scholarly journals Whole-cell microtiter plate screening assay for terminal hydroxylation of fatty acids by P450s

2016 ◽  
Vol 52 (36) ◽  
pp. 6158-6161 ◽  
Author(s):  
Martin J. Weissenborn ◽  
Sandra Notonier ◽  
Sarah-Luise Lang ◽  
Konrad B. Otte ◽  
Susanne Herter ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Microtiter Plate ◽  
Galactose Oxidase ◽  
Screening Assay ◽  
Whole Cell ◽  
Oxidase Enzyme

A whole cell screening assay for terminal hydroxylases was developed based on a readily available galactose oxidase enzyme.

scholarly journals A Mechanism-Based Whole-Cell Screening Assay to Identify Inhibitors of Protein Export in Escherichia coli by the Sec Pathway

2012 ◽  
Vol 17 (4) ◽  
pp. 535-541 ◽  
Author(s):  
Gregory J. Crowther ◽  
S. Arshiya Quadri ◽  
Benjamin J. Shannon-Alferes ◽  
Wesley C. Van Voorhis ◽  
Henry Rosen
Keyword(s):  
Escherichia Coli ◽  
High Throughput Screening ◽  
Drug Target ◽  
Rapid Identification ◽  
Protein Synthesis Inhibitors ◽  
Screening Assay ◽  
Whole Cell ◽  
Sec Pathway ◽  
Associated Proteins ◽  
Pass Through

More than 20% of bacterial proteins are noncytoplasmic, and most of these pass through the SecYEG channel en route to the periplasm, cell membrane, or surrounding environment. The Sec pathway, encompassing SecYEG and several associated proteins (SecA, SecB, YidC, SecDFYajC), is of interest as a potential drug target because it is distinct from targets of current drugs, is essential for bacterial growth, and exhibits dissimilarities in eukaryotes and bacteria that increase the likelihood of selectively inhibiting the microbial pathway. As a step toward validating the pathway as a drug target, we have adapted a mechanism-based whole-cell assay in a manner suitable for high-throughput screening (HTS). The assay uses an engineered strain of Escherichia coli that accumulates beta-galactosidase (β-gal) in its cytoplasm if translocation through SecYEG is blocked. The assay should facilitate rapid identification of compounds that specifically block the Sec pathway because widely, toxic compounds and nonspecific protein synthesis inhibitors prevent β-gal production and thus do not register as hits. Testing of current antibiotics confirmed that they do not generally act through the Sec pathway. A mini-screen of 800 compounds indicated the assay’s readiness for larger screening projects.

scholarly journals Amycolatopsis taiwanensis sp. nov., from soil

2006 ◽  
Vol 56 (8) ◽  
pp. 1811-1815 ◽  
Author(s):  
Min Tseng ◽  
Shu-Feng Yang ◽  
Wen-Jun Li ◽  
Cheng-Lin Jiang
Keyword(s):  
Fatty Acids ◽  
Soil Sample ◽  
Type Strain ◽  
Novel Species ◽  
Diaminopimelic Acid ◽  
Whole Cell ◽  
Mycolic Acids ◽  
Cellular Fatty Acids ◽  
Actinomycete Strain

An actinomycete strain (0345M-7T) was isolated from a soil sample from Yilan county, Taiwan. The isolate displayed substrate mycelia, upon which were borne short spore chains. The spore chains were composed of non-motile, smooth-surfaced, oval spores. Strain 0345M-7T had meso-diaminopimelic acid in its peptidoglycan. Whole-cell sugars were galactose, glucose, arabinose and ribose. The only phospholipid found was phosphatidylethanolamine. The predominant menaquinone was MK-9(H4). Mycolic acids were not detected. Major cellular fatty acids were iso-C16 : 0 (38.1 %) and C17 : 1 (25.4 %). The DNA G+C content of strain 0345M-7T was 68.9 mol%. On the basis of phenotypic and genotypic data, it is proposed that strain 0345M-7T (=BCRC 16802T=KCTC 19116T) should be classified as the type strain of a novel species of the genus Amycolatopsis, Amycolatopsis taiwanensis sp. nov.

Altered sodium current response to intracellular fatty acids in halothane-hypersensitive skeletal muscle

1996 ◽  
Vol 271 (1) ◽  
pp. C347-C353 ◽  
Author(s):  
S. J. Wieland ◽  
Q. H. Gong ◽  
J. E. Fletcher ◽  
H. Rosenberg
Keyword(s):  
Skeletal Muscle ◽  
Fatty Acids ◽  
Fatty Acid ◽  
Sodium Current ◽  
Primary Cultures ◽  
Na Channel ◽  
Current Response ◽  
Whole Cell ◽  
Na Currents

Biopsies of human skeletal muscle were analyzed by an in vitro contracture test (IVCT) for responsiveness to a halothane challenge: noncontracting (nonresponsive; IVCT-) and contracting (IVCT+). A muscle biopsy that is IVCT+ indicates potential malignant hyperthermia (MH) susceptibility. Primary cultures were grown from portions of the skeletal muscle biopsies, and voltage-activated currents were measured by whole cell recording in the presence or absence of 2-5 microM intracellular arachidonic or oleic acids. In untreated IVCT- cells, Na+ currents were predominantly tetrodotoxin (TTX) insensitive, indicating that most of the current was carried through the embryonic SkM2 isoform of the Na+ channel. Inclusion of fatty acids in the recording pipette of IVCT- cells produced an increase in voltage-activated Na+ currents during 20 min of recording. Approximately 70% of currents in fatty acid-treated cells were TTX sensitive, indicating activation of the adult SkM1 isoform of the Na+ channel. In contrast to IVCT- cells, IVCT+ cells expressed Na+ currents that were predominantly TTX sensitive even in the absence of added fatty acid, thus showing a relatively large baseline functional expression of SkM1 channels. Addition of fatty acids to the recording pipette produced little further change in the magnitude or TTX sensitivity of the whole cell currents in IVCT+ cells, suggesting altered functional regulation of Na+ channels in MH muscle.

Whole cell catalyzed esterification of fatty acids to biodiesel usingAspergillussp.

2011 ◽  
Vol 29 (6) ◽  
pp. 354-358 ◽  
Author(s):  
Satnam S. Aulakh ◽  
Manmohan Chhibber ◽  
Rasika Mantri ◽  
Ranjana Prakash
Keyword(s):  
Fatty Acids ◽  
Whole Cell
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