In Vitro Investigation of Crosstalk between Fatty Acid and Polyketide Synthases in the Andrimid Biosynthetic Assembly Line

ChemBioChem ◽  
2016 ◽  
Vol 17 (22) ◽  
pp. 2137-2142 ◽  
Author(s):  
Fumihiro Ishikawa ◽  
Hiroyasu Sugimoto ◽  
Hideaki Kakeya
Keyword(s):  
Fatty Acid ◽  
Assembly Line ◽  
Polyketide Synthases ◽  
In Vitro Investigation

scholarly journals An in-vitro investigation into the prebiotic potential of xylan derived from the edible red seaweed Palmaria palmata

2020 ◽  
Vol 79 (OCE2) ◽  
Author(s):  
Philip Allsopp ◽  
Cherry Paul ◽  
Conall Strain ◽  
Supriya Yadav ◽  
Thomas Smyth ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Short Chain Fatty Acid ◽  
National Development ◽  
Chain Fatty Acid ◽  
Short Chain ◽  
Palmaria Palmata ◽  
Red Seaweed ◽  
Positive Effects ◽  
In Vitro Investigation

AbstractPrebiotics are considered beneficial to health owing to positive effects upon the gut microbiota (GM). These effects on the GM include stimulating the growth of beneficial species and increasing short chain fatty acid (SCFA) production (1). Accumulating evidence suggests that the putative health benefits associated with seaweed consumption may be, in part, owing to their effects on the GM(2). The red seaweed Palmaria palmata is a source of xylan, a β(1–3) and β(1–4) D-xylose polysaccharide. Given that xylo-oligosaccharides are a recently accepted prebiotic (3), the aim of this investigation was to assess the prebiotic potential of xylan from Palmaria palmata using an in-vitro fermentation gut model. Fibres were subjected to an in-vitro digestion and underwent in-vitro batch culture fermentation (MicroMatrix) over 24 hours. Fermentation vessels were inoculated using a pooled faecal slurry (5% v/v), prepared from six healthy volunteers. Xylan fibre (n = 4) was compared to Cellulose (negative control, n = 8) and Synergy 1 (positive control, n = 8). Changes to GM composition was determined using qPCR (total bacteria, Lactobacilli, and Bifidobacteria), and MiSeq 16S rRNA sequencing. Short chain fatty acid analysis was conducted using gas chromatography-mass spectrometry. The differential abundance of taxa between fermentation substrates was determined using linear discriminant analysis (LDA) effect size (LEfSe). A permutational multivariate analysis of variance (PerMANOVA) was used to determine statistical differences of beta diversity whilst treatment associated differences of short chain fatty acids were determined using an unpaired Mann-Whitney U Test. Xylan altered GM composition at Phylum, Family and Genus taxonomic levels, notably a significant reduction in the Firmicutes/Bacteroides ratio (p = 0.004). Both 16S sequencing data and qPCR analysis revealed a significant increase in Bifidobacteria relative to cellulose, where the effect was comparable to Synergy 1. No significant differences in microbiota diversity were noted for either Xylan or Synergy 1 in comparison to the cellulose control. Xylan was shown to significantly modulate GM activity through increased short chain fatty acid production with increased acetate, propionate and butyrate. The evidence gained from this study suggests that Xylan from Palmaria palmata is a fermentable fibre with potential prebiotic characteristics, and therefore warrants further investigation in humans.This research was funded under the National Development Plan, through the Food Institutional Research Measure, administered by the Department of Agriculture, Food and the Marine, Ireland (13/F/511) and a Northern Ireland Department of Education and Learning PhD scholarship to Paul Cherry.

scholarly journals An in vitro platform for engineering and harnessing modular polyketide synthases

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Takeshi Miyazawa ◽  
Melissa Hirsch ◽  
Zhicheng Zhang ◽  
Adrian T. Keatinge-Clay
Keyword(s):  
Assembly Line ◽  
Polyketide Synthases ◽  
Assembly Lines ◽  
Dihydroxybenzoic Acid ◽  
Coa Ligase ◽  
Malonyl Coa ◽  
Aromatic Product ◽  
Order Of Magnitude ◽  
Line Engineering

AbstractTo harness the synthetic power of modular polyketide synthases (PKSs), many aspects of their biochemistry must be elucidated. A robust platform to study these megadalton assembly lines has not yet been described. Here, we in vitro reconstitute the venemycin PKS, a short assembly line that generates an aromatic product. Incubating its polypeptides, VemG and VemH, with 3,5-dihydroxybenzoic acid, ATP, malonate, coenzyme A, and the malonyl-CoA ligase MatB, venemycin production can be monitored by HPLC and NMR. Multi-milligram quantities of venemycin are isolable from dialysis-based reactors without chromatography, and the enzymes can be recycled. Assembly line engineering is performed using pikromycin modules, with synthases designed using the updated module boundaries outperforming those using the traditional module boundaries by over an order of magnitude. Using combinations of VemG, VemH, and their engineered derivatives, as well as the alternate starter unit 3-hydroxybenzoic acid, a combinatorial library of six polyketide products is readily accessed.

scholarly journals Strategies in megasynthase engineering – fatty acid synthases (FAS) as model proteins

2017 ◽  
Vol 13 ◽  
pp. 1204-1211 ◽  
Author(s):  
Manuel Fischer ◽  
Martin Grininger
Keyword(s):  
Fatty Acid ◽  
Bioactive Compounds ◽  
Assembly Line ◽  
Structural Information ◽  
Natural Compounds ◽  
Polyketide Synthases ◽  
Therapeutic Applications ◽  
Rational Engineering ◽  
Fatty Acid Synthases

