia (B-CLL) have been studied by radioactive labelling with impermeable probes and with MoAb. Several features not found in normal B cells have been observed. The abnormalities found in the expression of polypeptides of the leucocyte common (L-C) antigen, identified by appropriate MoAb, have been examined in detail. It has been shown by both biochemical analysis and MoAb binding that this group of polypeptides can, within a panel of B-CLL patients, range from a typical B cell pattern to the pattern resembling that normally found in T cells. The T lymphocyte profile is correlated with a poor prognosis (MVA C rating) and in the one patient where a change in the glycoprotein profile towards that of the T cell was observed, the change coincided with a clinical deterioration. The biological significance of the molecular diversity is discussed.

Antigen-Antibody Reactions↗
PubMed1985

Surface glycoproteins as markers of the cellular status of B chronic lymphocytic leukaemia lymphocytes.

The phenotype of B CLL cells is investigated with respect to their surface glycoproteins. These glycoproteins are identified by vectorial tritiation followed by 1 and 2 dimensional gel electrophoresis, and by lectin and MoAb binding using immunoprecipitation and flow cytometry. The profiles of the CLL cells are compared with those of normal B cells, prepared from tonsils, and T cells from peripheral blood. The CLL cells show many similarities with T cells, particularly the expression of glycoproteins which bind the MoAbs gpL 115, F10-44-2 and EZB 52, and a complex set of binding sites for Helix pomatia lectin. The significance of these observations in terms of the cellular origins of the leukaemic lymphocytes is discussed.

Antibodies, Monoclonal↗
PubMed1984

Abnormal pattern of erythrocyte ageing in hereditary spherocytosis as shown by Percoll density gradient centrifugation.

Erythrocytes from individuals with Hereditary Spherocytosis (HS) were fractionated on Percoll density gradients. The osmotic fragility, mean cell volume, mean cell haemoglobin concentration, reticulocyte count and cell morphology of the fractions showed that the gradients separate the erythrocytes according to age, the older small spherocytes appearing in the dense fractions. Comparison of the well established parameters of erythrocyte ageing, pyruvate kinase activity, acetylcholinesterase activity and cell potassium content in normal and HS erythrocyte fractions of similar age revealed abnormalities in the senescence of HS erythrocytes both before and after splenectomy.

Acetylcholinesterase↗
PubMed1981

The abnormal phosphorylation of spectrin in human hereditary spherocytosis.

The phosphorylation of the proteins of the erythrocyte membrane of patients suffering from hereditary spherocytosis is investigated in intact erythrocytes by their incubation in the presence of radioactive inorganic phosphate. Examination of the phosphorylated components by high-resolution two-dimensional gel electrophoresis reveals only one defect in the pathological membranes, a depressed phosphorylation of the smaller polypeptide of spectrin; band 2. The phosphorylation of band 2 is measured with reference to the phosphorylation of syndein (2.1 + 2.2 + 2.3). In patients showing overt clinical symptoms and for whom splenectomy is advocated the phosphorylation of band 2 is depressed by approx. 70%. After splenectomy the phosphorylation of membrane proteins is restored to normal levels.

Adolescent↗
PubMed1981

The molecular basis of the defect in phosphorylation of spectrin in human hereditary spherocytosis.

The molecular basis for the depressed phosphorylation of the smaller polypeptide of spectrin (band 2) in the erythrocytes of patients suffering from hereditary spherocytosis is investigated. Comparison of healthy and spherocytic spectrin polypeptides by controlled proteolysis reveals no abnormality in the degradation pattern or in the sites of phosphorylation. It is concluded that the lesion is a consequence of a defective control of phosphorylation. The defect can be mimicked in healthy cells by the introduction of calcium into the erythrocyte and the possibility that the primary pathological lesion is a deficient control of the calcium content of the erythrocyte is discussed.

Adult↗
PubMed1980

A re-evaluation of the surface complexity of the intact erythrocyte.

Surface proteins and glycoproteins of intact human red blood cells were labelled with 125I by the lactoperoxidase method. The radioactive proteins were then separated in each of the Fairbanks and Laemmli one-dimensional polyacrylamide gel electrophoresis systems. The radioactive polypeptides had different mobilities in the two systems, largely due to the anomalous migration of glycoproteins in polyacrylamide gels. A two-dimensional system was therefore developed using the Fairbanks and Laemmli buffer systems to exploit these anomalies. This procedure clearly resolved radioactive glycoproteins and proteins and enabled the identification of many more surface components than had previously proved possible.

Adult↗
PubMed1980

The polypeptides of rat liver nuclear envelope. I. Examination by nuclear pore complex polypeptides by solid-state lactoperoxidase labelling.

Purified nuclei retaining a high degree of ultrastructural integrity were isolated by conventional centrifugation techniques. The cytoplasmic surface of these nuclei was iodinated using lactoperoxidase immobilized onto giant Sepharose beads; thus the outer nuclear membrane and the cytoplasmic surface of nuclear pore complexes were selectively labelled. Pore complexes in association with a fibrous lamina were isolated from these nuclei by removal of the nucleoplasm and extraction with Triton X-100. The chemical composition of the pore-lamina fraction was 93.6% protein, 6% RNA, 0.4% phospholipid. The labelling suggests that major polypeptides N1 (70 000) and N2 (67 000) and more than 10 other more minor polypeptides, ranging from 33 000 to 200 000 mol. wt, as being components of the nuclear pore complex. Polypeptide N3 (58 000) is shown to be present only on the nucleoplasmic face of nuclear envelopes, probably in the fibrous lamina.

Animals↗
PubMed1980

The polypeptides of rat liver nuclear envelope. II. Comparison of rat liver nuclear membrane polypeptides with those of the rough endoplasmic reticulum.

Nuclear envelopes are separated into pore-lamina and membrane sub-fractions by extraction in 2.0% Triton X-100 followed by pelleting of the pore-laminae. The polypeptides of these subfractions are then compared with those from isolated rough endoplasmic reticulum. The dispositions of individual polypeptides in the cytoplasmic surface of nuclear envelopes and rought endoplasmic reticulum were studied by lactoperoxidase-catalysed iodination. These studies show that although the nuclear membranes exhibit several homologies with the Triton-soluble polypeptides of the rough endoplasmic reticulum the relative proportion of individual polypeptides within the two systems are very largely different. The cytoplasmic surfaces of the 2 membrane systems show only 2 obvious homologies at 105 000 and 15 000 mol. wt and the overall impression is that, at least in rat liver, the outer nuclear membrane is very substantially differentiated from rough endoplasmic reticulum. It is concluded that the nuclear membranes may not be regarded as a mere continuum of the endoplasmic reticulum, but should be seen as a highly specialized membrane system in their own right.

Animals↗
PubMed1978

The similarity of the two high-molecular-weight polypeptides of erythrocyte spectrin.

1. The two major polypeptides (P1 and P2) of erythrocyte-membrane spectrin were isolated by preparative polyacrylamide-gel electrophoresis. 2. The two polypeptides were shown to possess similar amino acid compositions, both with the characteristically high glutamate and leucine contents of the parent spectrin. 3. The tryptic-peptide 'maps' of the two polypeptides were prepared by a combination of t.l.c. and electrophoresis. 4. Radioactive peptides were prepared by [14C]carboxymethylation and chloramine-T-catalysed [125I]iodination. 5. 'Maps' of both sets of peptides demonstrate a marked similarity between the two polypeptides. 6. These new data confirm earlier evidence for the similarity of the two chains. 7. The number of peptides in the 'maps' of carboxymethylated peptides suggest that polypeptides P1 and P2 are not aggregates.

Amino Acids↗