scholarly journals Expanding the Use of Zymography by the Chemical Linkage of Small, Defined Substrates to the Gel Matrix

2003 ◽  
Vol 13 (8) ◽  
pp. 1961-1965
Author(s):  
Vladimir R. Kaberdin ◽  
Kenneth J. McDowall
Keyword(s):  
High Throughput Screening ◽  
Large Scale ◽  
Standard Procedure ◽  
Laboratory Method ◽  
Enzymatic Activities ◽  
Animal Origin ◽  
Clinical Samples ◽  
Separation Of Proteins ◽  
Identification And Characterization

In the postgenomic era, the comprehensive proteomic analysis of metabolic and signaling pathways is inevitably faced with the challenge of large-scale identification and characterization of polypeptides with a particular enzymatic activity. Previous work has shown that a wide variety of enzymatic activities of microbial, plant, and animal origin can be assigned to individual polypeptides using in-gel activity staining (zymography). However, a number of limitations, such as special substrate requirements, the lack of a standard procedure, and difficulties in distinguishing enzymes with overlapping activities have precluded the widespread use of zymography as a routine laboratory method. Here we demonstrate that, by employing small-defined substrates that are covalently attached to the gel matrix, we can largely overcome the aforementioned problems and assay readily a number of different classes of enzymatic activities within gels after standard SDS-polyacrylamide electrophoresis. Moreover, this development is compatible with the two-dimensional separation of proteins and thus has great potential in the high-throughput screening and characterization of complex biological and clinical samples.

scholarly journals Identification and Characterization of New Inhibitors of the Escherichia coli MurA Enzyme

2001 ◽  
Vol 45 (11) ◽  
pp. 3182-3188 ◽  
Author(s):  
Ellen Z. Baum ◽  
Deborah A. Montenegro ◽  
Lisa Licata ◽  
Ignatius Turchi ◽  
Glenda C. Webb ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
High Throughput Screening ◽  
Chemical Library ◽  
Peptidoglycan Synthesis ◽  
Bacterial Enzyme ◽  
Purine Analog ◽  
Rna And Protein Synthesis ◽  
Molecular Modeling Studies ◽  
Identification And Characterization

ABSTRACT The bacterial enzyme MurA catalyzes the transfer of enolpyruvate from phosphoenolpyruvate (PEP) to uridine diphospho-N-acetylglucosamine (UNAG), which is the first committed step of bacterial cell wall biosynthesis. From high-throughput screening of a chemical library, three novel inhibitors of the Escherichia coli MurA enzyme were identified: the cyclic disulfide RWJ-3981, the purine analog RWJ-140998, and the pyrazolopyrimidine RWJ-110192. When MurA was preincubated with inhibitor, followed by addition of UNAG and PEP, the 50% inhibitory concentrations (IC50s) were 0.2 to 0.9 μM, compared to 8.8 μM for the known MurA inhibitor, fosfomycin. The three compounds exhibited MICs of 4 to 32 μg/ml against Staphylococcus aureus; however, the inhibition of DNA, RNA, and protein synthesis in addition to peptidoglycan synthesis by all three inhibitors indicated that antibacterial activity was not due specifically to MurA inhibition. The presence of UNAG during the MurA and inhibitor preincubation lowered the IC50 at least fivefold, suggesting that, like fosfomycin, the three compounds may interact with the enzyme in a specific fashion that is enhanced by UNAG. Ultrafiltration and mass spectrometry experiments suggested that the compounds were tightly, but not covalently, associated with MurA. Molecular modeling studies demonstrated that the compounds could fit into the site occupied by fosfomycin; exposure of MurA to each compound reduced the labeling of MurA by tritiated fosfomycin. Taken together, the evidence indicates that these inhibitors may bind noncovalently to the MurA enzyme, at or near the site where fosfomycin binds.

scholarly journals Genome-Wide Identification and Characterization of Long Non-Coding RNAs in Peanut

Genes ◽  
2019 ◽  
Vol 10 (7) ◽  
pp. 536 ◽  
Author(s):  
Xiaobo Zhao ◽  
Liming Gan ◽  
Caixia Yan ◽  
Chunjuan Li ◽  
Quanxi Sun ◽  
...  
Keyword(s):  
Large Scale ◽  
Target Genes ◽  
Sequencing Data ◽  
Regulatory Processes ◽  
Genome Wide ◽  
Non Coding Rnas ◽  
Identification And Characterization ◽  
Lower Expression ◽  
Weighted Correlation

