In vitro effects of L-arginine on lysosomal cysteine proteases activity in isolated experiment and in the state of oxidative stress

Aim. Assessment of direct influence of arginine on lysosomal cysteine proteases activity in vitro, in isolation as well as the stimulation of oxidative stress. Methods. The study was conducted on the 72 female conventional mature Wistar rats 280-320 g divided into 6 series of 12 rats each. Lysosome slurries were isolated from the liver of intact animals with a subsequent in vitro incubation in a sucrose solution, in the presence of L-arginine, as well as in the presence of L-arginine accompanied by the stimulation of oxidative stress. Samples of control groups were exposed in vitro with the addition of isolate and oxidant, respectively. Each batch was reproduced three times, incubation was performed at 37 °C in a water bath for 1, 2 and 4 hours. The activity of cathepsins B, L and H was studied using spectrofluorimetric method in two fractions - intra- and extralysosomal. Acid phosphatase activity was used as the main marker of membrane labialization. Results. One hour Incubation with 5 mM arginine in vitro led to inhibition of the cathepsin H activity and lysosomal membrane damage, however, further increase in incubation time led to its stabilization. In vitro exposure to 5 mM H2O2 caused an increase in activity of cathepsines B and L and the drop in the cathepsin H activity without obvious changes in the distribution of enzymes between extra and intralysosomal fractions. In a state of oxidative stress 2-hour in vitro incubation with 5 mM arginine reduced the permeability of lysosomal membranes for cathepsines B, H and L; while 4-hour incubation led to the destabilization of lysosomal membranes. Conclusion. The direct effect of arginine at a concentration of 5 mM within the 1,2 and 4-hour time intervals leads to a distinct change as a lysosomal cysteine protease activity and stability of lysosomal membranes.

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IN VITRO EFFECT OF L-CARNITINE ON THE ACTIVITY OF LYSOSOMAL CYSTEINE PROTEASES AND THE STATE OF LYSOSOMAL MEMBRANE

I P Pavlov Russian Medical Biological Herald ◽

10.23888/pavlovj2017114-20 ◽

2017 ◽

Vol 25 (1) ◽

pp. 14-20 ◽

Cited By ~ 1

Author(s):

M A. Fomina ◽

A M. Kudlaeva ◽

A N. Ryabkov

Keyword(s):

Acid Phosphatase ◽

Cysteine Proteinase ◽

Cathepsin L ◽

Cysteine Proteases ◽

Lysosomal Membrane ◽

Female Rats ◽

In Vitro Incubation ◽

Cathepsin H ◽

Vitro Effect

The influence of L-carnitine in vitro on the lysosomal cysteine proteinase activity and stability of the lysosomal membrane of the liver homogenates of intact sexually Mature female rats of Wistar line weighing 280-330 g were studied. In the experimental groups isolated lysosomes were incubated in vitro in a solution of L-carnitine during 1, 2 and 4 hours, in the control groups in vitro incubation was carried out in a medium of isolating solution. The activity of ca-thepsins B, L and H was investigated by spectrofluorimetric method of Barrett & Kirschke in two fractions - lysosomal and outside of lysosomes. The activity of acid phosphatase was used as the main marker of a membrane labilization. In vitro incubation of lysosomes showed that carnitine at a concentration of 5 mM increases the total activity of cathepsin B in a one-hour incubation at 73,2% (p=0,008), cathepsin L in a two- and four-hour incubation - at 77,7% (p=0,005) and 42,3% (p=0,013) respectively, and reduces the overall activity of the cathepsin H in a one-hour incubation at 200,0% (p=0,008), in a two-hour - by 67,9% (p=0,05), in a four-hour -27,1% (p=0,02). In addition, incubation in 5 mM L-carnitine solution leads to an increase of unsedimentable activity and fall sedimentaries activity for cathepsin L in a two-hour, and for acid phosphatase - in a two - and four-hour exposure. 5 mM L-carnitine in one - and two-hour incubation stabilizes lysosomal membrane (whereas increase in incubation time up to 4 hours leads to its damage) and increases the selective permeability of the lysosomal membrane for the studied cathepsins, to the greatest extent - for cathepsin H.

