scholarly journals Detection and functional characterization of sigma class GST in Phlebotomus argentipes and its role in stress tolerance and DDT resistance

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Faizan Hassan ◽  
Krishn Pratap Singh ◽  
Vahab Ali ◽  
Sachidananda Behera ◽  
Pushkar Shivam ◽  
...  
Keyword(s):  
Stress Tolerance ◽  
Specific Activity ◽  
Functional Characterization ◽  
Cumene Hydroperoxide ◽  
Physiological Role ◽  
Immunoblot Analysis ◽  
Highly Active ◽  
Phlebotomus Argentipes ◽  
Ddt Resistance ◽  
Stress Dependent

AbstractSeveral Glutathione S-transferases (GSTs) enzymes, in insects, have previously been implicated in resistance developed against DDT and other insecticides. The GST enzyme particularly sigma class have important physiological role in detoxification of lipid peroxidation by-products in insects. Phlebotomus argentipes has been intensely exposed to DDT over years due to Indoor Residual Spray (IRS) programme for Kala-azar elimination in Bihar, India. However, in P. argentipes, role of GSTs in DDT resistance have not been elucidated. Here, sigma class GST of P. argentipes (Parg-GSTσ) was successfully cloned, expressed and purified by affinity chromatography. The recombinant Parg-GSTσ was found to be highly active towards cumene hydroperoxide and 4-HNE having specific activity 92.47 & 203.92 µM/min/mg of protein, respectively and exhibited low activity towards universal substrate CDNB i.e., 8.75 µM/min/mg of protein. RT-PCR and immunoblot analysis showed at least 2 and 1.8 fold overexpression of Parg-GSTσ in the single exposed and non exposed DDT resistant P. argentipes as compared to susceptible, implicating Parg-GSTσ also involved in DDT resistance probably by imparting enhanced stress tolerance. The DDT, H2O2 and temperature induction assays demonstrated stress-dependent induction of Parg-GSTσ expression indicating its important role in oxidative stress redressal.

Highly Active Modified Variants of Recombinant Phospholipase А2 from Streptomyces violaceoruber for Effective Biosynthesis in Yeasts

2019 ◽  
pp. 30-41 ◽  
Author(s):  
E.P. Sannikova ◽  
A.V. Malysheva ◽  
F.A. Klebanov ◽  
D.G. Kozlov
Keyword(s):  
New Technologies ◽  
Specific Activity ◽  
Point Mutations ◽  
Egg Yolk ◽  
Enzyme Preparations ◽  
Highly Active ◽  
High Calcium ◽  
Pla2 Enzyme ◽  
Streptomyces Violaceoruber ◽  
The Cost

The capacity of yeast to produce the highly active variants of PLA2 has been confirmed. The high-active variants were based on the original enzyme from the strain А-2688 of Streptomyces violaceoruber. To reduce the enzyme toxicity and to increase its expression, various approaches were tested including point mutations, construction of artificial N- and/or C-end pro-regions, hybridization with other proteins and engineering or inactivation of glycosylation sites. As a main result, the modified PLA2 enzymes were obtained which have the same secretion level as their low-active predecessors, but specific activity of which was at least tenfold higher. As the main feature, the selected mutants were characterized by a lower affinity for Ca2+ that probably accounts for their low toxicity (and high expression capacity) at the stage of biosynthesis and their ability to activate under special conditions, e.g. during the egg yolk fermentation. The data obtained can provide a basis for the cost reduction of highly active PLA2 enzyme preparations in industries where the application of high calcium concentrations is allowed. recombinant phospholipase А2, Streptomyces violaceoruber, yeasts, secretion, producer strain The work was initiated by the Innovation Center Biriuch - New Technologies, Ltd., and was supported within the framework of the State Assignment no. 595-00004-18 PR.

scholarly journals Down-Regulation of SlGRAS10 in Tomato Confers Abiotic Stress Tolerance

Genes ◽  
2021 ◽  
Vol 12 (5) ◽  
pp. 623
Author(s):  
Sidra Habib ◽  
Yee Yee Lwin ◽  
Ning Li
Keyword(s):  
Salt Stress ◽  
Abiotic Stress ◽  
Plant Growth ◽  
Stress Tolerance ◽  
Abiotic Stresses ◽  
Abiotic Stress Tolerance ◽  
Functional Characterization ◽  
Flavonoid Biosynthesis ◽  
Ros Scavenging ◽  
Down Regulation