Long non-coding RNAs (lncRNAs) are involved in various regulatory processes although they do not encode protein. Presently, there is little information regarding the identification of lncRNAs in peanut (Arachis hypogaea Linn.). In this study, 50,873 lncRNAs of peanut were identified from large-scale published RNA sequencing data that belonged to 124 samples involving 15 different tissues. The average lengths of lncRNA and mRNA were 4335 bp and 954 bp, respectively. Compared to the mRNAs, the lncRNAs were shorter, with fewer exons and lower expression levels. The 4713 co-expression lncRNAs (expressed in all samples) were used to construct co-expression networks by using the weighted correlation network analysis (WGCNA). LncRNAs correlating with the growth and development of different peanut tissues were obtained, and target genes for 386 hub lncRNAs of all lncRNAs co-expressions were predicted. Taken together, these findings can provide a comprehensive identification of lncRNAs in peanut.

Large-scale identification and characterization of phenolic compounds and their marker–trait association in wheat

Euphytica ◽  
2020 ◽  
Vol 216 (8) ◽  
Author(s):  
Monica Sharma ◽  
Mohammed Saba Rahim ◽  
Pankaj Kumar ◽  
Ankita Mishra ◽  
Himanshu Sharma ◽  
...  
Keyword(s):  
Phenolic Compounds ◽  
Large Scale ◽  
Trait Association ◽  
Identification And Characterization

Identification and Characterization of Novel Human Glucose-6-Phosphate Dehydrogenase Inhibitors

2012 ◽  
Vol 18 (3) ◽  
pp. 286-297 ◽  
Author(s):  
Janina Preuss ◽  
Adam D. Richardson ◽  
Anthony Pinkerton ◽  
Michael Hedrick ◽  
Eduard Sergienko ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Basic Research ◽  
Pentose Phosphate ◽  
Screening Assay ◽  
High Throughput Screening Assay ◽  
Key Enzyme ◽  
Small Molecule Compound ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Identification And Characterization

Glucose-6-phosphate dehydrogenase (G6PD) is the key enzyme of the pentose phosphate pathway, converting glucose-6-phosphate to 6-phosphoglucono-δ-lactone with parallel reduction of NADP+. Several human diseases, including cancer, are associated with increased G6PD activity. To date, only a few G6PD inhibitors have been available. However, adverse side effects and high IC50 values hamper their use as therapeutics and basic research probes. In this study, we developed a high-throughput screening assay to identify novel human G6PD (hG6PD) inhibitors. Screening the LOPAC (Sigma-Aldrich; 1280 compounds), Spectrum (Microsource Discovery System; 1969 compounds), and DIVERSet (ChemBridge; 49 971 compounds) small-molecule compound collections revealed 139 compounds that presented ≥50% hG6PD inhibition. Hit compounds were further included in a secondary and orthogonal assay in order to identify false-positives and to determine IC50 values. The most potent hG6PD inhibitors presented IC50 values of <4 µM. Compared with the known hG6PD inhibitors dehydroepiandrosterone and 6-aminonicotinamide, the inhibitors identified in this study were 100- to 1000-fold more potent and showed different mechanisms of enzyme inhibition. One of the newly identified hG6PD inhibitors reduced viability of the mammary carcinoma cell line MCF10-AT1 (IC50 ~25 µM) more strongly than that of normal MCF10-A cells (IC50 >50 µM).

scholarly journals Identification and characterization of growing large-scale en-echelon fractures in a salt mine

2013 ◽  
Vol 196 (2) ◽  
pp. 1092-1105 ◽  
Author(s):  
Samira Maghsoudi ◽  
Sebastian Hainzl ◽  
Simone Cesca ◽  
Torsten Dahm ◽  
Diethelm Kaiser
Keyword(s):  
Large Scale ◽  
Salt Mine ◽  
Identification And Characterization

scholarly journals Transferrin-a modulates hepcidin expression in zebrafish embryos

Blood ◽  
2009 ◽  
Vol 113 (12) ◽  
pp. 2843-2850 ◽  
Author(s):  
Paula G. Fraenkel ◽  
Yann Gibert ◽  
Jason L. Holzheimer ◽  
Victoria J. Lattanzi ◽  
Sarah F. Burnett ◽  
...  
Keyword(s):  
Large Scale ◽  
Critical Role ◽  
Hypochromic Anemia ◽  
Zebrafish Embryos ◽  
Genetic Screens ◽  
Hepcidin Expression ◽  
Morpholino Knockdown ◽  
Low Levels ◽  
Identification And Characterization

