Dissection of the bovine epidermal desmosome into cytoplasmic protein and membrane glycoprotein domains

1987 ◽  
Vol 87 (3) ◽  
pp. 411-421
Author(s):  
C.J. Skerrow ◽  
I. Hunter ◽  
D. Skerrow
Keyword(s):  
Morphological Changes ◽  
Complex Structure ◽  
Major Protein ◽  
Citrate Buffer ◽  
Sds Page ◽  
Keratin Filament ◽  
Cytoplasmic Compartment ◽  
Extracellular Molecules ◽  
Membrane Components ◽  
Alpha Keratin

Epidermal desmosomes contain two main regions. The core consists of a pair of membranes, one on either side of a cross-striated intercellular space bisected by a denser midline. The cytoplasmic compartment comprises a dense plaque deposited on the cytoplasmic surface of each membrane and a diffuse layer occupying the zone between the plaque and attached alpha-keratin filaments. Analysis of isolated desmosomes by SDS-PAGE has shown the presence of four major protein (dpl-4) and three major glycoprotein (dgl-3) bands, which have been allocated to the cytoplasmic and core compartments, respectively. In the present paper, we report the use of urea to fractionate this complex structure, both in situ and following isolation with citrate buffer, pH2.6. Extraction of the living layers of bovine epidermis with 9M-urea, pH7.5, resulted in rapid removal of the dense desmosomal plaques, followed by separation and vesiculation of desmosomal membranes. The resistance of the plaque to urea increased abruptly at the transition between living epidermis and dead, dehydrated horny layer. A similar sequence of morphological changes accompanied the extraction of isolated desmosomes with urea. Analysis of residues and extracts of isolated desmosomes by SDS-PAGE confirmed the selectivity of 9 M-urea, pH7.5, for the cytoplasmic compartment. The four major desmosomal proteins, dpl-4 (Mr240, 215, 90 and 83 (X 10(3)), respectively) predominated in the extracts. Desmosomal membranes, both paired and vesiculated, consisted almost entirely of the three desmosomal glycoproteins dgl-3 (Mr150, 120 and 110 (X 10(3)), respectively). These results provide evidence that all three desmosomal glycoproteins are integral membrane proteins. The separation of desmosomal membranes by urea, which is not accompanied by additional loss of proteins, further suggests that desmosomal adhesion is based on interactions between membrane components with no separate extracellular molecules being involved. The dissection of the desmosome by urea into two topographically and biochemically distinct domains should facilitate further studies on the molecular basis of desmosomal adhesion and alpha-keratin filament binding.

A metalloproteinase extracellularly released byCrithidia deanei

2003 ◽  
Vol 49 (10) ◽  
pp. 625-632 ◽  
Author(s):  
Claudia Masini d'Avila-Levy ◽  
Rodrigo F Souza ◽  
Rosana C Gomes ◽  
Alane B Vermelho ◽  
Marta H Branquinha
Keyword(s):  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Residual Activity ◽  
Major Protein ◽  
Motile Cells ◽  
Sds Page ◽  
Triton X

Actively motile cells from a cured strain of Crithidia deanei released proteins in phosphate buffer (pH 7.4). The molecular mass of the released polypeptides, which included some proteinases, ranged from 19 to 116 kDa. One of the major protein bands was purified to homogeneity by a combination of anion-exchange and gel filtration chromatographs. The apparent molecular mass of this protein was estimated to be 62 kDa by sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE). The incorporation of gelatin into SDS–PAGE showed that the purified protein presented proteolytic activity in a position corresponding to a molecular mass of 60 kDa. The enzyme was optimally active at 37 °C and pH 6.0 and showed 25% of residual activity at 28 °C for 30 min. The proteinase was inhibited by 1,10-phenanthroline and EDTA, showing that it belonged to the metalloproteinase class. A polyclonal antibody to the leishmanial gp63 reacted strongly with the released C. deanei protease. After Triton X-114 extraction, an enzyme similar to the purified metalloproteinase was detected in aqueous and detergent-rich phases. The detection of an extracellular metalloproteinase produced by C. deanei and some other Crithidia species suggests a potential role of this released enzyme in substrate degradation that may be relevant to the survival of trypanosomatids in the host.Key words: endosymbiont, trypanosomatid, extracellular, proteinase.

scholarly journals The State of the Hemomycocirculatory Bed of Adventitia of Varicose Veins of the Small Pelvis in Women with Chronic Inflammatory Diseases of the Internal Genital Organs

2017 ◽  
Vol 23 (2) ◽  
Author(s):  
Natalya Drohomyretska
Keyword(s):  
Inflammatory Diseases ◽  
Varicose Veins ◽  
Morphological Changes ◽  
Complex Structure ◽  
The State ◽  
Reproductive Age ◽  
Ovarian Vein ◽  
Women Of Reproductive Age ◽  
Vein Wall ◽  
Inflammatory Processes

