Alfred E. Thumser

The fatty acid transport function of fatty acid-binding proteins.

The intracellular fatty acid-binding proteins (FABPs) comprise a family of 14-15 kDa proteins which bind long-chain fatty acids. A role for FABPs in fatty acid transport has been hypothesized for several decades, and the accumulated indirect and correlative evidence is largely supportive of this proposed function. In recent years, a number of experimental approaches which more directly examine the transport function of FABPs have been taken. These include molecular level in vitro modeling of fatty acid transfer mechanisms, whole cell studies of fatty acid uptake and intracellular transfer following genetic manipulation of FABP type and amount, and an examination of cells and tissues from animals engineered to lack expression of specific FABPs. Collectively, data from these studies have provided strong support for defining the FABPs as fatty acid transport proteins. Further studies are necessary to elucidate the fundamental mechanisms by which cellular fatty acid trafficking is modulated by the FABPs.

Tissue-specific Functions in the Fatty Acid-binding Protein Family

The intracellular fatty acid-binding proteins (FABPs) are abundantly expressed in almost all tissues. They exhibit high affinity binding of a single long-chain fatty acid, with the exception of liver FABP, which binds two fatty acids or other hydrophobic molecules. FABPs have highly similar tertiary structures consisting of a 10-stranded antiparallel β-barrel and an N-terminal helix-turn-helix motif. Research emerging in the last decade has suggested that FABPs have tissue-specific functions that reflect tissue-specific aspects of lipid and fatty acid metabolism. Proposed roles for FABPs include assimilation of dietary lipids in the intestine, targeting of liver lipids to catabolic and anabolic pathways, regulation of lipid storage and lipid-mediated gene expression in adipose tissue and macrophages, fatty acid targeting to β-oxidation pathways in muscle, and maintenance of phospholipid membranes in neural tissues. The regulation of these diverse processes is accompanied by the expression of different and sometimes multiple FABPs in these tissues and may be driven by protein-protein and protein-membrane interactions.

Effect of sleep deprivation on the human metabolome

Significance Sleep restriction and circadian clock disruption are associated with metabolic disorders including obesity and diabetes; this association can be studied by using the powerful tool of metabolomics. By using liquid chromatography/MS metabolomics, we have characterized plasma metabolites that were significantly affected by acute sleep deprivation (mainly lipids and acylcarnitines), all increasing during sleep deprivation. Observed increased levels of serotonin, tryptophan, and taurine may explain the antidepressive effect of sleep deprivation and deserve further study. Clear daily rhythms were observed in most metabolites, with 24 h wakefulness mainly reducing the amplitude of these rhythms. Our results further the understanding of sleep/wake regulation and the associated metabolic processes, and will be vital when using metabolic profiling to identify robust biomarkers for disease states and drug efficacy. Sleep restriction and circadian clock disruption are associated with metabolic disorders such as obesity, insulin resistance, and diabetes. The metabolic pathways involved in human sleep, however, have yet to be investigated with the use of a metabolomics approach. Here we have used untargeted and targeted liquid chromatography (LC)/MS metabolomics to examine the effect of acute sleep deprivation on plasma metabolite rhythms. Twelve healthy young male subjects remained in controlled laboratory conditions with respect to environmental light, sleep, meals, and posture during a 24-h wake/sleep cycle, followed by 24 h of wakefulness. Two-hourly plasma samples collected over the 48 h period were analyzed by LC/MS. Principal component analysis revealed a clear time of day variation with a significant cosine fit during the wake/sleep cycle and during 24 h of wakefulness in untargeted and targeted analysis. Of 171 metabolites quantified, daily rhythms were observed in the majority (n = 109), with 78 of these maintaining their rhythmicity during 24 h of wakefulness, most with reduced amplitude (n = 66). During sleep deprivation, 27 metabolites (tryptophan, serotonin, taurine, 8 acylcarnitines, 13 glycerophospholipids, and 3 sphingolipids) exhibited significantly increased levels compared with during sleep. The increased levels of serotonin, tryptophan, and taurine may explain the antidepressive effect of acute sleep deprivation and deserve further study. This report, to our knowledge the first of metabolic profiling during sleep and sleep deprivation and characterization of 24 h rhythms under these conditions, offers a novel view of human sleep/wake regulation.

