Jelly plug dissolution in Discoglossus pictus eggs (Anura) involves peroxidase-like activity and oxidative opening of disulphide bonds

Zygote ◽  
1993 ◽  
Vol 1 (1) ◽  
pp. 61-69 ◽  
Author(s):  
G. Pitari ◽  
S. Dupré ◽  
C. Fusco ◽  
G. Maurizi ◽  
C. Campanella
Keyword(s):  
Hydrogen Peroxide ◽  
Peroxidase Activity ◽  
Electrophoretic Analysis ◽  
Ringer Solution ◽  
Cysteic Acid ◽  
Jelly Coat ◽  
Discoglossus Pictus ◽  
Disulphide Bonds ◽  
Sds Page

SummaryIn amphibian eggs the formation of a capsular chamber is one of the most striking events occurring either upon oviposition or after fertilisation. In the egg of the anuran Discoglossus pictus a capsular chamber forms following fertilisation or activation; the egg with its vitelline envelope rotates in this chamber according to gravity. Previous work showed that the chamber is the product of plug dissolution. The plug is a lens-shaped jelly coat, typical of Discoglossus, covering only part of the animal hemisphere. Its dissolution is caused by material released from the egg about 15 min after fertilisation through exocytosis of at least two types of vacuoles. Liquefaction of the plug correlates with the reduction of disulphide bonds present in the jelly matrix. In this study we investigated the nature of the substances released from the egg and some changes occurring in the plug during liquefaction. SDS-PAGE showed that the proteic profile of the plug changes dramatically after fertilisation, confirming proteic cleavage in the plug matrix during its dissolution. Through in vitro tests and electrophoretic analysis of the Ringer solution in which the egg exudate was collected, an increase in the activity of the solution was determined in the presence of hydrogen peroxide, and peroxidase activity was depicted in the egg exudate. The presence of free thiol groups and cysteic acid residues (or cysteine sulphinic acid) in the plugs of activated eggs was established, suggesting that during plug dissolution some disulphide bonds are oxidatively opened. This suggests that enzyme(s) with peroxidase activity are released following fertilisation. We surmise that such enzymes are contained in the intraovular vacuoles the exocytosis of which triggers the onset of plug liquefaction. The possible release of hydrogen peroxide from the egg is discussed.

scholarly journals A New Bevacizumab Carrier for Intravitreal Administration: Focus on Stability

Pharmaceutics ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 560
Author(s):  
Daniela Chirio ◽  
Elena Peira ◽  
Simona Sapino ◽  
Giulia Chindamo ◽  
Simonetta Oliaro-Bosso ◽  
...  
Keyword(s):  
Electrophoretic Analysis ◽  
Vascular Endothelial ◽  
Invasive Treatment ◽  
Efficient Procedure ◽  
Cell Compatibility ◽  
Zeta Potentials ◽  
Sustained Drug Delivery ◽  
Sds Page ◽  
Endothelial Growth Factor

Bevacizumab (BVZ) is a monoclonal antibody that binds to human vascular endothelial growth factor A (VEGF-A) and inhibits the interaction between VEGF-A and VEGF receptors, thus blocking the angiogenesis. Repeated intravitreal injections of BVZ for the treatment of ocular pathologies that present an excessive proliferation results in a low patience compliance. BVZ is specially indicated for the treatment of diabetic and degenerative retinopathy. In the present study, we designed lipid nanoparticles (NPs) as a BVZ sustained drug delivery system for reducing the frequency of administration. We used a simple and highly efficient procedure, “Cold dilution of microemulsions”, to obtain spherical NPs with mean diameters of 280–430 nm, Zeta potentials between −17 and −31 mV, and drug entrapment efficiencies between 50 to 90%. This study focused on the biochemical and biophysical stabilities of BVZ after entrapment in NPs. SDS-PAGE electrophoretic analysis and circular dichroism, dynamic light scattering, and scanning electron microscopy were used to characterize BVZ-loaded NPs. The biocompatibility was assessed by in vitro cell compatibility studies using the ARPE-19 cell line. Thus, in this work, a stable BVZ-loaded system was obtained. In addition, several studies have shown that BVZ is released slowly from the lipid matrix and that this system is biocompatible. The results are promising and the developed NPs could be exploited to create a new, potentially effective and minimally invasive treatment of intraocular diseases.

scholarly journals Differential modification of cyclo-oxygenase and peroxidase activities of prostaglandin endoperoxidase synthase by proteolytic digestion and hydroperoxides

1990 ◽  
Vol 269 (3) ◽  
pp. 603-607 ◽  
Author(s):  
A Raz ◽  
P Needleman
Keyword(s):  
Peroxidase Activity ◽  
Enzymic Activity ◽  
Peptide Fragments ◽  
Structural Domains ◽  
Thrombin Cleavage ◽  
Terminal Fragment ◽  
Oxygenase Activity ◽  
Sds Page ◽  
Prostaglandin Endoperoxide Synthase

