scholarly journals Assembly polypeptides from coated vesicles mediate reassembly of unique clathrin coats.

1983 ◽  
Vol 97 (5) ◽  
pp. 1339-1347 ◽  
Author(s):  
S Zaremba ◽  
J H Keen
Keyword(s):  
Sedimentation Coefficient ◽  
Coated Vesicles ◽  
Buffer System ◽  
Protein Activity ◽  
Uniform Size ◽  
Molar Ratios ◽  
Golgi Region ◽  
Coated Membranes

A protein activity has been identified in extracts of coated vesicles that enables purified clathrin triskelions to reassemble in vitro into coat structures of uniform size. Coats formed in the presence of this preparation, regardless of the buffer system employed, are uniform in size with a mean diameter of 78 nm (+/- 5 nm SD) and a sedimentation coefficient (S20,w) of approximately 250S. Analysis of the reassembled coats on dodecyl sulfate acrylamide gels reveals that they have specifically incorporated three polypeptides from the preparation: those of Mr congruent to 52,000, 100,000, and 110,000. The 52,000-, 100,000-, and 110,000-mol-wt polypeptides are incorporated in molar ratios of 0.85, 1.11, and 0.26, respectively, per three clathrin monomers (equivalent to one triskelion). We therefore designate these as assembly polypeptides (AP). In contrast, coats formed from clathrin alone, under permissive buffer conditions, are larger (400S), more heterogeneous in size (101 nm +/- 15 nm SD), and are composed only of clathrin and its associated light chains. These biochemical and biophysical characteristics distinguish AP-reassembled coats from coats formed by triskelions alone. AP-reassembled coats can be isolated, dissociated, then reassembled in the absence of any other factors. This recycling indicates that all the information needed for reassembly is present in the coat-incorporated polypeptides themselves. Reassembly is stoichiometric and saturable with respect to both clathrin and AP concentration. In the presence of AP, significant coat reassembly occurs at clathrin concentrations as low as 0.06 mg/ml. AP-mediated reassembly proceeds at 4 degrees, 22 degrees, and 37 degrees C. Coat formation also proceeds efficiently at intracellular pH values (7.2-7.5) in the presence of AP. In its absence, reassembly does not occur at all above pH 6.7. In summary, AP promotes clathrin reassembly into coat structures of uniform size and distinctive composition under physiologically relevant salt, temperature, and pH conditions. In addition, the close similarity in size between AP-reassembled coats in vitro and coated membranes in the Golgi region in vivo raises the possibility that AP in the cell may be associated with this subpopulation of coat structures.

scholarly journals COPII-coated membranes function as transport carriers of intracellular procollagen-1

2017 ◽  
Author(s):  
Amita Gorur ◽  
Lin Yuan ◽  
Samuel J Kenny ◽  
Satoshi Baba ◽  
Ke Xu ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Imaging Techniques ◽  
Coated Vesicles ◽  
Intact Cells ◽  
Vesicle Budding ◽  
Coated Membranes ◽  
Copii Vesicles ◽  
Optical Reconstruction

AbstractThe coat protein complex II (COPII) is essential for the secretion of large cargo, such as the 300 nm precursor fibrils of procollagen I (PC1). Previous work has shown that the CUL3-KLHL12 complex increases the size of COPII vesicles to over 300 nm in diameter and accelerates the secretion of PC1; however, the role of large COPII vesicles as PC1 transport carriers was not unambiguously demonstrated. In this study, using stochastic optical reconstruction microscopy (STORM), correlated light electron microscopy (CLEM), and live cell imaging we report the existence of mobile COPII-coated vesicles that completely encapsulate the cargo PC1 and are physically separated from ER. We have also developed a cell-free COPII vesicle budding reaction that reconstitutes the capture of PC1 into large COPII vesicles. This process requires COPII proteins and the GTPase activity of the COPII subunit SAR1. We conclude from in vivo and in vitro evidence that large COPII vesicles are bona fide carriers of PC1.SummaryCOPII may play a direct or indirect role in the traffic of large protein complexes such as procollagen. Using high resolution imaging techniques in intact cells and in vitro reconstituted vesicles, Gorur et al. show that COPII coated vesicles carry procollagen1.

