Radiolabelled Substrates Research Articles

Luciferase-based bioluminescence revealed the facilitated diffusion of D-luciferin mediated by SLC17A3.

SLC17A3 localized to the apical membrane of the renal proximal tubules has been implicated in the urinary excretion of drugs and endogenous/exogenous metabolites transported into the tubules by OAT1 and OAT3. Because SLC17A3 mediates the facilitated diffusion of organic anions, which requires a sensitive and rapid assay, no system has been established to evaluate its transport activity in mammalian cells. In this study, we demonstrated that the exposure of cells expressing click beetle luciferase (bLuc) and SLC17A3 to D-luciferin produces marked bioluminescence, which enables the evaluation of SLC17A3 function. The bioluminescence intensity increased under depolarized membrane potential conditions, consistent with the unique feature of SLC17A3 as a voltage-dependent organic anion transporter. SLC17A3-mediated bioluminescence was saturable with an apparent Michaelis-Menten constant (Km) of 8.1μM. The inhibitory effects of various compounds including OAT1/OAT3 substrates and inhibitors on bioluminescence were in good agreement with those reported in SLC17A3-expressing Xenopus oocytes using radio-labeled substrates. Interestingly, we found that sulfinpyrazone and lesinurad, uricosuric drugs that inhibit SLC22A12/URAT1, are potent SLC17A3 inhibitors, suggesting the possibility that they alter the pharmacokinetics of OAT1/OAT3-substrate drugs and urate. Taken together, the bioluminescence-based SLC17A3 functional assay is robust and reliable. This strategy enables the study of its transport activity and the identification of potential SLC17A3-mediated drug-drug interactions. This approach also provides an opportunity to elucidate the molecular mechanisms involved in the urinary excretion of organic anions.

Detection Strategies for Sialic Acid and Sialoglycoconjugates.

Glycoconjugates are a vast class of biomolecules implicated in biological processes important for human health and disease. The structural complexity of glycoconjugates remains a challenge to deciphering their precise biological roles and for their development as biomarkers and therapeutics. Human glycoconjugates on the outside of the cell are modified with sialic (neuraminic) acid residues at their termini. The enzymes that install sialic acids are sialyltransferases (SiaTs), a family of 20 different isoenzymes. The removal and degradation of sialic acids is mediated by neuraminidase (NEU; sialidase) enzymes, of which there are four isoenzymes. In this review, we discuss chemical and biochemical approaches for the detection and analysis of sialoglycoconjugate (SGC) structures and their enzymatic products. The most common methods include affinity probes and synthetic substrates. Fluorogenic and radiolabelled substrates are also important tools for many applications, including screening for enzyme inhibitors. Strategies that give insight into the native substrate-specificity of enzymes that regulate SGCs (SiaT & NEU) are necessary to improve our understanding of the role of sialic acid metabolism in health and disease.

Effects of sub-chronic, in vivo administration of sigma-1 receptor ligands on platelet and aortic arachidonate cascade in streptozotocin-induced diabetic rats.

Diabetes mellitus is a chronic metabolic disorder which induces endothelial dysfunction and platelet activation. Eicosanoids produced from arachidonic acid regulate cellular and vascular functions. Sigma-1 receptors (S1R) are expressed in platelets and endothelial cells and S1R expression is protective in diabetes. Our aim was to examine the influence of sub-chronic, in vivo administered S1R ligands PRE-084, (S)-L1 (a new compound) and NE-100 on the ex vivo arachidonic acid metabolism of platelets and aorta in streptozotocin-induced diabetic rats. The serum level of the S1R ligands was detected by LC-MS/MS before the ex vivo analysis. Sigma-1 receptor and cyclooxygenase gene expression in platelets were determined by RT-qPCR. The eicosanoid synthesis was examined with a radiolabelled arachidonic acid substrate and ELISA. One month after the onset of STZ-induced diabetes, in vehicle-treated, diabetic rat platelet TxB2 and aortic 6-k-PGF1α production dropped. Sub-chronic in vivo treatment of STZ-induced diabetes in rats for one week with PRE-084 enhanced vasoconstrictor and platelet aggregator and reduced vasodilator and anti-aggregator cyclooxygenase product formation. (S)-L1 reduced the synthesis of vasodilator and anti-aggregator cyclooxygenase metabolites and promoted the recovery of physiological platelet function in diabetic rats. The S1R antagonist NE-100 produced no significant changes in platelet arachidonic acid metabolism. (S)-L1 decreased the synthesis of vasoconstrictor and platelet aggregator cyclooxygenase metabolites, whereas NE-100 increased the quantity of aortic vasodilator and anti-aggregator cyclooxygenase products and promoted the recovery of diabetic endothelial dysfunction in the aorta. The novel S1R ligand, (S)-L1 had similar effects on eicosanoid synthesis in platelets as the agonist PRE-084 and in aortas as the antagonist NE-100. S1R ligands regulate cellular functions and local blood circulation by influencing arachidonic acid metabolism. In diabetes mellitus, the cell-specific effects of S1R ligands have a compensatory role and aid in restoring physiological balance between the platelet and vessel.