Factors affecting the performance of microbial fuel cells for sulfur pollutants removal

A microbial fuel cell (MFC) has been developed for removal of sulfur-based pollutants and can be used for simultaneous wastewater treatment and electricity generation. This fuel cell uses an activated carbon cloth+carbon fibre veil composite anode, air-breathing dual cathodes and the sulfate-reducing species Desulfovibrio desulfuricans. 1.16gdm(-3) sulfite and 0.97gdm(-3) thiosulfate were removed from the wastewater at 22 degrees C, representing sulfite and thiosulfate removal conversions of 91% and 86%, respectively. The anode potential was controlled by the concentration of sulfide in the compartment. The performance of the cathode assembly was affected by the concentration of protons in the cation-exchanging ionomer with which the electrocatalyst is co-bound at the three-phase (air, catalyst and support) boundary.

Decreased expression of the vitamin C transporter SVCT1 by ascorbic acid in a human intestinal epithelial cell line

Vitamin C (ascorbic acid) is an essential nutrient that is involved in a number of cellular processes. However, unlike most mammals, man is unable to synthesize vitamin C and it must therefore be acquired from the diet. Absorption of vitamin C is achieved by two transporters, SVCTI and SVCT2, recently cloned from rat and human kidney. SVCT1 is thought to be the predominant transporter in the intestine. Vitamin C supplements are increasingly common, thus contributing to an increased dietary load, and therefore the aim of the present study was to investigate the effect of high doses of ascorbic acid on SVCT1 expression. Using the Caco-2 TC7 cell model of small intestinal enterocytes, we measured the effects of ascorbic acid (4.5 mg/ml culture medium) on L-[14C]ascorbic acid uptake and SVCT1 expression (determined by reverse transcription-polymerase chain reaction). Ascorbic acid uptake was decreased significantly in Caco-2 TC7 cells exposed to ascorbate for 24 h (-50%, P<0.0005). Expression of SVCT1 was also significantly reduced by exposure to elevated levels of ascorbate for 24h (-77%, P<0.005). Taken together these results suggest that high-dose supplements might not be the most efficient way of increasing the body pool of vitamin C.

Characterization of Rhodamine-123 as a Tracer Dye for Use In In vitro Drug Transport Assays

Fluorescent tracer dyes represent an important class of sub-cellular probes and allow the examination of cellular processes in real-time with minimal impact upon these processes. Such tracer dyes are becoming increasingly used for the examination of membrane transport processes, as they are easy-to-use, cost effective probe substrates for a number of membrane protein transporters. Rhodamine 123, a member of the rhodamine family of flurone dyes, has been used to examine membrane transport by the ABCB1 gene product, MDR1. MDR1 is viewed as the archetypal drug transport protein, and is able to efflux a large number of clinically relevant drugs. In addition, ectopic activity of MDR1 has been associated with the development of multiple drug resistance phenotype, which results in a poor patient response to therapeutic intervention. It is thus important to be able to examine the potential for novel compounds to be MDR1 substrates. Given the increasing use rhodamine 123 as a tracer dye for MDR1, a full characterisation of its spectral properties in a range of in vitro assay-relevant media is warranted. Herein, we determine λmax for excitation and emission or rhodamine 123 and its metabolite rhodamine 110 in commonly used solvents and extraction buffers, demonstrating that fluorescence is highly dependent on the chemical environment: Optimal parameters are 1% (v/v) methanol in HBSS, with λex = 505 nm, λem = 525 nm. We characterise the uptake of rhodamine 123 into cells, via both passive and active processes, and demonstrate that this occurs primarily through OATP1A2-mediated facilitated transport at concentrations below 2 µM, and via micelle-mediated passive diffusion above this. Finally, we quantify the intracellular sequestration and metabolism of rhodamine 123, demonstrating that these are both cell line-dependent factors that may influence the interpretation of transport assays.

Direct electron transfer of glucose oxidase immobilized in an ionic liquid reconstituted cellulose-carbon nanotube matrix

Conductive cellulose-multiwalled carbon nanotube (MWCNT) matrix with a porous structure and good biocompatibility has been prepared using a room temperature ionic liquid (1-ethyl-3-methylimidazolium acetate) as solvent. Glucose oxidase (GOx) was encapsulated in this matrix and thereby immobilized on a glassy carbon surface. The direct electron transfer and electrocatalysis of the encapsulated GOx has been investigated using cyclic voltammetry and chronoamperometry. The GOx exhibited a pair of stable, well defined and nearly symmetric reversible redox peaks. The experimental results also demonstrate that the immobilized GOx retains its biocatalytic activity toward the oxidation of glucose and therefore can be employed in a glucose biosensor. The results show that the bioelectrode modified by the cellulose-MWCNT matrix has potential for use in biosensors and other bioelectronics devices.