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Structural and Functional Dynamics of Lysosomal Cysteine Proteases with Particular Reference to Cathepsin B and Cathepsin H

Frontiers in Protein Structure, Function, and Dynamics ◽

10.1007/978-981-15-5530-5_16 ◽

2020 ◽

pp. 391-424

Author(s):

Sudhir K. Agarwal ◽

Shalini Singh ◽

Samir Sharma

Keyword(s):

Cathepsin B ◽

Cysteine Proteases ◽

Functional Dynamics ◽

Cathepsin H ◽

Lysosomal Cysteine Proteases

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146 Quercetin supplementation during boar semen thawing and incubation improves the in vitro production of pig embryos

Reproduction Fertility and Development ◽

10.1071/rdv31n1ab146 ◽

2019 ◽

Vol 31 (1) ◽

pp. 198

Author(s):

E. Hicks ◽

E. Winn ◽

B. Whitaker

Keyword(s):

Oxidative Stress ◽

Embryonic Development ◽

Developmental Stages ◽

Membrane Damage ◽

In Vitro Production ◽

Cell Stage ◽

Stage Of Development ◽

Boar Semen ◽

Morphological Deformities

Elevated levels of reactive oxygen species in the in vitro environment cause oxidative stress, which leads to membrane damage, decreased fertility, and morphological deformities of spermatozoa. Antioxidants, such as quercetin (a polyphenol flavonoid), are often supplemented to reduce the effects of oxidative stress on spermatozoa. Supplementing frozen-thawed boar semen with quercetin improves sperm forward progressive motility, viability and lipid peroxidation up to 10h after thawing. However, the effects of fertilizing with quercetin-supplemented sperm are unknown. Therefore, the objective of this study was to determine the effects of supplementing quercetin (0.25, 0.50, 0.75mM) during the thawing and incubation of frozen-thawed boar semen on oocyte fertilization characteristics (n=400) and subsequent embryonic development (n=1340) at 48 and 144h for cleavage and blastocyst formation, respectively. Oocytes from aspired aspirated mature follicles (3-6mm diameter) were obtained from a local abattoir and matured in medium 199 for 40 to 44h at 38.5°C in an atmosphere of 5% CO2. Fertilization was performed using pooled frozen-thawed semen from 3 different boars, and co-incubation of the sperm (2×105 sperm mL−1) and oocytes (30 oocytes/well) lasted for 6 to 8h at 38.5°C in an atmosphere of 5% CO2. Data were analysed using ANOVA with the main effects including treatment, well and replicate. Chi-squared analysis was used to determine percentages of embryos reaching the different developmental stages for each treatment. There were no differences in penetration rates and male pronuclear formation between treatment groups; however, supplementation of 0.25 (18.18±10.63%), 0.50 (20.93±9.89%) and 0.75mM (18.07±12.02%) quercetin significantly decreased (P<0.05) polyspermic penetration rates compared with no supplementation (40.00±11.34%). Embryos produced from frozen-thawed boar sperm supplemented with 0.25 and 0.50mM quercetin had a significantly higher percentage (P<0.05) of embryos reaching the 2-cell stage of development by 48h after IVF (75.00±7.89%, 68.75±2.23%, respectively) compared with 0.75mM quercetin supplementation (64.62±3.88%) and no supplementation (62.97±4.11%). Supplementation of 0.25 (44.12±6.23%), 0.50 (43.75±7.02%) and 0.75mM (43.08±2.98%) quercetin to the sperm significantly increased (P<0.05) the percentage of embryos reaching the blastocyst stage of development by 144h after IVF compared with no supplementation (28.27±8.07%). These results indicate that supplementing frozen-thawed boar semen with quercetin decreases the incidence of polyspermic penetration and improves early embryonic development in pigs.

