scholarly journals Fibrinogenolytic activity of protease from the culture fluid of Pleurotus ostreatus

2020 ◽  
Vol 93 (2) ◽  
Author(s):  
Yevhenii M. Stohnii ◽  
Valeriia V. Sakovich ◽  
Volodymyr O. Chernyshenko ◽  
Volodymyr I. Chernishov ◽  
Tamara M. Chernyshenko ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Pleurotus Ostreatus ◽  
Culture Fluid ◽  
Molecular Forms ◽  
Reducing Conditions ◽  
Transmission Electron ◽  
New Information ◽  
Sds Page ◽  
Microplate Reader ◽  
Fungal Protease

The use of proteases makes it possible to obtain partially hydrolyzed forms of macromolecules with unique properties. The importance of proteases for studying the structure and functions of fibrinogen forces scientists to search for new sources of highly specific proteases. Thus, the aim of this work was to study the content of the Pleurotus ostreatus culture fluid in search of fibrinogen- specific proteases. P. ostreatus was cultured for 14 days at 27°C. The culture fluid was collected and the protein fraction was salted out with NaCl and then dialyzed. Fibrinogen hydrolysis products by P. ostreatus protease were characterized using SDS PAGE under reducing conditions followed by immunoprobing using murine monoclonal antibodies I-5A (anti-Aα505-610) and 2d2a (anti-Bβ26-42). The study of turbidity and platelet aggregation was performed using a Multiskan FC spectrophotometric microplate reader and a SOLAR-2110 aggregometer, respectively. Electron microscopy of fibrils formed by truncated compared with native fibrins was performed using a transmission electron microscope N-600. Analysis of the products of fibrinogen hydrolysis with a fungal protease using SDS-PAGE demonstrated the cleavage of the alpha chain of fibrinogen exclusively with the formation of a truncated form of fibrinogen in which there are no C-terminal portions of αC regions with a molecular weight of 25 kDa. A study of turbidity showed that the polymerization of truncated fibrin is significantly impaired. The rate of lateral association of protofibrils significantly decreased from 1.5 to 2.2 times in the case of truncated fibrinogen compared to the native one depending on the initial concentration of fibrinogen. It was shown that platelet aggregation in the presence of fibrinogen without 25 kDa fragments of αC regions was less effective than in the presence of native fibrinogen. Application of the preparation of the fungal protease allows us to obtain high molecular forms of the fibrinogen molecule with cleaved 25 kDa peptides, which provide new information on the role of these peptides in the fibrinogen functioning.

scholarly journals Fibrin(ogen)olytic and platelet modulating properties of a novel protease from the culture fluid of Pleurotus osreatus

2020 ◽  
Author(s):  
Yevhenii Stohnii ◽  
Valeriia Sakovich ◽  
Volodymyr Chernyshenko ◽  
Volodymyr Chernishov ◽  
Tamara Chernyshenko ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Culture Fluid ◽  
Molecular Forms ◽  
Reducing Conditions ◽  
Transmission Electron ◽  
New Information ◽  
Sds Page ◽  
Microplate Reader ◽  
Fungal Protease