Hemomicrocirculatory system – is a complex structure that reacts in every pathological process even before the clinical period and takes the first blow. The study of microhemocirculation will provide an opportunity to solve the important for practical medicine questions of pathogenesis of many diseases, as for the prevention and treatment of regional disorders of blood circulation.The objective of the research is to study the state of the hemomicrocirculatory bed (HMCB) of adventitia of varicose veins of the small pelvis (VVSP) in women with chronic inflammatory processes of the organs of the small pelvis (CIPOSP).Materials and methods of research. To evaluate the restructuring of the HMCB of adventitia of VVSP, the operating material of 12 women of reproductive age was used. Mainly, there were pieces of the ovarian vein. The study of the HMCB in the vein wall was performed by the non-injecting method of silver impregnation according to V.V. Kupriyanov. To standardize the results, the condition of the HMCB of adventitia of the venous wall in norm was studied in 5 women of reproductive age, who died as a result of various traumas.Results of the research. After the performed studies, the structural-morphological changes of the HMCB of the adventitia of the small pelvis veins were revealed. The dilation of capillaries, postcapillaries, postcapillary venules was observed. The diameter of the vessels of the HMCB of the ovarian vein adventitia was: venule – 94.21 ± 1.38 μM in comparison with the norm – 48.78 ± 1.60 μM (p<0.001); post-capillary venules – 46.76 ± 1.04 μM in comparison with the norm – 28.29 ± 1.1.01 μM (p<0.001); the capillaries were 11.22 ± 0.14 μM in comparison with the norm – 8.24 ± 0.16 μM (p<0.05), arterioles – 29.02 ± 0.76 μM in comparison with the norm – 25.19 ± 1.15 μM (p<0.01). The architectonics of the arterioles is almost unchanged. Lumen of venules is filled with formed elements. The structure of capillaries is polymorphic. The capillary net was localized and concentrated or was formed as a thick planar net, the capillaries were expanded. There were arterio-venulous anastomoses. Endothelial nuclei are shortened. In some preparations, the diameter of the arterioles corresponded to the diameter of the collection venules.  Conclusions:1. The first discovered by us changes in HMCB of adventitia of varicose veins of the small pelvis in women with CIPOSP can be one of the pathogenetic links of the development and progression of the varicose vein itself, which in turn aggravates the course of chronic inflammation.      2. The timely appointment of drugs that improve microcirculation will enable to prevent the development of dystrophic changes in the vein wall, improve the course of chronic inflammatory processes and reduce or completely eliminate the syndrome of “chronic pelvic pain”.

Isolation and biochemical characterization of septate junctions: differences between the proteins in smooth and pleated varieties

1989 ◽  
Vol 93 (1) ◽  
pp. 123-131
Author(s):  
NANCY J. LANE ◽  
STEPHEN M. DILWORTH
Keyword(s):  
Periplaneta Americana ◽  
Biochemical Characterization ◽  
Septate Junction ◽  
Septate Junctions ◽  
Molecular Weights ◽  
Thin Sectioning ◽  
Sds Page ◽  
High Concentrations ◽  
Membrane Components

Septate junctions are found only in invertebrate tissues, and are almost ubiquitous within them. In arthropods, the two major types are the ‘pleated’ and the ‘smooth’ varieties. Using tissues from different species, including the cockroach Periplaneta americana, procedures have been established for obtaining membrane fractions selectively enriched in septate junctions. The junctions have been identified in pellets of these fractions by both thin sectioning and freeze-fracturing. SDS-PAGE of these membrane fractions reveals two major polypeptide species with apparent molecular weights of 22000–24000 and 17000–18000. Consistent differences in these apparent molecular weights are observed between the pleated and smooth varieties of septate junction. These polypeptides are probably integral membrane components, as they remain associated after treatment with high concentrations of urea. Evidence suggests a plane of weakness in the mid-line of the extracellular septal ribbons.

