Amiloride-sensitive channels in type I fungiform taste cells in mouse

<p>Abstract</p> <p>Background</p> <p>Taste buds are the sensory organs of taste perception. Three types of taste cells have been described. Type I cells have voltage-gated outward currents, but lack voltage-gated inward currents. These cells have been presumed to play only a support role in the taste bud. Type II cells have voltage-gated Na⁺and K⁺current, and the receptors and transduction machinery for bitter, sweet, and umami taste stimuli. Type III cells have voltage-gated Na⁺, K⁺, and Ca²⁺currents, and make prominent synapses with afferent nerve fibers. Na⁺salt transduction in part involves amiloride-sensitive epithelial sodium channels (ENaCs). In rodents, these channels are located in taste cells of fungiform papillae on the anterior part of the tongue innervated by the chorda tympani nerve. However, the taste cell type that expresses ENaCs is not known. This study used whole cell recordings of single fungiform taste cells of transgenic mice expressing GFP in Type II taste cells to identify the taste cells responding to amiloride. We also used immunocytochemistry to further define and compare cell types in fungiform and circumvallate taste buds of these mice.</p> <p>Results</p> <p>Taste cell types were identified by their response to depolarizing voltage steps and their presence or absence of GFP fluorescence. TRPM5-GFP taste cells expressed large voltage-gated Na⁺and K⁺currents, but lacked voltage-gated Ca²⁺currents, as expected from previous studies. Approximately half of the unlabeled cells had similar membrane properties, suggesting they comprise a separate population of Type II cells. The other half expressed voltage-gated outward currents only, typical of Type I cells. A single taste cell had voltage-gated Ca²⁺current characteristic of Type III cells. Responses to amiloride occurred only in cells that lacked voltage-gated inward currents. Immunocytochemistry showed that fungiform taste buds have significantly fewer Type II cells expressing PLC signalling components, and significantly fewer Type III cells than circumvallate taste buds.</p> <p>Conclusion</p> <p>The principal finding is that amiloride-sensitive Na⁺channels appear to be expressed in cells that lack voltage-gated inward currents, likely the Type I taste cells. These cells were previously assumed to provide only a support function in the taste bud.</p

Comparison of hemodynamic, biochemical and hematological parameters of healthy pregnant women in the third trimester of pregnancy and the active labor phase

<p>Abstract</p> <p>Background</p> <p>Pregnancy is accompanied by several hemodynamic, biochemical and hematological changes which revert to normal values after labor. The mean values of these parameters have been reported for developed countries, but not for Mexican women. Furthermore, labor constitutes a stress situation, in which these factors may be altered. It is known that serologic increase of heat shock protein (Hsp) 70 is associated with abnormal pregnancies, presenting very low level in normal pregnant women. Nevertheless, there are no studies where these measurements are compared in healthy pregnant women at their third trimester of pregnancy (3TP) and the active labor phase (ActLP).</p> <p>Methods</p> <p>Seventy five healthy Mexican pregnant women were included. Hemodynamic, biochemical and hematological parameters were obtained in all cases, and serum Hsp70 levels were measured in a sample of 15 women at 3TP and at ActLP.</p> <p>Results</p> <p>Significant differences were found in most analysis performed and in Hsp70 concentration at 3TP as compared to ActLP, however all were within normal range in both conditions, supporting that only in pathological pregnancies Hsp70 is drastically increased.</p> <p>Conclusion</p> <p>Results obtained indicate that 3TP and ActLP have clinical similarities in normal pregnancies, therefore if abnormalities are found during 3TP, precautions should be taken before ActLP.</p

Expression of Genes Encoding Multi-Transmembrane Proteins in Specific Primate Taste Cell Populations

BACKGROUND: Using fungiform (FG) and circumvallate (CV) taste buds isolated by laser capture microdissection and analyzed using gene arrays, we previously constructed a comprehensive database of gene expression in primates, which revealed over 2,300 taste bud-associated genes. Bioinformatics analyses identified hundreds of genes predicted to encode multi-transmembrane domain proteins with no previous association with taste function. A first step in elucidating the roles these gene products play in gustation is to identify the specific taste cell types in which they are expressed. METHODOLOGY/PRINCIPAL FINDINGS: Using double label in situ hybridization analyses, we identified seven new genes expressed in specific taste cell types, including sweet, bitter, and umami cells (TRPM5-positive), sour cells (PKD2L1-positive), as well as other taste cell populations. Transmembrane protein 44 (TMEM44), a protein with seven predicted transmembrane domains with no homology to GPCRs, is expressed in a TRPM5-negative and PKD2L1-negative population that is enriched in the bottom portion of taste buds and may represent developmentally immature taste cells. Calcium homeostasis modulator 1 (CALHM1), a component of a novel calcium channel, along with family members CALHM2 and CALHM3; multiple C2 domains; transmembrane 1 (MCTP1), a calcium-binding transmembrane protein; and anoctamin 7 (ANO7), a member of the recently identified calcium-gated chloride channel family, are all expressed in TRPM5 cells. These proteins may modulate and effect calcium signalling stemming from sweet, bitter, and umami receptor activation. Synaptic vesicle glycoprotein 2B (SV2B), a regulator of synaptic vesicle exocytosis, is expressed in PKD2L1 cells, suggesting that this taste cell population transmits tastant information to gustatory afferent nerve fibers via exocytic neurotransmitter release. CONCLUSIONS/SIGNIFICANCE: Identification of genes encoding multi-transmembrane domain proteins expressed in primate taste buds provides new insights into the processes of taste cell development, signal transduction, and information coding. Discrete taste cell populations exhibit highly specific gene expression patterns, supporting a model whereby each mature taste receptor cell is responsible for sensing, transmitting, and coding a specific taste quality

