Gas-chromatography mass-spectrometry determination of phthalic acid in human urine as a biomarker of folpet exposure.

Agricultural workers are exposed to folpet, but biomonitoring data are limited. Phthalimide (PI), phthalamic acid (PAA), and phthalic acid (PA) are the ring metabolites of this fungicide according to animal studies, but they have not yet been measured in human urine as metabolites of folpet, only PA as a metabolite of phthalates. The objective of this study was thus to develop a reliable gas chromatography-tandem mass spectrometry (GC-MS) method to quantify the sum of PI, PAA, and PA ring-metabolites of folpet in human urine. Briefly, the method consisted of adding p-methylhippuric acid as an internal standard, performing an acid hydrolysis at 100 °C to convert ring-metabolites into PA, purifying samples by ethyl acetate extraction, and derivatizing with N,O-bis(trimethylsilyl)trifluoro acetamide prior to GC-MS analysis. The method had a detection limit of 60.2 nmol/L (10 ng/mL); it was found to be accurate (mean recovery, 97%), precise (inter- and intra-day percentage relative standard deviations <13%), and with a good linearity (R (2) > 0.98). Validation was conducted using unexposed peoples urine spiked at concentrations ranging from 4.0 to 16.1 μmol/L, along with urine samples of volunteers dosed with folpet, and of exposed workers. The method proved to be (1) suitable and accurate to determine the kinetic profile of PA equivalents in the urine of volunteers orally and dermally administered folpet and (2) relevant for the biomonitoring of exposure in workers.

Effect of abscisic acid on maize root gravireaction

Primary roots of intact maize seedlings (cv. LG11) were pretreated when kept vertically (1 or 2 in the dark) with a droplet of buffer solution containing ABA (at different concentrations) applied to the root tip. Then, apical root segments were prepared and placed horizontally in both light and darkness. The downward curvature was measured. In the light, curvature was greater than in darkness and ABA (5×10−5 mol·dm−3) significantly enhanced the curvature in both darkness and light. The amplitude of the increase in the gravireaction was found to be dependent on the ABA concentration and the duration of the pretreatment.

International union of basic and clinical pharmacology. XCI. structure, function, and pharmacology of acid-sensing ion channels and the epithelial Na+ channel.

The epithelial Na(+) channel (ENaC) and the acid-sensing ion channels (ASICs) form subfamilies within the ENaC/degenerin family of Na(+) channels. ENaC mediates transepithelial Na(+) transport, thereby contributing to Na(+) homeostasis and the maintenance of blood pressure and the airway surface liquid level. ASICs are H(+)-activated channels found in central and peripheral neurons, where their activation induces neuronal depolarization. ASICs are involved in pain sensation, the expression of fear, and neurodegeneration after ischemia, making them potentially interesting drug targets. This review summarizes the biophysical properties, cellular functions, and physiologic and pathologic roles of the ASIC and ENaC subfamilies. The analysis of the homologies between ENaC and ASICs and the relation between functional and structural information shows many parallels between these channels, suggesting that some mechanisms that control channel activity are shared between ASICs and ENaC. The available crystal structures and the discovery of animal toxins acting on ASICs provide a unique opportunity to address the molecular mechanisms of ENaC and ASIC function to identify novel strategies for the modulation of these channels by pharmacologic ligands.

Mice with an adipocyte-specific lipin 1 separation-of-function allele reveal unexpected roles for phosphatidic acid in metabolic regulation.

