scholarly journals VIP21-caveolin, a membrane protein constituent of the caveolar coat, oligomerizes in vivo and in vitro.

1995 ◽  
Vol 6 (7) ◽  
pp. 911-927 ◽  
Author(s):  
S Monier ◽  
R G Parton ◽  
F Vogel ◽  
J Behlke ◽  
A Henske ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Molecular Mass ◽  
Sodium Dodecyl ◽  
High Molecular Mass ◽  
Hydrophobic Domain ◽  
Cytoplasmic Surface ◽  
Triton X ◽  
Flanking Regions

VIP21-caveolin is a membrane protein, proposed to be a component of the striated coat covering the cytoplasmic surface of caveolae. To investigate the biochemical composition of the caveolar coat, we used our previous observation that VIP21-caveolin is present in large complexes and insoluble in the detergents CHAPS or Triton X-114. The mild treatment of these insoluble structures with sodium dodecyl sulfate leads to the detection of high molecular mass complexes of approximately 200, 400, and 600 kDa. The 400-kDa complex purified to homogeneity from dog lung is shown to consist exclusive of the two isoforms of VIP21-caveolin. Pulse-chase experiments indicate that the oligomers form early after the protein is synthesized in the endoplasmic reticulum (ER). VIP21-caveolin does indeed insert into the ER membrane through the classical translocation machinery. Its hydrophobic domain adopts an unusual loop configuration exposing the N- and C-flanking regions to the cytoplasm. Similar high molecular mass complexes can be produced from the in vitro-synthesized VIP21-caveolin. The complex formation occurs only if VIP21-caveolin isoforms are properly inserted into the membrane; formation is cytosol-dependent and does not involve a vesicle fusion step. We propose that high molecular mass oligomers of VIP21-caveolin represent the basic units forming the caveolar coat. They are formed in the ER and later, between the ER and the plasma membrane, these oligomers could associate into larger detergent-insoluble structures.

scholarly journals Towards uterus tissue engineering: a comparative study of sheep uterus decellularisation

2020 ◽  
Vol 26 (3) ◽  
pp. 167-178 ◽  
Author(s):  
T T Tiemann ◽  
A M Padma ◽  
E Sehic ◽  
H Bäckdahl ◽  
M Oltean ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Stem Cells ◽  
Sodium Dodecyl ◽  
Sodium Deoxycholate ◽  
Vascular Perfusion ◽  
Uterus Transplantation ◽  
Live Donors ◽  
Triton X

Abstract Uterus tissue engineering may dismantle limitations in current uterus transplantation protocols. A uterine biomaterial populated with patient-derived cells could potentially serve as a graft to circumvent complicated surgery of live donors, immunosuppressive medication and rejection episodes. Repeated uterine bioengineering studies on rodents have shown promising results using decellularised scaffolds to restore fertility in a partially impaired uterus and now mandate experiments on larger and more human-like animal models. The aim of the presented studies was therefore to establish adequate protocols for scaffold generation and prepare for future in vivo sheep uterus bioengineering experiments. Three decellularisation protocols were developed using vascular perfusion through the uterine artery of whole sheep uteri obtained from slaughterhouse material. Decellularisation solutions used were based on 0.5% sodium dodecyl sulphate (Protocol 1) or 2% sodium deoxycholate (Protocol 2) or with a sequential perfusion of 2% sodium deoxycholate and 1% Triton X-100 (Protocol 3). The scaffolds were examined by histology, extracellular matrix quantification, evaluation of mechanical properties and the ability to support foetal sheep stem cells after recellularisation. We showed that a sheep uterus can successfully be decellularised while maintaining a high integrity of the extracellular components. Uteri perfused with sodium deoxycholate (Protocol 2) were the most favourable treatment in our study based on quantifications. However, all scaffolds supported stem cells for 2 weeks in vitro and showed no cytotoxicity signs. Cells continued to express markers for proliferation and maintained their undifferentiated phenotype. Hence, this study reports three valuable decellularisation protocols for future in vivo sheep uterus bioengineering experiments.

