Low incidence of polyploids and high uniformity of karyotypes displayed by Delphinium (Ranunculaceae) in the Hengduan Mountains region of south‐west China

The chromosome numbers and morphology in 92 populations belonging to 49 species and three varieties in the genus Delphinium L. (Ranunculaceae), mostly from the Hengduan Mountains region of south‐west China, were studied. Forty seven species and three varieties were diploid, with 2n = 16, one species was tetraploid, with 2n = 32, and one species had diploid and tetraploid cytotypes. Three species had B chromosomes, representing the first time the occurrence of B chromosomes has been reported in the genus. The karyotypes of all the diploid species were quite uniform, commonly bimodal, and usually consisted of one pair of large median‐centromeric (m), one pair of large submedian‐centromeric (sm), five pairs of medium‐sized subterminal‐centromeric (st), and one pair of smaller sm (rarely st) chromosomes. The low incidence of polyploids in Delphinium from the Hengduan Mountains region indicates that polyploidy has played a minor role in the speciation of this highly diversified genus in the region. © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society, 2008, 158 , 172–188.     

Homeostasis of extracellular ATP in human erythrocytes

We explored the intra- and extracellular processes governing the kinetics of extracellular ATP (ATPe) in human erythrocytes stimulated with agents that increase cAMP. Using the luciferin-luciferase reaction in off-line luminometry we found both direct adenylyl cyclase activation by forskolin and indirect activation through β-adrenergic stimulation with isoproterenol-enhanced [ATP]e in a concentration-dependent manner. A mixture (3V) containing a combination of these agents and the phosphodiesterase inhibitor papaverine activated ATP release, leading to a 3-fold increase in [ATP]e, and caused increases in cAMP concentration (3-fold for forskolin + papaverine, and 10-fold for 3V). The pannexin 1 inhibitor carbenoxolone and a pannexin 1 blocking peptide ((10)Panx1) decreased [ATP]e by 75-84%. The residual efflux of ATP resulted from unavoidable mechanical perturbations stimulating a novel, carbenoxolone-insensitive pathway. In real-time luminometry experiments using soluble luciferase, addition of 3V led to an acute increase in [ATP]e to a constant value of ～1 pmol × (10(6) cells)(-1). A similar treatment using a surface attached luciferase (proA-luc) triggered a rapid accumulation of surface ATP levels to a peak concentration of 2.4 pmol × (10(6) cells)(-1), followed by a slower exponential decay (t(½) = 3.7 min) to a constant value of 1.3 pmol × (10(6) cells)(-1). Both for soluble luciferase and proA-luc, ATP efflux was fully blocked by carbenoxolone, pointing to a 3V-induced mechanism of ATP release mediated by pannexin 1. Ecto-ATPase activity was extremely low (～28 fmol × (10(6) cells min)(-1)), but nevertheless physiologically relevant considering the high density of erythrocytes in human blood.     

Promoter hypermethylation using 24-gene array in early head and neck cancer: Better outcome in oral than in oropharyngeal cancer

Silencing of tumor suppressor genes (TSGs) by DNA promoter hypermethylation is an early event in carcinogenesis and a potential target for personalized cancer treatment. In head and neck cancer, little is known about the role of promoter hypermethylation in survival. Using methylation specific multiplex ligation-dependent probe amplification (MS-MLPA) we investigated the role of promoter hypermethylation of 24 well-described genes (some of which are classic TSGs), which are frequently methylated in different cancer types, in 166 HPV-negative early oral squamous cell carcinomas (OSCC), and 51 HPV-negative early oropharyngeal squamous cell carcinomas (OPSCC) in relation to clinicopathological features and survival. Early OSCC showed frequent promoter hypermethylation in RARB (31% of cases), CHFR (20%), CDH13 (13%), DAPK1 (12%), and APC (10%). More hypermethylation (≥ 2 genes) independently correlated with improved disease specific survival (hazard ratio 0.17, P = 0.014) in early OSCC and could therefore be used as prognostic biomarker. Early OPSCCs showed more hypermethylation of CDH13 (58%), TP73 (14%), and total hypermethylated genes. Hypermethylation of two or more genes has a significantly different effect on survival in OPSCC compared with OSCC, with a trend toward worse instead of better survival. This could have a biological explanation, which deserves further investigation and could possibly lead to more stratified treatment in the future.     

The UDP-sugar-sensing P2Y14 receptor promotes Rho-mediated signaling and chemotaxis in human neutrophils

The G(i)-coupled P2Y(14) receptor (P2Y(14)-R) is potently activated by UDP-sugars and UDP. Although P2Y(14)-R mRNA is prominently expressed in circulating neutrophils, the signaling pathways and functional responses associated with this receptor are undefined. In this study, we illustrate that incubation of isolated human neutrophils with UDP-glucose resulted in cytoskeleton rearrangement, change of cell shape, and enhanced cell migration. We also demonstrate that UDP-glucose promotes rapid, robust, and concentration-dependent activation of RhoA in these cells. Ecto-nucleotidases expressed on neutrophils rapidly hydrolyzed extracellular ATP, but incubation with UDP-glucose for up to 1 h resulted in negligible metabolism of the nucleotide-sugar. HL60 human promyelocytic leukemia cells do not express the P2Y(14)-R, but neutrophil differentiation of HL60 cells with DMSO resulted in markedly enhanced P2Y(14)-R expression. Accordingly, UDP-glucose, UDP-galactose, and UDP-N-acetylglucosamine promoted Rho activation in differentiated but not in undifferentiated HL60 cells. Stable expression of recombinant human P2Y(14)-R conferred UDP-sugar-promoted responses to undifferentiated HL60 cells. UDP-glucose-promoted RhoA activation also was accompanied by enhanced cell migration in differentiated HL60 cells, and these responses were blocked by Rho kinase inhibitors. These results support the notion that UDP-glucose is a stable and potent proinflammatory mediator that promotes P2Y(14)-R-mediated neutrophil motility via Rho/Rho kinase activation.     

