UPLC-MS-based urine metabolomics reveals indole-3-lactic acid and phenyllactic acid as conserved biomarkers for alcohol-induced liver disease in the Ppara-null mouse model

Since the development and prognosis of alcohol-induced liver disease (ALD) vary significantly with genetic background, identification of a genetic background-independent noninvasive ALD biomarker would significantly improve screening and diagnosis. This study explored the effect of genetic background on the ALD-associated urinary metabolome using the Ppara-null mouse model on two different backgrounds, C57BL/6 (B6) and 129/SvJ (129S), along with their wild-type counterparts. Reversed-phase gradient UPLC-ESI-QTOF-MS analysis revealed that urinary excretion of a number of metabolites, such as ethylsulfate, 4-hydroxyphenylacetic acid, 4-hydroxyphenylacetic acid sulfate, adipic acid, pimelic acid, xanthurenic acid, and taurine, were background-dependent. Elevation of ethyl-β-d-glucuronide and N-acetylglycine was found to be a common signature of the metabolomic response to alcohol exposure in wild-type as well as in Ppara-null mice of both strains. However, increased excretion of indole-3-lactic acid and phenyllactic acid was found to be a conserved feature exclusively associated with the alcohol-treated Ppara-null mouse on both backgrounds that develop liver pathologies similar to the early stages of human ALD. These markers reflected the biochemical events associated with early stages of ALD pathogenesis. The results suggest that indole-3-lactic acid and phenyllactic acid are potential candidates for conserved and pathology-specific high-throughput noninvasive biomarkers for early stages of ALD.

LeProT1, a Transporter for Proline, Glycine Betaine, and γ-Amino Butyric Acid in Tomato Pollen

During maturation, pollen undergoes a period of dehydration accompanied by the accumulation of compatible solutes.Solute import across the pollen plasma membrane, which occurs via proteinaceous transporters, is required to support pollen development and also for subsequent germination and pollen tube growth. Analysis of the free amino acid composition of various tissues in tomato revealed that the proline content in flowers was 60 times higher than in any other organ analyzed. Within the floral organs, proline was confined predominantly to pollen, where it represented >70 of total free amino acids. Uptake experiments demonstrated that mature as well as germinated pollen rapidly take up proline. To identify proline transporters in tomato pollen, we isolated genes homologous to Arabidopsis proline transporters. LeProT1 was specifically expressed both in mature and germinating pollen, as demonstrated by RNA in situ hybridization. Expression in a yeast mutant demonstrated that LeProT1 transports proline and γ-amino butyric acid with low affinity and glycine betaine with high affinity. Direct uptake and competition studies demonstrate that LeProT1 constitutes a general transporter for compatible solutes.

Oxidation of 3-hydroxyanthranilic acid to the phenoxazinone cinnabarinic acid by peroxyl radicals and by compound I of peroxidases or catalase

Since 3-hydroxyanthranilic acid (3HAA), an oxidation product of tryptophan metabolism, is a powerful radical scavenger [Christen, S., Peterhans, E., ; Stocker, R. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 2506], its reaction with peroxyl radicals was investigated further. Exposure to aqueous peroxyl radicals generated at constant rate under air from the thermolabile radical initiator 2,2'-azobis[2-amid-inopropane] hydrochloride (AAPH) resulted in rapid consumption of 3HAA with initial accumulation of its cyclic dimer, cinnabarinic acid (CA). The initial rate of formation of the phenoxazinone CA accounted for approximately 75% of the initial rate of oxidation of 3HAA, taking into account that 2 mol of 3HAA are required to form 1 mol of CA. Consumption of 3HAA under anaerobic conditions (where alkyl radicals are produced from AAPH) was considerably slower and did not result in detectable formation of CA. Addition of superoxide dismutase enhanced autoxidation of 3HAA as well as the initial rates of peroxyl radical-induced oxidation of 3HAA and formation of CA by approximately 40-50%, whereas inclusion of xanthine/xanthine oxidase decreased the rate of oxidation of 3HAA by approximately 50% and inhibited formation of CA almost completely, suggesting that superoxide anion radical (O2.-) was formed and reacted with reaction intermediate(s) to curtail formation of CA. Formation of CA was also observed when 3HAA was added to performed compound I of horseradish peroxidase (HRPO) or catalytic amounts of either HRPO, myeloperoxidase, or bovine liver catalase together with glucose/glucose oxidase.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of noninvasive biomarkers for alcohol-induced liver disease using urinary metabolomics and the Ppara-null mouse