Hydrolysis of Methylumbeliferyl Substrate Proxies for Esterase Activities as Indicator for Microbial Oil Degradation in the Ocean: Evidence from Observations in the Aftermath of the Deepwater Horizon Oil Spill (Gulf of Mexico)

Biological oil weathering facilitated by specialized heterotrophic microbial communities plays a key role in the fate of petroleum hydrocarbon in the ocean. The most common methods of assessing oil biodegradation involve (i) measuring changes in the composition and concentration of oil over time and/or (ii) biological incubations with stable or radio-labelled substrates. Both methods provide robust and invaluable information on hydrocarbon biodegradation pathways; however, they also require extensive sample processing and are expensive in nature. More convenient ways to assess activities within microbial oil degradation networks involve measuring extracellular enzyme activity. This perspective article synthesizes previously published results from studies conducted in the aftermath of the 2010 Deepwater Horizon (DwH) oil spill in the northern Gulf of Mexico (nGoM), to test the hypothesis that fluorescence assays of esterases, including lipase activity, are sensitive indicators for microbial oil degradation in the ocean. In agreement with the rates and patterns of enzyme activity in oil-contaminated seawater and sediments in the nGoM, we found close correlations between esterase activity measured by means of methylumbeliferyl (MUF) oleate and MUF butyrate hydrolysis, and the concentration of petroleum hydrocarbons in two separate laboratory incubations using surface (<1 m) and deep nGoM waters (>1200 m). Correlations between esterase activities and oil were driven by the presence of chemical dispersants, suggesting a connection to the degree of oil dissolution in the medium. Our results clearly show that esterase activities measured with fluorogenic substrate proxies are a good indicator for oil biodegradation in the ocean; however, there are certain factors as discussed in this study that need to be taken into consideration while utilizing this approach.

Metabolic maturation of differentiating cardiosphere-derived cells

Cardiosphere-derived cells (CDCs) can be expanded in vitro and induced to differentiate along the cardiac lineage. To recapitulate the phenotype of an adult cardiomyocyte, differentiating progenitors need to upregulate mitochondrial glucose and fatty acid oxidation. Here we cultured and differentiated CDCs using protocols aimed to maintain stemness or to promote differentiation, including triggering fatty acid oxidation using an agonist of peroxisome proliferator-activated receptor alpha (PPARα). Metabolic changes were characterised in undifferentiated CDCs and during differentiation towards a cardiac phenotype. CDCs from rat atria were expanded on fibronectin or collagen IV via cardiosphere formation. Differentiation was assessed using flow cytometry and qPCR and substrate metabolism was quantified using radiolabelled substrates. Collagen IV promoted proliferation of CDCs whereas fibronectin primed cells for differentiation towards a cardiac phenotype. In both populations, treatment with 5-Azacytidine induced a switch towards oxidative metabolism, as shown by changes in gene expression, decreased glycolytic flux and increased oxidation of glucose and palmitate. Addition of a PPARα agonist during differentiation increased both glucose and fatty acid oxidation and expression of cardiac genes. We conclude that oxidative metabolism and cell differentiation act in partnership with increases in one driving an increase in the other.