scholarly journals Solvolysis of the Tumor-Inhibiting Ru(III)-Complex trans-Tetrachlorobis(Indazole)Ruthenate(III)

2000 ◽  
Vol 7 (4) ◽  
pp. 225-232 ◽  
Author(s):  
Thomas Pieper ◽  
Wolfgang Peti ◽  
Bernhard K. Keppler
Keyword(s):  
Sodium Salt ◽  
Buffer System ◽  
Spectroscopic Techniques ◽  
Tumor Models ◽  
Solubility In Water ◽  
Aqueous Acetonitrile ◽  
Hydrolysis Of

The ruthenium(III) complex Hlnd trans-[RuCl4,(ind)2], with two trans-standing indazole (ind) ligands bound to ruthenium via nitrogen, shows remarkable activity in different tumor models in vitro and in vivo. The solvolysis of the complex trans-[RuCl4,(ind)2]- has been investigated by means of spectroscopic techniques (UV/vis, NMR)in different solvents. We investigated the indazolium as well as the sodium salt, the latter showing improved solubility in water. In aqueous acetonitrile and ethanol the solvolysis results in one main solvento complex. The hydrolysis of the complex is more complicated and depends on the pH of the solution as well as on the buffer system.

Kinetics of exsheathment of infective ovine and bovine strongylid larvae in vivo and in vitro

Parasitology ◽  
2002 ◽  
Vol 125 (1) ◽  
pp. 65-70 ◽  
Author(s):  
H. HERTZBERG ◽  
U. HUWYLER ◽  
L. KOHLER ◽  
St REHBEIN ◽  
M. WANNER
Keyword(s):  
Haemonchus Contortus ◽  
Buffer System ◽  
Bicarbonate Concentration ◽  
Infective Larvae ◽  
Nylon Mesh ◽  
Mesh Bags ◽  
Sheep Rumen ◽  
Kinetics Of

The aim of the study was to investigate the longitudinal changes of exsheathment of ovine and bovine 3rd-stage strongylid larvae in an artificial rumen (RUSITEC) and to compare the results with in vivo data obtained from rumen-fistulated sheep. Infective larvae were incubated in nylon mesh bags in the sheep rumen or the RUSITEC apparatus for periods of 1, 6 and 12 h, respectively. The 12 h exsheathment rates in the rumen and the RUSITEC apparatus (in parentheses) were as follows: Haemonchus contortus: 100% (100%), Ostertagia circumcincta: 100% (76%), O. leptospicularis: 100% (100%), O. ostertagi: 53% (59%), Trichostrongylus axei: 100% (100%), T. colubriformis: 37% (36%), Cooperia curticei: 94% (76%), C. oncophora: 95% (89%), Nematodirus filicollis: 0% (N.D.), N. spathiger: 11% (15%), N. battus: 7% (5%), Oesophagostomum venulosum: 17% (9%), Chabertia ovina: 7% (2%), Dictyocaulus filaria: 1% (N.D.). Larvae of Nematodirus spp. and T. colubriformis showed a quick rise of the exsheathment rate 2 h after transfer into the abomasum. These results confirm that exsheathment generally occurs in the part of the gastrointestinal tract immediately anterior to the habitat of the adult parasite. The overall similar course of exsheathment in both systems indicates that the essential stimuli for exsheathment were generated and maintained under in vitro conditions of the artificial rumen. In both systems, the bicarbonate concentration and the pH reflected a similar status of the H2CO3/HCO buffer system, which is known to provide the essential stimuli for larval exsheathment of the abomasal species. These results give evidence that the RUSITEC system represents a valid system for studying the kinetics of exsheathment of strongylid nematodes under in vitro conditions. For 7 of the species investigated the obtained results represent the first data on larval exsheathment in vivo and in vitro.