Alcohol-induced liver disease (ALD) is a leading cause of nonaccident-related deaths in the United States. Although liver damage caused by ALD is reversible when discovered at the earlier stages, current risk assessment tools are relatively nonspecific. Identification of an early specific signature of ALD would aid in therapeutic intervention and recovery. In this study, the metabolic changes associated with ALD were examined using alcohol-fed male Ppara-null mouse as a model of ALD. Principal components analysis of the mass spectrometry-based urinary metabolic profile showed that alcohol-treated wild-type and Ppara-null mice could be distinguished from control animals without information on history of alcohol consumption. The urinary excretion of ethyl-sulfate, ethyl-beta-d-glucuronide, 4-hydroxyphenylacetic acid, and 4-hydroxyphenylacetic acid sulfate was elevated and that of the 2-hydroxyphenylacetic acid, adipic acid, and pimelic acid was depleted during alcohol treatment in both wild-type and the Ppara-null mice albeit to different extents. However, indole-3-lactic acid was exclusively elevated by alcohol exposure in Ppara-null mice. The elevation of indole-3-lactic acid is mechanistically related to the molecular events associated with development of ALD in alcohol-treated Ppara-null mice. This study demonstrated the ability of a metabolomics approach to identify early, noninvasive biomarkers of ALD pathogenesis in Ppara-null mouse model.

The auxin influx carrier is essential for correct leaf positioning

Auxin is of vital importance in virtually every aspect of plant growth and development, yet, even after almost a century of intense study, major gaps in our knowledge of its synthesis, distribution, perception, and signal transduction remain. One unique property of auxin is its polar transport, which in many well-documented cases is a critical part of its mode of action. Auxin is actively transported through the action of both influx and efflux carriers. Inhibition of polar transport by the efflux inhibitor N-1-naphthylphthalamic acid (NPA) causes a complete cessation of leaf initiation, a defect that can be reversed by local application of the auxin, indole-3-acetic acid (IAA), to the responsive zone of the shoot apical meristem. In this study, we address the role of the auxin influx carrier in the positioning and outgrowth of leaf primordia at the shoot apical meristem of tomato. By using a combination of transport inhibitors and synthetic auxins, we demonstrate that interference with auxin influx has little effect on organ formation as such, but prevents proper localization of leaf primordia. These results suggest the existence of functional auxin concentration gradients in the shoot apical meristem that are actively set up and maintained by the action of efflux and influx carriers. We propose a model in which efflux carriers control auxin delivery to the shoot apical meristem, whereas influx and efflux carriers regulate auxin distribution within the meristem.

Auxin Regulates the Initiation and Radial Position of Plant Lateral Organs

Leaves originate from the shoot apical meristem, a small mound of undifferentiated tissue at the tip of the stem. Leaf formation begins with the selection of a group of founder cells in the so-called peripheral zone at the flank of the meristem, followed by the initiation of local growth and finally morphogenesis of the resulting bulge into a differentiated leaf. Whereas the mechanisms controlling the switch between meristem propagation and leaf initiation are being identified by genetic and molecular analyses, the radial positioning of leaves, known as phyllotaxis, remains poorly understood. Hormones, especially auxin and gibberellin, are known to influence phyllotaxis, but their specific role in the determination of organ position is not clear. We show that inhibition of polar auxin transport blocks leaf formation at the vegetative tomato meristem, resulting in pinlike naked stems with an intact meristem at the tip. Microapplication of the natural auxin indole-3-acetic acid (IAA) to the apex of such pins restores leaf formation. Similarly, exogenous IAA induces flower formation on Arabidopsis pin-formed1-1 inflorescence apices, which are blocked in flower formation because of a mutation in a putative auxin transport protein. Our results show that auxin is required for and sufficient to induce organogenesis both in the vegetative tomato meristem and in the Arabidopsis inflorescence meristem. In this study, organogenesis always strictly coincided with the site of IAA application in the radial dimension, whereas in the apical–basal dimension, organ formation always occurred at a fixed distance from the summit of the meristem. We propose that auxin determines the radial position and the size of lateral organs but not the apical–basal position or the identity of the induced structures.

Dynamic Precision Phenotyping Reveals Mechanism of Crop Tolerance to Root Herbivory

The western corn rootworm (WCR) is a major pest of maize that is well adapted to most crop management strategies. Breeding for tolerance is a promising alternative to combat WCR, but is currently constrained by a lack of physiological understanding and phenotyping tools. We developed dynamic precision phenotyping approaches using carbon-11 with positron emission tomography, root autoradiography and radiometabolite flux analysis to understand maize tolerance to WCR. Our results reveal that WCR attack induces specific patterns of lateral root growth which are associated with a shift in auxin biosynthesis from indole-3-pyruvic acid to indole-3-acetonitrile. WCR attack also increases transport of newly synthesized amino acids to the roots, including the accumulation of glutamine. Finally, the regrowth zones of WCR attacked roots show an increase in glutamine turnover which strongly correlates with the induction of indole-3-acetonitrile-dependent auxin biosynthesis. In summary, our findings identify local changes in the auxin flux network as a promising marker for induced WCR tolerance.

