scholarly journals In Vivo Assay Reveals Microbial OleA Thiolases Initiating Hydrocarbon and β-Lactone Biosynthesis

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Megan D. Smith ◽  
Serina L. Robinson ◽  
Mandkhai Molomjamts ◽  
Lawrence P. Wackett
Keyword(s):  
Metabolic Pathways ◽  
Xanthomonas Campestris ◽  
Membrane Lipids ◽  
Membrane Lipid ◽  
Limited Information ◽  
Content Type ◽  
Bacterial Phyla ◽  
Long Chain

ABSTRACT OleA, a member of the thiolase superfamily, is known to catalyze the Claisen condensation of long-chain acyl coenzyme A (acyl-CoA) substrates, initiating metabolic pathways in bacteria for the production of membrane lipids and β-lactone natural products. OleA homologs are found in diverse bacterial phyla, but to date, only one homodimeric OleA has been successfully purified to homogeneity and characterized in vitro. A major impediment for the identification of new OleA enzymes has been protein instability and time-consuming in vitro assays. Here, we developed a bioinformatic pipeline to identify OleA homologs and a new rapid assay to screen OleA enzyme activity in vivo and map their taxonomic diversity. The screen is based on the discovery that OleA displayed surprisingly high rates of p-nitrophenyl ester hydrolysis, an activity not shared by other thiolases, including FabH. The high rates allowed activity to be determined in vitro and with heterologously expressed OleA in vivo via the release of the yellow p-nitrophenol product. Seventy-four putative oleA genes identified in the genomes of diverse bacteria were heterologously expressed in Escherichia coli, and 25 showed activity with p-nitrophenyl esters. The OleA proteins tested were encoded in variable genomic contexts from seven different phyla and are predicted to function in distinct membrane lipid and β-lactone natural product metabolic pathways. This study highlights the diversity of unstudied OleA proteins and presents a rapid method for their identification and characterization. IMPORTANCE Microbially produced β-lactones are found in antibiotic, antitumor, and antiobesity drugs. Long-chain olefinic membrane hydrocarbons have potential utility as fuels and specialty chemicals. The metabolic pathway to both end products share bacterial enzymes denoted as OleA, OleC, and OleD that transform acyl-CoA cellular intermediates into β-lactones. Bacteria producing membrane hydrocarbons via the Ole pathway additionally express a β-lactone decarboxylase, OleB. Both β-lactone and olefin biosynthesis pathways are initiated by OleA enzymes that define the overall structure of the final product. There is currently very limited information on OleA enzymes apart from the single representative from Xanthomonas campestris. In this study, bioinformatic analysis identified hundreds of new, putative OleA proteins, 74 proteins were screened via a rapid whole-cell method, leading to the identification of 25 stably expressed OleA proteins representing seven bacteria phyla.

scholarly journals Phosphatidylcholine Synthesis Influences the Diacylglycerol Homeostasis Required for Sec14p-dependent Golgi Function and Cell Growth

2001 ◽  
Vol 12 (3) ◽  
pp. 511-520 ◽  
Author(s):  
Annette L. Henneberry ◽  
Thomas A. Lagace ◽  
Neale D. Ridgway ◽  
Christopher R. McMaster
Keyword(s):  
Cell Death ◽  
Cell Growth ◽  
Phosphatidic Acid ◽  
Metabolic Pathways ◽  
Eukaryotic Cells ◽  
Kennedy Pathway ◽  
Phosphatidylcholine Synthesis ◽  
Long Chain

Phosphatidylcholine and phosphatidylethanolamine are the most abundant phospholipids in eukaryotic cells and thus have major roles in the formation and maintenance of vesicular membranes. In yeast, diacylglycerol accepts a phosphocholine moiety through aCPT1-derived cholinephosphotransferase activity to directly synthesize phosphatidylcholine. EPT1-derived activity can transfer either phosphocholine or phosphoethanolamine to diacylglcyerol in vitro, but is currently believed to primarily synthesize phosphatidylethanolamine in vivo. In this study we report that CPT1- and EPT1-derived cholinephosphotransferase activities can significantly overlap in vivo such that EPT1 can contribute to 60% of net phosphatidylcholine synthesis via the Kennedy pathway. Alterations in the level of diacylglycerol consumption through alterations in phosphatidylcholine synthesis directly correlated with the level of SEC14-dependent invertase secretion and affected cell viability. Administration of synthetic di8:0 diacylglycerol resulted in a partial rescue of cells fromSEC14-mediated cell death. The addition of di8:0 diacylglycerol increased di8:0 diacylglycerol levels 20–40-fold over endogenous long-chain diacylglycerol levels. Di8:0 diacylglcyerol did not alter endogenous phospholipid metabolic pathways, nor was it converted to di8:0 phosphatidic acid.