Adverse environmental factors like salt stress, drought, and extreme temperatures, cause damage to plant growth, development, and crop yield. GRAS transcription factors (TFs) have numerous functions in biological processes. Some studies have reported that the GRAS protein family plays significant functions in plant growth and development under abiotic stresses. In this study, we demonstrated the functional characterization of a tomato SlGRAS10 gene under abiotic stresses such as salt stress and drought. Down-regulation of SlGRAS10 by RNA interference (RNAi) produced dwarf plants with smaller leaves, internode lengths, and enhanced flavonoid accumulation. We studied the effects of abiotic stresses on RNAi and wild-type (WT) plants. Moreover, SlGRAS10-RNAi plants were more tolerant to abiotic stresses (salt, drought, and Abscisic acid) than the WT plants. Down-regulation of SlGRAS10 significantly enhanced the expressions of catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) to reduce the effects of reactive oxygen species (ROS) such as O2− and H2O2. Malondialdehyde (MDA) and proline contents were remarkably high in SlGRAS10-RNAi plants. Furthermore, the expression levels of chlorophyll biosynthesis, flavonoid biosynthesis, and stress-related genes were also enhanced under abiotic stress conditions. Collectively, our conclusions emphasized the significant function of SlGRAS10 as a stress tolerate transcription factor in a certain variety of abiotic stress tolerance by enhancing osmotic potential, flavonoid biosynthesis, and ROS scavenging system in the tomato plant.

scholarly journals Structural and Functional Characterization of the ABCC6 Transporter in Hepatic Cells: Role on PXE, Cancer Therapy and Drug Resistance

2021 ◽  
Vol 22 (6) ◽  
pp. 2858
Author(s):  
Faustino Bisaccia ◽  
Prashant Koshal ◽  
Vittorio Abruzzese ◽  
Maria Antonietta Castiglione Morelli ◽  
Angela Ostuni
Keyword(s):  
Cancer Therapy ◽  
Functional Characterization ◽  
Physiological Role ◽  
Pseudoxanthoma Elasticum ◽  
Connective Tissues ◽  
Functional Studies ◽  
Extracellular Milieu ◽  
Hepatic Cells ◽  
Ectopic Mineralization ◽  
Anti Cancer

Pseudoxanthoma elasticum (PXE) is a complex autosomal recessive disease caused by mutations of ABCC6 transporter and characterized by ectopic mineralization of soft connective tissues. Compared to the other ABC transporters, very few studies are available to explain the structural components and working of a full ABCC6 transporter, which may provide some idea about its physiological role in humans. Some studies suggest that mutations of ABCC6 in the liver lead to a decrease in some circulating factor and indicate that PXE is a metabolic disease. It has been reported that ABCC6 mediates the efflux of ATP, which is hydrolyzed in PPi and AMP; in the extracellular milieu, PPi gives potent anti-mineralization effect, whereas AMP is hydrolyzed to Pi and adenosine which affects some cellular properties by modulating the purinergic pathway. Structural and functional studies have demonstrated that silencing or inhibition of ABCC6 with probenecid changed the expression of several genes and proteins such as NT5E and TNAP, as well as Lamin, and CDK1, which are involved in cell motility and cell cycle. Furthermore, a change in cytoskeleton rearrangement and decreased motility of HepG2 cells makes ABCC6 a potential target for anti-cancer therapy. Collectively, these findings suggested that ABCC6 transporter performs functions that modify both the external and internal compartments of the cells.

scholarly journals Targeting spectrin redox switches to regulate the mechanoproperties of red blood cells

2020 ◽  
Vol 0 (0) ◽  
Author(s):  
Frederik Barbarino ◽  
Lucas Wäschenbach ◽  
Virginia Cavalho-Lemos ◽  
Melissa Dillenberger ◽  
Katja Becker ◽  
...  
Keyword(s):  
Mechanical Properties ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Redox Regulation ◽  
Current Knowledge ◽  
Structural Characteristics ◽  
Functional Characterization ◽  
Physiological Role ◽  
Spectrin Cytoskeleton ◽  
Redox Switches