Simultaneous determination of 3-hydroxyanthranilic and cinnabarinic acid by high-performance liquid chromatography with photometric or electrochemical detection

A convenient and rapid method for the simultaneous determination by HPLC of 3-hydroxyanthranilic acid and the dimer derived by its oxidation, cinnabarinic acid, is described. Buffers or biological samples containing these two Trp metabolites were acidified to pH 2.0 and extracted with ethyl acetate with recoveries of 96.5 +/- 0.5 and 93.4 +/- 3.7% for 3-hydroxyanthranilic and cinnabarinic acid, respectively. The two compounds were separated on a reversed-phase (C18) column combined with ion-pair chromatography and detected photometrically or electrochemically. The method was applied successfully to biological systems in which formation of either 3-hydroxyanthranilic or cinnabarinic acid had been described previously. Thus, interferon-gamma-treated human peripheral blood mononuclear cells formed and released significant amounts of 3-hydroxyanthranilic acid into the culture medium and mouse liver nuclear fraction possessed high "cinnabarinic acid synthase" activity. In contrast, addition of 3-hydroxyanthranilic acid to human erythrocytes resulted in only marginal formation of cinnabarinic acid. We conclude that the method described is specific, sensitive, and suitable for the detection of the two Trp metabolites in biological systems.

Zoledronic acid efficacy and safety over five years in postmenopausal osteoporosis

In a 5-year study involving 119 postmenopausal women, zoledronic acid 4 mg given once-yearly for 2, 3 or 5 years was well tolerated with no evidence of excessive bone turnover reduction or any safety signals. BMD increased significantly. Bone turnover markers decreased from baseline and were maintained within premenopausal reference ranges. INTRODUCTION: After completion of the core study, two consecutive, 2-year, open-label extensions investigated the efficacy and safety of zoledronic acid 4 mg over 5 years in postmenopausal osteoporosis. METHODS: In the core study, patients received 1 to 4 mg zoledronic acid or placebo. In the first extension, most patients received 4 mg per year and then patients entered the second extension and received 4 mg per year or calcium only. Patients were divided into three subgroups according to years of active treatment received (2, 3 or 5 years). Changes in BMD and bone turnover markers (bone ALP and CTX-I) were assessed. RESULTS: All subgroups showed substantial increases in BMD and decreases in bone markers. By the end of the core study, 37.5% of patients revealed a suboptimal reduction (< 30%) of bone ALP levels. After subsequent study drug administration during the extensions, there was no evidence of progressive reduction of bone turnover markers. Furthermore, increased marker levels after treatment discontinuation demonstrates preservation of bone remodelling capacity. CONCLUSIONS: This study showed that zoledronic acid 4 mg once-yearly was well tolerated and effective in reducing biomarkers over 5 years. Detailed analysis of bone marker changes, however, suggests that this drug regimen causes insufficient reduction of remodelling activity in one third of patients.

Pharmacokinetics and excretion of gamma-hydroxybutyrate (GHB) in healthy subjects

In Europe and the United States, the recreational use of gamma-hydroxy butyric acid (GHB) at dance clubs and "rave" parties has increased substantially. In addition, GHB is used to assist in the commission of sexual assaults. The aim of this controlled clinical study was to acquire pharmacokinetic profiles, detection times, and excretion rates in human subjects. Eight GHB-naïve volunteers were administered a single 25-mg/kg body weight oral dose of GHB, and plasma, urine, and oral fluid specimens were analyzed by using gas chromatography-mass spectrometry (GC-MS). Liquid-liquid extraction was performed after acid conversion of GHB to gamma-butyrolactone. Limits of quantitation of 0.1 (oral fluid), 0.2 (urine), and 0.5 microg/mL (plasma) could be achieved in the selected ion monitoring mode. GHB plasma peaks of 39.4 +/- 25.2 microg/mL (mean +/- SEM) occurred 20-45 min after administration. The terminal plasma elimination half-life was 30.4 +/- 2.45 min, the distribution volume 52.7 +/- 15.0 L, and the total clearance 1228 +/- 233 microL/min. In oral fluid, GHB could be detected up to 360 min, with peak concentrations of 203 +/- 92.4 microg/mL in the 10-min samples. In urine, 200 +/- 71.8 and 230 +/- 86.3 microg/mL, were the highest GHB levels measured at 30 and 60 min, respectively. Only 1.2 +/- 0.2% of the dose was excreted, resulting in a detection window of 720 min. Common side-effects were confusion, sleepiness, and dizziness; euphoria and change of vital functions were not observed. GHB is extensively metabolized and rapidly eliminated in urine and oral fluid. Consequently, samples should be collected as soon as possible after ingestion.