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Evaluation of low-grade In Vitro Oxidative Stress in Goat Erythrocytes ‎Exposed to Hydrogen Peroxide and Dexamethasone ‎

Sahel Journal of Veterinary Sciences ◽

10.54058/saheljvs.v18i2.244 ◽

2021 ◽

Vol 18 (2) ◽

pp. 33-37

Author(s):

M. B. Monguno ◽

E. S. Philip ◽

I. C. Uku ◽

I. O. Igbokwe

Keyword(s):

Oxidative Stress ◽

Hydrogen Peroxide ◽

In Vitro Model ◽

Sucrose Solution ◽

Osmotic Fragility ◽

Low Grade ◽

Mean Values ◽

Red Blood Cell Count ◽

Vitro Model

Under conditions of oxidative stress, erythrocytes of goats could be predisposed to haemolysis. This study was aimed at evaluating the effect of oxidant exposure to goat erythrocytes using an in vitro model. Blood samples from 10 goats were incubated with 0.06 – 0.18 mM hydrogen peroxide (H2O2) either singly or in combination with 0.02 µM dexamethasone for 60 min, and erythrocyte parameters such as packed cell volume (PCV), red blood cell count (RBC), mean corpuscular volume (MCV) and percentage haemolysis in hypotonic sucrose solution (250 mOsmol/L) were determined thereafter. No significant changes in the mean values of all parameters were observed. The in vitro model indicated that erythrocyte parameters remained stable under low-grade oxidant exposure in goats. Therefore, plasma H2O2 concentration of ≤ 0.18 mM, whether in the presence or absence of 0.02 µM dexamethasone, may not induce apparent oxidative damage in goat erythrocytes that could be estimated by PCV, RBC, MCV and sucrose-based osmotic fragility at low hypotonicity.

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Protective effect of diosmetin on in vitro cell membrane damage and oxidative stress in cultured rat hepatocytes

Toxicology ◽

10.1016/0300-483x(92)90101-j ◽

1992 ◽

Vol 73 (2) ◽

pp. 179-189 ◽

Cited By ~ 21

Author(s):

Pia Villa ◽

Dario Cova ◽

Laura De Francesco ◽

Amalia Guaitani ◽

Giuseppina Palladini ◽

...

Keyword(s):

Oxidative Stress ◽

Cell Membrane ◽

Protective Effect ◽

Membrane Damage ◽

Rat Hepatocytes ◽

Cell Membrane Damage ◽

Cultured Rat Hepatocytes ◽

And Oxidative Stress

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Protective Effects of Fruit Wines against Hydrogen Peroxide—Induced Oxidative Stress in Rat Synaptosomes

Agronomy ◽

10.3390/agronomy11071414 ◽

2021 ◽

Vol 11 (7) ◽

pp. 1414

Author(s):

Uroš Čakar ◽

Mirjana Čolović ◽

Danijela Milenković ◽

Branislava Medić ◽

Danijela Krstić ◽

...

Keyword(s):

Oxidative Stress ◽

Hydrogen Peroxide ◽

Glutathione Peroxidase ◽

Membrane Damage ◽

Principal Component ◽

Antioxidant Enzyme Activities ◽

Protective Effects ◽

Sod Activity ◽

Black Chokeberry

This study aimed to evaluate, in vitro, the antioxidative potential of fruit wines produced from berry fruits (i.e., black chokeberry, blueberry, blackberry, and raspberry), cherry, and apple by different technological processes. For this purpose, the activities of antioxidant enzymes (catalase, glutathione peroxidase (GPx), and superoxide dismutase (SOD)) and malondialdehyde (MDA) content as a marker of membrane damage were determined in wine-treated synaptosomes with hydrogen peroxide-induced oxidative stress. All studied wines induced increased antioxidant enzyme activities and decreased MDA levels compared to hydrogen peroxide-treated synaptosomes (i.e., control). The highest SOD activity was observed in synaptosomes treated with blackberry wine (6.81 U/mg), whereas blueberry wine induced the highest catalase and glutathione peroxidase activities (0.058 U/mg and 0.017 U/mg, respectively). Black chokeberry proved to be the best in lipid peroxidation protection with the lowest MDA value (1.42 nmol/mg). Finally, principal component analysis and hierarchical cluster analysis additionally highlighted a higher antioxidant capacity of wines produced from dark-skinned fruits (i.e., blackberry, black chokeberry, and blueberry). The results suggest protective effects of the fruit wines against oxidative damage, and, accordingly, their promising application as functional food.