Cytoplasmic surface and intramembrane components of rat heart gap junctional proteins

1984 ◽  
Vol 246 (6) ◽  
pp. H865-H875 ◽  
Author(s):  
C. K. Manjunath ◽  
G. E. Goings ◽  
E. Page
Keyword(s):  
Electron Microscopy ◽  
Gap Junctions ◽  
Electron Micrographs ◽  
Rat Heart ◽  
Major Protein ◽  
Thin Sections ◽  
Cytoplasmic Surface ◽  
Sds Page ◽  
Junction Protein ◽  
Major Protein Band

Gap junctions were purified from rat hearts in the presence of absence of proteolysis inhibitors and examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electron microscopy of thin sections. In absence of proteolysis inhibitors or in presence of ethylenediaminetetraacetic acid or leupeptin, gap junctions contained a single major protein band at relative molecular weight (Mr) 29,500 and minor bands at Mr 44,000–47,000, 17,750, and 16,500 and showed smooth cytoplasmic surfaces in electron micrographs. SDS-PAGE of junctions prepared with phenylmethylsulfonylfluoride (PMSF) showed markedly decreased intensity of the Mr 29,500 band and increased intensity of bands at Mr 44,000, 45,500, and 47,000; electron microscopy of these gap junctions showed presence of a fuzzy layer on their cytoplasmic surfaces. Urea (8 M) could not remove this fuzzy layer. In electron micrographs of rat ventricular myocytes, cytoplasmic surfaces of gap junctions were fuzzy. We conclude that rat heart gap junction protein consists of an intramembrane component (Mr 29,500) that extends into the “gap” and a cytoplasmic surface component (Mr 14,500–17,500) that corresponds to the fuzzy layer and is hydrolyzable by a serine protease.

scholarly journals Purification of human gamma interferon receptors by sequential affinity chromatography on immobilized monoclonal antireceptor antibodies and human gamma interferon.

1987 ◽  
Vol 165 (4) ◽  
pp. 988-999 ◽  
Author(s):  
M Aguet ◽  
G Merlin
Keyword(s):  
Competitive Inhibition ◽  
Raji Cell ◽  
Gamma Interferon ◽  
Major Protein ◽  
Ifn Gamma ◽  
Gamma Receptor ◽  
Sds Page ◽  
Number Of Binding Sites ◽  
Direct Binding ◽  
Triton X

mAbs against human IFN-gamma (huIFN-gamma) receptors were obtained by immunizing a BALB/c mouse with eluates from immobilized recombinant huIFN-gamma (rhuIFN-gamma) on which lysates of enriched Raji cell membranes had been adsorbed. mAbs were selected for competitive inhibition of receptor binding of 125I-labeled rhuIFN-gamma. The following additional properties suggest that these antibodies are specific for huIFN-gamma receptors: they bind to the surface of human cells expressing IFN-gamma receptors but not to heterologous cells; this binding is inhibited competitively by addition of rhuIFN-gamma; the number of binding sites revealed by direct binding of 125I-labeled rhuIFN-gamma correlates with the amount of antigen recognized by the mAbs on different cell lines. A Triton X-100 extract of a membrane-enriched fraction of human Raji cells was affinity purified with these mAbs and the eluates from such columns were further purified on immobilized rhuIFN-gamma. As revealed by SDS-PAGE, the final eluate contained two major protein bands with approximate Mr of 90,000 (p90) and 50,000 (p50), respectively. Both proteins were able to specifically bind 125I-labeled rhuIFN-gamma upon electroblotting to nitrocellulose. This binding could be inhibited by the huIFN-gamma receptor mAbs, suggesting that the same epitopes are recognized on p90, p50, and on the cell surface. Therefore, these proteins most likely represent at least a part of huIFN-gamma receptors.

scholarly journals Activated Charcoal—A Potential Material in Glucoamylase Recovery

2011 ◽  
Vol 2011 ◽  
pp. 1-4 ◽  
Author(s):  
S. O. Kareem ◽  
I. Akpan ◽  
T. O. S. Popoola ◽  
L. O. Sanni
Keyword(s):  
Molecular Weight ◽  
Activated Charcoal ◽  
Contact Time ◽  
Enzyme Purification ◽  
Downstream Processing ◽  
Single Step ◽  
Major Protein ◽  
Rhizopus Oligosporus ◽  
Sds Page ◽  
Effects Of Ph

The potential of activated charcoal in the purification of fungal glucoamylase was investigated. Various concentrations of activated charcoal (1–4% w/v) were used to concentrate crude glucoamylase from Rhizopus oligosporus at different temperature values (30–50°C). Effects of pH (3.0–6.0) and contact time (0–60 min) on enzyme purification were also monitored. Activated charcoal (3% w/v) gave a 16-fold purification in a single-step purification at 50°C for 20 min and pH 5.5. The result of SDS-PAGE analysis of purified glucoamylase showed two major protein bands with corresponding molecular weight of 36 kDa and 50 kDa. The method is inexpensive, rapid, and simple which could facilitate downstream processing of industrial enzyme.