Identification of human plasma metabolites exhibiting time-of-day variation using an untargeted liquid chromatography-mass spectrometry metabolomic approach.

Although daily rhythms regulate multiple aspects of human physiology, rhythmic control of the metabolome remains poorly understood. The primary objective of this proof-of-concept study was identification of metabolites in human plasma that exhibit significant 24-h variation. This was assessed via an untargeted metabolomic approach using liquid chromatography–mass spectrometry (LC-MS). Eight lean, healthy, and unmedicated men, mean age 53.6 (SD ± 6.0) yrs, maintained a fixed sleep/wake schedule and dietary regime for 1 wk at home prior to an adaptation night and followed by a 25-h experimental session in the laboratory where the light/dark cycle, sleep/wake, posture, and calorific intake were strictly controlled. Plasma samples from each individual at selected time points were prepared using liquid-phase extraction followed by reverse-phase LC coupled to quadrupole time-of-flight MS analysis in positive ionization mode. Time-of-day variation in the metabolites was screened for using orthogonal partial least square discrimination between selected time points of 10:00 vs. 22:00 h, 16:00 vs. 04:00 h, and 07:00 (d 1) vs. 16:00 h, as well as repeated-measures analysis of variance with time as an independent variable. Subsequently, cosinor analysis was performed on all the sampled time points across the 24-h day to assess for significant daily variation. In this study, analytical variability, assessed using known internal standards, was low with coefficients of variation <10%. A total of 1069 metabolite features were detected and 203 (19%) showed significant time-of-day variation. Of these, 34 metabolites were identified using a combination of accurate mass, tandem MS, and online database searches. These metabolites include corticosteroids, bilirubin, amino acids, acylcarnitines, and phospholipids; of note, the magnitude of the 24-h variation of these identified metabolites was large, with the mean ratio of oscillation range over MESOR (24-h time series mean) of 65% (95% confidence interval [CI]: 49–81%). Importantly, several of these human plasma metabolites, including specific acylcarnitines and phospholipids, were hitherto not known to be 24-h variant. These findings represent an important baseline and will be useful in guiding the design and interpretation of future metabolite-based studies. (Author correspondence: Jooern.Ang@icr.ac.uk or Florence.Raynaud@icr.ac.uk)

A Role for Microbial Palladium Nanoparticles in Extracellular Electron Transfer

Herein we have demonstrated a DET mechanism used by D. desulfuricans; where the periplasmic cytochromes and hydrogenases play an important role, and Pd nanoparticles bound to the microbes may participate in the electron transfer process. The present work is of importance not only for the fundamental studies of electron transfer processes in microbial physiology and ecology, but also for increased understanding and improvement of the performance of bioelectrochemical techniques e.g. precious metals are extensively used and important catalysts, and therefore present in many industry processing wastewaters. Bio-nanoparticles can oxidize in situ metabolites e.g. H2, formate and ethanol in the anode chambers, while also acting as cathodic catalysts for the oxygen reduction reaction[23]. In addition, this study indicates the feasibility of using bioelectrochemical systems for metal immobilization, recovery or detoxification

Fatty acid binding proteins: tissue-specific functions in health and disease

Purpose of reviewThe purpose of this study is to review recent evidence for the role of the cytosolic fatty acid binding proteins (FABPs) as central regulators of whole-body metabolic control. Recent findingsDysregulated FABPs have been associated with a number of diseases, including obesity and nonalcoholic fatty liver disease (FABP1, FABP2, FABP4), cardiovascular risk (FABP3) and cancer (FABP5, FABP7). As underlying mechanisms become better understood, FABPs may represent novel biomarkers for therapeutic targets. In addition, the role of FABPs as important signalling molecules has also been highlighted in recent years; for example, FABP3 may act as a myokine, matching whole-body metabolism to muscular energy demands and FABP4 functions as an adipokine in regulating macrophage and adipocyte interactions during inflammation. SummaryIn addition to their traditional role as fatty acid trafficking proteins, increasing evidence supports the role of FABPs as important controllers of global metabolism, with their dysregulation being linked to a host of metabolic diseases.