Sources of Intravascular ATP During Exercise in Humans: Critical Role for Skeletal Muscle Perfusion

Exercise hyperemia is regulated by several factors and one factor known to increase with exercise that evokes powerful vasomotor action is extracellular ATP. The origination of ATP detectable in plasma from exercising muscle of humans is, however, a matter of debate and ATP has been suggested to arise from sympathetic nerves, blood sources (e.g. erythrocytes), endothelial cells, and skeletal myocytes, among others. Therefore, we tested the hypothesis that acute augmentation of sympathetic nervous system activity (SNA) results in elevated plasma ATP draining skeletal muscle, and that SNA superimposition during exercise further increases ATP vs exercise alone. We show that increased SNA via −40mmHg lower body negative pressure (LBNP) at rest does not increase plasma ATP (51±8 vs 58±7 nmol/L with LBNP), nor does it increase [ATP] above levels observed during rhythmic handgrip exercise (79±11 exercise alone vs 71±8 nmol/L with LBNP). Secondly, we tested the hypothesis that active perfusion of skeletal muscle is essential to observe increased plasma ATP during exercise. We identify that complete obstruction of blood flow to contracting muscle abolishes exercise-mediated increases in plasma ATP (90±19 to 49±12 nmol/L), and further, that cessation of blood flow prior to exercise completely inhibits the typical rise in ATP (3 vs 61%; obstructed vs intact perfusion). The lack of ATP change during occlusion occurred in the face of continued muscle work and elevated SNA, indicating the rise of intravascular ATP is not resultant from these extravascular sources. Our collective observations indicate that the elevation in extracellular ATP observed in blood during exercise is unlikely to originate from sympathetic nerves or the contacting muscle itself, but rather is dependent on intact skeletal muscle perfusion. We conclude that an intravascular source for ATP is essential and points toward an important role for blood sources (e.g. red blood cells) in augmenting and maintaining elevated plasma ATP during exercise

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Characterization of the transporterB0AT3 (Slc6a17) in the rodent central nervous system

Background: The vesicular B(0)AT3 transporter (SLC6A17), one of the members of the SLC6 family, is a transporter for neutral amino acids and is exclusively expressed in brain. Here we provide a comprehensive expression profile of B(0)AT3 in mouse brain using in situ hybridization and immunohistochemistry. Results: We confirmed previous expression data from rat brain and used a novel custom made antibody to obtain detailed co-labelling with several cell type specific markers. B(0)AT3 was highly expressed in both inhibitory and excitatory neurons. The B(0)AT3 expression was highly overlapping with those of vesicular glutamate transporter 2 (VGLUT2) and vesicular glutamate transporter 1 (VGLUT1). We also show here that Slc6a17mRNA is up-regulated in animals subjected to short term food deprivation as well as animals treated with the serotonin reuptake inhibitor fluoxetine and the dopamine/noradrenaline reuptake inhibitor bupropion. Conclusions: This suggests that the B(0)AT3 transporter have a role in regulation of monoaminergic as well as glutamatergic synapses

B(0)AT2 (SLC6A15) is localized to neurons and astrocytes, and is involved in mediating the effect of leucine in the brain

The B(0)AT2 protein is a product of the SLC6A15 gene belonging to the SLC6 subfamily and has been shown to be a transporter of essential branched-chain amino acids. We aimed to further characterize the B(0)AT2 transporter in CNS, and to use Slc6a15 knock out (KO) mice to investigate whether B(0)AT2 is important for mediating the anorexigenic effect of leucine. We used the Slc6a15 KO mice to investigate the role of B(0)AT2 in brain in response to leucine and in particular the effect on food intake. Slc6a15 KO mice show lower reduction of food intake as well as lower neuronal activation in the ventromedial hypothalamic nucleus (VMH) in response to leucine injections compared to wild type mice. We also used RT-PCR on rat tissues, in situ hybridization and immunohistochemistry on mouse CNS tissues to document in detail the distribution of SLC6A15 on gene and protein levels. We showed that B(0)AT2 immunoreactivity is mainly neuronal, including localization in many GABAergic neurons and spinal cord motor neurons. B(0)AT2 immunoreactivity was also found in astrocytes close to ventricles, and co-localized with cytokeratin and diazepam binding inhibitor (DBI) in epithelial cells of the choroid plexus. The data suggest that B(0)AT2 play a role in leucine homeostasis in the brain