Lipin 1 is a coregulator of DNA-bound transcription factors and a phosphatidic acid (PA) phosphatase (PAP) enzyme that catalyzes a critical step in the synthesis of glycerophospholipids. Lipin 1 is highly expressed in adipocytes, and constitutive loss of lipin 1 blocks adipocyte differentiation; however, the effects of Lpin1 deficiency in differentiated adipocytes are unknown. Here we report that adipocyte-specific Lpin1 gene recombination unexpectedly resulted in expression of a truncated lipin 1 protein lacking PAP activity but retaining transcriptional regulatory function. Loss of lipin 1-mediated PAP activity in adipocytes led to reduced glyceride synthesis and increased PA content. Characterization of the deficient mice also revealed that lipin 1 normally modulates cAMP-dependent signaling through protein kinase A to control lipolysis by metabolizing PA, which is an allosteric activator of phosphodiesterase 4 and the molecular target of rapamycin. Consistent with these findings, lipin 1 expression was significantly related to adipose tissue lipolytic rates and protein kinase A signaling in adipose tissue of obese human subjects. Taken together, our findings identify lipin 1 as a reciprocal regulator of triglyceride synthesis and hydrolysis in adipocytes, and suggest that regulation of lipolysis by lipin 1 is mediated by PA-dependent modulation of phosphodiesterase 4.

Deoxyribonucleic acid content as an indicator of progression of squamous cell carcinogenesis in the esophagus: comparative analysis on imprint-cytospin and tissue section preparation.

BACKGROUND: The purpose of this study was to explore the potential use of image analysis on tissue sections preparation as a predictive marker of early malignant changes during squamous cell (SC) carcinogenesis in the esophagus. Results of DNA ploidy quantification on formalin-fixed, paraffin-embedded tissue using two different techniques were compared: imprint-cytospin and 6 microm thick tissue sections preparation. METHODS: This retrospective study included 26 surgical specimens of squamous cell carcinoma (SCC) from patients who underwent surgery alone at the Department of Surgery in CHUV Hospital in Lausanne between January 1993 and December 2000. We analyzed 53 samples of healthy tissue, 43 tumors and 7 lymph node metastases. RESULTS: Diploid DNA histogram patterns were observed in all histologically healthy tissues, either distant or proximal to the lesion. Aneuploidy was observed in 34 (79%) of 43 carcinomas, namely 24 (75%) of 32 early squamous cell carcinomas and 10 (91%) of 11 advanced carcinomas. DNA content was similar in the different tumor stages, whether patients presented with single or multiple synchronous tumors. All lymph node metastases had similar DNA content as their primary tumor. CONCLUSIONS: Early malignant changes in the esophagus are associated with alteration in DNA content, and aneuploidy tends to correlate with progression of invasive SCC. A very good correlation between imprint-cytospin and tissue section analysis was observed. Although each method used here showed advantages and disadvantages; tissue sections preparation provided useful information on aberrant cell-cycle regulation and helped select the optimal treatment for the individual patient along with consideration of other clinical parameters.

Pitfalls in aminoacid and organic acid analysis: 3-hydroxypropionic aciduria.

Pitfalls in organic acid analysis can originate from inadequate methodology, analytical interferences, in vivo interactions and from pre-analytical conditions which often are unknown to the specialized analytical laboratory. Among the latter, ingested food and additives, metabolites of food processing or medications have to be considered. Bacterial metabolites from the gastrointestinal or urogenital system or formed after sample collection can lead to pitfalls as well. An example of such a patient whose urinary metabolites mimic at first glance inherited propionic aciduria is described.

Linking supply to demand: the neuronal monocarboxylate transporter MCT2 and the alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid receptor GluR2/3 subunit are associated in a common trafficking process.

MCT2 is the major neuronal monocarboxylate transporter (MCT) that allows the supply of alternative energy substrates such as lactate to neurons. Recent evidence obtained by electron microscopy has demonstrated that MCT2, like alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid (AMPA) receptors, is localized in dendritic spines of glutamatergic synapses. Using immunofluorescence, we show in this study that MCT2 colocalizes extensively with GluR2/3 subunits of AMPA receptors in neurons from various mouse brain regions as well as in cultured neurons. It also colocalizes with GluR2/3-interacting proteins, such as C-kinase-interacting protein 1, glutamate receptor-interacting protein 1 and clathrin adaptor protein. Coimmunoprecipitation of MCT2 with GluR2/3 and C-kinase-interacting protein 1 suggests their close interaction within spines. Parallel changes in the localization of both MCT2 and GluR2/3 subunits at and beneath the plasma membrane upon various stimulation paradigms were unraveled using an original immunocytochemical and transfection approach combined with three-dimensional image reconstruction. Cell culture incubation with AMPA or insulin triggered a marked intracellular accumulation of both MCT2 and GluR2/3, whereas both tumor necrosis factor alpha and glycine (with glutamate) increased their cell surface immunolabeling. Similar results were obtained using Western blots performed on membrane or cytoplasm-enriched cell fractions. Finally, an enhanced lactate flux into neurons was demonstrated after MCT2 translocation on the cell surface. These observations provide unequivocal evidence that MCT2 is linked to AMPA receptor GluR2/3 subunits and undergoes a similar translocation process in neurons upon activation. MCT2 emerges as a novel component of the synaptic machinery putatively linking neuroenergetics to synaptic transmission.