scholarly journals Characterization of Mediator Complexes from HeLa Cell Nuclear Extract

2001 ◽  
Vol 21 (14) ◽  
pp. 4604-4613 ◽  
Author(s):  
Gang Wang ◽  
Greg T. Cantin ◽  
Jennitte L. Stevens ◽  
Arnold J. Berk
Keyword(s):  
Hela Cell ◽  
Molecular Mass ◽  
Rna Polymerase Ii ◽  
Nuclear Extract ◽  
High Molecular Mass ◽  
Multiprotein Complexes ◽  
Hela Cell Nuclear Extract ◽  
Cell Nuclear Extract

ABSTRACT A number of mammalian multiprotein complexes containing homologs ofSaccharomyces cerevisiae Mediator subunits have been described recently. High-molecular-mass complexes (1 to 2 MDa) sharing several subunits but apparently differing in others include the TRAP/SMCC, NAT, DRIP, ARC, and human Mediator complexes. Smaller multiprotein complexes (∼500 to 700 kDa), including the murine Mediator, CRSP, and PC2, have also been described that contain subsets of subunits of the larger complexes. To evaluate whether these different multiprotein complexes exist in vivo in a single form or in multiple different forms, HeLa cell nuclear extract was directly resolved over a Superose 6 gel filtration column. Immunoblotting of column fractions using antisera specific for several Mediator subunits revealed one major size class of high-molecular-mass (∼2-MDa) complexes containing multiple mammalian Mediator subunits. No peak was apparent at ∼500 to 700 kDa, indicating that either the smaller complexes reported are much less abundant than the higher-molecular-mass complexes or they are subcomplexes generated by dissociation of larger complexes during purification. Quantitative immunoblotting indicated that there are about 3 × 105to 6 × 105 molecules of hSur2 Mediator subunit per HeLa cell, i.e., the same order of magnitude as RNA polymerase II and general transcription factors. Immunoprecipitation of the ∼2-MDa fraction with anti-Cdk8 antibody indicated that at least two classes of Mediator complexes occur, one containing CDK8 and cyclin C and one lacking this CDK-cyclin pair. The ∼2-MDa complexes stimulated activated transcription in vitro, whereas a 150-kDa fraction containing a subset of Mediator subunits inhibited activated transcription.

scholarly journals Towards a bioengineered uterus: bioactive sheep uterus scaffolds are effectively recellularized by enzymatic preconditioning

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Arvind Manikantan Padma ◽  
Laura Carrière ◽  
Frida Krokström Karlsson ◽  
Edina Sehic ◽  
Sara Bandstein ◽  
...  
Keyword(s):  
Sodium Dodecyl ◽  
Chorioallantoic Membrane ◽  
Sodium Deoxycholate ◽  
Matrix Metalloproteinase 2 ◽  
Uterus Transplantation ◽  
Fetal Stem Cells ◽  
Culturing Conditions ◽  
Triton X

AbstractUterine factor infertility was considered incurable until recently when we reported the first successful live birth after uterus transplantation. However, risky donor surgery and immunosuppressive therapy are factors that may be avoided with bioengineering. For example, transplanted recellularized constructs derived from decellularized tissue restored fertility in rodent models and mandate translational studies. In this study, we decellularized whole sheep uterus with three different protocols using 0.5% sodium dodecyl sulfate, 2% sodium deoxycholate (SDC) or 2% SDC, and 1% Triton X-100. Scaffolds were then assessed for bioactivity using the dorsal root ganglion and chorioallantoic membrane assays, and we found that all the uterus scaffolds exhibited growth factor activity that promoted neurogenesis and angiogenesis. Extensive recellularization optimization was conducted using multipotent sheep fetal stem cells and we report results from the following three in vitro conditions; (a) standard cell culturing conditions, (b) constructs cultured in transwells, and (c) scaffolds preconditioned with matrix metalloproteinase 2 and 9. The recellularization efficiency was improved short-term when transwells were used compared with standard culturing conditions. However, the recellularization efficiency in scaffolds preconditioned with matrix metalloproteinases was 200–300% better than the other strategies evaluated herein, independent of decellularization protocol. Hence, a major recellularization hurdle has been overcome with the improved recellularization strategies and in vitro platforms described herein. These results are an important milestone and should facilitate the production of large bioengineered grafts suitable for future in vivo applications in the sheep, which is an essential step before considering these principles in a clinical setting.