scholarly journals Modulation Effects of Curcumin on Erythrocyte Ion-Transporter Activity

2015 ◽  
Vol 2015 ◽  
pp. 1-8 ◽  
Author(s):  
Prabhakar Singh ◽  
Syed Ibrahim Rizvi
Keyword(s):  
Oxidative Stress ◽  
Membrane Lipids ◽  
Curcuma Longa ◽  
Membrane Lipid ◽  
Membrane Transporters ◽  
Protective Mechanism ◽  
Lipid Hydroperoxides ◽  
Beneficial Effects

Curcumin ((1E,6E)-1,7-Bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione), the yellow biphenolic pigment isolated from turmeric (Curcuma longa), has various medicinal benefits through antioxidation, anti-inflammation, cardiovascular protection, immunomodulation, enhancing of the apoptotic process, and antiangiogenic property. We explored the effects of curcuminin vitro(10−5 M to 10−8 M) andin vivo(340 and 170 mg/kg b.w., oral) on Na+/K+ATPase (NKA), Na+/H+exchanger (NHE) activity, and membrane lipid hydroperoxides (ROOH) in control and experimental oxidative stress erythrocytes of Wistar rats. As a result, we found that curcumin potently modulated the membrane transporters activity with protecting membrane lipids against hydro-peroxidation in control as well as oxidatively challenged erythrocytes evidenced by stimulation of NKA, downregulation of NHE, and reduction of ROOH in the membrane. The observed results corroborate membrane transporters activity with susceptibility of erythrocyte membrane towards oxidative damage. Results explain the protective mechanism of curcumin against oxidative stress mediated impairment in ions-transporters activity and health beneficial effects.

scholarly journals Membrane Interactivity of Non-steroidal Anti-inflammatory Drugs: A Literature Review

2020 ◽  
pp. 1-30
Author(s):  
Hironori Tsuchiya ◽  
Maki Mizogami
Keyword(s):  
Membrane Fluidity ◽  
Membrane Lipids ◽  
Direct Interaction ◽  
Membrane Lipid ◽  
Lipid Phase ◽  
Lipid Bilayer Membranes ◽  
Inflammatory Drugs ◽  
Anti Inflammatory

Background: Although the mode of action of non-steroidal anti-inflammatory drugs (NSAIDs) has been exclusively referred to as inhibition of cyclooxygenase, their broad pharmacological and toxicological spectra are not necessarily interpreted by the direct interaction with such enzyme proteins. Aims: Since NSAIDs have the common amphiphilic structure, they have the possibility of acting on membrane-constituting lipids. In order to gain insights into the additional mechanism of NSAIDs, we reviewed their membrane interactivity to modify the physicochemical properties of membranes. Methodology: We retrieved scientific articles from PubMed/MEDLINE, Google Scholar and ACS Publications by searching databases from 1990 to 2019. Research papers published in English in the internationally recognized journals and on-line journals were cited with preference to more recent publications. Collected articles were reviewed by title, abstract and text for relevance. Results: Results of the literature search indicated that NSAIDs structure-specifically cause the in vitro and in vivo interactions with artificial and biological membranes to change membrane fluidity, lipid phase transition and permeability. The features and potencies of their membrane interactivity vary depending on drug concentration, medium pH and membrane lipid composition. In addition to membrane proteins, NSAIDs act on membrane lipids to exhibit the anti-inflammatory and anti-tumor activity by interacting with lipid bilayer membranes at relatively low concentrations to decrease membrane fluidity and thereby affect the enzymatic activity of membrane-associated proteins and to exhibit the gastrointestinal and cardiovascular toxicity by interacting with membranous phospholipids at relatively high concentrations to increase membrane fluidity and thereby impair the membrane-relevant biofunctions. Other diverse effects of NSAIDs may also be related to their membrane interactions. Conclusion: NSAIDs share the membrane interactivity common to them as one of possible pharmacological and toxicological mechanisms.            