The biographical perspectives in youth migration

Jauniešu mobilitāte ir nozīmīgs pētījumu virziens migrācijas literatūrā. Jaunieši ir ģeogrāfiski ļoti mobila iedzīvotāju grupa. Migrācijas izpētē iedzīvotāju pārvietošanās bieži tiek skatīta cilvēka dzīves gājuma kontekstā. Šajā darbā pētītas vienas Rīgas ģimnāzijas klases absolventu migrācijas biogrāfijas, lai labāk izprastu jauniešu migrācijas komplekso raksturu Latvijā. Pētījumā izmantota mikro analītiskā pieeja jauniešu migrācijas izpētē, bet nepieciešamie dati iegūti ar padziļinātu un strukturētu interviju palīdzību. Darba rezultāti atklāj jauniešu privātās un profesionālās dzīves notikumu saikni ar ģeogrāfiskās mobilitātes procesiem. Jauniešu dzīvesvietas izvēli visbiežāk ietekmē studijas universitātē, darba gaitu uzsākšana un kopdzīves veidošana, kas ir svarīgi notikumi ikviena cilvēka dzīvē. Savukārt ikdienas pārvietošanās raksturu pārsvarā nosaka mācību un darbavietas izvēle. Kopumā rezultāti liecina, ka pat vienā sociālajā grupā jauniešu mobilitāte ir ļoti daudzpusīgs process.Mobility of young people is a relatively new line of research in migration studies revealing that young people are the most mobile section of the population. In most studies, information about human movement is presented in connection of their life course. This study researches biographical information of a specific class of Riga State Gymnasium graduates to understand the complex migration behaviour of young people in Latvia. It also uses micro-analytical approach to achieve understanding of young people`s migration patterns and uses data critical for this research acquired from detailed and structured interviews. The outcome of this study reveals linkage of private and professional circumstances of subjects’ lives with the processes connected to geographical mobility. In general, the outcome of this study provides us with the evidence that mobility of young people belonging to the same social class is a many-sided and complex process

Chemical Probes to Study ADP-Ribosylation: Synthesis and Biochemical Evaluation of Inhibitors of the Human ADP-Ribosyltransferase ARTD3/PARP3

The racemic 3-(4-oxo-3,4-dihydroquinazolin-2-yl)-<i>N</i>-[1-(pyridin-2-yl)­ethyl]­propanamide, <b>1</b>, has previously been identified as a potent but unselective inhibitor of diphtheria toxin-like ADP-ribosyltransferase 3 (ARTD3). Herein we describe synthesis and evaluation of 55 compounds in this class. It was found that the stereochemistry is of great importance for both selectivity and potency and that substituents on the phenyl ring resulted in poor solubility. Certain variations at the meso position were tolerated and caused a large shift in the binding pose. Changes to the ethylene linker that connects the quinazolinone to the amide were also investigated but proved detrimental to binding. By combination of synthetic organic chemistry and structure-based design, two selective inhibitors of ARTD3 were discovered

Cellular localization of the B⁰AT2 protein in mouse brain and spinal cord.

<p>Immunohistochemistry on free floating adult mouse brain sections using the polyclonal B⁰AT2 antibody (red), cell nucleus marker DAPI (blue), and antibody markers (green). Thin arrows indicate B⁰AT2 expressing cells and thick arrows cells labelled with markers. (<b>A</b>) The B⁰AT2 antibody stained cells in the cell membrane and in the axons, asterisk indicate a labelled axon. The B⁰AT2 protein and neuronal marker NeuN had similar expression patterns in cortex and a number of cells showed co-localization. (<b>B</b>) B⁰AT2 staining in cells surrounding and within the lateral ventricle (LV) as well as in a few cells in the inner layer of cortex. Co-localization of B⁰AT2 and NeuN was shown only in a few cells in the cortex. (<b>C</b>) High overlap of B⁰AT2 and GABAergic neuronal marker GAD67 in cortex. (<b>D</b>) B⁰AT2 protein localized to the cytoplasm around the nuclei in cells surrounding the third ventricle (3V) and in other hypothalamic cells, and high overlap was shown between B⁰AT2 and the epithelial marker pan-Cytokeratin. (<b>E</b>) High overlap between the B⁰AT2 protein and pan-Cytokeratin within the dorsal third ventricle (D3V). (<b>F</b>) B⁰AT2 showed expression in both cells surrounding the 3V and other hypothalamic cells, while the astrocyte marker GFAP expressing cells were located more close to the 3V. A number of B⁰AT2 expressing cells co-localized with GFAP. (<b>G</b>) B⁰AT2 expression in motor neurons in spinal cord (L2), with B⁰AT2 labelling the cytosol and the neuronal axons. B⁰AT2-labelled cells did not co-localize with the vesicle marker synaptophysin. (<b>H</b>) B⁰AT2 was found in cells following the edge of the LV and in cells within the ventricle, while the DBI positive cells was only found in ependymocytes bordering the ventricle. High overlap was seen for B⁰AT2 and the product of the DBI gene. (<b>I</b>) B⁰AT2 expression in hypothalamus, with a number of cells stained close to the upper part of the 3V. The DBI positive cells were located in the epithelial cells surrounding the ventricle and spread though the hypothalamus. Co-localization of B⁰AT2 and DBI positive cells was only found in the epithelial cells. The A, C, D and G images are photographed with 40× magnification and 20× magnification was used for the B, E, F, H and I images. Row A and B are at Bregma −0.46, row C–E at −1.58 and row F, H and I at −0.82.</p