Abstract 186: Hdac7-derived 7aa Peptide May Function as a Phosphorylation Carrier

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Junyao Yang ◽  
Wen Wang ◽  
Qian Wang ◽  
Lingfang Zeng
Keyword(s):  
Endothelial Cell ◽  
Vascular Endothelial Cell ◽  
Blood Perfusion ◽  
Open Reading Frames ◽  
In Vivo Studies ◽  
Buffer System ◽  
Vascular Progenitor Cells ◽  
Upstream Kinase

Background: Histone deacetylase 7 (HDAC7) belongs to class II HDAC family, playing a pivotal role in the maintenance of endothelium integrity. There are 8 splicing variants in mouse HDAC7 mRNAs. Within the 5’ terminal non-coding area of some variants, there exist some short open reading frames (sORFs). Whether these sORFs can be translated and their potential roles in cellular physiology remain unclear. Method and results: Our previous studies suggested that one mouse HDAC7 produced a 7aa peptide from the non-coding area. In this study, we demonstrated that one sORF encoding a 7 amino acids (aa)-peptide could be translated in response to vascular endothelial cell growth factor (VEGF) in vascular progenitor cells (VPCs). The 7aa-peptide (7A) could be phosphorylated at serine residue via MEKK1. Importantly, the phosphorylated 7aa-peptide (7Ap) could transfer the phosphorylation group to the Thr residue of the 14-3-3γ protein in a cell free in-gel buffer system. The in vitro functional analyses revealed that 7A enhanced VEGF-induced VPC migration and differentiation toward endothelial cell (EC) lineage, in which MEKK1 and 14-3-3γ served as upstream kinase and downstream effector respectively. Knockdown of either MEKK1 or 14-3-3γ attenuated VEGF-induced VPC migration and differentiation. Exogenous 7Ap could rescue VEGF effect in MEKK1 but not in 14-3-3γ knockdown cells. The in vivo studies showed that 7A especially 7Ap induced capillary vessel formation within matrigel plug assays, increased re-endothelialization and suppressed neointima formation in the femoral artery injury model, and promoted the foot blood perfusion recovery in the hindlimb ischemia model. Conclusion: These results indicate that the sORFs within the non-coding area can be translated under some circumstances and that the 7aa-peptide may play an important role in cellular processes like migration and differentiation via acting as a phosphorylation carrier. Significance: As a phosphorylation carrier, 7aa possesses therapeutic potentials in tackling angiogenesis related diseases.

Abstract 319: Dalcetrapib-Mediated Modulation of Cholesteryl Ester Transfer Protein Activity Increases HDL-Cholesterol, Apolipoprotein A-I Levels and Cholesterol Efflux Capacity in Chow-Fed Rabbits

2012 ◽  
Vol 32 (suppl_1) ◽  
Author(s):  
Mathieu R Brodeur ◽  
David Rhainds ◽  
Daniel Charpentier ◽  
Téodora Mihalache-Avram ◽  
Cyrille Maugeais ◽  
...  
Keyword(s):  
Cholesteryl Ester ◽  
Cholesteryl Ester Transfer Protein ◽  
Cholesterol Efflux ◽  
Hdl Cholesterol ◽  
Lipid Transfer ◽  
Protein Activity ◽  
Cholesterol Efflux Capacity ◽  
Transfer Protein

