scholarly journals In Vitro Reconstitution of an NADPH-Dependent Superoxide Reduction Pathway from Pyrococcus furiosus

2005 ◽  
Vol 71 (3) ◽  
pp. 1522-1530 ◽  
Author(s):  
Amy M. Grunden ◽  
Francis E. Jenney ◽  
Kesen Ma ◽  
Mikyoung Ji ◽  
Michael V. Weinberg ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Pyrococcus Furiosus ◽  
Expression System ◽  
Anaerobic Bacteria ◽  
Flavin Mononucleotide ◽  
Superoxide Reductase ◽  
Recombinant Enzymes ◽  
Significant Similarity ◽  
Midpoint Potential

ABSTRACT A scheme for the detoxification of superoxide in Pyrococcus furiosus has been previously proposed in which superoxide reductase (SOR) reduces (rather than dismutates) superoxide to hydrogen peroxide by using electrons from reduced rubredoxin (Rd). Rd is reduced with electrons from NAD(P)H by the enzyme NAD(P)H:rubredoxin oxidoreductase (NROR). The goal of the present work was to reconstitute this pathway in vitro using recombinant enzymes. While recombinant forms of SOR and Rd are available, the gene encoding P. furiosus NROR (PF1197) was found to be exceedingly toxic to Escherichia coli, and an active recombinant form (rNROR) was obtained via a fusion protein expression system, which produced an inactive form of NROR until cleavage. This allowed the complete pathway from NAD(P)H to the reduction of SOR via NROR and Rd to be reconstituted in vitro using recombinant proteins. rNROR is a 39.9-kDa protein whose sequence contains both flavin adenine dinucleotide (FAD)- and NAD(P)H-binding motifs, and it shares significant similarity with known and putative Rd-dependent oxidoreductases from several anaerobic bacteria, both mesophilic and hyperthermophilic. FAD was shown to be essential for activity in reconstitution assays and could not be replaced by flavin mononucleotide (FMN). The bound FAD has a midpoint potential of −173 mV at 23°C (−193 mV at 80°C). Like native NROR, the recombinant enzyme catalyzed the NADPH-dependent reduction of rubredoxin both at high (80°C) and low (23°C) temperatures, consistent with its proposed role in the superoxide reduction pathway. This is the first demonstration of in vitro superoxide reduction to hydrogen peroxide using NAD(P)H as the electron donor in an SOR-mediated pathway.

scholarly journals Production and Application of a Soluble Hydrogenase fromPyrococcus furiosus

Archaea ◽  
2015 ◽  
Vol 2015 ◽  
pp. 1-8 ◽  
Author(s):  
Chang-Hao Wu ◽  
Patrick M. McTernan ◽  
Mary E. Walter ◽  
Michael W. W. Adams
Keyword(s):  
Hydrogen Production ◽  
Alternative Energy ◽  
Pyrococcus Furiosus ◽  
Hydrogen Gas ◽  
Energy Carrier ◽  
Biotechnological Applications ◽  
Biological Mechanisms ◽  
Recombinant Enzymes ◽  
Energy Needs

Hydrogen gas is a potential renewable alternative energy carrier that could be used in the future to help supplement humanity’s growing energy needs. Unfortunately, current industrial methods for hydrogen production are expensive or environmentally unfriendly. In recent years research has focused on biological mechanisms for hydrogen production and specifically on hydrogenases, the enzyme responsible for catalyzing the reduction of protons to generate hydrogen. In particular, a better understanding of this enzyme might allow us to generate hydrogen that does not use expensive metals, such as platinum, as catalysts. The soluble hydrogenase I (SHI) from the hyperthermophilePyrococcus furiosus, a member of the euryarchaeota, has been studied extensively and used in various biotechnological applications. This review summarizes the strategies used in engineering and characterizing three different forms of SHI and the properties of the recombinant enzymes. SHI has also been used inin vitrosystems for hydrogen production and NADPH generation and these systems are also discussed.

scholarly journals Secreted Aspartic Proteinase Family ofCandida tropicalis

2001 ◽  
Vol 69 (1) ◽  
pp. 405-412 ◽  
Author(s):  
Christophe Zaugg ◽  
Margarete Borg-von Zepelin ◽  
Utz Reichard ◽  
Dominique Sanglard ◽  
Michel Monod
Keyword(s):  
Southern Blotting ◽  
Genomic Library ◽  
Expression System ◽  
Methylotrophic Yeast ◽  
Sole Source ◽  
Gene Products ◽  
Related Gene ◽  
Recombinant Enzymes ◽  
Basic Level

