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Biomedical subjects

B M Turner

Publications and source records attributed to B M Turner.

At least 91 records · Page 5Linked to original sources
PubMed1985

Characterization of a family of nuclear and chromosomal proteins identified by a monoclonal antibody.

A monoclonal antibody (3C5) isolated from a mouse immunized with human chromatin stained the nuclei of all cultured cell types tested by indirect immunofluorescence. Experiments with HeLa and PtK1 cells demonstrated striking cell-cycle-related changes in the staining properties of the target antigen. A rapid increase in nuclear fluorescence was seen in prophase, with antigen located between the condensing chromosomes. In metaphase and anaphase cells antigen was present throughout the cytoplasm with the chromosomes apparently unstained. However, isolated metaphase chromosomes showed intense, peripheral staining. In telophase cells immunofluorescent staining was most intense among the decondensing chromosomes and by early G1 staining was predominantly nuclear. Nuclear fluorescence faded as cells progressed through interphase. By protein blotting and immunostaining, 3C5 recognized protein bands with subunit molecular weights of 130, 73, 50, 38, 32 and 22 to 25 kDa. These bands were present in all human and rodent cultured cell types tested. All bands were extracted by 6 M urea or 1% sodium dodecyl sulfate (SDS) but not by Triton X-100. Our results provide evidence against the involvement of a common carbohydrate moiety, in vitro proteolysis or non-specific cross reaction in this multi-banded pattern. The same family of proteins was detected in mitotic and interphase cells, suggesting that the changes in immunofluorescent staining through mitosis are due to changes in antigen accessibility. Subcellular fractionation experiments showed that all major bands were present in the nuclear fraction. Only two (50 and 32 kDa) were detected also in the post-nuclear membrane fraction and none were present in the soluble cytoplasmic fraction.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

A monoclonal antibody identifies a 215 000-dalton nuclear envelope protein restricted to certain cell types.

The monoclonal antibody, AGF2.3, was isolated from mice immunised with the human promyeloid cell line HL60. By immunofluorescence and immunoelectron microscopy the antibody was shown to bind to the nuclear envelope in uninduced HL60 cells. Immunofluorescent staining was reduced to very low levels in HL60 cells induced to mature to monocytes or neutrophils by addition of 12-0-tetradecanoylphorbol-13-acetate or dimethyl sulfoxide respectively. Blood neutrophils did not express the antigen. Weak immunofluorescent staining of cell nuclei was observed in peripheral blood lymphocytes and in sections of normal human kidney, tonsil and skin epithelium. The AGF2.3 antigen was strongly expressed on the nuclei of 21/21 haemopoietic cell lines and 21/25 permanent non-haemopoietic cell lines representing various cell types. In contrast, the antigen was not expressed by any of six primary (untransformed) cell cultures. These included fibroblasts, endothelial cells and keratinocytes. The antigen was expressed in the Q10 SV-40 transformed cell line derived from a non-expressing primary fibroblast culture. AGF2.3 antibody precipitated a protein with an apparent subunit molecular weight of approximately 215 kDa from Triton X-100 extracts of HL60 and HeLa cells labelled with 35S-methionine. This protein was not detectable in extracts of primary skin fibroblasts prepared in parallel. We conclude that AGF2.3 antibody recognises a previously undescribed protein associated with the nuclear envelope which is expressed at high levels in most transformed cell lines but which is weakly expressed or absent in normal tissues and primary cell cultures.

Animals↗
PubMed1985

Immunofluorescent localisation of cytokeratin antigens in mitotic HeLa cells using monoclonal antibodies.

The organisation of cytokeratin filaments in mitotic HeLa cells has been analysed by immunofluorescence microscopy using a monoclonal antibody which recognises proteins with apparent subunit molecular weights of 52 kDa and 57 kDa and which binds exclusively to cytokeratin-type filaments. Mitotic cells were prepared for microscopic analysis by hypotonic swelling, centrifugation onto glass slides, brief pre-extraction with 0.1% Triton X-100 and fixation in 80% ethanol. This procedure gave particularly good resolution of intermediate filaments and preservation of chromosome morphology. In prometaphase-metaphase cells the antigen was present in an anastomosing filament network which completely or partially enclosed the chromosomes, in filament fragments and in cytoplasmic aggregates. The epichromosomal filament network was absent from cells in anaphase or later stages of mitosis. In these cells non-filamentous antigen was often located in a narrow band defining the periphery of individual chromosomes and in variable numbers of cytoplasmic filaments or fragments. The results suggest that extensive disaggregation and reformation of cytokeratin filaments occurs during mitosis and that disaggregated cytokeratin proteins are frequently located adjacent to mitotic chromosomes.

