scholarly journals Demonstration and characterization of phosphate transport in mammalian peroxisomes

2005 ◽  
Vol 389 (3) ◽  
pp. 717-722 ◽  
Author(s):  
Wouter F. Visser ◽  
Carlo W. van Roermund ◽  
Lodewijk Ijlst ◽  
Klaas J. Hellingwerf ◽  
Ronald J. A. Wanders ◽  
...  
Keyword(s):  
Small Molecules ◽  
Adenine Nucleotide ◽  
Phosphate Transport ◽  
Significant Extent ◽  
Peroxisomal Membrane ◽  
Chain Acyl ◽  
Long Chain

It is now well established that the peroxisomal membrane is not freely permeable to small molecules in vivo, which implies the existence of metabolite transporters in the peroxisomal membrane. A few putative peroxisomal metabolite transporters have indeed been identified, but the function of these proteins has remained largely unresolved so far. The only peroxisomal transporter characterized to a significant extent is the adenine nucleotide transporter, which is presumably required to sustain the activity of the intraperoxisomal very-long-chain-acyl-CoA synthetase. In addition to AMP, this acyl-CoA synthetase also produces pyrophosphate, which must be exported from the peroxisome. In the present study, we demonstrate that the peroxisomal membrane contains a transporter activity that facilitates the passage of phosphate and possibly pyrophosphate across the peroxisomal membrane. By reconstitution of peroxisomal membrane proteins in proteoliposomes, some kinetic parameters of the transporter could be established in vitro. The transporter can be distinguished from the mitochondrial phosphate transporter by its different sensitivity to inhibitors.

scholarly journals Effect of long-chain fatty acyl-CoA on mitochondrial and cytosolic ATP/ADP ratios in the intact liver cell

1984 ◽  
Vol 220 (2) ◽  
pp. 371-376 ◽  
Author(s):  
S Soboll ◽  
H J Seitz ◽  
H Sies ◽  
B Ziegler ◽  
R Scholz
Keyword(s):  
Liver Cell ◽  
Adenine Nucleotide ◽  
Intact Cell ◽  
Inhibitory Effect ◽  
Fatty Acyl ◽  
Fed State ◽  
Physiological Relevance ◽  
Chain Acyl ◽  
Long Chain

The effect of long-chain acyl-CoA on subcellular adenine nucleotide systems was studied in the intact liver cell. Long-chain acyl-CoA content was varied by varying the nutritional state (fed and starved states) or by addition of oleate. Starvation led to an increase in the mitochondrial and a decrease in the cytosolic ATP/ADP ratio in liver both in vivo and in the isolated perfused organ as compared with the fed state. The changes were reversed on re-feeding glucose in liver in vivo or on infusion of substrates (glucose, glycerol) in the perfused liver, respectively. Similar changes in mitochondrial and cytosolic ATP/ADP ratios occurred on addition of oleate, but, importantly, not with a short-chain fatty acid such as octanoate. It is concluded that long-chain acyl-CoA exerts an inhibitory effect on mitochondrial adenine nucleotide translocation in the intact cell, as was previously postulated in the literature from data obtained with isolated mitochondria. The physiological relevance with respect to pyruvate metabolism, i.e. regulation of pyruvate carboxylase and pyruvate dehydrogenase by the mitochondrial ATP/ADP ratio, is discussed.

Substrate preferences of long-chain acyl-CoA synthetase and diacylglycerol acyltransferase contribute to enrichment of flax seed oil with α-linolenic acid

2018 ◽  
Vol 475 (8) ◽  
pp. 1473-1489 ◽  
Author(s):  
Yang Xu ◽  
Roman Holic ◽  
Darren Li ◽  
Xue Pan ◽  
Elzbieta Mietkiewska ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Linolenic Acid ◽  
Seed Oil ◽  
Diacylglycerol Acyltransferase ◽  
Acyl Donor ◽  
Flax Seed ◽  
Chain Acyl ◽  
Long Chain