The use of proteases makes it possible to obtain partially hydrolyzed forms of macromolecules with unique properties. The importance of proteases for studying the structure and functions of fibrinogen forces scientists to search for new sources of highly specific proteases. Thus, the aim of this work was to study the content of the Pleurotus ostreatus culture fluid in search of fibrinogen-specific proteases. P. ostreatus was cultured for 14 days at 27 ° C. The culture fluid was collected and the protein fraction was salted out with NaCl and then dialyzed. Fibrinogen hydrolysis products by P. ostreatus protease were characterized using SDS PAGE under reducing conditions followed by immunoprobing using murine monoclonal antibodies I-5A (anti-Aα505-610) and 2d2a (anti-Bβ26-42). The study of turbidity and platelet aggregation was performed using a Multiskan FC spectrophotometric microplate reader and a SOLAR-2110 aggregometer, respectively. Electron microscopy of fibrils formed by truncated compared with native fibrins was performed using a transmission electron microscope N-600.Analysis of the products of fibrinogen hydrolysis with a fungal protease using SDS-PAGE demonstrated the cleavage of the alpha chain of fibrinogen exclusively with the formation of a truncated form of fibrinogen in which there are no C-terminal portions of αC regions with a molecular weight of 25 kDa. A study of turbidity showed that the polymerization of truncated fibrin is significantly impaired. The rate of lateral association of protofibrils significantly decreased from 1.5 to 2.2 times in the case of truncated fibrinogen compared to the native one depending on the initial concentration of fibrinogen. It was shown that platelet aggregation in the presence of fibrinogen without 25 kDa fragments of αC regions was less effective than in the presence of native fibrinogen. Application of the preparation of the fungal protease allows us to obtain high molecular forms of the fibrinogen molecule with cleaved 25 kDa peptides, which provide new information on the role of these peptides in the fibrinogen functioning.

Purification and Characterization of a Novel Metalloproteinase, Acurhagin, from Agkistrodon acutus Venom

2002 ◽  
Vol 87 (04) ◽  
pp. 641-650 ◽  
Author(s):  
Wen-Jeng Wang ◽  
Tur-Fu Huang
Keyword(s):  
Platelet Aggregation ◽  
Molecular Mass ◽  
Hydrophobic Interaction Chromatography ◽  
Inhibitory Effect ◽  
High Molecular Mass ◽  
Dependent Manner ◽  
Peptide Fragments ◽  
Crude Venom ◽  
Reducing Conditions ◽  
Sds Page

SummaryAcurhagin, a high-molecular mass hemorrhagic metalloproteinase, was purified from the crude venom of Agkistrodon acutus using anionexchange and hydrophobic interaction chromatography. Acurhagin is a monomer with a molecular mass of 51.4 kDa under non-reducing conditions on SDS-PAGE and 48,133 Da by mass spectrometry. Partial amino acid sequence of its metalloproteinase domain is homologous to other high-molecular mass metalloproteinases from snake venoms. It preferentially cleaved Aα. chain of fibrinogen, followed by Bβ chain, while γ chains was minimally affected. Monitored by RP-HPLC, it extensively degraded fibrinogen into various peptide fragments. In aqueous solution, acurhagin autoproteolyzed to a 30 kDa fragment at 37° C. The N-terminal sequence of the 30 kDa fragment of acurhagin showed a high homology to those proteins consisting of disintegrinlike and cysteine-rich domains. Caseinolytic assay showed that the proteinase activity of acurhagin was slightly enhanced by Ca2+ and Mg2+, but completely inhibited by Zn2+. When treated with metal chelators, acurhagin was completely inactivated. Furthermore, acurhagin exerts an inhibitory effect on ADP-induced platelet aggregation of plateletrich plasma in an incubation-time dependent manner. It also impairs collagen- and ristocetin-induced platelet aggregation by cleaving collagen and vWF, respectively.

Immunogold labeling of Pneumocystis carinii for Electron microscopy

1991 ◽  
Vol 49 ◽  
pp. 270-271
Author(s):  
Michael P. Goheen ◽  
Marilyn S. Bartlett ◽  
James W. Smith
Keyword(s):  
Electron Microscopy ◽  
Pneumocystis Carinii ◽  
Severe Pneumonia ◽  
Culture Fluid ◽  
Immunogold Labeling ◽  
Antigenic Sites ◽  
Transmission Electron ◽  
Response To Chemotherapy ◽  
Acquired Immunodeficiency ◽  
Immunodeficiency Syndrome