Antibodies, Monoclonal↗
PubMed1983

The use of alkaline-phosphatase-conjugated second antibody for the visualization of electrophoretically separated proteins recognized by monoclonal antibodies.

An immunoenzyme procedure is described for detection of proteins separated by SDS-polyacrylamide gel electrophoresis. Proteins were electrophoretically transferred to nitrocellulose filters which were then sequentially incubated with mouse monoclonal antibody, alkaline-phosphatase-conjugated antibody to mouse immunoglobulins and a substrate mixture containing beta-naphthyl phosphate and Fast Blue B. The procedure has been successfully applied to several monoclonal antibodies of various heavy chain classes and can detect protein antigens at concentrations down to 0.5 ng/mm2.

Alkaline Phosphatase↗
PubMed1983

Immunocytochemical localization of cytoskeletal proteins and histone 2B in isolated membrane-depleted nuclei, metaphase chromatin, and whole Chinese hamster ovary cells.

Immunocytochemical techniques employing protein A-gold labeling were used to locate actin, tubulin, and histone 2B in thin sections of embedded, isolated membrane-depleted nuclei, metaphase chromosomes, and whole Chinese hamster ovary (CHO) cells. Actin and tubulin were detected in significant amounts in the cytoplasm and the nucleus of whole cells. The isolated membrane-depleted nuclei and metaphase chromosomes showed levels of actin and tubulin labeling either comparable to or sometimes lower than the levels of labeling in whole interphase cells. The results suggest that actin and tubulin are normal components of nuclei throughout the cell cycle. A mouse monoclonal antibody to histone 2B gave relatively weak labeling of nuclei and chromosomes in whole cells but intense labeling of isolated nucleoids and chromosomes. An increased concentration of antibody at the periphery of interphase nucleoids was noted.

Actins↗
PubMed1982

Immunofluorescent staining of human metaphase chromosomes with monoclonal antibody to histone H2B.

A mouse monoclonal IgM antibody against the core histone H2B has been shown, by indirect immunofluorescence, to stain metaphase chromosomes from a variety of cultured cell types. Experiments carried out with human HeLa cells showed that the intensity of staining varied along the length of chromosome arms giving in some cases a rudimentary banded staining pattern. Considerable variation in staining intensity was noted between individual chromosomes and between different metaphase spreads. It was noted that chromosomes having a more swollen appearance stained more intensely than those with a more compact structure, which were often unstained. Preincubation of unfixed metaphase chromosomes in buffered salt solutions virtually eliminated the cell to cell and chromosome to chromosome variation in staining, even when no visible effect on chromosome morphology was caused by such treatment. It is concluded that the determinant recognised by antibody HBC-7 is ubiquitous but is inaccessible in some chromosomes or chromosome regions. Digestion of purified chromatin (primarily interphase) with DNAase 1 or micrococcal nuclease resulted in a several-fold increase in the binding of antibody HBC-7 measured by solid-phase radioimmunoassay. This increase was abolished by subsequent treatment with trypsin, which suggests that the antigenic determinant recognised by antibody HBC-7 lies in the trypsin-sensitive N-terminal region of nucleosomal H2B. As the cationic N-terminal regions of the core histones are involved in DNA binding, it is likely that the accessibility of the determinant recognised by antibody HBC-7 is influenced by the relationship between the core histones and their associated DNA.

Antibodies, Monoclonal↗
PubMed1981

Human beta-glucosidase: inhibition by sulphates and purification by affinity chromatography on Dextran-sulphate-Sepharose.