Prostaglandin endoperoxide synthase (PES, EC 1.14.99.1) catalyse the conversion of arachidonic acid into prostaglandin H2. The enzyme is a 140 kDa homodimer which contains both a cyclo-oxygenase activity (converting arachidonate into prostaglandin G2) and peroxidase activity (reducing prostaglandin G2 to H2). PES undergoes rapid self-inactivation during oxygenation of arachidonate to prostaglandin H2 in vitro. The previously reported cDNA-derived amino acid sequence indicates numerous sites for trypsin or thrombin cleavage. Most of these sites must be inaccessible, since these enzymes cleave only at Arg253. The enzyme appears to be a self-adherent and highly folded molecule, since after cleavage it retains its functional assembly and its homodimer size of 140 kDa, as well as its overall enzymic activity. Only under denaturing conditions (e.g. SDS/PAGE) can the proteolytic peptides be demonstrated: a 38 kDa C-terminal fragment containing the aspirin-derived-acetyl-binding ability, and a 33 kDa N-terminal fragment. In the present studies we investigated whether the two enzymic activities of PES can be differentially manipulated by proteolytic cleavage or by substrate (arachidonate) self-inactivation. The results indicated that, during arachidonate oxygenation by PES, the cyclooxygenase activity is selectively inactivated, whereas the peroxidase activity is essentially retained. By contrast, thrombin or trypsin cleavage of pure PES or microsomal PES (to yield the 38 and 33 kDa peptide fragments) inactivated the peroxidase, but not the cyclo-oxygenase. Taken together, these results suggest the presence of separate cyclo-oxygenase and peroxidase structural domains on the enzyme.

The Anticoagulant Properties of a Modified Form of Protein S

1988 ◽  
Vol 60 (02) ◽  
pp. 298-304 ◽  
Author(s):  
C A Mitchell ◽  
S M Kelemen ◽  
H H Salem
Keyword(s):  
Monoclonal Antibody ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Factor Xa ◽  
Protein S ◽  
Human Neutrophil Elastase ◽  
Factor X ◽  
Sds Page

SummaryProtein S (PS) is a vitamin K-dependent anticoagulant that acts as a cofactor to activated protein C (APC). To date PS has not been shown to possess anticoagulant activity in the absence of APC.In this study, we have developed monoclonal antibody to protein S and used to purify the protein to homogeneity from plasma. Affinity purified protein S (PSM), although identical to the conventionally purified protein as judged by SDS-PAGE, had significant anticoagulant activity in the absence of APC when measured in a factor Xa recalcification time. Using SDS-PAGE we have demonstrated that prothrombin cleavage by factor X awas inhibited in the presence of PSM. Kinetic analysis of the reaction revealed that PSM competitively inhibited factor X amediated cleavage of prothrombin. PS preincubated with the monoclonal antibody, acquired similar anticoagulant properties. These results suggest that the interaction of the monoclonal antibody with PS results in an alteration in the protein exposing sites that mediate the observed anticoagulant effect. Support that the protein was altered was derived from the observation that PSM was eight fold more sensitive to cleavage by thrombin and human neutrophil elastase than conventionally purified protein S.These observations suggest that PS can be modified in vitro to a protein with APC-independent anticoagulant activity and raise the possibility that a similar alteration could occur in vivo through the binding protein S to a cellular or plasma protein.

In Vitro Stability of a Tissue-Type Plasminogen Activator Mutant, BM 06.022, in Human Plasma

1994 ◽  
Vol 72 (06) ◽  
pp. 906-911 ◽  
Author(s):  
D C Rijken ◽  
E Groeneveld ◽  
M M Barrett-Bergshoeff
Keyword(s):  
Plasminogen Activator ◽  
Half Life ◽  
Polyacrylamide Gel Electrophoresis ◽  
Tissue Type ◽  
Single Chain ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Sds Page ◽  
Chain Form