Studies of the biology of Pneumocystis carinii (PC) are of increasing importance because this extracellular pathogen is a frequent source of severe pneumonia in patients with acquired immunodeficiency syndrome (AIDS) and is a leading cause of mortality in these patients. Immunoelectron microscopic localization of antigenic sites on the surface of PC would improve the understanding of these sites and their role in pathenogenisis of the disease and response to chemotherapy. The purpose of this study was to develop a methodology for visualizing immunoreactive sites on PC with transmission electron microscopy (TEM) using immunogold labeled probes.Trophozoites of PC were added to spinner flask cultures and allowed to grow for 7 days, then aliquots of tissue culture fluid were centrifuged at 12,000 RPM for 30 sec. Pellets of organisims were fixed in either 1% glutaraldehyde, 0.1% glutaraldehyde-4% paraformaldehyde, or 4% paraformaldehyde for 4h. All fixatives were buffered with 0.1M Na cacodylate and the pH adjusted to 7.1. After fixation the pellets were rinsed in 0.1M Na cacodylate (3X), dehydrated with ethanol, and immersed in a 1:1 mixture of 95% ethanol and LR White resin.

Correlation Between Cellular Functions and Morphological Changes of Human Trophoblast in Vitro

1975 ◽  
Vol 33 ◽  
pp. 600-601
Author(s):  
John C. Garancis ◽  
Robert O. Hussa ◽  
Michael T. Story ◽  
Donald Yorde ◽  
Roland A. Pattillo
Keyword(s):  
Electron Microscopy ◽  
Cellular Proliferation ◽  
Morphological Changes ◽  
Culture Fluid ◽  
Cellular Functions ◽  
Human Trophoblast ◽  
Transmission Electron ◽  
Marked Activity ◽  
Malignant Trophoblast

Human malignant trophoblast cells in continuous culture were incubated for 3 days in medium containing 1 mM N6-O2'-dibutyryl cyclic adenosine 3':5'-monophosphate (dibutyryl cyclic AMP) and 1 mM theophylline. The culture fluid was replenished daily. Stimulated cultures secreted many times more chorionic gonadotropin and estrogens than did control cultures in the absence of increased cellular proliferation. Scanning electron microscopy revealed remarkable surface changes of stimulated cells. Control cells (not stimulated) were smooth or provided with varying numbers of microvilli (Fig. 1). The latter, usually, were short and thin. The surface features of stimulated cells were considerably different. There was marked increase of microvilli which appeared elongated and thick. Many cells were covered with confluent polypoid projections (Fig. 2). Transmission electron microscopy demonstrated marked activity of cytoplasmic organelles. Mitochondria were increased in number and size; some giant forms with numerous cristae were observed.

Modern TEM systems architecture

1986 ◽  
Vol 44 ◽  
pp. 602-605
Author(s):  
U. Gross ◽  
P. Hagemann
Keyword(s):  
Electron Microscope ◽  
Information Gathering ◽  
Systems Architecture ◽  
Control Functions ◽  
Transmission Electron ◽  
New Information ◽  
Comprehensive Information ◽  
Scanning Transmission ◽  
Diffraction Patterns ◽  
Analytical Equipment

By addition of analytical equipment, scanning transmission accessories and data processing equipment the basic transmission electron microscope (TEM) has evolved into a comprehensive information gathering system. This extension has led to increased complexity of the instrument as compared with the straightforward imaging microscope, since in general new information capacity has required the addition of new control hardware. The increased operational complexity is reflected in a proliferation of knobs and buttons.In the conventional electron microscope design the operating panel of the instrument has distinct control elements to alter optical conditions of the microscope column in different modes. As a consequence a multiplicity of control functions has been inevitable. Examples of this are the three pairs of focus and magnification controls needed for TEM imaging, diffraction patterns, and STEM images.