Seed oil from flax (Linum usitatissimum) is enriched in α-linolenic acid (ALA; 18:3Δ9cis,12cis,15cis), but the biochemical processes underlying the enrichment of flax seed oil with this polyunsaturated fatty acid are not fully elucidated. Here, a potential process involving the catalytic actions of long-chain acyl-CoA synthetase (LACS) and diacylglycerol acyltransferase (DGAT) is proposed for ALA enrichment in triacylglycerol (TAG). LACS catalyzes the ATP-dependent activation of free fatty acid to form acyl-CoA, which in turn may serve as an acyl-donor in the DGAT-catalyzed reaction leading to TAG. To test this hypothesis, flax LACS and DGAT cDNAs were functionally expressed in Saccharomyces cerevisiae strains to probe their possible involvement in the enrichment of TAG with ALA. Among the identified flax LACSs, LuLACS8A exhibited significantly enhanced specificity for ALA over oleic acid (18:1Δ9cis) or linoleic acid (18:2Δ9cis,12cis). Enhanced α-linolenoyl-CoA specificity was also observed in the enzymatic assay of flax DGAT2 (LuDGAT2-3), which displayed ∼20 times increased preference toward α-linolenoyl-CoA over oleoyl-CoA. Moreover, when LuLACS8A and LuDGAT2-3 were co-expressed in yeast, both in vitro and in vivo experiments indicated that the ALA-containing TAG enrichment process was operative between LuLACS8A- and LuDGAT2-3-catalyzed reactions. Overall, the results support the hypothesis that the cooperation between the reactions catalyzed by LACS8 and DGAT2 may represent a route to enrich ALA production in the flax seed oil.

scholarly journals A multiplexed, automated evolution pipeline enables scalable discovery and characterization of biosensors

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Brent Townshend ◽  
Joy S. Xiang ◽  
Gabriel Manzanarez ◽  
Eric J. Hayden ◽  
Christina D. Smolke
Keyword(s):  
Small Molecules ◽  
Biosynthetic Pathway ◽  
De Novo ◽  
Living Cells ◽  
Regulate Gene Expression ◽  
Liquid Handling ◽  
Regulate Gene

AbstractBiosensors are key components in engineered biological systems, providing a means of measuring and acting upon the large biochemical space in living cells. However, generating small molecule sensing elements and integrating them into in vivo biosensors have been challenging. Here, using aptamer-coupled ribozyme libraries and a ribozyme regeneration method, de novo rapid in vitro evolution of RNA biosensors (DRIVER) enables multiplexed discovery of biosensors. With DRIVER and high-throughput characterization (CleaveSeq) fully automated on liquid-handling systems, we identify and validate biosensors against six small molecules, including five for which no aptamers were previously found. DRIVER-evolved biosensors are applied directly to regulate gene expression in yeast, displaying activation ratios up to 33-fold. DRIVER biosensors are also applied in detecting metabolite production from a multi-enzyme biosynthetic pathway. This work demonstrates DRIVER as a scalable pipeline for engineering de novo biosensors with wide-ranging applications in biomanufacturing, diagnostics, therapeutics, and synthetic biology.

scholarly journals A multiplexed, automated evolution pipeline enables scalable discovery and characterization of biosensors

2020 ◽  
Author(s):  
Brent Townshend ◽  
Joy Xiang ◽  
Gabriel Manzanarez ◽  
Eric Hayden ◽  
Christina Smolke
Keyword(s):  
Small Molecules ◽  
Biosynthetic Pathway ◽  
De Novo ◽  
Living Cells ◽  
Regulate Gene Expression ◽  
Liquid Handling ◽  
Regulate Gene

AbstractBiosensors are key components in engineered biological systems, providing a means of measuring and acting upon the large biochemical space in living cells. However, generating small molecule sensing elements and integrating them into in vivo biosensors have been challenging. Using aptamer-coupled ribozyme libraries and a novel ribozyme regeneration method, we developed de novo rapid in vitro evolution of RNA biosensors (DRIVER) that enables multiplexed discovery of biosensors. With DRIVER and high-throughput characterization (CleaveSeq) fully automated on liquid-handling systems, we identified and validated biosensors against six small molecules, including five for which no aptamers were previously found. DRIVER-evolved biosensors were applied directly to regulate gene expression in yeast, displaying activation ratios up to 33-fold. DRIVER biosensors were also applied in detecting metabolite production from a multi-enzyme biosynthetic pathway. This work demonstrates DRIVER as a scalable pipeline for engineering de novo biosensors with wide-ranging applications in biomanufacturing, diagnostics, therapeutics, and synthetic biology.

scholarly journals Amiodarone and Bepridil Inhibit Anthrax Toxin Entry into Host Cells

2007 ◽  
Vol 51 (7) ◽  
pp. 2403-2411 ◽  
Author(s):  
Ana M. Sanchez ◽  
Diane Thomas ◽  
Eugene J. Gillespie ◽  
Robert Damoiseaux ◽  
Joseph Rogers ◽  
...  
Keyword(s):  
Small Molecules ◽  
Lethal Toxin ◽  
Host Cells ◽  
Anthrax Lethal Toxin ◽  
Raw 264.7 Macrophages ◽  
Endosomal Acidification ◽  
A Cell