The acid beta-glucosidase (D-glucosyl-N-acylsphingosine glucohydrolase, EC 3.2.1.45) from human placenta is inhibited by sulphated macromolecules such as Dextran sulphate or chondroitin sulphate. This inhibition is alleviated by compounds such as crude taurocholate or phospholipids, which are known activators of acid beta-glucosidase. Partially-purified human beta-glucosidase will bind to Dextran sulphate linked to Sepharose 4B and can be eluted with low concentrations of crude sodium taurocholate. This procedure gives a 10-15 fold purification with good yield and has been included in a scheme giving an approx. 4000-fold purification of placental beta-glucosidase. Evidence is presented which suggests that phospholipids bind to beta-glucosidase by both ionic and hydrophobic interactions. The inhibition of enzyme activity caused by sulphated compounds and non-ionic detergents may be attributed to interference with, respectively, the ionic and hydrophobic binding of phospholipid to the enzyme.

Chromatography, Affinity↗
PubMed1981

Isolation of monoclonal antibodies to chromatin and preliminary characterization of target antigens.

This paper describes the isolation of monoclonal antibodies to chromatin-associated protein antigens and their use in the characterization of such proteins by indirect immunofluorescence. Hybridomas were derived by fusion of the mouse myeloma Ag8653 with spleen cells from mice immunized with chromatin from human liver, rat liver or a human lymphoblastoid cell line. Hybrids were screened by solid-phase radioimmunoassay. The proportion of positive hybrids varied with the immunizing chromatin as follows: human liver 55/83, human lymphoblast 8/183 and rat liver 2/82. Fifteen antibodies derived from these fusions (7, 7 and 1 respectively) were subjected to further analysis. Most of these (11/13) were IgM and recognized both human and rat chromatin (12/15). Most of the target antigens were protease sensitive (8/13) and nuclease resistant. In fact the binding of five antibodies to lymphoblast chromatin was more than doubled by preincubation with DNAase I. The subcellular location of target antigens was examined by indirect immunofluorescence. Seven antibodies stained at least one of several cultured cell lines tested. Three gave staining patterns consistent with the in vivo association of the target antigen with chromatin recognizing, respectively, the interphase nucleus and metaphase chromosomes, the nuclear periphery and the mitotic spindle and other microtubule-containing structures. The remaining four all recognized antigens associated with the intermediate filament network.

Animals↗
PubMed1980

Secretion of alpha 1-antitrypsin by an established human hepatoma cell line and by human/mouse hybrids.

The human hepatoma cell line SK-HEP-1 has been shown by radioimmunoelectrophoresis to synthesize and secrete a protein which coprecipitates with human alpha 1-antitrypsin. This protein was indistinguishable from serum alpha 1-antitrypsin in terms of electrophoretic mobility, apparent subunit molecular weight (47,000), and binding to concanavalin-A. The protein identified as alpha 1-antitrypsin (alpha 1 AT) was secreted by seven clones derived from SK-HEP-1 and by twelve out of eighteen hybrid clones derived from the fusion of SK-HEP-1 with mouse RAG cells. There was no correlation between the expression of alpha 1 AT and that of human enzymes assigned to sixteen different autosomes. There was an imperfect correlation between the expression of alpha 1 AT and of the two chromosome 9 marker enzymes AK1 and AK3 (two discordant clones).

Adenocarcinoma↗
PubMed1979

Human inosine triphosphatase: catalytic properties and population studies.

Using a reliable 2-step colorimetric assay, some catalytic properties of human erythrocyte ITPase (inosine triphosphate pyrophosphohydrolase) (EC 3.6.1.19) were determined. The enzyme hydrolyses ITP, dITP (deoxyinosine triphosphate) and XTP (xanthine triphosphate) but not other nucleotides. IDP is a competitive inhibitor. Subcellular fractionation of cultured lymphoid-line cells showed ITPase activity to be in the cytosol. ATP and GTP hydrolysing activities were not enriched in the cytosol fraction. The range of activity was investigated in erythrocytes, peripheral leukocytes, amniotic fluid cells, skin fibroblasts, long term lymphoid lines and bone marrow fibroblasts. The mean ITPase activity was found to vary in different cell types from 4.9 to 294 units per mg protein. Fractionation of red blood cells by age revealed that the enzyme decays exponentially with erythrocyte age with a half-life of 35 days. The enzyme had a wide range of specific activity in erythrocytes from 150 normal randomly selected individuals (15--722 pmol/h/g Hb). Within the low range of erythrocyte specific activities (from 15 to 200) a correlation was found between an individual's erythrocyte activity and the activity in peripheral leukocytes. This correlation was not found at higher erythrocyte ITPase activities. Two individuals were detected who had erythrocyte ITPase activities which were close to the lowest ITPase activities of all samples tested. Thus, low or absent ITPase activity is an individual characteristic which may be expressed both in erythrocytes and in other cell types.