Functional Characterization of PM6/13, a β3-specific (GPIIIa/CD61) Monoclonal Antibody that Shows Preferential Inhibition of Fibrinogen Binding over Fibronectin Binding to Activated Human Platelets

1998 ◽  
Vol 79 (01) ◽  
pp. 177-185 ◽  
Author(s):  
Ashia Siddiqua ◽  
Michael Wilkinson ◽  
Vijay Kakkar ◽  
Yatin Patel ◽  
Salman Rahman ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Aggregation ◽  
Platelet Rich Plasma ◽  
Functional Characterization ◽  
Human Platelets ◽  
Western Blots ◽  
Activated Platelets ◽  
Reducing Conditions ◽  
Fibronectin Binding

SummaryWe report the characterization of a monoclonal antibody (MAb) PM6/13 which recognises glycoprotein IIIa (GPIIIa) on platelet membranes and in functional studies inhibits platelet aggregation induced by all agonists examined. In platelet-rich plasma, inhibition of aggregation induced by ADP or low concentrations of collagen was accompanied by inhibition of 5-hydroxytryptamine secretion. EC50 values were 10 and 9 [H9262]g/ml antibody against ADP and collagen induced responses respectively. In washed platelets treated with the cyclooxygenase inhibitor, indomethacin, PM6/13 inhibited platelet aggregation induced by thrombin (0.2 U/ml), collagen (10 [H9262]g/ml) and U46619 (3 [H9262]M) with EC50 = 4, 8 and 4 [H9262]g/ml respectively, without affecting [14C]5-hydroxytryptamine secretion or [3H]arachidonate release in appropriately labelled cells. Studies in Fura 2-labelled platelets revealed that elevation of intracellular calcium by ADP, thrombin or U46619 was unaffected by PM6/13 suggesting that the epitope recognised by the antibody did not influence Ca2+ regulation. In agreement with the results from the platelet aggregation studies, PM6/13 was found to potently inhibit binding of 125I-fibrinogen to ADP activated platelets. Binding of this ligand was also inhibited by two other MAbs tested, namely SZ-21 (also to GPIIIa) and PM6/248 (to the GPIIb-IIIa complex). However when tested against binding of 125I-fibronectin to thrombin stimulated platelets, PM6/13 was ineffective in contrast with SZ-21 and PM6/248, that were both potent inhibitors. This suggested that the epitopes recognised by PM6/13 and SZ-21 on GPIIIa were distinct. Studies employing proteolytic dissection of 125I-labelled GPIIIa by trypsin followed by immunoprecipitation with PM6/13 and analysis by SDS-PAGE, revealed the presence of four fragments at 70, 55, 30 and 28 kDa. PM6/13 did not recognize any protein bands on Western blots performed under reducing conditions. However Western blotting analysis with PM6/13 under non-reducing conditions revealed strong detection of the parent GP IIIa molecule, of trypsin treated samples revealed recognition of an 80 kDa fragment at 1 min, faint recognition of a 60 kDa fragment at 60 min and no recognition of any product at 18 h treatment. Under similar conditions, SZ-21 recognized fragments at 80, 75 and 55 kDa with the 55kDa species persisting even after 18 h trypsin treatment. These studies confirm the epitopes recognised by PM6/13 and SZ-21 to be distinct and that PM6/13 represents a useful tool to differentiate the characteristics of fibrinogen and fibronectin binding to the GPIIb-IIIa complex on activated platelets.

Molecular Forms of Human Protein C: Comparison and Distribution in Human Adult Plasma

1989 ◽  
Vol 62 (03) ◽  
pp. 902-905 ◽  
Author(s):  
Brian S Greffe ◽  
Marilyn J Manco-Johnson ◽  
Richard A Marlar
Keyword(s):  
Heavy Chain ◽  
Protein C ◽  
Molecular Forms ◽  
Post Translational Modification ◽  
Single Chain ◽  
Beta Form ◽  
Sds Page ◽  
Dependent Protein ◽  
Adult Plasma ◽  
The Difference