Non-radioactive Assay to Determine Product Profile of Short-chain Isoprenyl Diphosphate Synthases

Isoprenoids represent the largest class of metabolites with amazing diversities in structure and function. They are involved in protecting plants against pathogens or herbivores or involved in attracting pollinators. Isoprenoids are derived from geranyl diphosphate (GPP; C10), farnesyl diphosphate (FPP; C15), geranylgeranyl diphosphate (GGPP; C20), and geranylfarnesyl diphosphate (GFPP; C25) that are in turn formed by sequential condensations of isopentenyl diphosphate (IPP; C5) with an allylic acceptor such as dimethylallyl diphosphate (DMAPP; C5), GPP, FPP, or GGPP in a reaction catalyzed by isoprenyl diphosphate synthases (IDSs). IDS enzyme assay for determination of prenyl diphosphate products is generally performed using radiolabelled substrates, and the products formed are identified by employing expensive instruments such as phosphor imager, radio-GC, or radio-HPLC. Though a non-radioactive assay for measuring IDS activity in crude plant extract has been reported, it requires a complex methodology utilizing chromatography coupled with tandem mass spectrometry (LC/MS-MS). Here, we describe a non-radioactive and simple inexpensive assay for determining the IDS assay products using non-radiolabeled IPP and its co-allylic substrates DMAPP, GPP, and FPP. The detection of prenyl diphosphate products generated in the assay was highly efficient and spots corresponding to prenyl alcohols were visible at >40 µM concentrations of IPP and DMAPP/GPP/FPP substrates. The protocol described here is sensitive, reliable, and technically simple, which could be used for functional characterization of IDS candidates.

Imaging antigen-specific T cells

Immunology Positron emission tomography (PET) has previously been exploited to trace immune responses in living animals. However, its use has been largely limited to cells expressing surrogate markers recognized by radiolabeled antibodies or cells genetically modified to take up radio-labeled substrates. Woodham et al. now report a nonintrusive, in vivo method to track antigen-specific T cells that recognize known antigens presented by major histocompatibility complex (MHC) molecules. The authors made a class of radiolabeled peptide-bound MHC dimers linked to antibody Fc regions, which they called “synTacs.” In a mouse model of cancer, they were able to follow CD8+ T cells within tumors that recognized a particular oncoprotein. They could also detect influenza virus (IAV) nucleoprotein–specific CD8+ T cells in the lungs of IAV-infected mice. This work may help pave the way for future tools to noninvasively track human T cell responses during interventions such as vaccination and cancer immunotherapy. Nat. Methods 17 , 1025 (2020).

Studying the Metabolism of Epithelial-Mesenchymal Plasticity Using the Seahorse XFe96 Extracellular Flux Analyzer.

The critical role of metabolism in facilitating cancer cell growth and survival has been demonstrated by a combination of methods including, but not limited to, genomic sequencing, transcriptomic and proteomic analyses, measurements of radio-labelled substrate flux and the high throughput measurement of oxidative metabolism in unlabelled live cells using the Seahorse Extracellular Flux (XF) technology. These studies have revealed that tumour cells exhibit a dynamic metabolic plasticity, using numerous pathways including both glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) to support cell proliferation, energy production and the synthesis of biomass. These advanced technologies have also demonstrated metabolic differences between cancer cell types, between molecular subtypes within cancers and between cell states. This has been exemplified by examining the transitions of cancer cells between epithelial and mesenchymal phenotypes, referred to as epithelial-mesenchymal plasticity (EMP). A growing number of studies are demonstrating significant metabolic alterations associated with these transitions, such as increased use of glycolysis by triple negative breast cancers (TNBC) or glutamine addiction in lung cancer. Models of EMP, including invasive cell lines and xenografts, isolated circulating tumour cells and metastatic tissue have been used to examine EMP metabolism. Understanding the metabolism supporting molecular and cellular plasticity and increased metastatic capacity may reveal metabolic vulnerabilities that can be therapeutically exploited. This chapter describes protocols for using the Seahorse Extracellular Flux Analyzer (XFe96), which simultaneously performs real-time monitoring of oxidative phosphorylation and glycolysis in living cells. As an example, we compare the metabolic profiles generated from two breast cancer sublines that reflect epithelial and mesenchymal phenotypes, respectively. We use this example to show how the methodology described can generate bioenergetic results that in turn can be correlated to EMP phenotypes. Normalisation of bioenergetic studies should be considered with respect to cell number, and to potential differences in mitochondrial mass, itself being an important bioenergetics endpoint.