scholarly journals Destabilizing missense mutations in the tumour suppressor protein p53 enhance its ubiquitination in vitro and in vivo

2006 ◽  
Vol 397 (2) ◽  
pp. 355-367 ◽  
Author(s):  
Harumi Shimizu ◽  
David Saliba ◽  
Maura Wallace ◽  
Lee Finlan ◽  
Patrick R. R. Langridge-Smith ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Covalent Modification ◽  
High Molecular Mass ◽  
Mutant Form ◽  
Dependent Manner ◽  
Ubiquitination System ◽  
Covalent Adducts ◽  
In Cells

p53 ubiquitination catalysed by MDM2 (murine double minute clone 2 oncoprotein) provides a biochemical assay to dissect stages in E3-ubiquitin-ligase-catalysed ubiquitination of a conformationally flexible protein. A mutant form of p53 (p53F270A) containing a mutation in the second MDM2-docking site in the DNA-binding domain of p53 (F270A) is susceptible to modification of long-lived and high-molecular-mass covalent adducts in vivo. Mutant F270A is hyperubiquitinated in cells as defined by immunoprecipitation and immunoblotting with an anti-ubiquitin antibody. Transfection of His-tagged ubiquitin along with p53R175H or p53F270A also results in selective hyperubiquitination in cells under conditions where wild-type p53 is refractory to covalent modification. The extent of mutant p53R175H or p53F270A unfolding in cells as defined by exposure of the DO-12 epitope correlates with the extent of hyperubiquitination, suggesting a link between substrate conformation and E3 ligase function. The p53F270A:6KR chimaeric mutant (where 6KR refers to the simultaneous mutation of lysine residues at positions 370, 372, 373, 381, 382 and 386 to arginine) maintains the high-molecular-mass covalent adducts and is modified in an MDM2-dependent manner. Using an in vitro ubiquitination system, mutant p53F270A and the p53F270A:6KR chimaeric mutant is also subject to hyperubiquitination outwith the C-terminal domain, indicating direct recognition of the mutant p53 conformation by (a) factor(s) in the cell-free ubiquitination system. These data identify an in vitro and in vivo assay with which to dissect how oligomeric protein conformational alterations are linked to substrate ubiquitination in cells. This has implications for understanding the recognition of misfolded proteins during aging and in human diseases such as cancer.

scholarly journals Properties of the yeast nuclear histone deacetylase

1994 ◽  
Vol 303 (3) ◽  
pp. 723-729 ◽  
Author(s):  
M M Sanchez del Pino ◽  
G Lopez-Rodas ◽  
R Sendra ◽  
V Tordera
Keyword(s):  
Molecular Mass ◽  
Histone Deacetylase ◽  
Trichostatin A ◽  
High Mobility ◽  
Inhibitory Effect ◽  
High Molecular Mass ◽  
Core Histones ◽  
Nuclear Histone