ABSTRACT Medically important yeasts of the genus Candida secrete aspartic proteinases (Saps), which are of particular interest as virulence factors. Like Candida albicans, Candida tropicalis secretes in vitro one dominant Sap (Sapt1p) in a medium containing bovine serum albumin (BSA) as the sole source of nitrogen. Using the gene SAPT1 as a probe and under low-stringency hybridization conditions, three new closely related gene sequences, SAPT2 to SAPT4, encoding secreted proteinases were cloned from a C. tropicalis λEMBL3 genomic library. All bands identified by Southern blotting ofEcoRI-digested C. tropicalis genomic DNA withSAPT1 could be assigned to a specific SAP gene. Therefore, the SAPT gene family of C. tropicalis is likely to contain only four members. Interestingly, the SAPT2 and SAPT3 gene products, Sapt2p and Sapt3p, which have not yet been detected in C. tropicaliscultures in vitro, were produced as active recombinant enzymes with the methylotrophic yeast Pichia pastoris as an expression system. As expected, reverse transcriptase PCR experiments revealed a strong SAPT1 signal with RNA extracted from cells grown in BSA medium. However, a weak signal was obtained with all otherSAPT genes under several conditions tested, showing that these SAPT genes could be expressed at a basic level. Together, these experiments suggest that the gene products Sapt2p, Sapt3p, and Sapt4p could be produced under conditions yet to be described in vitro or during infection.

scholarly journals Kaposi's sarcoma-associated herpesvirus K8 protein interacts with hSNF5

2003 ◽  
Vol 84 (3) ◽  
pp. 665-676 ◽  
Author(s):  
Seungmin Hwang ◽  
Daeyoup Lee ◽  
Yousang Gwack ◽  
Hyesun Min ◽  
Joonho Choe
Keyword(s):  
Kaposi's Sarcoma ◽  
Expression System ◽  
Kaposi’S Sarcoma ◽  
Chromatin Remodelling ◽  
Significant Similarity ◽  
Early Protein ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

Kaposi's sarcoma-associated herpesvirus (KSHV) is a human gammaherpesvirus related to Epstein–Barr virus (EBV) and herpesvirus saimiri. KSHV open reading frame K8 encodes a basic region-leucine zipper protein of 237 aa that homodimerizes. K8 shows significant similarity to the EBV immediate-early protein Zta, a key regulator of EBV reactivation and replication. In this study, a carboxyl-terminal deletion mutant of K8, K8(1–115), that had strong transactivating properties was found. Screening using transcriptionally inactive K8(1–75) showed that K8 interacts and co-localizes with hSNF5, a cellular chromatin-remodelling factor, both in vivo and in vitro. This interaction requires aa 48–183 of hSNF5 and 1–75 of K8. In a yeast expression system, the ability of K8 and K8(1–115) to activate transcription requires the presence of SNF5, the yeast homologue of hSNF5. These data suggest a mechanism by which the SWI–SNF complex is recruited to specific genes. They also suggest that K8 functions as a transcriptional activator under specific conditions and that its transactivation activity requires its interaction with the cellular chromatin remodelling factor hSNF5.

Non-Viral Vectors for Gene Delivery

2018 ◽  
Vol 9 (1) ◽  
pp. 4-11 ◽  
Author(s):  
Aparna Bansal ◽  
Himanshu
Keyword(s):  
Gene Therapy ◽  
Viral Vectors ◽  
Transfection Efficiency ◽  
Gene Transfection ◽  
Expression System ◽  
Target Cells ◽  
Specific Expression ◽  
Naked Dna

Introduction: Gene therapy has emerged out as a promising therapeutic pave for the treatment of genetic and acquired diseases. Gene transfection into target cells using naked DNA is a simple and safe approach which has been further improved by combining vectors or gene carriers. Both viral and non-viral approaches have achieved a milestone to establish this technique, but non-viral approaches have attained a significant attention because of their favourable properties like less immunotoxicity and biosafety, easy to produce with versatile surface modifications, etc. Literature is rich in evidences which revealed that undoubtedly, non–viral vectors have acquired a unique place in gene therapy but still there are number of challenges which are to be overcome to increase their effectiveness and prove them ideal gene vectors. Conclusion: To date, tissue specific expression, long lasting gene expression system, enhanced gene transfection efficiency has been achieved with improvement in delivery methods using non-viral vectors. This review mainly summarizes the various physical and chemical methods for gene transfer in vitro and in vivo.

In Vitro Activity of New Quinolones against Anaerobic Bacteria

Drugs ◽  
1995 ◽  
Vol 49 (Supplement 2) ◽  
pp. 233-234
Author(s):  
Carl Erik Nord ◽  
Ann Lindmark ◽  
Ingela Persson
Keyword(s):  
Anaerobic Bacteria ◽  
Vitro Activity ◽  
In Vitro Activity ◽  
New Quinolones

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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