Lipid Metabolism as a Site of Herbicide Action

Weed Science ◽  
1973 ◽  
Vol 21 (5) ◽  
pp. 477-480 ◽  
Author(s):  
J. B. St. John ◽  
J. L. Hilton
Keyword(s):  
Membrane Lipids ◽  
Lipid Synthesis ◽  
Membrane Lipid ◽  
Cell Membrane Permeability ◽  
Glyceride Synthesis ◽  
Membrane Structure And Function ◽  
And Function ◽  
A Site

Dinoseb (2-sec-butyl-4,6-dinitrophenol) and MBR 8251 [1,1 1-trifluoro-4′-(phenylsulfonyl)-methanesulfono-o-toluidide] inhibited enzymic synthesis of glycerides in vitro. The physiological significance of this inhibition was confirmed in intact wheat [Triticum aestivumL., ‘Mediterranean’ (C.I. 5303)] seedlings; dinoseb and MBR 8251 inhibition of glyceride synthesis in vivo was evidenced by a buildup in free fatty acids and a decrease in neutral and polar lipids. Glyceride synthesis and growth were reduced approximately equally by dinoseb and MBR 8251. However, polar (membrane) lipids were reduced more drastically than growth. It is suggested that dinoseb and MBR 8251 alter membrane structure and function through an inhibition of membrane lipid synthesis. DNP (dinitrophenol) was only slightly inhibitory in either the in vitro or in vivo system. Dinoseb was more effective than MBR 8251 in destruction of cell membrane permeability of intact roots immediately after treatment.

scholarly journals In VitroActivity of Glucosinolates and Their Degradation Products against Brassica-Pathogenic Bacteria and Fungi

2014 ◽  
Vol 81 (1) ◽  
pp. 432-440 ◽  
Author(s):  
T. Sotelo ◽  
M. Lema ◽  
P. Soengas ◽  
M. E. Cartea ◽  
P. Velasco
Keyword(s):  
Pseudomonas Syringae ◽  
Xanthomonas Campestris ◽  
Pathogenic Bacteria ◽  
Degradation Products ◽  
Allyl Isothiocyanate ◽  
Leaf Extracts ◽  
Soil Pathogens ◽  
Content Type ◽  
Positive Effects

ABSTRACTGlucosinolates (GSLs) are secondary metabolites found inBrassicavegetables that confer on them resistance against pests and diseases. Both GSLs and glucosinolate hydrolysis products (GHPs) have shown positive effects in reducing soil pathogens. Information about theirin vitrobiocide effects is scarce, but previous studies have shown sinigrin GSLs and their associated allyl isothiocyanate (AITC) to be soil biocides. The objective of this work was to evaluate the biocide effects of 17 GSLs and GHPs and of leaf methanolic extracts of different GSL-enrichedBrassicacrops on suppressingin vitrogrowth of two bacterial (Xanthomonas campestrispv. campestris andPseudomonas syringaepv. maculicola) and two fungal (AlternariabrassicaeandSclerotiniascletoriorum)Brassicapathogens. GSLs, GHPs, and methanolic leaf extracts inhibited the development of the pathogens tested compared to the control, and the effect was dose dependent. Furthermore, the biocide effects of the different compounds studied were dependent on the species and race of the pathogen. These results indicate that GSLs and their GHPs, as well as extracts of differentBrassicaspecies, have potential to inhibit pathogen growth and offer new opportunities to study the use ofBrassicacrops in biofumigation for the control of multiple diseases.

scholarly journals Glucose Oligosaccharide and Long-Chain Glucomannan Feed Additives Induce Enhanced Activation of Intraepithelial NK Cells and Relative Abundance of Commensal Lactic Acid Bacteria in Broiler Chickens

2021 ◽  
Vol 8 (6) ◽  
pp. 110
Author(s):  
Nathalie Meijerink ◽  
Jean E. de Oliveira ◽  
Daphne A. van Haarlem ◽  
Guilherme Hosotani ◽  
David M. Lamot ◽  
...  
Keyword(s):  
Immune System ◽  
Nk Cells ◽  
Relative Abundance ◽  
Nk Cell ◽  
Feed Additives ◽  
Microbiota Composition ◽  
In Ovo ◽  
Long Chain