Cellular co-localization of B⁰AT2, GFAP and DBI positive cells.

<p>Immunohistochemistry on free floating adult mouse brain sections using the polyclonal B⁰AT2 antibody (red), cell nucleus marker DAPI (blue), DBI marker (green), and GFAP marker (white). Thin arrows indicate B⁰AT2 expressing cells, thick arrows DBI labelled cells and star-like arrows GFAP expressing cells. <b>A row</b>; Hypothalamic expression of B⁰AT2 both close to the third ventricle and in the surrounding areas. The expression of the two markers DBI and GFAP is seen in cells surrounding the ventricle and the border of the brain section. <b>B row</b>; Co-localization of B⁰AT2 and GFAP and the co-localization of B⁰AT2 and DBI expressing cells only in cells close to the ventricle and in epithelial cells surrounding the third ventricle. The section is at Bregma −1.06 and 20× magnification was used for the image.</p

<i>Slc6a15</i> mRNA expression in mouse brain and spinal cord.

<p>Floating <i>in situ</i> hybridization using 500 ng digoxigenin labelled mouse <i>Slc6a15</i> probe to detect cells and nuclei populations expressing the mRNA, with overview image of coronal mouse brain sections (<b>A–E</b>), close up images (<b>F–L</b>) and spinal cord (<b>M</b>). The Bregma coordinates, abbreviations and described brain regions is depicted using Franklin and Paxinos 2007 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058651#pone.0058651-Franklin1" target="_blank">[18]</a>. <i>Slc6a15</i> mRNA brain expression is found in following brain regions. (<b>F</b>) Patches of caudate putamen (striatum, CPu). (<b>G</b>) Supraoptic nucleus (SO), rhomboid thalamic nucleus (Rh), zona incerta (ZI), paraventricular hypothalamic nucleus dorsal cap (PaDC), paraventricular hypothalamic nucleus lateral magnocellular part (PaLM), paraventricular hypothalamic nucleus medial mag nocellular part (PaMM) and periventricular hypothalamic nucleus (Pe). (<b>H</b>) Cortical layer 2–6, granule cell layer of the dentate gyrus (GrDG) and pyramidal cell layer of the hippocampus (Py). (<b>I</b>) Rhomboid thalamic nucleus (Rh), zona incerta (ZI), paraventricular hypothalamic nucleus posterior part (PaPo), anterior hypothalamic area, posterior part (AHP), anterior hypothalamic area, central part (AHC) and ventromedial hypothalamic nucleus (VMH). (<b>J</b>) Lateral amygdaloid nucleus dorsolateral part (LaDL), lateral amygdaloid nucleus ventrolateral part (LaVL), dorsal endopiriform claustrum (DEn), basolateral amygdaloid nucleus anterior part (BLA), basolateral amygdaloid nucleus posterior part (BLP), basomedial amygdaloid nucleus posterior part (BMP), piriform cortex (Pir), posterolateral cortical amygdaloid area (PLCo), medial amygdaloid nucleus posterodorsal part (MePD) and medial amygdaloid nucleus posteroventral part (MePV). (<b>K</b>) Zona incerta ventral part (ZIV), dorsomedial hypothalamic nucleus (DM), ventromedial hypothalamic nucleus dorsomedial part (VMHDM), ventromedial hypothalamic nucleus central part (VMHC), ventromedial hypothalamic nucleus ventrolateral part (VMHVL) and arcuate hypothalamic nucleus (Arc). (<b>L</b>) Locus coeruleus (LC) and Barrington's nucleus (Bar). (<b>M</b>) The spinal cord mRNA expression of <i>Slc6a15</i> is found in subsets of somatic motor neurons and interneurons in the upper vertebrae L2 lumbar.</p