On conditional skewness with applications to environmental data

The statistical literature contains many univariate and multivariate skewness measures that allow two datasets to be compared, some of which are defined in terms of quantile values. In most situations, the comparison between two random vectors focuses on univariate comparisons of conditional random variables truncated in quantiles; this kind of comparison is of particular interest in the environmental sciences. In this work, we describe a new approach to comparing skewness in terms of the univariate convex transform ordering proposed by van Zwet (Convex transformations of random variables. Mathematical Centre Tracts, Amsterdam, 1964), associated with skewness as well as concentration. The key to these comparisons is the underlying dependence structure of the random vectors. Below we describe graphical tools and use several examples to illustrate these comparisons.The research of Félix Belzunce, Julio Mulero and José María Ruíz is partially funded by the Ministerio de Economía y Competitividad (Spain) under Grant MTM2012-34023-FEDER. Alfonso Suárez-Llorens acknowledges support received from the Ministerio de Economía y Competitividad (Spain) under Grant MTM2014-57559-P

Insights from a national survey into why substance abuse treatment units add prevention and outreach services

BACKGROUND: Previous studies have found that even limited prevention-related interventions can affect health behaviors such as substance use and risky sex. Substance abuse treatment providers are ideal candidates to provide these services, but typically have little or no financial incentive to do so. The purpose of this study was therefore to explore why some substance abuse treatment units have added new prevention and outreach services. Based on an ecological framework of organizational strategy, three categories of predictors were tested: (1) environmental, (2) unit-level, and (3) unit leadership. RESULTS: A lagged cross-sectional logistic model of 450 outpatient substance abuse treatment units revealed that local per capita income, mental health center affiliation, and clinical supervisors' graduate degrees were positively associated with likelihood of adding prevention-related education and outreach services. Managed care contracts and methadone treatment were negatively associated with addition of these services. No hospital-affiliated agencies added prevention and outreach services during the study period. CONCLUSION: Findings supported the study's ecological perspective on organizational strategy, with factors at environmental, unit, and unit leadership levels associated with additions of prevention and outreach services. Among the significant predictors, ties to managed care payers and unit leadership graduate education emerge as potential leverage points for public policy. In the current sample, units with managed care contracts were less likely to add prevention and outreach services. This is not surprising, given managed care's emphasis on cost control. However, the association with this payment source suggests that public managed care programs might affects prevention and outreach differently through revised incentives. Specifically, government payers could explicitly compensate substance abuse treatment units in managed care contracts for prevention and outreach. The effects of supervisor graduate education on likelihood of adding new prevention and outreach programs suggests that leaders' education can affect organizational strategy. Foundation and government officials may encourage prevention and outreach by funding curricular enhancements to graduate degree programs demonstrating the importance of public goods. Overall, these findings suggest that both money and professional education affect substance abuse treatment unit additions of prevention and outreach services, as well as other factors less amenable to policy intervention

Effect of hypoxia and Beraprost sodium on human pulmonary arterial smooth muscle cell proliferation: the role of p27kip1

