scholarly journals Protective Effect of Transfection with Secretable Superoxide Dismutase (SOD) (a Signal Sequence-SOD Fusion Protein Coding cDNA) Expression Vector on Superoxide Anion-Induced Cytotoxicity in Vitro.

1997 ◽  
Vol 20 (5) ◽  
pp. 530-536 ◽  
Author(s):  
Fusao KOMADA ◽  
Kohshi NISHIGUCHI ◽  
Yasuke TANIGAWA ◽  
Mariko ISHIDA ◽  
Xiao-Ying WU ◽  
...  
Keyword(s):  
Superoxide Dismutase ◽  
Fusion Protein ◽  
Protective Effect ◽  
Superoxide Anion ◽  
Expression Vector ◽  
Signal Sequence ◽  
Protein Coding ◽  
Cdna Expression

Immunoprotective Effect of Cu/Zn Superoxide Dismutase on Myeloid Graffi Tumor-Bearing Hamsters

2000 ◽  
Vol 55 (7-8) ◽  
pp. 649-656 ◽  
Author(s):  
Reneta A. Toshkova ◽  
Petia A. Dimitrova ◽  
Emilia H. Ivanova ◽  
Pavlina A. Dolashka ◽  
Maria B. Angelova ◽  
...  
Keyword(s):  
Superoxide Dismutase ◽  
Tumor Growth ◽  
Protective Effect ◽  
Phagocytic Activity ◽  
Tumor Antigens ◽  
Early Stage ◽  
Tumor Development ◽  
Peritoneal Macrophages ◽  
Tumor Bearing

Abstract Investigation on the immunoprotective activity of Cu/Zn superoxide dismutase from Humicola lutea 103 AL (HLSOD) in hamsters with transplanted myeloid tumor was performed. Survivability, tumor growth and tumor transplantability were followed. The immune status of tumor-bearing animals, injected with the optimal protective HLSOD dose, was examined during 27 days after tumor transplantation by the following parameters: (i) the number, migration and phagocytic activity of peritoneal macrophages, (ii) the phagocytic activity of blood polymorphonuclear leukocytes (PMNs), (iii) the responsibility in vitro of spleen lymphocytes to T and B cell mitogens. It was established that intraperitoneal inoculation of HLSOD produced a protective effect on the development of tumors. Elongation of the latent time for tumor appearance and inhibition of the tumor growth were observed. The decreased percentage of mortality in early stage of tumor progression was established. Immunological studies on tumor-bearing hamsters (TBH) induced a tem porary immunorestoring effect on the suppressed phagocytic activities of peritoneal macrophages and blood PMNs during the first 14 days of tumor development. Moreover, HLSOD showed an expressed stimulating effect on proliferative activity in vitro of spleen B lymphocytes from healthy and TBH as well. The immunorestoring and protective effect of the enzyme was probably due to improve of the oxidant-antioxidant balance in peritoneal phagocytes. The tem porary character of the effect can be explained with the interference of immunosuppressing factors produced by tumor tissue as well as by the presence of tumor antigens, tumor cells and antigen-antibody complexes in the circulation.

scholarly journals Environment and Male Fertility: Effects of Benzo-α-Pyrene and Resveratrol on Human Sperm Function In Vitro

2019 ◽  
Vol 8 (4) ◽  
pp. 561 ◽  
Author(s):  
Angela Alamo ◽  
Rosita A. Condorelli ◽  
Laura M. Mongioì ◽  
Rossella Cannarella ◽  
Filippo Giacone ◽  
...  
Keyword(s):  
Protective Effect ◽  
Sperm Motility ◽  
Dose Response ◽  
Superoxide Anion ◽  
Male Fertility ◽  
Negative Impact ◽  
Sperm Parameters ◽  
Mitochondrial Superoxide ◽  
Chromatin Compactness