ABSTRACT Anthrax lethal toxin is one of the fundamental components believed to be responsible for the virulence of Bacillus anthracis. In order to find novel compounds with anti-lethal toxin properties, we used a cell-based assay to screen a collection of approximately 500 small molecules. Nineteen compounds that blocked lethal toxin-mediated killing of RAW 264.7 macrophages were identified, and we report here on the characterization of the two most potent antitoxic compounds, amiodarone and bepridil. These drugs are used to treat cardiac arrhythmia or angina in humans at doses similar to those that provide protection against lethal toxin in vitro. Our results support a model whereby the antitoxic properties of both drugs result from their ability to block endosomal acidification, thereby blocking toxin entry. Amiodarone was tested in vivo and found to significantly increase survival of lethal toxin-challenged Fischer rats.

Rumen hydrogenation of long-chain fatty acids from fish meal

1997 ◽  
Vol 1997 ◽  
pp. 129-129
Author(s):  
M.D. Carro ◽  
E.L. Miller ◽  
O.C. Fabb
Keyword(s):  
Fatty Acids ◽  
Fish Meal ◽  
Long Chain Fatty Acids ◽  
Flow Data ◽  
Significant Extent ◽  
Rumen Microorganisms ◽  
Long Chain

In one in vitro study Ashes et al. (1992) reported that C20 and C22 fatty acids (FA) from fish oil were not hydrogenated to any significant extent by rumen microorganisms. However, to our knowledge, no measurement on hydrogenation has been performed on fishmeal (FM) FA. The aim of this experiment was to study the in vivo and in situ rumen hydrogenation of long-chain FA of two different FM: FMl (60 g FA/g DM) and FM2 (85 g FA/g DM).Six sheep fitted with rumen cannulae and single duodenal cannulae were fed every 2 hours, receiving 1 kg/d of a 60:40 hay:concentrate diet, either alone (Control; C) or supplemented with 40 g FM/d (FMl and FM2). The experiment was carried out over four periods (two sheep received one of the diets in each period) and Cr-NDF was used as a marker to estimate duodenal flow. Data were subjected to analysis of variance using the ANOVA procedure of the Statistical Analysis Systems (SAS, 1994).

scholarly journals PPARδ activation induces hepatic long-chain acyl-CoA synthetase 4 expression in vivo and in vitro

2015 ◽  
Vol 1851 (5) ◽  
pp. 577-587 ◽  
Author(s):  
Chin Fung Kelvin Kan ◽  
Amar Bahadur Singh ◽  
Bin Dong ◽  
Vikram Ravindra Shende ◽  
Jingwen Liu
Keyword(s):  
Chain Acyl ◽  
Long Chain

In vitro characterization and in vivo expression of human very-long chain acyl-CoA dehydrogenase

2006 ◽  
Vol 88 (4) ◽  
pp. 351-358 ◽  
Author(s):  
J. Lawrence Merritt ◽  
Dietrich Matern ◽  
Jerry Vockley ◽  
Jan Daniels ◽  
Tien V. Nguyen ◽  
...  
Keyword(s):  
In Vitro Characterization ◽  
In Vivo Expression ◽  
Chain Acyl ◽  
Long Chain

Fatty acid oxidation by isolated perfused working hearts of aged rats.

1977 ◽  
Vol 232 (3) ◽  
pp. E258 ◽  
Author(s):  
G M Abu-Erreish ◽  
J R Neely ◽  
J T Whitmer ◽  
V Whitman ◽  
D R Sanadi
Keyword(s):  
Adenine Nucleotide ◽  
Acid Oxidation ◽  
Tissue Mass ◽  
Chain Acyl ◽  
Pressure Development ◽  
Heart Preparation ◽  
Old Rats ◽  
Total Carnitine ◽  
Long Chain

It has been reported that mitochondria isolated from hearts of old rats have lower respiratory activity than mitochondria from young rats. In order to determine the physiological correlates of these changes, the metabolism of hearts from young and old rats has been compared in a perfused working heart preparation. The oxidation of [14C]palmitate to 14CO2, oxygen consumption, and nucleotide levels were measured under different cardiac workloads. The hearts from old animals performed less cardiac work and utilized less oxygen and palmitate in proportion to tissue mass, but the ratio of oxygen consumed to pressure developed was unaltered. There was a small but significant decrease in cardiac efficiency expressed as the ratio between the rate of oxygen consumed and ventricular pressure development. Tissue levels of total carnitine and long-chain acylcarnitine derivatives were greatly reduced in the older heart without significant change in free CoA, acetyl-CoA, or long-chain acyl-CoA. The adenine nucleotide levels were not significantly different in the two groups. The results appear consistent with the in vitro studies on isolated mitochondria.

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