Megasynthases are large multienzyme proteins that produce a plethora of important natural compounds by catalyzing the successive condensation and modification of precursor units. Within the class of megasynthases, polyketide synthases (PKS) are responsible for the production of a large spectrum of bioactive polyketides (PK), which have frequently found their way into therapeutic applications. Rational engineering approaches have been performed during the last 25 years that seek to employ the “assembly-line synthetic concept” of megasynthases in order to deliver new bioactive compounds. Here, we highlight PKS engineering strategies in the light of the newly emerging structural information on megasynthases, and argue that fatty acid synthases (FAS) are and will be valuable objects for further developing this field.

In vitro investigation of Varizella zoster virus as an oncolytic virus in glioblastoma therapy

2008 ◽  
Vol 35 (S 01) ◽  
Author(s):  
H Leske ◽  
A Baiker ◽  
C Schichor ◽  
J.C Tonn ◽  
R Goldbrunner ◽  
...  
Keyword(s):  
Oncolytic Virus ◽  
In Vitro Investigation ◽  
Varizella Zoster Virus

In Vitro Incorporation and Metabolism of Icosapentaenoic and Docosahexaenoic Acids in Human Platelets - Effect on Aggregation

1986 ◽  
Vol 56 (01) ◽  
pp. 057-062 ◽  
Author(s):  
Martine Croset ◽  
M Lagarde
Keyword(s):  
Arachidonic Acid ◽  
Fatty Acid ◽  
Platelet Aggregation ◽  
Fatty Acid Distribution ◽  
Thromboxane A2 ◽  
Human Platelets ◽  
Aggregation Response ◽  
Docosahexaenoic Acids ◽  
Membrane Level

SummaryWashed human platelets were pre-loaded with icosapentaenoic acid (EPA), docosahexaenoic acid (DHA) or EPA + DHA and tested for their aggregation response in comparison with control platelets. In fatty acid-rich platelets, an inhibition of the aggregation could be observed when induced by thrombin, collagen or U-46619. The strongest inhibition was observed with DHA-rich platelets and it was reduced when DHA was incorporated in the presence of EPA.Study of fatty acid distribution in cell lipids after loading showed that around 90% of EPA or DHA taken up was acylated into phospholipids and a very small amount (less than 2%) remained in their free and hydroxylated forms. DHA was more efficiently acylated into phosphatidylethanolamine (PE) than into phosphatidylinositol (PI) in contrast to what observed with EPA, and both acids were preferentially incorporated into phosphatidylcholine (PC). EPA inhibited total incorporation of DHA and increased its relative acylation into PE at the expense of PC. In contrast, DHA did not affect the acylation of EPA. Upon stimulation with, thrombin, EPA was liberated from phospholipids and oxygenated (as judged by the formation of its monohydroxy derivative) whereas DHA was much less metabolized, although consistently transferred into PE.It is concluded that EPA and DHA might affect platelet aggregation via different mechanisms when pre-loaded in phospholipids. Whereas EPA is known to alter thromboxane A2 metabolism from endogenous arachidonic acid, by competing with it, DHA might act directly at the membrane level for inhibiting aggregation.

Coumarin-Fatty Acid Conjugates as Potential ERα/AKT-1 Antagonists for ER Positive Breast Cancer

2020 ◽  
Vol 20 (4) ◽  
pp. 437-449
Author(s):  
Jubie Selvaraj ◽  
Jameera B.A. John ◽  
Nanjan M. Joghee ◽  
Justin Antony ◽  
Ashish Wadhwani ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Fatty Acid ◽  
Binding Assay ◽  
Uterine Cancer ◽  
Design Development ◽  
Structure Based Drug Design ◽  
Ic50 Values ◽  
Cancer Activity

Background: : Current drugs used for the treatment of hormone-dependent breast cancer function as anti-estrogens in the breast, in addition to Estrogen Receptor (ER) agonists in the uterus, thus elevate a woman’s risk of developing uterine cancer. This is due to the lack of selective binding and partial agonistic effect of these drugs towards estrogen receptors. In recent years, therefore, researchers have turned their attention towards antiestrogens devoid of these agonist properties and thus have a mechanism of action different from the existing drugs. Objective:: In this context, we report here the design, development and in vitro evaluation of some novel pharmacophores containing coumarin and fatty acid scaffolds for their anti-breast cancer activity. Methods: : A library of coumarin-fatty acid conjugates was designed using structure-based drug design approach. The conjugates which have shown good in silico results were then synthesized, characterized and evaluated for their anti-breast cancer activity by MTT assay, Apoptotic assay, Cell proliferation assay, Estrogen binding assay and Gene expression study. Results: Out of the fifteen compounds screened, two compounds, SAC-2 and LNAC-2, showed good activity with IC50 values 22µg/ml, 25μg/ml, respectively. These compounds suppressed the proliferation of ER overexpressed MCF-7 cells, increased ERα degradation and hence inactivate the ERα pathway. ER binding assay and gene expression RT-PCR study reveal that SAC-2 downregulated the expression of ERα receptor and AKT-1 gene. Conclusion:: Compound SAC-2 is a good antagonist to ER and hence has a potential for treating breast cancer and other cancers where AKT plays an important role.

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