Introduction: A potential approach to reduce CV risk is to increase HDL-C levels. This could be achieved by reducing cholesteryl ester transfer protein (CETP) activity. Dalcetrapib, which modulates CETP activity by changing its conformation and raises HDL-C without inhibiting CETP-induced pre-β-HDL formation in humans, was shown to decrease progression of atherosclerosis in rabbits. Hypothesis: Investigate the modifications of HDL particle size distribution and cholesterol efflux capacity of serum produced by dalcetrapib in normocholesterolemic rabbits. Methods: New Zealand white rabbits were treated with dalcetrapib (300 mg/kg as food admix) or placebo for 14 days. We evaluated CETP conformation and mass by ELISAs (including antibodies sensitive to conformational change), CETP activity by fluorescent lipid transfer, lipid profile and apoA-I distribution in HDL subclasses by 2D-non denaturing gradient gels (2D-NDGGE). Cholesterol efflux capacity of rabbit sera was determined after loading cells with 3 H-free cholesterol, using HepG2 hepatocytes to measure SR-BI-dependent efflux and by inducing ABCA1 or ABCG1 expression in BHK cells. Results: Dalcetrapib modified the conformation of rabbit CETP in vitro and in vivo and, after 14 days, this was associated with increased CETP mass (+50%, p<0.001) and reduced CETP activity (-86%, p<0.001). Total cholesterol was increased with dalcetrapib (+178%, p<0.001), due to a higher HDL-C level. In contrast, dalcetrapib reduced LDL-C and triglycerides by 41% (p<0.01) and 48% (p<0.001). Serum analysis by 2D-NDGGE showed that total rabbit apoA-I was increased 1.7- fold in animals treated with dalcetrapib. This was associated with an increase in large HDL but also in small α-migrating HDL with pre-β-HDL size. Cholesterol efflux assays showed that ABCA1-, ABCG1- and SR-BI-dependent efflux were all increased in dalcetrapib-treated rabbits (+24%, p=0.038; +21%, p=0.021; +44%, p<0.001). Conclusion: Modulation of CETP activity and conformation by dalcetrapib increases HDL-C and apoA-I levels and affects apoA-I distribution in HDL subclasses. These changes are associated with increased cholesterol efflux capacity, suggesting that HDL functionality is preserved in dalcetrapib-treated chow-fed rabbits.

ON THE MECHANISM OF THE IN VIVO SYNERGISM BETWEEN TISSUE-TYPE PLASMINOGEN ACTIVATOR (t-PA) AND SINGLE CHAIN UROKINASE-TYPE PLASMINOGEN ACTIVATOR (scu-PA)

1987 ◽  
Author(s):  
J M Stassen ◽  
D Collen
Keyword(s):  
Plasminogen Activator ◽  
Rabbit Model ◽  
Sequential Therapy ◽  
Tissue Type ◽  
Single Chain ◽  
Molar Ratios ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator

t-PA and scu-PA, in molar ratios between 1:4 and 4:1 do not act synergically in vitro (Thromb. Haemost. 56,35,1986) but display marked synergism in a rabbit model (Circulation 74, 838, 1986) and in man (Am. Heart J. 112, 1083, 1986). To investigate the mechanism of in vivo synergism in the rabbit model (J. Clin. Invest. 71, 368, 1983), t-PA and scu-PA were infused 1) simultaneously over 4 hrs, 2) t-PA over 1 hr, then 15 min later scu-PA over 2 hrs and 3) scu-PA over 1 hr, then 15 min later t-PA over 2 hrs.Significant synergism on thrombolysis is observed when t-PA and scu-PA are infused simultaneously or when t-PA is followed by scu-PA but not when scu-PA is followed by t-PA. These results suggest that low dose t-PA induces some plasminogen activation, sufficient to partially degrade fibrin, exposing COOH-terminal lysines with high affinity for plasminogen (Eur. J. Biochem. 140, 513, 1984). scu-PA might then activate surface-bound Glu-pla-minogen more efficiently.Sequential therapy with t-PA (or any other agent which "predigests" the thrombus), followed by scu-PA might constitute an alternative to simultaneous infusion of synergistic thrombolytic agents.

scholarly journals Engineered anti-inflammatory peptides inspired by mapping an evasin–chemokine interaction