Lifestyle, cigarette smoking and environmental pollution have a negative impact on male fertility. Therefore, the aim of this study was to evaluate the in-vitro effects of benzo-α-pyrene (BaP) and aryl hydrocarbon receptor (AHR) agonists on motility and bio-functional sperm parameters. We further assessed whether resveratrol (RES), an AHR antagonist and antioxidant molecule, had any protective effect. To accomplish this, 30 normozoospermic, healthy, non-smoker men not exposed to BaP were enrolled. Spermatozoa of 15 men were incubated with increasing concentrations of BaP to evaluate its effect and to establish its dose response. Then, spermatozoa of the 15 other men were incubated with BaP (15 µM/mL), chosen according to the dose-response and/or RES to evaluate its antagonistic effects. The effects of both substances were evaluated after 3 h of incubation on total and progressive sperm motility and on the following bio-functional sperm parameters evaluated by flow cytometry: Degree of chromatin compactness, viability, phosphatidylserine externalization (PS), late apoptosis, mitochondrial membrane potential (MMP), DNA fragmentation, degree of lipoperoxidation (LP), and concentrations of mitochondrial superoxide anion. Benzo-α-pyrene decreased total and progressive sperm motility, impaired chromatin compactness, and increased sperm lipoperoxidation and mitochondrial superoxide anion levels. All these effects were statistically significant at the lowest concentration tested (15 µM/mL) and they were confirmed at the concentration of 45 µM/mL. In turn, RES was able to counteract the detrimental effects of BaP on sperm motility, abnormal chromatin compactness, lipid peroxidation, and mitochondrial superoxide. This study showed that BaP alters sperm motility and bio-functional sperm parameters and that RES exerts a protective effect on BaP-induced sperm damage.

ADAMTS13 Binds VWF Via its C-Terminal CUB2 Domain.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 126-126 ◽  
Author(s):  
Weirui Zhang ◽  
David Motto ◽  
David Ginsburg
Keyword(s):  
Fusion Protein ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Expression Vector ◽  
Full Length ◽  
Mammalian Expression ◽  
Partial Denaturation

Abstract Thrombotic thrombocytopenic purpura (TTP) is a life threatening illness due to a deficiency of the VWF-cleaving protease, ADAMTS13. The ADAMTS13 protein is composed of a propeptide, followed by a typical zinc metalloprotease domain. The C-terminal 2/3 of the molecule contains disintegrin-like, cystine-rich, and spacer domains, as well as a total of eight TSP1 motifs and two CUB domains. The function of this C-terminal portion of the molecule and its composite motifs is unknown, though TSP1 and CUB domains of other proteins have been shown to mediate protein-protein interactions. To further explore the interaction between ADAMTS13 and VWF, we cloned full length human cDNAs for both ADAMTS13 and VWF into the mammalian expression vector pcDNA3.1. These constructs were transiently transfected into 293T cells and COS cells respectively, and conditioned media collected for analysis. Using an anti-myc antibody, myc-tagged VWF co-immunoprecipitated (co-IP) with ADAMTS13, as demonstrated by western blot analysis using antisera raised against a C-terminal peptide derived from the predicted ADAMTS13 sequence. This direct interaction required partial denaturation of VWF in 1M urea, with no co-IP observed in the absence of urea. To map the segment within ADAMTS13 responsible for VWF binding, we cloned a series of overlapping ADAMTS13 fragments into the bacterial expression vector, Pet44b. Fusion proteins were purified by binding of the included His-tag to Ni-NTA beads and incubated with recombinant myc-VWF in the presence of 1M urea. Association with VWF was analyzed by co-IP with anti-myc followed by western blot analysis using an antibody to the C-terminal HSV-tag present in each fusion protein. The CUB2 (Glu1298- Thr1427) fusion protein co-IP’d with full-length VWF and also demonstrated concentration-dependent competition with full-length ADAMTS13 for VWF binding. In summary, we have demonstrated a direct protein-protein interaction between VWF and ADAMTS13. Binding requires partial denaturation of VWF and appears to be mediated primarily through contacts with the ADAMTS13 CUB2 domain. This interaction may account for the previously observed co-purification of VWF and ADAMTS13 from human plasma. Furthermore, the requirement for 1M urea suggests that this interaction may only occur physiologically under conditions of high shear. Though others have shown that the C-terminal domains of ADAMTS13, including CUB2, are not required for VWF cleavage in vitro, our data, together with several C-terminal mutations previously reported in TTP patients, suggest that interactions between VWF and the ADAMTS13 CUB2 domain may be important in vivo.

scholarly journals BIOMIMETIC APPROACHES TO STUDY OF PROPERTIES OF MEDICINAL SUBSTANCES

2019 ◽  
Vol 62 (10) ◽  
pp. 4-29
Author(s):  
Nina B. Melnikova ◽  
Olga N. Solovyova ◽  
Evgeni N. Evgeni N. Kochetkov
Keyword(s):  
Antioxidant Activity ◽  
Superoxide Dismutase ◽  
Lipid Bilayers ◽  
Superoxide Anion ◽  
Molecular Form ◽  
Biologically Active ◽  
New Drugs ◽  
In Vitro Experiments ◽  
Medicinal Substances