Cell Line↗
PubMed1979

Purification and characterisation of alpha-L-fucosidase from human placenta. pH-dependent changes in molecular size.

alpha-L-Fucosidase has been purified 12 000 fold from human placenta. The enzyme is a glycoprotein containing, by weight: 0.9% galactose; 1.9% mannose, 1.9% N-acetylglucosamine and 1.9% N-acetylneuraminic acid. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate separated proteins with molecular weights ot 55 000, 51 400 and 25 000. Resolution of the two larger protein bands varied with the gel system and these proteins may differ only in carbohydrate content. Gel filtration of te purified enzyme failed to separate the three proteins. Treatments with the cross-linking reagent dimethyl suberimidate prior to electrophoresis, resulted in a diminution of the original protein bands and the formation of oligomers with molecular weights of 80 000, 100 000, 130 000, and 144 000. These results suggest that the heavy (55 000 and 51 400) and light (25 000) proteins are structurally associated. The molecular weight of the native enzyme, measured by gel filtration, was dependent on the pH of the eluting buffer. At pH 5.0 or 6.0 a catalytically active peak was observed, with a molecular weight of 305 000. At pH 7.5 this peak was completely absent and the enzyme eluted as an asymmetrical peak with an apparent molecular weight of about 60 000. The reduction in apparent molecular weight at pH 7.5 was reversible by dialysis of isolated fractions at pH 6.0. In agreement with these findings the sedimentation coefficient was 8.5 S at pH 5.0 but only 3.6 S at pH 7.5. The results can be accounted for by the existence of a pH-dependent equilibrium between aggregated and dissociated forms of the enzyme or by pH-depedent conformational changes.

Carbohydrates↗
PubMed1979

Metabolic correction of fucosidosis fibroblasts by human alpha-L-fucosidase.

Human alpha-L-fucosidase, purified from placenta, was taken up from the culture medium by skin fibroblasts from patients with fucosidosis (alpha-L-fucosidase deficiency). The rate of uptake was low (uptake coefficient = 6 X 10(-4) ml.mg-1.h-1). Intracellular alpha-L-fucosidase activity was directly proportional to enzyme in the medium up to an activity of at least 40 nmoles/min/ml. No evidence for saturation of specific cell-surface receptors was seen. However, uptake was reduced by 75% by 1 mM mannose-6-phosphate and by 50% by 1mM glucose-6-phosphate, suggesting that uptake may be mediated by a receptor recognising a phosphorylated sugar or an analagous compound. Enzyme taken up by the cells was most active in subcellular fractions enriched with lysosomes and had an isozyme pattern, by isoelectric focusing, identical to that of the original enzyme preparation. Fucosidosis fibroblasts were shown to accumulate low molecular-weight, fucose-containing compounds to a level several times greater than control cells. This stored material was eluted from Sephadex G-25 as an asymmetrical peak with an elution volume of approximately twice the void volume of the column. Addition of placental alpha-L-fucosidase to the culture medium of fucosidosis fibroblasts prevented excessive accumulation of fucose-containing material and accelerated the breakdown of material accumulated prior to enzyme uptake.

Carbohydrate Metabolism, Inborn Errors↗
PubMed1978

Assignment of the gene locus for human alpha-L-fucosidase to chromosome 1 by analysis of somatic cell hybrids.

The alpha-L-fucosidases (EC 3.2.1.51) from human and mouse cells could be separated by isoelectric focusing of neuraminidase-treated cell extracts in acrylamide slab gels. Fourteen hybrid clones derived from the fusion of mouse and human cultured fibroblasts and 37 hybrid clones derived from the fusion of human long-term lymphoid lines with mouse RAG cells were tested for expression of human alpha-L-fucosidase. A strong correlation between the expression of the human enzyme and the presence or absence of human chromosome 1 was found. The presence of human alpha-L-fucosidase in clones scored as positive by isoelectric focusing was confirmed by Ouchterlony double immunodiffusion against IgG from rabbits immunized with purified human alpha-L-fucosidase. It is concluded that the structural gene locus for human alpha-L-fucosidase is located on chromosome 1.

Animals↗