2020 ◽  
Vol 295 (32) ◽  
pp. 10926-10939 ◽  
Author(s):  
Benoit Darlot ◽  
James R. O. Eaton ◽  
Lucia Geis-Asteggiante ◽  
Gopala K. Yakala ◽  
Kalimuthu Karuppanan ◽  
...  
Keyword(s):  
Inflammatory Diseases ◽  
Titration Calorimetry ◽  
Protein Activity ◽  
Binding Interface ◽  
Chemokine Ligand ◽  
Therapeutic Development ◽  
Hydrogen Deuterium ◽  
Anti Inflammatory

Chemokines mediate leukocyte migration and homeostasis and are key targets in inflammatory diseases including atherosclerosis, cytokine storm, and chronic autoimmune disease. Chemokine redundancy and ensuing network robustness has frustrated therapeutic development. Salivary evasins from ticks bind multiple chemokines to overcome redundancy and are effective in several preclinical disease models. Their clinical development has not progressed because of concerns regarding potential immunogenicity, parenteral delivery, and cost. Peptides mimicking protein activity can overcome the perceived limitations of therapeutic proteins. Here we show that peptides possessing multiple chemokine-binding and anti-inflammatory activities can be developed from the chemokine-binding site of an evasin. We used hydrogen–deuterium exchange MS to map the binding interface of the evasin P672 that physically interacts with C–C motif chemokine ligand (CCL) 8 and synthesized a 16-mer peptide (BK1.1) based on this interface region in evasin P672. Fluorescent polarization and native MS approaches showed that BK1.1 binds CCL8, CCL7, and CCL18 and disrupts CCL8 homodimerization. We show that a BK1.1 derivative, BK1.3, has substantially improved ability to disrupt P672 binding to CCL8, CCL2, and CCL3 in an AlphaScreen assay. Using isothermal titration calorimetry, we show that BK1.3 directly binds CCL8. BK1.3 also has substantially improved ability to inhibit CCL8, CCL7, CCL2, and CCL3 chemotactic function in vitro. We show that local as well as systemic administration of BK1.3 potently blocks inflammation in vivo. Identification and characterization of the chemokine-binding interface of evasins could thus inspire the development of novel anti-inflammatory peptides that therapeutically target the chemokine network in inflammatory diseases.

scholarly journals In Vitro Interactions of Artemisinin with Atovaquone, Quinine, and Mefloquine against Plasmodium falciparum

2002 ◽  
Vol 46 (5) ◽  
pp. 1510-1515 ◽  
Author(s):  
S. Gupta ◽  
M. M. Thapar ◽  
W. H. Wernsdorfer ◽  
A. Björkman
Keyword(s):  
Plasmodium Falciparum ◽  
Drug Interaction ◽  
In Vitro Culture ◽  
Effective Concentration ◽  
Interstrain Differences ◽  
Molar Ratios ◽  
The Mean ◽  
In Vitro Interactions

ABSTRACT The interactions of artemisinin with atovaquone, quinine, and mefloquine were investigated in three Plasmodium falciparum strains (strains F-32, FCR-3, and K-1) by an in vitro culture assay. The parasites were cultured for 48 h in the presence of different concentrations and proportions of two drugs at a time in a checkerboard design. The response parameters were determined, and the sums of the fractional inhibitory concentrations (ΣFICs) of the drug combinations were calculated for different degrees of inhibition (50% effective concentration [EC50], EC90, and EC99). Within therapeutically relevant molar ratios (19 to 200), the combination of quinine and artemisinin showed mean ΣFICs of 1.71 at the EC50, 0.36 at the EC90, and 0.13 at the EC99, indicating increasing synergism. Within the range of molar ratios of 4.3 to 50, the combination of mefloquine and artemisinin yielded mean ΣFCIs of 0.93, 0.44, and 0.31 at the EC50, EC90, and EC99, respectively, indicating synergism. The atovaquone combination showed additive activity to synergism at atovaquone/artemisinin proportions considered relevant to the in vivo situation, i.e., between 4.3 and 200, with the mean ΣFICs decreasing from 1.34 at the EC50 to 0.85 and 0.23 at the EC90 and EC99, respectively. Interstrain differences in the degree of drug interaction were seen with the three strains for all combinations. Synergism was most consistent with quinine.