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Bioallethrin enhances generation of ROS, damages DNA, impairs the redox system and causes mitochondrial dysfunction in human lymphocytes

Scientific Reports ◽

10.1038/s41598-021-87799-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Amin Arif ◽

Ruhul Quds ◽

Riaz Mahmood

Keyword(s):

Oxidative Stress ◽

Nuclear Dna ◽

Insect Pests ◽

Human Lymphocytes ◽

Membrane Damage ◽

Tail Length ◽

Redox System ◽

Ros Generation ◽

Apoptosis And Necrosis

AbstractBioallethrin is a synthetic pesticide that is widely used to control insect pests. The wide use of bioallethrin has resulted in inevitable human exposure. In this study we report the effect of different concentrations of bioallethrin (10 to 200 µM, 2 h at 37 °C) on human lymphocytes under in vitro conditions. Bioallethrin treatment resulted in loss of cell viability (> 30% at 200 µM bioallethrin). Oxidative stress markers like lipid peroxidation and protein oxidation were significantly increased accompanied by lower ratio of reduced to oxidized glutathione. Enhanced ROS generation was observed through fluorescence spectroscopy and microscopy. Bioallethrin-induced oxidative stress also compromised the antioxidant defence as it reduced antioxidant capacity of cells and inhibited major antioxidant enzymes. Biomolecular modifications and systemic toxicity by bioallethrin resulted in plasma membrane damage with mitochondrial depolarization. Comet assay showed nuclear DNA fragmentation and strand scission with significant increase in tail length and olive tail moment. Apoptosis and necrosis of cells was confirmed through acridine orange/ethidium bromide dual staining and visualization under fluorescence microscope. Thus, bioallethrin causes oxidative damage and compromises the antioxidant system leading to DNA damage, cellular and organelle toxicity, resulting in apoptosis and necrosis of human lymphocytes.

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Effect of L-arginine and L-name on lysosomal cysteine proteases activity and lysosomal membranes permeability in rat aorta

Kazan medical journal ◽

10.17750/kmj2016-250 ◽

2016 ◽

Vol 97 (2) ◽

pp. 250-255 ◽

Cited By ~ 1

Author(s):

A I Arapova ◽

M A Fomina

Keyword(s):

Acid Phosphatase ◽

Cathepsin B ◽

Cysteine Proteases ◽

Total Activity ◽

Rat Aorta ◽

Lysosomal Fraction ◽

Lysosomal Membranes ◽

Cathepsin H ◽

Male Wistar Rats ◽

Combined Drugs

Aim. To study the effect of L-arginine and its analogue N-nitro-L-arginine methyl ester (L-NAME) alone and in combination on lysosomal cysteine proteolysis and lysosomal membranes state in rat aorta.Methods. The study was performed on male Wistar rats kept under standard vivarium conditions and divided into three control and three experimental groups of 8 animals each. The experimental samples included groups with L-arginine and/or L-NAME administration. The indicators were studied in the rat aorta homogenate precipitating and non precipitating fractions. Acid phosphatase activity was determined by a standardized method of «end point», the cathepsins B, L and H activity was studied by spectrofluorimetric method.Results. When simulating the changes of nitric oxide synthesis level using L-arginine, the increase of the total cathepsins activity was detected, acid phosphatase lability coefficient was reduced, what is characterized by general lysosomal membranes stabilization. L-NAME group, in contrast, is characterized by a decrease in the cathepsin B and H activity indicators, differences from arginine group were observed in the cathepsin H in lysosomal and general fractions, lysosomal membrane is labile. Combined drugs administration reduces the total cathepsins activity, while there is an increase of the acid phosphatase total activity, all indicators suggest lysosomal membranes labilization.Conclusion. L-arginine at a dose of 500 mg/kg causes increase in the total cathepsins B, L and H activity in rat aorta due to lysosomal fraction; L-arginine action leads to lysosomal membranes stabilization; L-NAME group in cathepsin H shows a decrease in the cathepsins secretion level with decreased total activity due to both factions; combined administration of arginine + L-NAME group in cathepsin B is characterized by an increase in secretion due to lysosomes membrane labilization.