The review is devoted to an assessment of the current level of use of biomimetic approaches to the study of the properties of known drugs and the development of new drugs. In this review, we consider the main biological functions of superoxide dismutase, namely the catalytic decomposition of toxic superoxide anion of oxygen to the molecular form of oxygen and protection against induced apoptosis. The biomimetic enzymes-Mn- and TEMPO-containing equivalents of superoxide dismutase SOD with antitumor and antioxidant activity were discussed more detail. The relationship between the properties and activity of SOD mimetics with their structure among them the nature of the anion and ligands, the coordination number, the geometry of the presence of conjugated bonds, and other parameters of the molecules. The study of the properties of Mn-SOD mimetics makes it possible to develop a new class of drugs successfully tested by in vivo and in vitro experiments and which are at the stages of clinical trials. Stable TEMPO radicals containing compounds are able to perform SOD functions, exhibiting antioxidant activity in relation not only to superoxide-anion, but also to peroxynitrile, and moreover to act as a spin label. The biomimetic membrane systems (monolayers, planar lipid bilayers, liposomes and other nano-sized objects) are discussed too for studying properties in in vitro experiments and for delivering potent and medicinal substances. The biomimetic approach combination allows to create the new promising drugs, including those based on SOD mimetics, and to develop the synthetic analogues of biologically active substances and methods of their delivery. The advantages of such dosage forms are lower toxicity of the preparations, lack of immunogenicity and a decrease in the dose of potent drugs.

Studies on the Possible Mechanism of Inactivation of Phenylalanine Hydroxylase by Destructive Oxygen Species

1984 ◽  
Vol 39 (7-8) ◽  
pp. 734-737 ◽  
Author(s):  
Rita M. Fink ◽  
Erich F. Elstner
Keyword(s):  
Hydrogen Peroxide ◽  
Superoxide Dismutase ◽  
Protective Effect ◽  
Active Site ◽  
Phenylalanine Hydroxylase ◽  
Oxygen Species ◽  
Hydroxylase Activity

Abstract The enzymic hydroxylation of phenylalanine by phenylalanine hydroxylase (E.C. 1.14.16.1.) in vitro is dependent on the presence of hydrogen peroxide removing processes. The loss of phenylalanine hydroxylase activity can be prevented to the same extent by catalase as well as the presence of optimized amounts of both peroxidase and superoxide dismutase. Peroxidase alone exhibited only two third of the maximal protective effect of catalase whereas superoxide dismutase alone was not able to exert any protective influence on phenylalanine hydroxylase. These findings suggest that the termination of phenylalanine hydroxylation in the absence of hydrogen peroxide removing reactions is probably due to destructive oxygen species generated at the active site iron of phenylalanine hydroxylase in the presence of H2O2 and the tetrahydropterin cofactor.

scholarly journals Vaccination with Live Escherichia coli ExpressingBrucella abortus Cu/Zn Superoxide Dismutase Protects Mice against Virulent B. abortus

1999 ◽  
Vol 67 (2) ◽  
pp. 986-988 ◽  
Author(s):  
Angel A. Oñate ◽  
Ramesh Vemulapalli ◽  
Edilia Andrews ◽  
Gerhardt G. Schurig ◽  
Stephen Boyle ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Superoxide Dismutase ◽  
Fusion Protein ◽  
Vaccine Strain ◽  
Brucella Abortus ◽  
Proliferative Response ◽  
E Coli ◽  
The One

ABSTRACT Vaccination of mice with Escherichia coli expressingBrucella Cu/Zn superoxide dismutase (SOD) [E. coli(pBSSOD)] induced a significant level of protection against virulent Brucella abortus challenge, although this level was not as high as the one reached with B. abortusvaccine strain RB51. In addition, vaccination with E. coli(pBSSOD) induced antibodies to Cu/Zn SOD and a strong proliferative response of splenocytes when stimulated in vitro with a thioredoxin-Cu/Zn SOD fusion protein.

scholarly journals Staurosporine Induces Caspase-Dependent Proteolysis of MEF2D-Fusion Protein and Cell Death Selective to MEF2D-Fusion-Positive ALL Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1349-1349
Author(s):  
Naoyuki Tange ◽  
Fumihiko Hayakawa ◽  
Takahiko Yasuda ◽  
Hideyuki Yamamoto ◽  
Daiki Hirano ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Cell Lines ◽  
Research Funding ◽  
Cytotoxic Effect ◽  
Expression Vector ◽  
Caspase 3 ◽  
Micro Rna ◽  
Protein Expression Level ◽  
Caspase 7