A nuclear histone deacetylase from yeast was partially purified and some of its characteristics were studied. Histone deacetylase activity was stimulated in vitro by high-mobility-group nonhistone chromatin proteins 1 and 2 and ubiquitin and inhibited by spermine and spermidine, whereas n-butyrate had no significant inhibitory effect. Like the mammalian enzyme, partially purified histone deacetylase from yeast was strongly inhibited by trichostatin A. However, in crude extract preparations the yeast enzyme was not inhibited and treatment with trichostatin in vivo did not show any effect, either on the histone acetylation level or on cell viability. At low ionic strength, the enzyme can be isolated as a complex of high molecular mass that is much less inhibited by trichostatin A than is partially purified histone deacetylase activity. Furthermore, radiolabelled oligonucleosomes were more efficiently deacetylated by the complex than by the low-molecular-mass form of the enzyme. The histone deacetylase activity was separated from a polyamine deacetylase activity and its specificity studied. Using h.p.l.c.-purified core histone species as substrate, histone deacetylase from yeast is able to deacetylate all core histones with a slight preference for H3. Our results support the idea that the yeast histone deacetylase may act as a high-molecular-mass complex in vivo.

scholarly journals Effective decellularisation of human saphenous veins for biocompatible arterial tissue engineering applications: Bench optimisation and feasibility in vivo testing

2021 ◽  
Vol 12 ◽  
pp. 204173142098752
Author(s):  
Nadiah S Sulaiman ◽  
Andrew R Bond ◽  
Vito D Bruno ◽  
John Joseph ◽  
Jason L Johnson ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Mechanical Strength ◽  
Vascular Tissue ◽  
Sodium Dodecyl ◽  
Host Cells ◽  
Replacement Model ◽  
In Vivo Testing ◽  
Vascular Scaffolds

Human saphenous vein (hSV) and synthetic grafts are commonly used conduits in vascular grafting, despite high failure rates. Decellularising hSVs (D-hSVs) to produce vascular scaffolds might be an effective alternative. We assessed the effectiveness of a detergent-based method using 0% to 1% sodium dodecyl sulphate (SDS) to decellularise hSV. Decellularisation effectiveness was measured in vitro by nuclear counting, DNA content, residual cell viability, extracellular matrix integrity and mechanical strength. Cytotoxicity was assessed on human and porcine cells. The most effective SDS concentration was used to prepare D-hSV grafts that underwent preliminary in vivo testing using a porcine carotid artery replacement model. Effective decellularisation was achieved with 0.01% SDS, and D-hSVs were biocompatible after seeding. In vivo xeno-transplantation confirmed excellent mechanical strength and biocompatibility with recruitment of host cells without mechanical failure, and a 50% patency rate at 4-weeks. We have developed a simple biocompatible methodology to effectively decellularise hSVs. This could enhance vascular tissue engineering toward future clinical applications.

scholarly journals Antimicrobial Photodynamic Inactivation Inhibits Candida albicans Virulence Factors and ReducesIn VivoPathogenicity

2012 ◽  
Vol 57 (1) ◽  
pp. 445-451 ◽  
Author(s):  
Ilka Tiemy Kato ◽  
Renato Araujo Prates ◽  
Caetano Padial Sabino ◽  
Beth Burgwyn Fuchs ◽  
George P. Tegos ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Virulence Factors ◽  
Sodium Dodecyl ◽  
Systemic Infection ◽  
Tube Formation ◽  
Photodynamic Inactivation ◽  
Content Type ◽  
Daughter Cells

ABSTRACTThe objective of this study was to evaluate whetherCandida albicansexhibits altered pathogenicity characteristics following sublethal antimicrobial photodynamic inactivation (APDI) and if such alterations are maintained in the daughter cells.C. albicanswas exposed to sublethal APDI by using methylene blue (MB) as a photosensitizer (0.05 mM) combined with a GaAlAs diode laser (λ 660 nm, 75 mW/cm2, 9 to 27 J/cm2).In vitro, we evaluated APDI effects onC. albicansgrowth, germ tube formation, sensitivity to oxidative and osmotic stress, cell wall integrity, and fluconazole susceptibility.In vivo, we evaluatedC. albicanspathogenicity with a mouse model of systemic infection. Animal survival was evaluated daily. Sublethal MB-mediated APDI reduced the growth rate and the ability ofC. albicansto form germ tubes compared to untreated cells (P< 0.05). Survival of mice systemically infected withC. albicanspretreated with APDI was significantly increased compared to mice infected with untreated yeast (P< 0.05). APDI increasedC. albicanssensitivity to sodium dodecyl sulfate, caffeine, and hydrogen peroxide. The MIC for fluconazole forC. albicanswas also reduced following sublethal MB-mediated APDI. However, none of those pathogenic parameters was altered in daughter cells ofC. albicanssubmitted to APDI. These data suggest that APDI may inhibit virulence factors and reducein vivopathogenicity ofC. albicans. The absence of alterations in daughter cells indicates that APDI effects are transitory. The MIC reduction for fluconazole following APDI suggests that this antifungal could be combined with APDI to treatC. albicansinfections.