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Acquired Bortezomib (Velcade®)-Resistance Is Accompanied by Downmodulation of Proteasome Activity and Upregulation of Tripeptidyl Peptidase II (TPPII) and a Ubiquitin-Specific Protease.

Blood ◽

10.1182/blood.v106.11.3375.3375 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3375-3375

Author(s):

Christoph Driessen ◽

Jeanette Gogel ◽

Marianne Kraus ◽

Huib Ovaa ◽

Alexander Beck ◽

...

Keyword(s):

Cell Lines ◽

Cysteine Proteases ◽

Proteasome Activity ◽

Parental Line ◽

Fluorogenic Substrate ◽

Synergistic Cytotoxicity ◽

Tripeptidyl Peptidase ◽

Lysosomal Cysteine Proteases ◽

Tripeptidyl Peptidase Ii

Abstract Because cell lines can be adapted to proteasome inhibition in vitro (Pfeifer G. et al., Science 283: 978–981, 1999.), secondary resistance of malignant cells towards Bortezomib (Velcade®) in vivo is a likely scenario. We have succeeded to adapt the human AML cell line HL-60 to Bortezomib in vitro, so that the adapted subline (HL-60a) shows normal viability and growth rate at 40 nM Velcade®, while cell death is induced above 10nM in the parental line. We hypothesized that alternative proteolytic pathways might allow the continued proliferation and survival of Bortezomib-adapted cells, and assessed the activity-profiles of proteasomal subunits, ubiquitin-specific proteases and lysosomal cysteine proteases in a functional proteomics approach, using recently developed synthetic affinity probes. These tools for the first time allow semiquantitative visualization of the individual members of these protease families, based on their activity, in contrast to western blot which lacks activity information or to the turnover of fluorogenic substrates, which is not truly protease-specific. After 72h of culture in Bortezomib-free medium (wash out phase), HL-60a cells contained significantly reduced levels of active proteasomal β1,β2, and β5 subunits, compared to the parental line, as confirmed by a reduced turnover of the β5-selective fluorogenic substrate Suc-LLVY-AMC. A panel of 7 different ubiquitin-specific proteases (USP) was visualized in both types of cells, using the affinity probe HAUbVS. Of these, a 97 kD USP that we have identified as USP14 by mass spectrometry-based sequencing, was significantly upregulated in HL-60a cells. By contrast, the activity of lysosomal cysteine proteases remained unchanged in HL-60a cells. Because the cytosolic protease tripeptidyl peptidase II (TPPII) can partially substitute for proteasome activity in lactacystine-treated cell lines, we assessed TPPII-activity using a fluorogenic substrate. We observed a significant upregulation of TPPII-activity in HL-60a cells, compared to non-adapted controls. Inhibition of TPPII, however, was not sufficient to restore Bortezomib-sensitivity in HL-60a cells. Interestingly, the combination of Bortezomib with the HIV protease inhibitor Ritonavir induced synergistic cytotoxicity both in HL-60 and HL-60a cells. Thus, Bortezomib-resistance is accompabied by upregulation of protease activities in alternative pathways. Combining different protease inhibitors like Bortezomib and Ritonavir might be a promising option to overcome primary or secondary Bortezomib resistance.

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