SummaryProtein C (PC) is a vitamin K-dependent protein which functions as both an anticoagulant and profibrinolytic. It is synthesized as a single chain protein (SC-PC) and post-transla-tionally modified into a two chain form (2C-PC). Two chain PC consists of a light chain (LC) and a heavy chain (HC). The present study was undertaken to determine the composition of the molecular forms of PC in plasma. PC was immunoprecipitated, subjected to SDS-PAGE and Western blotting. The blots were scanned by densitometry to determine the distribution of the various forms. The percentage of SC-PC and 2C-PC was found to be 10% and 90% respectively. This is in agreement with previous work. SC-PC and the heavy chain of 2C-PC consisted of three molecular forms (“alpha”, “beta”, and “gamma”). The “alpha” form of HC is the standard 2C form with a MW of 40 Kd. The “beta” form of HC has also been described and has MW which is 4 Kd less than the “alpha” form. The “gamma” species of the SC and 2C-PC has not been previously described. However, its 3 Kd difference from the “beta” form could be due to modification of the “beta” species or to a separate modification of the alpha-HC. The LC of PC was shown to exist in two forms (termed form 1 and form 2). The difference between these two forms is unknown. The molecular forms of PC are most likely due to a post-translational modification (either loss of a carbohydrate or a peptide) rather than from plasma derived degradation.

scholarly journals Lopinavir and Nelfinavir Induce the Accumulation of Crystalloid Lipid Inclusions within the Reservosomes of Trypanosoma cruzi and Inhibit Both Aspartyl-Type Peptidase and Cruzipain Activities Detected in These Crucial Organelles

2021 ◽  
Vol 6 (3) ◽  
pp. 120
Author(s):  
Leandro S. Sangenito ◽  
Miria G. Pereira ◽  
Thais Souto-Padron ◽  
Marta H. Branquinha ◽  
André L. S. Santos
Keyword(s):  
Trypanosoma Cruzi ◽  
Opportunistic Infections ◽  
Lipid Inclusion ◽  
Transmission Electron ◽  
New Information ◽  
Lipid Inclusions ◽  
Immunodeficiency Virus ◽  
Parasite Viability ◽  
Aspartyl Peptidases ◽  
New Evidence

Several research groups have explored the repositioning of human immunodeficiency virus aspartyl peptidase inhibitors (HIV-PIs) on opportunistic infections caused by bacteria, fungi and protozoa. In Trypanosoma cruzi, HIV-PIs have a high impact on parasite viability, and one of the main alterations promoted by this treatment is the imbalance in the parasite’s lipid metabolism. However, the reasons behind this phenomenon are unknown. In the present work, we observed by transmission electron microscopy (TEM) that the treatment of T. cruzi epimastigotes with the HIV-PIs lopinavir and nelfinavir induced a huge accumulation of crystalloid-shaped lipids within the reservosomes, most of them deforming these key organelles. As previously reported, those structures are characteristic of lipid inclusions formed mostly of cholesterol and cholesterol-esters. The fractionation of nontreated epimastigotes generated two distinct fractions enriched in reservosomes: one mostly composed of lipid inclusion-containing reservosomes (Fraction B1) and one where lipid inclusions were much less abundant (Fraction B2). Interestingly, the extract of Fraction B2 presented enzymatic activity related to aspartyl-type peptidases 3.5 times higher than that found in the extract obtained from Fraction B1. The cleavage of cathepsin D substrate by this class of peptidases was strongly impaired by pepstatin A, a prototypical aspartyl PI, and the HIV-PIs lopinavir and nelfinavir. In addition, both HIV-PIs also inhibited (to a lesser extent) the cruzipain activity present in reservosomes. Finally, our work provides new evidence concerning the presence and supposed participation of aspartyl peptidases in T. cruzi, even as it adds new information about the mechanisms behind the alterations promoted by lopinavir and nelfinavir in the protozoan.