Metabolic Maturation of Differentiating Cardiosphere-Derived Cells

Cardiosphere-derived cells (CDCs) can be expanded in vitro and induced to differentiate along the cardiac lineage. To recapitulate the phenotype of an adult cardiomyocyte, differentiating progenitors need to upregulate mitochondrial glucose and fatty acid oxidation. Here we cultured and differentiated CDCs using protocols aimed to maintain stemness or to promote differentiation, including triggering fatty acid oxidation using an agonist of peroxisome proliferation receptor α (PPARα). Metabolic changes were characterised as the CDCs differentiated towards a cardiac phenotype. CDCs from rat atria were expanded on fibronectin or collagen IV via cardiosphere formation. Differentiation was characterised using flow cytometry and qPCR and substrate metabolism was measured using radiolabelled substrates. Collagen IV promoted proliferation of CDCs, with a greater proportion of cells expressing progenitor-cell markers, whereas fibronectin primed cells for differentiation towards a cardiac phenotype. In both populations, differentiation induced a switch towards oxidative metabolism, as shown by changes in gene expression, decreased glycolytic flux and increased oxidation of glucose and palmitate. Addition of a PPARα agonist increased both glucose and fatty acid oxidation and expression of cardiac genes. We conclude that oxidative metabolism and cell differentiation act in partnership with increases in one driving an increase in the other.

Innervation and electrical pulse stimulation - in vitro effects on human skeletal muscle cells.

Contraction-induced adaptations in skeletal muscles are well characterized in vivo, but the underlying cellular mechanisms are still not completely understood. Cultured human myotubes represent an essential model system for human skeletal muscle that can be modulated ex vivo, but they are quiescent and do not contract unless being stimulated. Stimulation can be achieved by innervation of human myotubes in vitro by co-culturing with embryonic rat spinal cord, or by replacing motor neuron activation by electrical pulse stimulation (EPS). Effects of these two in vitro approaches, innervation and EPS, were characterized with respects to the expression of myosin heavy chains (MyHCs) and metabolism of glucose and oleic acid in cultured human myotubes. Adherent human myotubes were either innervated with rat spinal cord segments or exposed to EPS. The expression pattern of MyHCs was assessed by quantitative polymerase chain reaction, immunoblotting, and immunofluorescence, while the metabolism of glucose and oleic acid were studied using radiolabelled substrates. Innervation and EPS promoted differentiation towards different fiber types in human myotubes. Expression of the slow MyHC-1 isoform was reduced in innervated myotubes, whereas it remained unaltered in EPS-treated cells. Expression of both fast isoforms (MyHC-2A and MyHC-2X) tended to decrease in EPS-treated cells. Both approaches induced a more oxidative phenotype, reflected in increased CO2 production from both glucose and oleic acid. Novelty: Innervation and EPS favour differentiation into different fiber types in human myotubes. Both innervation and EPS promote a metabolically more oxidative phenotype in human myotubes.

Refining the use of PET imaging to detect moderate changes in P-gp function at the blood-brain barrier

In the clinical practice, complete repression of P-glycoprotein (P-gp) function at blood-brain barrier (BBB) is not likely to occur. However, partial inhibition/deficit is suspected in many pathophysiological situations. PET imaging using radiolabelled substrates of P-glycoprotein (P-gp) is the most advanced technique to study P-gp function at the human BBB. However, the sensitivity of current radiolabelled P-gp substrates to detect moderate changes in P-gp function at the BBB remained to be compared. In vitro and in vivo studies were conducted to compare 11C-verapamil (11C-V), 11C-N-desmethyl-loperamide (11C-N) and 11C-metoclopramide (11C-M). In vitro uptake assays were performed in MDCKII-hMDR1 cells to compare the transport capacity and the 50% inhibitory concentration (IC50) toward the P-gp inhibitors tariquidar (TQD) and cyclosporine A (CsA) for each radiolabelled substrate. In vivo, increasing I.V. doses of TQD on the brain distribution of 11C-M were first studied by microPET imaging in rats. In vitro modelling results suggest that the transport capacity and IC50 are highly dependent of the substrate/inhibitor couples. With both inhibitors, 11C-M showed the best sensitivity (1.4 to 6 fold lower IC50) and the lowest transport capacity. In vivo, IC50 was reached for the 1 mg/kg TQD dose. In vivo experiments using 11C-V and 11C-N in the same conditions are ongoing. Compared with 11C-M, avid P-gp substrates (11C-V and 11C-N) may lack sensitivity to detect partial P-gp inhibition/deficit and may underestimate the impact of fine regulation of P-gp function at the human BBB.