MEF2D fusion (M-fusion) genes are newly discovered recurrent gene abnormalities that are detected in approximately 5% of acute lymphoblastic leukemia (ALL) cases. We previously found that the loss of the micro RNA target site in wild-type MEF2D gene by translocation led to strong expression of M-fusion protein in ALL cells by evasion from micro RNA and that M-fusion protein inhibited the transcriptional activity of PAX5, a B-cell differentiation regulator, in a dose-dependent manner. These findings prompted us to explore drugs that induced proteolysis of M-fusion protein as possible therapeutic agents for M-fusion-positive ALL. We developed a high-throughput screening system to find compounds that reduced protein expression level of MEF2D. The expression vector of the fusion protein of N-terminal half of MEF2D (MEF2D N) and luciferase (MEF2D N-Luc) was stably transfected to 293T cells (MEF2D N-Luc/293T). Stable transfectant of the expression vector of luciferase was also established (Luc/293T). We could easily measure protein expression level in these cells by luciferase assay. We screened 3766 compounds with known pharmaceutical activities with this system and selected staurosporine, a multi-kinase inhibitor, as a possible proteolysis-inducer of MEF2D. Staurosporine strongly reduced the luciferase value in MEF2D N-Luc/293T but not in Luc/293T (Figure 1A). Staurosporine induced proteolysis of MEF2D-HNRNPUL1 (M-H) and MEF2D-DAZAP1 (M-D) in M-fusion-positive ALL cell lines within 6 h. Proteolysis of M-fusion proteins were inhibited not by MG-132, a proteasome inhibitor, but by Z-VAD FMK, a caspase inhibitor, indicating that these proteolyses were caspase-dependent (Figure 1B). Consistent with this, Z-VAD-FMK blocked apoptosis by staurosporine in M-H positive ALL cell lines . We confirmed the cleavage of M-H by caspase 3 and caspase 7 in vitro and identified the cleavage site (Figure 1C). Furthermore, staurosporine demonstrated stronger cytotoxic effect on M-fusion-positive ALL cell lines than M-fusion-negative ones (Figure 1D). These results indicated that staurosporine induced apoptosis of M-fusion-positive ALL cells through caspase-dependent proteolysis of M-fusion protein at least in part. Luciferase-based proteolysis screening provided a novel strategy for the development of anti-cancer drugs. Figure legends Figure 1. A. Staurosporine strongly reduced the luciferase value in MEF2D N-Luc/293T but not in Luc/293T. MEF2D N-Luc/293T were treated with 3766 compounds (2uM each) for 24 h. Then luciferase assays were performed to estimate the amount of MEF2D N-Luc protein. Top 15 compounds which reduced the relative luciferase value were selected for the second screening where compounds were added to MEF2D N-Luc/293T and Luc/293T, then we estimated their effect on the expression of MEF2D N-Luc and luciferase. Results of the second screening were plotted on a scattergram, on which the relative luciferase value in Luc/293T and MEF2D N-Luc/293T were set on the Y-axis and X-axis, respectively. Relative luciferase values are relative values to those of control cells treated with vehicle (DMSO). Staurosporine and K252a, an analog of staurosporine, were selected as hit compounds which reduced MEF2D N-Luc but not luciferase. B. Staurosporine reduced the expression of M-H and M-D, which inhibited by Z-VAD FMK. M-H-positive ALL cell lines, Kasumi-7 and Kasumi-9 and a M-D-positive ALL cell line, TS-2 were treated with 1uM staurosporine, 20uM Z-VAD-FMK, or both for 6 h. Cells were lysed and subjected to immunoblotting with indicated antibodies. C. Caspase-3 and caspase-7 cleaved M-H in vitro. M-H and its mutants with aspartate substitutions at possible caspase cleavage sites were synthesized with [35S]-methionine-labeled in vitro translation and were incubated with purified caspase-3 or -7. Cleaved fragments were resolved on SDS-PAGE and visualized by autoradiography. Mutant 4 was resistant to the cleavage by both caspases. D. Staurosporine demonstrated stronger cytotoxic effect on M-fusion-positive ALL cell lines than M-fusion-negative ones. ALL cell lines were treated with staurosporine at the indicated doses for 24 h. Cell viabilities were measured using MTT assay. Figure 1 Disclosures Kiyoi: Takeda Pharmaceutical Co., Ltd.: Research Funding; Pfizer Japan Inc.: Honoraria; Astellas Pharma Inc.: Honoraria, Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; FUJIFILM Corporation: Research Funding; Eisai Co., Ltd.: Research Funding; Bristol-Myers Squibb: Research Funding; Daiichi Sankyo Co., Ltd: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co.,Ltd.: Research Funding; Perseus Proteomics Inc.: Research Funding.