AbstractThe mechanical properties of red blood cells (RBCs) are fundamental for their physiological role as gas transporters. RBC flexibility and elasticity allow them to survive the hemodynamic changes in the different regions of the vascular tree, to dynamically contribute to the flow thereby decreasing vascular resistance, and to deform during the passage through narrower vessels. RBC mechanoproperties are conferred mainly by the structural characteristics of their cytoskeleton, which consists predominantly of a spectrin scaffold connected to the membrane via nodes of actin, ankyrin and adducin. Changes in redox state and treatment with thiol-targeting molecules decrease the deformability of RBCs and affect the structure and stability of the spectrin cytoskeleton, indicating that the spectrin cytoskeleton may contain redox switches. In this perspective review, we revise current knowledge about the structural and functional characterization of spectrin cysteine redox switches and discuss the current lines of research aiming to understand the role of redox regulation on RBC mechanical properties. These studies may provide novel functional targets to modulate RBC function, blood viscosity and flow, and tissue perfusion in disease conditions.

scholarly journals Biosynthesis and secretion of functional protein S by a human megakaryoblastic cell line (MEG-01)

Blood ◽  
1987 ◽  
Vol 70 (1) ◽  
pp. 301-306
Author(s):  
M Ogura ◽  
N Tanabe ◽  
J Nishioka ◽  
K Suzuki ◽  
H Saito
Keyword(s):  
Cell Line ◽  
Plasma Protein ◽  
Culture Medium ◽  
De Novo ◽  
Specific Activity ◽  
Calf Serum ◽  
Protein S ◽  
Immunoblot Analysis ◽  
Functional Assay ◽  
Functional Protein

A human megakaryoblastic cell line (MEG-01) was investigated for the presence of protein S in culture medium and cell lysates using a specific enzyme-linked immunoassay (ELISA) and a functional assay. When 5 X 10(5) MEG-01 cells/mL was subcultured in RPMI 1640 medium with 10% fetal calf serum (FCS), the concentration of protein S antigen in the culture medium increased progressively with time from less than 8 ng/mL on day 0 to 105.6 +/- 6.0 ng/mL on day 13. Vitamin K2(1 microgram/mL) increased the production of functional protein S, whereas warfarin (1 microgram/mL) profoundly decreased the quantity and the specific activity of secreted protein S. By an indirect immunofluorescent technique, protein S antigen was detected in both MEG-01 cells and human bone marrow megakaryocytes. Immunoblot analysis of culture medium revealed two distinct bands (mol wt 84,000 and 78,000) that are identical to the doublets of purified plasma protein S. De novo synthesis of protein S was demonstrated by the presence of specific immunoprecipitable radioactivity in the medium after 5 hours of labeling of the cells with [35S]-methionine as a 84,000 mol wt protein. Plasma protein S levels of nine patients with severe aplastic anemia were not significantly different from those of normal controls. These results suggest that megakaryocytes produce functional protein S and contain the enzymes required for the carboxylation of selected glutamic acid residues, and that protein S synthesized by megakaryocytes does not represent a main source of plasma protein S.

scholarly journals Identification of an Oligosaccharide Dehydrogenase from Pycnoporus Cinnabarinus Provides Insights into Fungal Breakdown of Lignocellulose

2021 ◽  
Author(s):  
Gabriele Cerutti ◽  
Elena Gugole ◽  
Linda Celeste Montemiglio ◽  
Annick Turbé-Doan ◽  
Dehbia Chena ◽  
...  
Keyword(s):  
Function Analysis ◽  
Sequence Similarity ◽  
Functional Characterization ◽  
Physiological Role ◽  
Free Form ◽  
Lignocellulose Degradation ◽  
Carbohydrate Active Enzymes ◽  
Recognition Mechanism ◽  
Pycnoporus Cinnabarinus ◽  
Enzymatic Function