Ryanodine receptor-targeting small molecule fluorescent probes enables non-isotopic labeling and efficient drug screening for green insecticides

Ryanodine receptors (RyRs) are calcium release channels located on endoplasmic reticulum (ER) membrane, which play important role in excitation-contraction coupling in muscular response. Flubendiamide represents a novel chemical family of green insecticides which selectively activate invertebrate RyR by interacting with the receptor distinct from the ryanodine binding site and has almost no effect on mammalian ryanodine receptors. Traditional methods to screen RyR modulators involve either radio-labeled RyR substrates or calcium signal-based indirect approaches. However, there is lack of RyR-directed non-isotope molecular tools for RyR agonists/antagonists screening and bioimaging. Here we developed a series of fluorescent probes based on the pharmacophore of flubendiamide with the aims to elucidate the mechanism of diamide insecticides and screen novel RyR-targeting insecticides. These probes revealed the specific RyR staining and in vivo RyR targeting properties in diamondback moth RyR transfected Sf9 cells (Sf9-RyR) and RyR enriched insect tissues. The designed fluorescent probes could induce an effective calcium release from ER membrane of Sf9-RyR cells and also showed competitive RyR binding effect with flubendiamide in cell-based fluorometric assay. Having the non-isotope RyR recognition probes will not only accelerate the screening process of new green agrochemicals but also enables deciphering molecular mechanisms of the high selectivity and the drug resistance associated with the diamides.

Virtual screening approach and biochemical evaluation on MurB

Abstract We present a modern in silico virtual screening workflow towards novel MurB inhibitors. Namely, the field of antibacterial research is in crucial need of novel lead compounds on new or underexplored therapeutic targets such as MurB. We identified three structurally distinct compounds that indicated promising micromolar inhibitory activity on E. coli MurB. All three compounds did not display cytotoxicity on HepG2 cell line, but also did not possess in vitro antimicrobial activity against S. aureus and E. coli. After the second biological evaluation using radio-labelled substrates in a MurA-MurB coupled test, only marginal inhibitory potency could be reproduced on MurB. As no compound in the series was able to significantly decrease the residual activity of the enzyme, we decided to report the complete inactive compound set to be used as decoys.

Postprandial Skeletal Muscle Metabolism Following a High Fat Diet in Sedentary and Endurance Trained Males

ObjectiveFasting and postprandial metabolism are adversely affected by a sedentary lifestyle. However, the role of the prevailing diet remains unclear. The objective of the present study was to determine the influence of a high fat diet (HFD) on fasting and postprandial skeletal muscle substrate metabolism in sedentary compared with endurance trained (ET) humans.MethodsMetabolically healthy sedentary (n=17) and ET (n=7) males were control‐fed a 10‐day isocaloric moderate fat diet (30% fat [<10% saturated fat], 55% carbohydrate) followed by a 5‐day isocaloric HFD (55% fat [25% saturated fat], 30% carbohydrate). Before and after the HFD participants completed a high fat meal challenge (820 kcals; 63% fat [26% saturated fat], 25% carbohydrate) in which skeletal muscle biopsies were taken in the fasted and 4‐hour fed states. Complete fat, glucose, and pyruvate oxidation were assessed by measuring 14CO2 production in homogenized skeletal muscle incubated with radio‐labeled substrates. Incomplete fat oxidation was assessed by measuring 14C‐labeled acid soluble metabolites. Total fat oxidation was comprised of the sum of complete and incomplete fat oxidation. A substrate preference assay was performed by assessing the free fatty acid (FFA)‐induced suppression of [1‐14C]‐pyruvate oxidation. The meal effect for each substrate oxidation measure was calculated as the percent change from the fasted to 4‐hour fed state.ResultsThe meal effect for glucose oxidation was not altered by the HFD in the ET group (128 ± 92% vs. 41 ± 15%, P=0.375) but was reduced in the sedentary group (78 ± 26% vs. −21 ± 6%, P=0.002). The meal effect for pyruvate oxidation was similar between ET and sedentary groups before the HFD (34 ± 13% vs. 14 ± 17%, respectively; P=0.145) but was lower in the sedentary compared with the ET group after the HFD (−16 ± 6% vs. 24 ± 17%, P=0.047). The meal effect for substrate preference as indicated by FFA‐induced suppression of pyruvate oxidation was retained after the HFD in the ET group (20 ± 4% vs. 41 ± 21%, P=0.342) but was reduced in the sedentary group (34 ± 7% vs. 4 ± 5%, P=0.003). The meal effects for incomplete and total fat oxidation tended to decrease as a result of the HFD in the overall sample (P=0.075 and P=0.062, respectively). There were no physical activity group or HFD effects on meal effects for complete fat oxidation or the ratio of complete‐to‐incomplete fat oxidation.ConclusionsThese findings suggest skeletal muscle from sedentary individuals displays reduced postprandial glucose oxidation, yet pyruvate is preferred over fatty acids as an oxidative substrate following consumption of a HFD. These data highlight the need to better understand key regulatory nodes in skeletal muscle substrate metabolism between ET and sedentary humans.Support or Funding InformationADA‐07‐12 (MWH); Virginia Tech Translational Obesity Research IGEPThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Role of neutral amino acid transporter LAT4 in mouse epithelia