SummaryBM 06.022 is a non-glycosylated mutant of human tissue-type plasminogen activator (t-PA) comprising only the kringle-2 and proteinase domains. The in vivo half-life of BM 06.022 antigen is 4- to 5-fold longer than that of t-PA antigen. The in vitro half-life of the activity of BM 06.022 at therapeutic concentrations in plasma is shorter than that of t-PA. In this study the inactivation of BM 06.022 in plasma was further investigated.Varying concentrations of BM 06.022 were incubated in plasma for 0-150 min. Activity assays on serial samples showed a dose-dependent decline of BM 06.022 activity with a half-life from 72 min at 0.3 μg/ml to 38 min at 10 μg/ml. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by fibrin autography showed the generation of several BM 06.022-complexes. These complexes could be completely precipitated with antibodies against Cl-inactivator, α2-antiplasmin and α1-antitrypsin.During the incubation of BM 06.022 in plasma, plasmin was generated dose-dependently as revealed by varying degrees of a2-anti-plasmin consumption and fibrinogen degradation. SDS-PAGE and immunoblotting showed that single-chain BM 06.022 was rapidly (i. e. within 45 min) converted into its two-chain form at concentrations of 5 μg/ml BM 06.022 and higher.In conclusion, BM 06.022 at therapeutic concentrations in plasma was inactivated by Cl-inactivator, a2-antiplasmin and a j-antitrypsin. The half-life of the activity decreased at increasing BM 06.022 concentrations, probably as a result of the generation of two-chain BM 06.022 which may be inactivated faster than the single-chain form.

An ESR study of the peroxidase reaction catalyzed by human methaemoglobin and methaemoglobin-haptoglobin complex

1991 ◽  
Vol 56 (4) ◽  
pp. 923-932
Author(s):  
Jana Stejskalová ◽  
Pavel Stopka ◽  
Zdeněk Pavlíček
Keyword(s):  
Hydrogen Peroxide ◽  
Ascorbic Acid ◽  
Amino Acid ◽  
Peroxidase Activity ◽  
Amino Acid Analysis ◽  
Superoxide Radical ◽  
Esr Spectra ◽  
The Other ◽  
Delocalized Electron

The ESR spectra of peroxidase systems of methaemoglobin-ascorbic acid-hydrogen peroxide and methaemoglobin-haptoglobin complex-ascorbic acid-hydrogen peroxide have been measured in the acetate buffer of pH 4.5. For the system with methaemoglobin an asymmetrical signal with g ~ 2 has been observed which is interpreted as the perpendicular region of anisotropic spectrum of superoxide radical. On the other hand, for the system with methaemoglobin-haptoglobin complex the observed signal with g ~ 2 is symmetrical and is interpreted as a signal of delocalized electron. After realization of three repeatedly induced peroxidase processes the ESR signal of the perpendicular part of anisotropic spectrum of superoxide radical is distinctly diminished, whereas the signal of delocalized electron remains practically unchanged. An amino acid analysis of methaemoglobin along with results of the ESR measurements make it possible to derive a hypothesis about the role of haptoglobin in increasing of the peroxidase activity of methaemoglobin.

An in vitro senescence model of gingival epithelial cell induced by hydrogen peroxide treatment

Odontology ◽  
2021 ◽  
Author(s):  
Sarita Giri ◽  
Ayuko Takada ◽  
Durga Paudel ◽  
Koki Yoshida ◽  
Masae Furukawa ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Epithelial Cell ◽  
Gingival Epithelial Cell ◽  
Hydrogen Peroxide Treatment

scholarly journals Chromatic intervention and biocompatibility assay for biosurfactant derived from Balanites aegyptiaca (L.) Del

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Vishal Panchariya ◽  
Vishal Bhati ◽  
Harishkumar Madhyastha ◽  
Radha Madhyastha ◽  
Jagdish Prasad ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Skin Fibroblast ◽  
Mouse Skin ◽  
Fibroblast Cells ◽  
Toxicity Assay ◽  
Balanites Aegyptiaca ◽  
Hemolysis Assay ◽  
Full Factorial ◽  
Human Rbcs

AbstractExtraction of biosurfactants from plants is advantageous than from microbes. The properties and robustness of biosurfactant derived from the mesocarp of Balanites aegyptiaca have been reported. However, the dark brown property of biosurfactant and lack of knowledge of its biocompatibility limits its scope. In the present work, the decolorization protocol for this biosurfactant was optimized using hydrogen peroxide. The hemolytic potential and biocompatibility based on cell toxicity and proliferation were also investigated. This study is the first report on the decolorization and toxicity assay of this biosurfactant. For decolorization of biosurfactant, 34 full factorial design was used, and the data were subjected to ANOVA. Results indicate that 1.5% of hydrogen peroxide can decolorize the biosurfactant most efficiently at 40 °C in 70 min at pH 7. Mitochondrial reductase (MTT) and reactive oxygen species (ROS) assays on M5S mouse skin fibroblast cells revealed that decolorized biosurfactant up to 50 µg/mL for 6 h had no significant toxic effect. Hemolysis assay showed ~ 2.5% hemolysis of human RBCs, indicating the nontoxic effect of this biosurfactant. The present work established a decolorization protocol making the biosurfactant chromatically acceptable. Biocompatibility assays confirm its safer use as observed by experiments on M5S skin fibroblast cells under in vitro conditions.

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