Characterization of columns grown during KrF laser micromachining of Al2O3–TiC ceramics

2003 ◽  
Vol 18 (5) ◽  
pp. 1123-1130 ◽  
Author(s):  
V. Oliveira ◽  
R. Vilar
Keyword(s):  
Electron Microscopy ◽  
Photoelectron Spectroscopy ◽  
Laser Pulses ◽  
Formation Mechanisms ◽  
X Ray ◽  
Reducing Conditions ◽  
Transmission Electron ◽  
Column Formation ◽  
Krf Laser

This paper aims to contribute to the understanding of column formation mechanisms in Al2O3–TiC ceramics micromachined using excimer lasers. Chemical and structural characterization of columns grown in Al2O3–TiC composite processed with 200 KrF laser pulses at 10 J/cm2 was carried out by scanning electron microscopy, transmission electron microscopy, x-ray photoelectron spectroscopy, and x-ray diffraction analysis. Fully developed columns consist of a core of unprocessed material surrounded by an outer layer of Al2TiO5, formed in oxidizing conditions, and an inner layer, formed in reducing conditions, composed of TiC and Al3Ti or an AlTi solid solution. Possible mechanisms of column formation are discussed.

scholarly journals ROS Promote Ox-LDL-Induced Platelet Activation by Up-Regulating Autophagy Through the Inhibition of the PI3K/AKT/mTOR Pathway

2018 ◽  
Vol 50 (5) ◽  
pp. 1779-1793 ◽  
Author(s):  
Xiang Wang ◽  
Yun-Feng Fu ◽  
Xiao Liu ◽  
Guo Feng ◽  
Dan Xiong ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Platelet Aggregation ◽  
Platelet Activation ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Mtor Pathway ◽  
Enhanced Chemiluminescence ◽  
Transmission Electron ◽  
Related Proteins

Background/Aims: Oxidized low-density lipoprotein (oxLDL) promotes unregulated platelet activation in patients with dyslipidemic disorders. Although oxLDL stimulates activating signaling, researchers have not clearly determined how these events drive accelerated thrombosis. Here, we describe the mechanism by which ROS regulate autophagy during ox-LDL-induced platelet activation by modulating the PI3K/AKT/mTOR signaling pathway. Methods: For in vitro experiments, ox-LDL, the ROS scavenger N-acetylcysteine (NAC), the mTOR inhibitor rapamycin and the autophagy inhibitor 3-MA were used alone or in combination with other compounds to treat platelets. Then, platelet aggregation was evaluated on an aggregometer and platelet adhesion was measured under shear stress. The levels of a platelet activation marker (CD62p) were measured by flow cytometry, reactive oxygen species (ROS) levels were then quantified by measuring DCFH-DA fluorescence intensity via flow cytometry. Nitric oxide (NO) and superoxide (O2·-) levels were determined by the nitric acid deoxidize enzyme method and lucigenin-enhanced chemiluminescence (CL), respectively. Transmission electron microscopy was used to observe the autophagosome formation, immunofluorescence staining was employed to detect LC3 expression and western blotting was used to measure the levels of PI3K/AKT/mTOR pathway- and autophagy-related proteins. Results: Ox-LDL-induced platelets showed a significant increase in platelet aggregation and adhesion, CD62p expression, ROS level and O2·- content, with an elevated LC3II/LC3I ratio and Beclin1 expression, but a dramatic reduction in the levels of p62 and pathway-related proteins (all P < 0.05). However, platelet activation and autophagy were aggravated by the Rapamycin treatment, and decreased following treatment with NAC, 3-MA, or NAC and 3-MA, together with increased activity of the PI3K/AKT/mTOR pathway. Additionally, decreased platelet activation and autophagy were observed in platelets treated with NAC and Rapamycin or Rapamycin and 3-MA compared with platelets treated with Rapamycin alone, suggesting that both NAC and 3-MA reversed the effects of Rapamycin. Conclusion: Inhibition of ROS production may reduce autophagy to suppress ox-LDL-induced platelet activation by activating PI3K/AKT/mTOR pathway.

Sign in / Sign up

Export Citation Format

Share Document