scholarly journals Biocompatibility and Immune Response of a Newly Developed Volume-Stable Magnesium-Based Barrier Membrane in Combination with a PVD Coating for Guided Bone Regeneration (GBR)

Biomedicines ◽  
2020 ◽  
Vol 8 (12) ◽  
pp. 636
Author(s):  
Larissa Steigmann ◽  
Ole Jung ◽  
Wolfgang Kieferle ◽  
Sanja Stojanovic ◽  
Annica Proehl ◽  
...  
Keyword(s):  
Immune Response ◽  
Collagen Membrane ◽  
Promising Alternative ◽  
Barrier Membrane ◽  
Pvd Coating ◽  
Coated Membranes ◽  
Gas Cavity ◽  
Inflammatory Macrophages

To date, there are no bioresorbable alternatives to non-resorbable and volume-stable membranes in the field of dentistry for guided bone or tissue regeneration (GBR/GTR). Even magnesium (Mg) has been shown to constitute a favorable biomaterial for the development of stabilizing structures. However, it has been described that it is necessary to prevent premature degradation to ensure both the functionality and the biocompatibility of such Mg implants. Different coating strategies have already been developed, but most of them did not provide the desired functionality. The present study analyses a new approach based on ion implantation (II) with PVD coating for the passivation of a newly developed Mg membrane for GBR/GTR procedures. To demonstrate comprehensive biocompatibility and successful passivation of the Mg membranes, untreated Mg (MG) and coated Mg (MG-Co) were investigated in vitro and in vivo. Thereby a collagen membrane with an already shown biocompatibility was used as control material. All investigations were performed according to EN ISO 10993 regulations. The in vitro results showed that both the untreated and PVD-coated membranes were not cytocompatible. However, both membrane types fulfilled the requirements for in vivo biocompatibility. Interestingly, the PVD coating did not have an influence on the gas cavity formation compared to the uncoated membrane, but it induced lower numbers of anti-inflammatory macrophages in comparison to the pure Mg membrane and the collagen membrane. In contrast, the pure Mg membrane provoked an immune response that was fully comparable to the collagen membrane. Altogether, this study shows that pure magnesium membranes represent a promising alternative compared to the nonresorbable volume-stable materials for GBR/GTR therapy.

scholarly journals Clathrin structure characterized with monoclonal antibodies. II. Identification of in vivo forms of clathrin.

1985 ◽  
Vol 101 (6) ◽  
pp. 2055-2062 ◽  
Author(s):  
F M Brodsky
Keyword(s):  
Monoclonal Antibodies ◽  
Heavy Chain ◽  
Light Chain ◽  
Coated Vesicles ◽  
Cell Lysates ◽  
Over Time

Clathrin was isolated from detergent-solubilized, biosynthetically radiolabeled cells by immunoprecipitation with anti-clathrin monoclonal antibodies. Immunoprecipitates obtained after pulse-chase labeling demonstrated that after biosynthesis the LCa light chain of clathrin could be found either complexed to heavy chain or in a free pool (not associated with heavy chain) which decreased steadily over time. More than half of the free LCa disappeared within the first hour after biosynthesis, but some was still detectable after several hours. Incorporation of clathrin LCa light chain and heavy chain into coated vesicles was coordinate and increased up to 4 h after biosynthesis. Comparison of these kinetics suggested that once incorporated into coated vesicles, LCa and heavy chain did not dissociate, even during depolymerization of the vesicle. There was also little apparent degradation of clathrin found in coated vesicles for up to 22 h after biosynthesis. Immunoprecipitation with anti-clathrin monoclonal antibodies was carried out after fractionation of continuously radiolabeled cell lysates using two different sizing columns. These experiments indicated that the triskelion form of clathrin that has been isolated from coated vesicles in vitro also exists in vivo. They also confirmed the existence of a transient but detectable pool of newly synthesized free LCa light chain.

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