Voltage-dependent anion channel-1 (VDAC-1) contributes to ATP release and cell volume regulation in murine cells

Extracellular ATP regulates several elements of the mucus clearance process important for pulmonary host defense. However, the mechanisms mediating ATP release onto airway surfaces remain unknown. Mitochondrial voltage-dependent anion channels (mt-VDACs) translocate a variety of metabolites, including ATP and ADP, across the mitochondrial outer membrane, and a plasmalemmal splice variant (pl-VDAC-1) has been proposed to mediate ATP translocation across the plasma membrane. We tested the involvement of VDAC-1 in ATP release in a series of studies in murine cells. First, the full-length coding sequence was cloned from a mouse airway epithelial cell line (MTE7b-) and transfected into NIH 3T3 cells, and pl-VDAC-1-transfected cells exhibited higher rates of ATP release in response to medium change compared with mock-transfected cells. Second, ATP release was compared in cells isolated from VDAC-1 knockout [VDAC-1 (-/-)] and wild-type (WT) mice. Fibroblasts from VDAC-1 (-/-) mice released less ATP than WT mice in response to a medium change. Well-differentiated cultures from nasal and tracheal epithelia of VDAC-1 (-/-) mice exhibited less ATP release in response to luminal hypotonic challenge than WT mice. Confocal microscopy studies revealed that cell volume acutely increased in airway epithelia from both VDAC-1 (-/-) and WT mice after luminal hypotonic challenge, but VDAC-1 (-/-) cells exhibited a slower regulatory volume decrease (RVD) than WT cells. Addition of ATP or apyrase to the luminal surface of VDAC-1 (-/-) or WT cultures with hypotonic challenge produced similar initial cell height responses and RVD kinetics in both cell types, suggesting that involvement of VDAC-1 in RVD is through ATP release. Taken together, these studies suggest that VDAC-1, directly or indirectly, contributes to ATP release from murine cells. However, the observation that VDAC-1 knockout cells released a significant amount of ATP suggests that other molecules also play a role in this function.     

Relative accuracy of spatial predictive models for lynx Lynx canadensis derived using logistic regression-AIC,multiple criteria evaluation and Bayesian approaches

We compared probability surfaces derived using one set of environmental variables in three Geographic Information Systems (GIS) -based approaches: logistic regression and Akaike's Information Criterion (AIC),Multiple Criteria Evaluation (MCE),and Bayesian Analysis (specifically Dempster-Shafer theory). We used lynx Lynx canadensis as our focal species,and developed our environment relationship model using track data collected in Banff National Park,Alberta,Canada,during winters from 1997 to 2000. The accuracy of the three spatial models were compared using a contingency table method. We determined the percentage of cases in which both presence and absence points were correctly classified (overall accuracy),the failure to predict a species where it occurred (omission error) and the prediction of presence where there was absence (commission error). Our overall accuracy showed the logistic regression approach was the most accurate (74.51% ). The multiple criteria evaluation was intermediate (39.22%),while the Dempster-Shafer (D-S) theory model was the poorest (29.90%). However,omission and commission error tell us a different story: logistic regression had the lowest commission error,while D-S theory produced the lowest omission error. Our results provide evidence that habitat modellers should evaluate all three error measures when ascribing confidence in their model. We suggest that for our study area at least,the logistic regression model is optimal. However,where sample size is small or the species is very rare,it may also be useful to explore and/or use a more ecologically cautious modelling approach (e.g. Dempster-Shafer) that would over-predict,protect more sites,and thereby minimize the risk of missing critical habitat in conservation plans.     

Nucleotide release provides a mechanism for airway surface liquid homeostasis

Nucleotides within the airway surface liquid (ASL) regulate airway epithelial ion transport rates by Ca(2+) -and protein kinase C-dependent mechanisms via activation of specific P2Y receptors. Extracellular adenine nucleotides also serve as precursors for adenosine, which promotes cyclic AMP-mediated activation of the cystic fibrosis transmembrane regulator chloride channel via A(2b) adenosine receptors. A biological role for extracellular ATP in ASL volume homeostasis has been suggested by the demonstration of regulated ATP release from airway epithelia. However, nucleotide hydrolysis at the airway surface makes it difficult to assess the magnitude of ATP release and the relative abundance of adenyl purines and, hence, to define their biological functions. We have combined ASL microsampling and high performance liquid chromatography analysis of fluorescent 1,N(6)-ethenoadenine derivatives to measure adenyl purines in ASL. We found that adenosine, AMP, and ADP accumulated in high concentrations relative to ATP within the ASL covering polarized primary human normal or cystic fibrosis airway epithelial cells. By using immortalized epithelial cell monolndogenayers that eously express a luminal A(2b) adenosine receptor, we found that basal as well asforskolin-promoted cyclic AMP production was reduced by exogenous adenosine deaminase, suggesting that A(2b) receptors sense endogenous adenosine within the ASL. The physiological role of adenosine was further established by illustrating that adenosine removal or inhibition of adenosine receptors in primary cultures impaired ASL volume regulation. Our data reveal a complex pattern of nucleotides/nucleosides in ASL under resting conditions and suggest that adenosine may play a key role in regulating ASL volume homeostasis.