Abstract The iron regulatory hormone hepcidin is transcriptionally up-regulated in response to iron loading, but the mechanisms by which iron levels are sensed are not well understood. Large-scale genetic screens in the zebrafish have resulted in the identification of hypochromic anemia mutants with a range of mutations affecting conserved pathways in iron metabolism and heme synthesis. We hypothesized that transferrin plays a critical role both in iron transport and in regulating hepcidin expression in zebrafish embryos. Here we report the identification and characterization of the zebrafish hypochromic anemia mutant, gavi, which exhibits transferrin deficiency due to mutations in transferrin-a. Morpholino knockdown of transferrin-a in wild-type embryos reproduced the anemia phenotype and decreased somite and terminal gut iron staining, while coinjection of transferrin-a cRNA partially restored these defects. Embryos with transferrin-a or transferrin receptor 2 (TfR2) deficiency exhibited low levels of hepcidin expression, however anemia, in the absence of a defect in the transferrin pathway, failed to impair hepcidin expression. These data indicate that transferrin-a transports iron and that hepcidin expression is regulated by a transferrin-a–dependent pathway in the zebrafish embryo.

scholarly journals Hi-C detects novel structural variants in HL-60 and HL-60/S4 cell lines

2018 ◽  
Author(s):  
Elsie C. Jacobson ◽  
Ralph S. Grand ◽  
Jo K. Perry ◽  
Mark H. Vickers ◽  
Ada L. Olins ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Large Scale ◽  
Rna Seq ◽  
Structural Variants ◽  
The Stability ◽  
Different Sources ◽  
Identification And Characterization

AbstractCancer cell lines often have large structural variants (SVs) that evolve over time. There are many reported differences in large scale SVs between HL-60 and HL-60/S4, two cell lines derived from the same acute myeloid leukemia sample. However, the stability and variability of inter- and intra-chromosomal structural variants between different sources of the same cell line is unknown. Here, we used Hi-C and RNA-seq to identify and compare large SVs in HL-60 and HL-60/S4 cell lines. Comparisons with previously published karyotypes identified novel SVs in both cell lines. Hi-C was used to characterize the known expansion centered on the MYC locus. The MYC expansion was integrated into known locations in HL-60/S4, and a novel location (chr4) in HL-60. The HL-60 cell line has more within-line structural variation than the HL-60/S4 derivative cell line. Collectively we demonstrate the usefulness of Hi-C and with RNA-seq data for the identification and characterization of SVs.

scholarly journals An insight into the sequential changes in enzymatic activities during retting of jute (Corchorus spp. L.).

2021 ◽  
Vol 42 (3) ◽  
pp. 636-643
Author(s):  
B. Majumdar ◽  
◽  
A.R. Saha ◽  
S. Sarkar ◽  
S.K. Sarkar ◽  
...  
Keyword(s):  
Water Samples ◽  
Large Scale ◽  
Microbial Consortium ◽  
Standard Procedure ◽  
Sharp Decrease ◽  
Quality Parameters ◽  
Enzymatic Activities ◽  
Pectin Lyase ◽  
Lyase Activity ◽  
Last Stage

Aim: To study the dynamics of enzymes involved in biochemical process of jute (Corchorus spp.) retting with and without microbial retting consortium. Methodology: Two large scale retting trials were conducted with and without microbial retting consortium in triplicate. The retting water samples were collected every day at 24 hrs interval from both the trials. Polygalcturonase (PG), pectin lyase (PNL) and xylanase activities along with the pH were measured from the collected retting water samples following standard procedure. Fibre quality parameters were also studied from the resultant fibre obtained from both the retting trials. Results: There was a sharp decrease in pH of retting liquor by 1.35 units and that of pectin lyase activity by 97.9 Uml-1 within 24 hrs of inoculation of microbial retting consortium. Thereafter, higher pectin lyase (123.1 Uml-1), polygalacturonase (3.56 Iuml-1) and xylanase (0.818 IUml-1) activities were recorded during middle stage of retting. The enzyme activities were lower and non-significant at last stage of retting (11-14 days). The completion of retting without microbial consortium took longer time due to lower enzymatic activities as compared to microbial consortium mediated retting. Interpretation: The PG, PNL and xylanase enzymes released by the microbial consortium during retting of jute helped in faster biodegradation of pectin and xylan compared to control retting. Hence, the pre retting treatment of jute with microbial consortium is suggested for quick retting.

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