Restrictions on the use of antibiotics in the poultry industry stimulate the development of alternative nutritional solutions to maintain or improve poultry health. This requires more insight in the modulatory effects of feed additives on the immune system and microbiota composition. Compounds known to influence the innate immune system and microbiota composition were selected and screened in vitro, in ovo, and in vivo. Among all compounds, 57 enhanced NK cell activation, 56 increased phagocytosis, and 22 increased NO production of the macrophage cell line HD11 in vitro. Based on these results, availability and regulatory status, six compounds were selected for further analysis. None of these compounds showed negative effects on growth, hatchability, and feed conversion in in ovo and in vivo studies. Based on the most interesting numerical results and highest future potential feasibility, two compounds were analyzed further. Administration of glucose oligosaccharide and long-chain glucomannan in vivo both enhanced activation of intraepithelial NK cells and led to increased relative abundance of lactic acid bacteria (LAB) amongst ileum and ceca microbiota after seven days of supplementation. Positive correlations between NK cell subsets and activation, and relative abundance of LAB suggest the involvement of microbiota in the modulation of the function of intraepithelial NK cells. This study identifies glucose oligosaccharide and long-chain glucomannan supplementation as effective nutritional strategies to modulate the intestinal microbiota composition and strengthen the intraepithelial innate immune system.

scholarly journals Mechanistic physiology-based pharmacokinetic modeling to elucidate vincristine-induced peripheral neuropathy following treatment with novel kinase inhibitors

2021 ◽  
Author(s):  
Venkatesh Pilla Reddy ◽  
Adrian J. Fretland ◽  
Diansong Zhou ◽  
Shringi Sharma ◽  
Buyun Chen ◽  
...  
Keyword(s):  
Peripheral Neuropathy ◽  
Kinase Inhibitors ◽  
Pbpk Model ◽  
Model Development ◽  
Limited Information ◽  
Time Curve ◽  
Btk Inhibitor ◽  
Chop Therapy

Abstract Purpose Limited information is available regarding the drug–drug interaction (DDI) potential of molecular targeted agents and rituximab plus cyclophosphamide, doxorubicin (hydroxydaunorubicin), vincristine (Oncovin), and prednisone (R-CHOP) therapy. The addition of the Bruton tyrosine kinase (BTK) inhibitor ibrutinib to R-CHOP therapy results in increased toxicity versus R-CHOP alone, including higher incidence of peripheral neuropathy. Vincristine is a substrate of P-glycoprotein (P-gp, ABCB1); drugs that inhibit P-gp could potentially cause increased toxicity when co-administered with vincristine through DDI. While the combination of the BTK inhibitor acalabrutinib and R-CHOP is being explored clinically, the DDI potential between these therapies is unknown. Methods A human mechanistic physiology-based pharmacokinetic (PBPK) model of vincristine following intravenous dosing was developed to predict potential DDI interactions with combination therapy. In vitro absorption, distribution, metabolism, and excretion and in vivo clinical PK parameters informed PBPK model development, which was verified by comparing simulated vincristine concentrations with observed clinical data. Results While simulations suggested no DDI between vincristine and ibrutinib or acalabrutinib in plasma, simulated vincristine exposure in muscle tissue was increased in the presence of ibrutinib but not acalabrutinib. Extrapolation of the vincristine mechanistic PBPK model to other P-gp substrates further suggested DDI risk when ibrutinib (area under the concentration–time curve [AUC] ratio: 1.8), but not acalabrutinib (AUC ratio: 0.92), was given orally with venetoclax or digoxin. Conclusion Overall, these data suggest low DDI risk between acalabrutinib and P-gp substrates with negligible increase in the potential risk of vincristine-induced peripheral neuropathy when acalabrutinib is added to R-CHOP therapy.

scholarly journals Impact of Membrane Lipids on UapA and AzgA Transporter Subcellular Localization and Activity in Aspergillus nidulans

2021 ◽  
Vol 7 (7) ◽  
pp. 514
Author(s):  
Mariangela Dionysopoulou ◽  
George Diallinas
Keyword(s):  
Plasma Membrane ◽  
Subcellular Localization ◽  
Aspergillus Nidulans ◽  
Membrane Lipids ◽  
Membrane Lipid ◽  
Lipid Biosynthesis ◽  
Transport Kinetics ◽  
Negative Effect ◽  
And Function

Recent biochemical and biophysical evidence have established that membrane lipids, namely phospholipids, sphingolipids and sterols, are critical for the function of eukaryotic plasma membrane transporters. Here, we study the effect of selected membrane lipid biosynthesis mutations and of the ergosterol-related antifungal itraconazole on the subcellular localization, stability and transport kinetics of two well-studied purine transporters, UapA and AzgA, in Aspergillus nidulans. We show that genetic reduction in biosynthesis of ergosterol, sphingolipids or phosphoinositides arrest A. nidulans growth after germling formation, but solely blocks in early steps of ergosterol (Erg11) or sphingolipid (BasA) synthesis have a negative effect on plasma membrane (PM) localization and stability of transporters before growth arrest. Surprisingly, the fraction of UapA or AzgA that reaches the PM in lipid biosynthesis mutants is shown to conserve normal apparent transport kinetics. We further show that turnover of UapA, which is the transporter mostly sensitive to membrane lipid content modification, occurs during its trafficking and by enhanced endocytosis, and is partly dependent on autophagy and Hect-type HulARsp5 ubiquitination. Our results point out that the role of specific membrane lipids on transporter biogenesis and function in vivo is complex, combinatorial and transporter-dependent.