Amino acids (AAs) are not only building blocks of proteins, but also play key roles in energy metabolism and as (precursors of) neurotransmitters. To maintain amino acid homeostasis, small intestine (SI) and kidney play a major role by absorbing amino acids from food and preventing their loss in the urine, respectively. Given their zwitterionic nature, neutral AAs require transport proteins to cross cell membranes. LAT4 (Slc43a2), a facilitated diffusion uniporter specific for essential neutral amino acids (Leu, Ile, Val, Met and Phe) was first identified by Bodoy et al., 2005 and then shown by us to be highly expressed in the basolateral membranes of SI and kidney tubule epithelia. LAT4 global knockout caused a postnatal malnutrition‐like condition leading to death within 10 days, which was hypothesized to be due to a severe defect in intestinal absorption of AAs (Guetg et al. 2015). To test this hypothesis, we generated an intestine‐specific LAT4 knockout mouse model using Cre‐LoxP system. Intestine specific LAT4 knockout pups were viable and did not show any significant changes in body weight gain as compared to their control littermates even up to 2 months after birth. Quantitative PCR, western blot and immunofluorescence data showed a nearly 95% efficient knockout of LAT4 throughout the SI (n=4). However, these mice displayed no visible phenotype. Amino acid transport along the SI was then assessed by gavaging an AA mix containing radiolabelled LAT4 substrate 3H‐Leu. Blood samples were collected from the tail vein 10′ before and 2′,5′, 10′, 20′ and 60′ after gavage. Mice were then euthanized, SI divided into 4 equal parts and the intestinal tissue and its contents collected separately. Measuring the distribution of 3H‐Leu using a β‐counter revealed an accumulation of Leu in the initial segment of the SI of knockout mice (n=5, P<0.001 by ANOVA), accompanied by a delayed bolus movement. Thus, we show that LAT4 knockout in SI is not the sole or major reason for the lethal phenotype of LAT4 global knockout pups, although it could have contributed to the lethality. In parallel, we have generated a doxycycline‐inducible kidney tubule‐specific LAT4 knockout mouse model to assess the effects of LAT4 deletion in kidney, both in young and adult animals. Results obtained with this new model will allow us to determine whether the urinary loss of amino acids may have strongly contributed to the lethal phenotype of LAT4 global knockout mice or whether it was caused by the defect of LAT4 in other tissues.Support or Funding InformationThis project is funded by a grant of the Swiss National Foundation (SNF) to FV.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Label-Free Sensitive Detection of DNA Methyltransferase by Target-Induced Hyperbranched Amplification with Zero Background Signal.