Beta-oxidation in fatty acid degradation and beyond.

The degradation of fatty acids in plants occurs primarily in the peroxisomes through the beta-oxidation cycle. Enzymes that are involved in various aspects of beta-oxidation have been identified recently and shown to act biochemically on a diversity of fatty acids and derivatives. Analysis of several mutants has revealed essential roles for beta-oxidation in the breakdown of reserve triacylglycerols, seed development, seed germination and post-germinative growth before the establishment of photosynthesis. Beta-oxidation has also a considerable importance during the vegetative and reproductive growth phases, and plays a role in plant responses to stress, particularly in the synthesis of jasmonic acid.

Characterization of rapeseed (Brassica napus) oils by bulk C, O, H, and fatty acid C stable isotope analyses

Rapeseed (Brassica napus) oils differing in cultivar, sites of growth, and harvest year were characterized by fatty acid concentrations and carbon, hydrogen, and oxygen stable isotope analyses of bulk oils (delta(13)C(bulk), delta(2)H(bulk), delta(18)O(bulk) values) and individual fatty acids (delta(13)C(FA)). The delta(13)C(bulk), delta(2)H(bulk), and delta(18)O(bulk) values were determined by continuous flow combustion and high-temperature conversion elemental analyzer isotope ratio mass spectrometry (EA/IRMS, TC-EA/IRMS). The delta(13)C(FA) values were determined using gas chromatography-combustion isotope ratio mass spectrometry (GC/C/IRMS). For comparison, other C(3) vegetable oils rich in linolenic acid (flax and false flax oils) and rich in linoleic acid (poppy, sunflower, and safflower oils) were submitted to the same chemical and isotopic analyses. The bulk and molecular delta(13)C values were typical for C(3) plants. The delta(13)C value of palmitic acid (delta(13)C(16:0)) and n-3 alpha-linolenic acid (delta(13)C(18:3n-3)) differed (p < 0.001) between rape, flax, and poppy oils. Also within species, significant differences of delta(13)C(FA) were observed (p < 0.01). The hydrogen and oxygen isotope compositions of rape oil differed between cultivars (p < 0.05). Major differences in the individual delta(13)C(FA) values were found. A plant-specific carbon isotope fractionation occurs during the biosynthesis of the fatty acids and particularly during desaturation of C(18) acids in rape and flax. Bulk oil and specific fatty acid stable isotope analysis might be useful in tracing dietary lipids differing in their origin.

Auxin response factors ARF6 and ARF8 promote jasmonic acid production and flower maturation.

Pollination in flowering plants requires that anthers release pollen when the gynoecium is competent to support fertilization. We show that in Arabidopsis thaliana, two paralogous auxin response transcription factors, ARF6 and ARF8, regulate both stamen and gynoecium maturation. arf6 arf8 double-null mutant flowers arrested as infertile closed buds with short petals, short stamen filaments, undehisced anthers that did not release pollen and immature gynoecia. Numerous developmentally regulated genes failed to be induced. ARF6 and ARF8 thus coordinate the transition from immature to mature fertile flowers. Jasmonic acid (JA) measurements and JA feeding experiments showed that decreased jasmonate production caused the block in pollen release, but not the gynoecium arrest. The double mutant had altered auxin responsive gene expression. However, whole flower auxin levels did not change during flower maturation, suggesting that auxin might regulate flower maturation only under specific environmental conditions, or in localized organs or tissues of flowers. arf6 and arf8 single mutants and sesquimutants (homozygous for one mutation and heterozygous for the other) had delayed stamen development and decreased fecundity, indicating that ARF6 and ARF8 gene dosage affects timing of flower maturation quantitatively.