scholarly journals Protective role of tocopherol and ascorbic acid in taxol-treated human erythrocytes in vitro

2017 ◽  
Vol 1 ◽  
pp. 239784731770581
Author(s):  
Uzma Faridi ◽  
Fatema Alatawi ◽  
Magda Mostafa
Keyword(s):  
Ascorbic Acid ◽  
Superoxide Dismutase ◽  
Side Effects ◽  
Protective Effect ◽  
Haematological Toxicity ◽  
Osmotic Fragility ◽  
Natural Antioxidants ◽  
Drug Induced ◽  
Osmotic Lysis

Taxol is a well-known anticancer agent. It is used for the treatment of several kinds of cancer, including breast cancer, lung cancer and ovarian cancer. In spite of being a good chemotherapeutic agent, taxol has several side effects. Drug-induced haemolytic anaemia is one of the most common side effects of taxol. This study investigated the haemolytic effect of taxol on normal erythrocytes and the protective effect of natural antioxidants ascorbic acid and tocopherol in the presence of taxol. We evaluated the osmotic fragility and the activity of enzymes superoxide dismutase and catalase of erythrocytes in the presence of taxol alone and taxol in combination with tocopherol and ascorbic acid. Taxol-induced haematological perturbation significantly caused haemolysis and reduced the activities of superoxide dismutase and catalase in erythrocytes. The antioxidants tocopherol and ascorbic acid demonstrated a protective effect when added to taxol. The combination of tocopherol with taxol significantly protected the osmotic lysis of erythrocytes and increased the activities of superoxide dismutase but had less effect on catalase. Ascorbic acid showed significant protection of erythrocytes from osmotic lysis but didn’t show any significant effect on superoxide and catalase. The results suggest that both antioxidants, especially tocopherol, could exhibit a protective effect against taxol-induced haematological toxicity.

Silver Nanoparticle-Mediated Cellular Responses in Human Keratinocyte Cell Line HaCaT in Vitro

2019 ◽  
Vol 2 (2) ◽  
pp. 1-9 ◽  
Author(s):  
Khaled Habas ◽  
Lijun Shang
Keyword(s):  
Superoxide Dismutase ◽  
Antioxidant Enzymes ◽  
Glutathione Peroxidase ◽  
Superoxide Anion ◽  
Hacat Cells ◽  
Lactate Dehydrogenase Release ◽  
Skin Cell ◽  
Keratinocyte Cell Line Hacat ◽  
Endogenous Antioxidant

The interactions between cells and nanoparticles has been the focus of recent research in the area. The effects of AgNPs on skin cell lines for further potential biological applications are highlighted. This study aimed to investigate the mechanism of cytotoxic and genotoxic effects of AgNPs nanoparticles on human skin keratinocytes (HaCaT). Genocytotoxic effects of AgNPs was assessed using changes in various cellular parameters of HaCaT cells involving viability, superoxide anion radical production, lactate dehydrogenase release and the levels of the antioxidant enzymes, namely, Catalase, Glutathione peroxidase (GPX) and Superoxide Dismutase (SOD). Superoxide anion was detected using nitroblue tetrazolium NBT reduction assay. LDH levels was evaluated using the standard kit, and activity of antioxidant enzymes such as catalase (CAT), glutathione peroxidase 1 (GPX-1) and superoxide dismutase 1 (SOD-1) were quantified using qPCR. Our results indicated that AgNPs caused severe HaCaT oxidative damage, accompanied by increased the production of superoxide anion levels as well as significant decrease in endogenous antioxidant enzyme of SOD, CAT, GPX expression involved in HaCat cells in vitro. Our study suggests that AgNPs exposure increased oxidative stress levels. Moreover; the low cytotoxic effect observed on human HaCaT keratinocytes suggested that these nano-compounds have a potential toxic effect at the skin level only after long-term exposure.

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