Flagellin, a major protein present in SDS-PAGE profiles of Sarkosyl-OMP-enriched fractions from Bordetella bronchiseptica Bvg − or modulated Bvg + strains

1997 ◽  
Vol 56 (1-2) ◽  
pp. 65-77 ◽  
Author(s):  
B.N. Passerini de Rossi ◽  
L.E. Friedman ◽  
S. Darnaud ◽  
R.A. de Torres ◽  
M.A. Franco
Keyword(s):  
Bordetella Bronchiseptica ◽  
Major Protein ◽  
Sds Page

scholarly journals Isolation and characterization of the cytoplasmic and outer membranes of the Legionnaires' disease bacterium (Legionella pneumophila).

1985 ◽  
Vol 161 (2) ◽  
pp. 409-422 ◽  
Author(s):  
J E Gabay ◽  
M A Horwitz
Keyword(s):  
Outer Membrane ◽  
Legionella Pneumophila ◽  
Cytoplasmic Membrane ◽  
Mononuclear Phagocytes ◽  
Major Protein ◽  
Protein Species ◽  
Isolation And Characterization ◽  
Sds Page ◽  
Legionnaires Disease ◽  
Cell Envelopes

Legionella pneumophila, the etiologic agent of Legionnaires' disease, is phagocytized in an unusual way and multiplies in human mononuclear phagocytes in a novel phagosome. As a first step toward understanding these L. pneumophila-phagocyte interactions, we have studied the envelope of L. pneumophila Philadelphia 1 strain. We isolated cell envelopes by treating whole bacterial cells with lysozyme and EDTA to convert them to spheroplasts, then lysing the spheroplasts osmotically or sonically. We resolved the cell envelopes into two membrane fractions by isopycnic centrifugation. We localized NADH oxidase to the fraction of buoyant density 1.145, which we designated cytoplasmic membrane, and lipopolysaccharide (LPS) to the fraction of density 1.222, which we designated outer membrane. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed that the L. pneumophila outer membrane contains a single major protein species migrating at 28,000 mol wt; this is the major protein of the bacterium. The cytoplasmic membrane also contains a single major protein species migrating at 65,000 mol wt. Surface iodination of the bacteria and agglutination and immunofluorescence studies with rabbit antibody produced against the purified major outer membrane protein (MOMP) revealed that this protein is exposed at the cell surface. We isolated LPS from L. pneumophila membranes by SDS-EDTA treatment. The pattern obtained by subjecting the LPS to SDS-PAGE and staining the gel with silver nitrate suggests that L. pneumophila LPS might be atypical. We studied patient serologic responses to cell envelope components of L. pneumophila Philadelphia 1, a serogroup 1 organism. Sera from patients with evidence of infection with serogroup 1 L. pneumophila contained large amounts of antibody to this strain. Few of these antibodies recognized the MOMP of L. pneumophila. In contrast, greater than 98% of these antibodies were directed against the LPS. This indicates that LPS is the dominant serogroup antigen and the major antigen responsible for the reactivity of patient sera in the indirect fluorescent antibody assay, currently the principal diagnostic assay for Legionella infection.

Some morphological, anatomical, and physiological changes in the pear fruit (Pyrus communis var. Williams Bon Chrétien) during development and following harvest

1961 ◽  
Vol 9 (2) ◽  
pp. 99 ◽  
Author(s):  
JM Bain
Keyword(s):  
Cell Division ◽  
Morphological Changes ◽  
Complex Structure ◽  
Titratable Acidity ◽  
Dry Weight ◽  
Cell Number ◽  
Pyrus Communis ◽  
Physiological Changes ◽  
Pear Fruit ◽  
Protein Nitrogen

Morphological, anatomical, and physiological changes occurring in the developing fruit of Pyrus communis var. Williams Bon Chretien were studied at frequent intervals, from blossom until after commercial maturity, in three successive seasons. Morphological changes were shown by increase in measurements of volume, long and short axis, and the width of the cortex (flesh), the morphology of the fruit being interpreted by the receptacular theory, Anatomical changes were given by the duration and distribution of cell division, differentiation of tissues, cell size, and cell number. Physiological changes were expressed as changes in fresh weight, dry weight, and moisture content for the whole fruit, and separately for the flesh, peel, and core in the second and third seasons. Total and reducing sugars, starch, titratable acidity, and total and protein nitrogen were estimated per gram of dried flesh at each sampling. Respiration rates for whole fruit were measured by the Pettenkofer method. Physiological changes could not be expressed on a per cell basis because of the complex structure of pear tissue. Data presented on the basis of the number of days from blossom showed two distinct stages in fruit growth. Stage I, the first 42-56 days of development, corresponded to the main cell division period and was characterized by more rapid morphological but slower physiological changes (except for increase in protein nitrogen) than Stage 11, the remainder of the time on the tree. Comparable trends were found in the three seasons, but drought reduced growth rate in the first season. Some ripening changes were followed on removal from the tree and after periods of cold storage at 0°C.

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