<p>Abstract</p> <p>Background</p> <p>Hypoxia induces the proliferation of pulmonary arterial smooth muscle cell (PASMC) <it>in vivo </it>and <it>in vitro</it>, and prostacyclin analogues are thought to inhibit the growth of PASMC. Previous studies suggest that p27^kip1, a kind of cyclin-dependent kinase inhibitor, play an important role in the smooth muscle cell proliferation. However, the mechanism of hypoxia and the subcellular interactions between p27^kip1and prostacyclin analogues in human pulmonary arterial smooth muscle cell (HPASMC) are not fully understood.</p> <p>Methods</p> <p>We investigated the role of p27^kip1in the ability of Beraprost sodium (BPS; a stable prostacyclin analogue) to inhibit the proliferation of HPASMC during hypoxia. To clarify the biological effects of hypoxic air exposure and BPS on HPASMC, the cells were cultured in a hypoxic chamber under various oxygen concentrations (0.1–21%). Thereafter, DNA synthesis was measured as bromodeoxyuridine (BrdU) incorporation, the cell cycle was analyzed by flow cytometry with propidium iodide staining. The p27^kip1mRNA and protein expression and it's stability was measured by real-time RT-PCR and Western blotting. Further, we assessed the role of p27^kip1in HPASMC proliferation using p27^kip1gene knockdown using small interfering RNA (siRNA) transfection.</p> <p>Results</p> <p>Although severe hypoxia (0.1% oxygen) suppressed the proliferation of serum-stimulated HPASMC, moderate hypoxia (2% oxygen) enhanced proliferation in accordance with enhanced p27^kip1protein degradation, whereas BPS suppressed HPASMC proliferation under both hypoxic and normoxic conditions by suppressing p27^kip1degradation with intracellular cAMP-elevation. The 8-bromo-cyclic adenosine monophosphate (8-Br-cAMP), a cAMP analogue, had similar action as BPS in the regulation of p27^kip1. Moderate hypoxia did not affect the stability of p27^kip1protein expression, but PDGF, known as major hypoxia-induced growth factors, significantly decreased p27^kip1protein stability. We also demonstrated that BPS and 8-Br-cAMP suppressed HPASMC proliferation under both hypoxic and normoxic conditions by blocking p27^kip1mRNA degradation. Furthermore, p27^kip1gene silencing partially attenuated the effects of BPS and partially restored hypoxia-induced proliferation.</p> <p>Conclusion</p> <p>Our study suggests that moderate hypoxia induces HPASMC proliferation, which is partially dependent of p27^kip1down-regulation probably <it>via </it>the induction of growth factors such as PDGF, and BPS inhibits both the cell proliferation and p27^kip1mRNA degradation through cAMP pathway.</p

Accelerated turnover of taste bud cells in mice deficient for the cyclin-dependent kinase inhibitor p27Kip1

Background: Mammalian taste buds contain several specialized cell types that coordinately respond to tastants and communicate with sensory nerves. While it has long been appreciated that these cells undergo continual turnover, little is known concerning how adequate numbers of cells are generated and maintained. The cyclin-dependent kinase inhibitor p27Kip1 has been shown to influence cell number in several developing tissues, by coordinating cell cycle exit during cell differentiation. Here, we investigated its involvement in the control of taste cell replacement by examining adult mice with targeted ablation of the p27Kip1 gene.Results: Histological and morphometric analyses of fungiform and circumvallate taste buds reveal no structural differences between wild-type and p27Kip1-null mice. However, when examined in functional assays, mutants show substantial proliferative changes. In BrdU incorporation experiments, more S-phase-labeled precursors appear within circumvallate taste buds at 1 day post-injection, the earliest time point examined. After 1 week, twice as many labeled intragemmal cells are present, but numbers return to wild-type levels by 2 weeks. Mutant taste buds also contain more TUNEL-labeled cells and 50% more apoptotic bodies than wild-type controls. In normal mice, p27 Kip1 is evident in a subset of receptor and presynaptic taste cells beginning about 3 days post-injection, correlating with the onset of taste cell maturation. Loss of gene function, however, does not alter the proportions of distinct immunohistochemically-identified cell types.Conclusions: p27Kip1 participates in taste cell replacement by regulating the number of precursor cells available for entry into taste buds. This is consistent with a role for the protein in timing cell cycle withdrawal in progenitor cells. The equivalence of mutant and wild-type taste buds with regard to cell number, cell types and general structure contrasts with the hyperplasia and tissue disruption seen in certain developing p27Kip1-null sensory organs, and may reflect a compensatory capability inherent in the regenerative taste system

Sarco/Endoplasmic Reticulum Ca2+-ATPases (SERCA) Contribute to GPCR-Mediated Taste Perception

The sense of taste is important for providing animals with valuable information about the qualities of food, such as nutritional or harmful nature. Mammals, including humans, can recognize at least five primary taste qualities: sweet, umami (savory), bitter, sour, and salty. Recent studies have identified molecules and mechanisms underlying the initial steps of tastant-triggered molecular events in taste bud cells, particularly the requirement of increased cytosolic free Ca2+ concentration ([Ca2+]c) for normal taste signal transduction and transmission. Little, however, is known about the mechanisms controlling the removal of elevated [Ca2+]c from the cytosol of taste receptor cells (TRCs) and how the disruption of these mechanisms affects taste perception. To investigate the molecular mechanism of Ca2+ clearance in TRCs, we sought the molecules involved in [Ca2+]c regulation using a single-taste-cell transcriptome approach. We found that Serca3, a member of the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) family that sequesters cytosolic Ca2+ into endoplasmic reticulum, is exclusively expressed in sweet/umami/bitter TRCs, which rely on intracellular Ca2+ release for signaling. Serca3-knockout (KO) mice displayed significantly increased aversive behavioral responses and greater gustatory nerve responses to bitter taste substances but not to sweet or umami taste substances. Further studies showed that Serca2 was mainly expressed in the T1R3-expressing sweet and umami TRCs, suggesting that the loss of function of Serca3 was possibly compensated by Serca2 in these TRCs in the mutant mice. Our data demonstrate that the SERCA family members play an important role in the Ca2+ clearance in TRCs and that mutation of these proteins may alter bitter and perhaps sweet and umami taste perception