scholarly journals Exogenous Alginate Protects Staphylococcus aureus from Killing by Pseudomonas aeruginosa

2019 ◽  
Vol 202 (8) ◽  
Author(s):  
Courtney E. Price ◽  
Dustin G. Brown ◽  
Dominique H. Limoli ◽  
Vanessa V. Phelan ◽  
George A. O’Toole
Keyword(s):  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
Physical Space ◽  
Late Time ◽  
Protective Effects ◽  
Content Type ◽  
Alginate Production ◽  
The Impact

ABSTRACT Cystic fibrosis (CF) patients chronically infected with both Pseudomonas aeruginosa and Staphylococcus aureus have worse health outcomes than patients who are monoinfected with either P. aeruginosa or S. aureus. We showed previously that mucoid strains of P. aeruginosa can coexist with S. aureus in vitro due to the transcriptional downregulation of several toxic exoproducts typically produced by P. aeruginosa, including siderophores, rhamnolipids, and HQNO (2-heptyl-4-hydroxyquinoline N-oxide). Here, we demonstrate that exogenous alginate protects S. aureus from P. aeruginosa in both planktonic and biofilm coculture models under a variety of nutritional conditions. S. aureus protection in the presence of exogenous alginate is due to the transcriptional downregulation of pvdA, a gene required for the production of the iron-scavenging siderophore pyoverdine as well as the downregulation of the PQS (Pseudomonas quinolone signal) (2-heptyl-3,4-dihydroxyquinoline) quorum sensing system. The impact of exogenous alginate is independent of endogenous alginate production. We further demonstrate that coculture of mucoid P. aeruginosa with nonmucoid P. aeruginosa strains can mitigate the killing of S. aureus by the nonmucoid strain of P. aeruginosa, indicating that the mechanism that we describe here may function in vivo in the context of mixed infections. Finally, we investigated a panel of mucoid clinical isolates that retain the ability to kill S. aureus at late time points and show that each strain has a unique expression profile, indicating that mucoid isolates can overcome the S. aureus-protective effects of mucoidy in a strain-specific manner. IMPORTANCE CF patients are chronically infected by polymicrobial communities. The two dominant bacterial pathogens that infect the lungs of CF patients are P. aeruginosa and S. aureus, with ∼30% of patients coinfected by both species. Such coinfected individuals have worse outcomes than monoinfected patients, and both species persist within the same physical space. A variety of host and environmental factors have been demonstrated to promote P. aeruginosa-S. aureus coexistence, despite evidence that P. aeruginosa kills S. aureus when these organisms are cocultured in vitro. Thus, a better understanding of P. aeruginosa-S. aureus interactions, particularly mechanisms by which these microorganisms are able to coexist in proximal physical space, will lead to better-informed treatments for chronic polymicrobial infections.

scholarly journals In Vitro Synergy and In Vivo Activity of Tigecycline-Ciprofloxacin Combination Therapy against Vibrio vulnificus Sepsis

2019 ◽  
Vol 63 (10) ◽  
Author(s):  
Seong Eun Kim ◽  
Hee Kyung Kim ◽  
Su-Mi Choi ◽  
Yohan Yu ◽  
Uh Jin Kim ◽  
...  
Keyword(s):  
Survival Rate ◽  
Mortality Rate ◽  
Combination Therapy ◽  
Combination Treatment ◽  
Vibrio Vulnificus ◽  
Antibiotic Regimen ◽  
Content Type ◽  
Time Kill

ABSTRACT The mortality rate associated with Vibrio vulnificus sepsis remains high. An in vitro time-kill assay revealed synergism between tigecycline and ciprofloxacin. The survival rate was significantly higher in mice treated with tigecycline plus ciprofloxacin than in mice treated with cefotaxime plus minocycline. Thus, combination treatment with tigecycline-ciprofloxacin may be an effective novel antibiotic regimen for V. vulnificus sepsis.

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