Partial purification of a boar sperm membrane protein inducing sperm agglutinating antibody

1990 ◽  
Vol 10 (2) ◽  
pp. 131-139
Author(s):  
Oyewole Adeyemo ◽  
E. O. Okegbile ◽  
O. O. Olorunsogo
Keyword(s):  
Molecular Weight ◽  
Membrane Protein ◽  
Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Partial Purification ◽  
Boar Sperm ◽  
Sperm Membrane ◽  
Triton X

For the development of immunological contraception, attention is being concentrated on the possibility of using a sperm membrane antigen. Boar sperm membrane was extracted with triton-X 100 and fractionated by Sephadex G-150 column chromatography. The glycosylated and nonglycosylated portions of protein peaks from the gel filtration were obtained by fractionating on concanavalin A-Sepharose and eluting the bound protein with 0.3 M methyl mannoside. A glycosylated fraction was found to induce sperm agglutinating antibodies in rabbit. The partially purified protein has a molecular weight of 30 kilodaltons, as determined by sodium dodecyl polyaccyrlamide gel electrophoresis. Further work is planned on the histochemical determination of the origin of this protein and species cross-activity of the antibody.

scholarly journals Protein synthesis in chloroplasts. Characteristics and products of protein synthesis in vitro in etioplasts and developing chloroplasts from pea leaves

1975 ◽  
Vol 146 (3) ◽  
pp. 675-685 ◽  
Author(s):  
S G Siddell ◽  
R J Ellis
Keyword(s):  
Energy Source ◽  
Protein Synthesis ◽  
Large Subunit ◽  
Sodium Dodecyl ◽  
Supernatant Fraction ◽  
Pisum Sativum L ◽  
Fraction I ◽  
Plastid Ribosomes

The function of plastid ribosomes in pea (Pisum sativum L.) was investigated by characterizing the products of protein synthesis in vitro in plastids isolated at different stages during the transition from etioplast to chloroplast. Etioplasts and plastids isolated after 24, 48 and 96h of greening in continuous white light, use added ATP to incorporate labelled amino acids into protein. Plastids isolated from greening leaves can also use light as the source of energy for protein synthesis. The labelled polypeptides synthesized in isolated plastids were analysed by electrophoresis in sodium dodecyl sulphate-ureapolyacrylamide gels. Six polypeptides are synthesized in etioplasts with ATP as energy source. Only one of these polypeptides is present in a 150 000g supernatant fraction. This polypeptide has been identified as the large subunit of Fraction I protein (3-phospho-D-glycerate carboxylyase EC 4.1.1.39) by comparing the tryptic ‘map’ of its L-(35S)methionine-labelled peptides with the tryptic ‘map’ of large subunit peptides from Fraction I labelled with L-(35S)methionine in vivo. The same gel pattern of six polypeptides is seen when plastids isolated from greening leaves are incubated with either added ATP or light as the energy source. However, the rates of synthesis of particular polypeptides are different in plastids isolated at different stages of the etioplast to chloroplast transition. The results support the idea that plastid ribosomes synthesize only a small number of proteins, and that the number and molecular weight of these proteins does not alter during the formation of chloroplasts from etioplasts.

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.

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