Prefrontal GABA and glutathione imbalance in posttraumatic stress disorder: Preliminary findings.

Although posttraumatic stress disorder (PTSD) is associated with a variety of structural and functional brain changes, the molecular pathophysiological mechanisms underlying these macroscopic alterations are unknown. Recent studies support the existence of an altered excitation-inhibition balance in PTSD. Further, there is preliminary evidence from blood-sample studies suggesting heightened oxidative stress in PTSD, potentially leading to neural damage through excessive brain levels of free radicals. In this study we investigated PTSD (n=12) and non-PTSD participants (n=17) using single-voxel proton magnetic resonance spectroscopy (MRS) in dorsolateral prefrontal cortex (DLPFC) and anterior cingulate cortex (ACC). We found significantly higher levels of γ-amino butyric acid (GABA) (a primary inhibitory neurotransmitter) and glutathione (a marker for neuronal oxidative stress) in PTSD participants. Atypically high prefrontal inhibition as well as oxidative stress may be involved in the pathogenesis of PTSD.

Identification of selective norbornane-type aspartate analogue inhibitors of the glutamate transporter 1 (GLT-1) from the chemical universe generated database (GDB)

A variety of conformationally constrained aspartate and glutamate analogues inhibit the glutamate transporter 1 (GLT-1, also known as EAAT2). To expand the search for such analogues, a virtual library of aliphatic aspartate and glutamate analogues was generated starting from the chemical universe database GDB-11, which contains 26.4 million possible molecules up to 11 atoms of C, N, O, F, resulting in 101026 aspartate analogues and 151285 glutamate analogues. Virtual screening was realized by high-throughput docking to the glutamate binding site of the glutamate transporter homologue from Pyrococcus horikoshii (PDB code: 1XFH ) using Autodock. Norbornane-type aspartate analogues were selected from the top-scoring virtual hits and synthesized. Testing and optimization led to the identification of (1R*,2R*,3S*,4R*,6R*)-2-amino-6-phenethyl-bicyclo[2.2.1]heptane-2,3-dicarboxylic acid as a new inhibitor of GLT-1 with IC(50) = 1.4 ?M against GLT-1 and no inhibition of the related transporter EAAC1. The systematic diversification of known ligands by enumeration with help of GDB followed by virtual screening, synthesis, and testing as exemplified here provides a general strategy for drug discovery.

Discovering imaging endophenotypes for major depression

Psychiatry research lacks an in-depth understanding of mood disorders phenotypes, leading to limited success of genetics studies of major depressive disorder (MDD). The dramatic progress in safe and affordable magnetic resonance-based imaging methods has the potential to identify subtle abnormalities of neural structures, connectivity and function in mood disordered subjects. This review paper presents strategies to improve the phenotypic definition of MDD by proposing imaging endophenotypes derived from magnetic resonance spectroscopy measures, such as cortical gamma-amino butyric acid (GABA) and glutamate/glutamine concentrations, and from measures of resting-state activity and functional connectivity. The proposed endophenotypes are discussed regarding specificity, mood state-independence, heritability, familiarity, clinical relevance and possible associations with candidate genes. By improving phenotypic definitions, the discovery of new imaging endophenotypes will increase the power of candidate gene and genome-wide associations studies. It will also help to develop and evaluate novel therapeutic treatments and enable clinicians to apply individually tailored therapeutic approaches. Finally, improvements of the phenotypic definition of MDD based on neuroimaging measures will contribute to a new classification system of mood disorders based on etiology and pathophysiology.

Membrane disrupting antimicrobial peptide dendrimers with multiple amino termini

Antimicrobial peptide dendrimer H1 Leu8(Lys-Leu)4(Lys-Phe)2Lys-LysNH2 (Lys = branching lysine) was identified by screening a 6750-membered combinatorial library by the bead-diffusion assay. Sequence variations also revealed dendrimer bH1 Leu8(Dap-Leu)4(Dap-Phe)2Dap-LysNH2 (Dap = branching 2,3-diaminopropanoic acid) as a more potent analog. H1 and bH1 showed good antimicrobial activities mediated by membrane disruption (MIC = 2–4 μg mL−1 on Bacillus subtilis and Escherichia coli) but low hemolytic activity (MHC = 310 μg mL−1 respectively >2000 μg mL−1).