DNA methyltransferases (MTases) may specifically recognize the short palindromic sequences and transfer a methyl group from S-adenosyl-l-methionine to target cytosine/adenine. The aberrant DNA methylation is linked to the abnormal DNA MTase activity, and some DNA MTases have become promising targets of anticancer/antimicrobial drugs. However, the reported DNA MTase assays often involve laborious operation, expensive instruments, and radio-labeled substrates. Here, we develop a simple and label-free fluorescent method to sensitively detect DNA adenine methyltransferase (Dam) on the basis of terminal deoxynucleotidyl transferase (TdT)-activated Endonuclease IV (Endo IV)-assisted hyperbranched amplification. We design a hairpin probe with a palindromic sequence in the stem as the substrate and a NH2-modified 3' end for the prevention of nonspecific amplification. The substrate may be methylated by Dam and subsequently cleaved by DpnI, producing three single-stranded DNAs, two of which with 3'-OH termini may be amplified by hyperbranched amplification to generate a distinct fluorescence signal. Because high exactitude of TdT enables the amplification only in the presence of free 3'-OH termini and Endo IV only hydrolyzes the intact apurinic/apyrimidinic sites in double-stranded DNAs, zero background signal can be achieved. This method exhibits excellent selectivity and high sensitivity with a limit of detection of 0.003 U/mL for pure Dam and 9.61 × 10-6 mg/mL for Dam in E. coli cells. Moreover, it can be used to screen the Dam inhibitors, holding great potentials in disease diagnosis and drug development.

Manipulation of Amyloid Precursor Protein Processing Impacts Brain Bioenergetics and Glucose Metabolism

The amyloid precursor protein (APP) and one of its terminal cleavage products, amyloid beta (Aβ) are proposed to play a central role in the pathogenesis of Alzheimer's disease (AD). These proteins have also been shown to co-localise within mitochondria with Aβ directly inhibiting the electron transport chain. Cleavage of APP occurs down distinct pathways. Initial cleavage of the full-length protein is mediated by either α- or β-secretase (BACE1). Subsequent cleavage by γ-secretase releases p3 or Aβ protein fragments into the extracellular milieu. Whilst these interactions with mitochondria have been noted, the impact of manipulating APP processing by these distinct pathways on cellular metabolism and bioenergetics has yet to be elucidated. We utilised the human SH-SY5Y neuronal cell line stably overexpressing BACE1 and cortical slices from mice bearing inducible overexpression of APP harbouring the Swedish and Indiana mutations, favouring Aβ production. Protein expression and enzyme activity were determined via western blotting and enzyme-linked immunosorbance assays (ELISA). A Seahorse extracellular flux analyser and radio-labelled substrate assays were used to determine cellular bioenergetics in real-time. All data are expressed as mean ± standard error of the mean and statistical significance determined by Student's t-test. Manipulation of APP cleavage resulted in alterations of 2-deoxyglucose uptake into cells. Chronic elevation in BACE1 resulted in impaired functioning of a key fuel-partitioning enzyme, pyruvate dehydrogenase (PDH, activity reduced to 69 ± 8%, p < 0.05, n=6). This reduced substrate delivery to the mitochondria and increased reliance upon aerobic glycolysis for ATP generation (oxygen consumption rate (OCR) reduced to 65 ± 9%, p < 0.01, n=7 and extracellular acidification rate (ECAR) increased to 165 ± 16%, p < 0.01, n=7). Thus BACE1 overexpression impairs neuronal glucose oxidation resulting in an increased reliance upon aerobic glycolysis for ATP generation. Acute cortical overexpression of APP favouring amyloidogenic processing resulted in a significant reduction in PDH protein expression (reduced to 62 ± 6%, p < 0.01, n = 9-10) and activity of key tricarboxylic acid cycle enzymes, isocitrate dehydrogenase and alpha-ketoglutarate dehydrogenase (reduced to 62 ± 7%, p < 0.01, n=6 and 88 ± 2%, p < 0.001, n = 6). Therefore, manipulation of APP processing towards Aβ production in cells and tissue significantly impacts cellular metabolic pathways at the level of glucose uptake, substrate entry to the mitochondria and the TCA cycle.

Effects of frequently used pharmaceutical excipients on the organic cation transporters 1–3 and peptide transporters 1/2 stably expressed in MDCKII cells