Abstract Background: Fungal glucose dehydrogenases (GDHs) are FAD-dependent enzymes belonging to the glucose-methanol-choline oxidoreductase superfamily. These enzymes are classified in the “Auxiliary Activity” family 3 (AA3) of the Carbohydrate-Active enZymes database, and more specifically in subfamily AA3_2, that also includes the closely related flavoenzymes aryl-alcohol oxidase and glucose 1-oxidase. Based on sequence similarity to known fungal GDHs, an AA3_2 enzyme active on glucose was identified in the genome of Pycnoporus cinnabarinus, a model Basidiomycete able to completely degrade lignin.Results: In our work, substrate screening and functional characterization showed an unexpected preferential activity of this enzyme toward oligosaccharides containing a b(1à3) glycosidic bond, with the highest efficiency observed for the disaccharide laminaribiose. Despite its sequence similarity to GDHs, we defined a novel enzymatic activity, namely oligosaccharide dehydrogenase (ODH), for this enzyme. The crystallographic structures of ODH in the sugar-free form and in complex with glucose and laminaribiose unveiled a peculiar saccharide recognition mechanism which is not shared with previously characterized AA3 oxidoreductases and accounts for ODH preferential activity toward oligosaccharides. The sugar molecules in the active site of ODH are mainly stabilized through CH-p interactions with aromatic residues rather than through hydrogen bonds with highly conserved residues, as observed instead for the fungal glucose dehydrogenases and oxidases characterized to date. Finally, three sugar-binding sites were identified on ODH external surface, which were not previously observed and might be of importance in the physiological scenario.Conclusions: Structure-function analysis of ODH is consistent with its role as an auxiliary enzyme in lignocellulose degradation and unveils yet another enzymatic function within the AA3 family of the Carbohydrate-Active enZymes database. Our findings allow deciphering the molecular determinants of substrate binding and provide insight into the physiological role of ODH, opening new perspectives to exploit biodiversity for lignocellulose transformation into fuels and chemicals.

scholarly journals Identification and Functional Characterization of the CVOMT and EOMT Genes Promoters from Ocimum Basilicum L

2021 ◽  
Author(s):  
Fatemeh Khakdan ◽  
Zahra Shirazi ◽  
Mojtaba Ranjbar
Keyword(s):  
Water Deficit ◽  
Transcriptional Control ◽  
Functional Characterization ◽  
Pcr Analysis ◽  
Water Deficit Stress ◽  
Specific Expression ◽  
Stress Dependent ◽  
First Time ◽  
Dependent Mechanism

Abstract Methyl chavicol and methyl eugenol are important phenylpropanoid compounds previously purified from basil. These compounds are significantly enhanced by the water deficit stress-dependent mechanism. Here, for the first time, pObCVOMT and pObEOMT promoters were extracted by the genome walking method. They were then cloned into the upstream of the β-glucuronidase (GUS) reporter gene to identify the pattern of GUS water deficit stress-specific expression. Histochemical GUS assays showed in transgenic tobacco lines bearing the GUS gene driven by pObCVOMT and pObEOMT promoters, GUS was strongly expressed under water deficit stress. qRT-PCR analysis of pObCVOMT and pObEOMT transgenic plants confirmed the histochemical assays, indicating that the GUS expression is also significantly induced and up-regulated by increasing density of water deficit stress. This indicates these promoters are able to drive inducible expression. The cis-acting elements analysis showed that the pObCVOMT and pObEOMT promoters contained dehydration or water deficit-related transcriptional control elements.

scholarly journals Functional Characterization of Cholinergic Receptors in Melanoma Cells

Cancers ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 3141
Author(s):  
Anna Maria Lucianò ◽  
Ada Maria Tata
Keyword(s):  
Nervous System ◽  
Acetylcholine Receptors ◽  
Epithelial Mesenchymal Transition ◽  
Functional Characterization ◽  
Physiological Role ◽  
Melanoma Cells ◽  
Cholinergic Receptors ◽  
Mesenchymal Transition ◽  
Relevant Role

In the last two decades, the scientific community has come to terms with the importance of non-neural acetylcholine in light of its multiple biological and pathological functions within and outside the nervous system. Apart from its well-known physiological role both in the central and peripheral nervous systems, in the autonomic nervous system, and in the neuromuscular junction, the expression of the acetylcholine receptors has been detected in different peripheral organs. This evidence has contributed to highlight new roles for acetylcholine in various biological processes, (e.g., cell viability, proliferation, differentiation, migration, secretion). In addition, growing evidence in recent years has also demonstrated new roles for acetylcholine and its receptors in cancer, where they are involved in the modulation of cell proliferation, apoptosis, angiogenesis, and epithelial mesenchymal transition. In this review, we describe the functional characterization of acetylcholine receptors in different tumor types, placing attention on melanoma. The latest set of data accessible through literature, albeit limited, highlights how cholinergic receptors both of muscarinic and nicotinic type can play a relevant role in the migratory processes of melanoma cells, suggesting their possible involvement in invasion and metastasis.

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