Uric acid and cardio-metabolic risk factors : an epidemiological approach

The role of serum uric acid (SUA) in cardio-metabolic conditions has long been contentious. It is still unclear if SUA is an independent risk factor or marker of cardio-metabolic conditions and most observed associations are not necessarily causal. This study aimed to further understand and explore the causal role of SUA in cardio-metabolic conditions using genetic and non-genetic epidemiological methods in population-based data. In the first part of this study, we found moderate to high heritability estimates for SUA and fractional excretion of urate (FEUA) suggesting the role of genetic factors in the etiology of hyperuricemia. With regards to the role of SUA on inflammatory markers (IMs), a strong positive association of SUA with C-reactive protein (CRP) and a weaker positive association with tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) was observed, which was in part mediated by body mass index (BMI). These findings suggest that SUA may have a role in sterile inflammation. In view of the inconsistency surrounding the causal nature and direction of the relation between SUA and adiposity, we applied a bidirectional Mendelian randomization approach using genetic variants to decipher the association. The finding that elevated SUA is a consequence rather than a cause of adiposity was not totally unexpected and is compatible with the hypothesis that hyperinsulinemia, accompanying obesity, enhances renal proximal tubular reabsorption of uric acid. The fourth part of this study examined the relationship between SUA and blood pressure (BP) in young adults. The association between SUA and BP, significant only in females, was strongly attenuated upon adjustment for BMI. The possibility that BMI lies in the causal pathway may explain the attenuation observed in the associations of SUA with BP and IMs. Finally, a significant hockey-stick shaped association of SUA with social phobia in our data suggests a protective effect of SUA only up to a certain concentration. Although our study findings have shed some light on the uncertainty underlying the pathophysiology of SUA, more compelling evidence using longitudinal designs, randomized controlled trials and the use of robust genetic tools is warranted to increase our understanding of the clinical significance of SUA.

Dinor-oxo-phytodienoic acid: a new hexadecanoid signal in the jasmonate family.

Jasmonic acid and its precursors are potent regulatory molecules in plants. We devised a method for the simultaneous extraction of these compounds from plant leaves to quantitate changes in the levels of jasmonate family members during health and on wounding. During our study, we identified a novel 16-carbon cyclopentenoic acid in leaf extracts from Arabidopsis and potato. The new compound, a member of the jasmonate family of signals, was named dinor-oxo-phytodienoic acid. Dinor-oxo-phytodienoic acid was not detected in the Arabidopsis mutant fad5, which is incapable of synthesizing 7Z,10Z, 13Z-hexadecatrienoic acid (16:3), suggesting that the metabolite is derived directly from plastid 16:3 rather than by beta-oxidation of the 18-carbon 12-oxo-phytodienoic acid. Simultaneous quantitation of jasmonate family members in healthy leaves of Arabidopsis and potato suggest that different plant species have different relative levels of jasmonic acid, oxo-phytodienoic acid, and dinor-oxo-phytodienoic acid. We term these profiles "oxylipin signatures." Dinor-oxo-phytodienoic acid levels increased dramatically in Arabidopsis and potato leaves on wounding, suggesting roles in wound signaling. Treatment of Arabidopsis with micromolar levels of dinor-oxo-phytodienoic acid increased the ability of leaf extracts to transform linoleic acid into the alpha-ketol 13-hydroxy-12-oxo-9(Z) octadecenoic acid indicating that the compound can regulate part of its own biosynthetic pathway. Tightly regulated changes in the relative levels of biologically active jasmonates may permit sensitive control over metabolic, developmental, and defensive processes in plants.