There is ample evidence that pharmaceutical excipients, which are supposed to be pharmacologically inactive, have an impact on drug metabolism and efflux transport. So far, little is known whether they also modulate uptake transporter proteins. We have recently shown that commonly used solubilizing agents exert significant effects on the function of organic anion uptake transporting polypeptides. Therefore, we investigated in this study the influence of frequently used pharmaceutical excipients on the transport activity of organic cation transporters OCT1, OCT2 and OCT3 and the peptide transporters PEPT1 and PEPT2.Inhibition of the OCTs and PEPTs by the excipients polyethylene glycol 400 (PEG), hydroxypropyl-β-cyclodextrin (HPCD), Solutol® HS15 (SOL), Cremophor® EL (CrEL), Tween® 20 (Tw20), Tween® 80 (Tw80), Kolliphor® P188 (P188) and Kolliphor® P407 (P407) was evaluated using stably transfected MDCKII cells with radio-labeled reference substrates and established inhibitors as controls. Intracellular accumulation of [3H]-1-methyl-4-phenylpyridinium (MPP+) for the OCTs and [3H]-glycyl-sarcosine (Gly-Sar) for the PEPTs was measured by liquid scintillation counting after cell lysis.Our studies revealed that PEG, HPCD, SOL, CrEL, Tw20 and Tw80 were potent inhibitors of OCT1-3 (e.g., Tw20 IC50 values<0.04%). Cellular uptake of Gly-Sar by PEPT1 and PEPT2 was strongly inhibited by both Tw20 and Tw80. SOL was also a strong inhibitor of PEPT1 and PEPT2 (e.g., SOL IC50 values<0.02%), while CrEL showed significantly inhibition of only PEPT2. The substantial inhibitory effects of certain solubilizing agents on OCTs and PEPTs should be considered if they are to be used in dosage forms for new chemical entities and registered drugs to avoid misinterpretation of pharmacokinetic data and undesired drug interactions.

The Role of Lysophospholipid Acyltransferases in the Golgi Complex.

Determining the abundance of phospholipids and neutral lipids in cellular membranes is paramount to understanding their biological functions. Many lipid-modifying enzymes have yet to be characterized due to limitations in substrate-product measurements and purification of membrane-bound enzymes. The method described here uses radiolabeled phospholipid substrates and cell-purified organelles to quantify phospholipid metabolism using thin-layer chromatography. This assay has the benefits of being specific and adaptable for numerous applications and systems.

Increased oxidative metabolism following hypoxia in the type 2 diabetic heart, despite normal hypoxia signalling and metabolic adaptation.

Adaptation to hypoxia makes the heart more oxygen efficient, by metabolising more glucose. In contrast, type 2 diabetes makes the heart metabolise more fatty acids. Diabetes increases the chances of the heart being exposed to hypoxia, but whether the diabetic heart can adapt and respond is unknown. In this study we show that diabetic hearts retain the ability to adapt their metabolism in response to hypoxia, with functional hypoxia signalling pathways. However, the hypoxia-induced changes in metabolism are additive to abnormal baseline metabolism, resulting in hypoxic diabetic hearts metabolising more fat and less glucose than controls. This stops the diabetic heart being able to recover its function when stressed. These results demonstrate that the diabetic heart retains metabolic flexibility to adapt to hypoxia, but is hindered by the baseline effects of the disease. This increases our understanding of how the diabetic heart is affected by hypoxia-associated complications of the disease. Hypoxia activates the hypoxia-inducible factor (HIF), promoting glycolysis and suppressing mitochondrial respiration. In the type 2 diabetic heart, glycolysis is suppressed whereas fatty acid metabolism is promoted. The diabetic heart experiences chronic hypoxia as a consequence of increased obstructive sleep apnoea and cardiovascular disease. Given the opposing metabolic effects of hypoxia and diabetes, we questioned whether diabetes affects cardiac metabolic adaptation to hypoxia. Control and type 2 diabetic rats were housed for 3weeks in normoxia or 11% oxygen. Metabolism and function were measured in the isolated perfused heart using radiolabelled substrates. Following chronic hypoxia, both control and diabetic hearts upregulated glycolysis, lactate efflux and glycogen content and decreased fatty acid oxidation rates, with similar activation of HIF signalling pathways. However, hypoxia-induced changes were superimposed on diabetic hearts that were metabolically abnormal in normoxia, resulting in glycolytic rates 30% lower, and fatty acid oxidation 36% higher, in hypoxic diabetic hearts than hypoxic controls. Peroxisome proliferator-activated receptor α target proteins were suppressed by hypoxia, but activated by diabetes. Mitochondrial respiration in diabetic hearts was divergently activated following hypoxia compared with controls. These differences in metabolism were associated with decreased contractile recovery of the hypoxic diabetic heart following an acute hypoxic insult. In conclusion, type 2 diabetic hearts retain metabolic flexibility to adapt to hypoxia, with normal HIF signalling pathways. However, they are more dependent on oxidative metabolism following hypoxia due to abnormal normoxic metabolism, which was associated with a functional deficit in response to stress.