Expression of a truncated tobacco NtCBP4 channel in transgenic plants and disruption of the homologous Arabidopsis CNGC1 gene confer Pb2+ tolerance

Recently we reported on a plasma membrane tobacco protein (designated NtCBP4) that binds calmodulin. When overexpressed in transgenic plants, NtCBP4 confers Pb2+ hypersensitivity associated with enhanced accumulation of this toxic metal. To further investigate possible modulation of Pb2+ tolerance in plants, we prepared transgenic plants that express a truncated version of this protein (designated NtCBP4DeltaC) from which its C-terminal, with the calmodulin-binding domain and part of the putative cyclic nucleotide-binding domain, was removed. In contrast to the phenotype of transgenic plants expressing the full-length gene, transgenic plants expressing the truncated gene showed improved tolerance to Pb2+, in addition to attenuated accumulation of this metal. Furthermore, disruption by T-DNA insertion mutagenesis of the Arabidopsis CNGC1 gene, which encodes a homologous protein, also conferred Pb2+ tolerance. We suggest that NtCBP4 and AtCNGC1 are components of a transport pathway responsible for Pb2+ entry into plant cells.     

Phylogenetic relationships within cation transporter families of Arabidopsis

Uptake and translocation of cationic nutrients play essential roles in physiological processes including plant growth, nutrition, signal transduction, and development. Approximately 5% of the Arabidopsis genome appears to encode membrane transport proteins. These proteins are classified in 46 unique families containing approximately 880 members. In addition, several hundred putative transporters have not yet been assigned to families. In this paper, we have analyzed the phylogenetic relationships of over 150 cation transport proteins. This analysis has focused on cation transporter gene families for which initial characterizations have been achieved for individual members, including potassium transporters and channels, sodium transporters, calcium antiporters, cyclic nucleotide-gated channels, cation diffusion facilitator proteins, natural resistance-associated macrophage proteins (NRAMP), and Zn-regulated transporter Fe-regulated transporter-like proteins. Phylogenetic trees of each family define the evolutionary relationships of the members to each other. These families contain numerous members, indicating diverse functions in vivo. Closely related isoforms and separate subfamilies exist within many of these gene families, indicating possible redundancies and specialized functions. To facilitate their further study, the PlantsT database (http://plantst.sdsc.edu) has been created that includes alignments of the analyzed cation transporters and their chromosomal locations.     

Energization of potassium uptake in Arabidopsis thaliana

Plant roots accumulate K⁺ from micromolar external concentrations. However, the absence of a firm determination of the trans-plasma-membrane electrochemical gradient for K⁺ in these conditions has precluded an assessment of whether K⁺-accumulation requires energization in addition to the driving force provided by the inside-negative membrane electrical potential (E_m). To address this question unequivocally, we measured E_m, and the cytosolic and external K⁺-activities in root cells of Arabidopsis thaliana (L.) Heynh. cv. Columbia in conditions in which net K⁺-accumulation occurs at low external K⁺ (10 M). In these conditions, net K⁺-uptake was about 0.1 mol · (g FW)^-1 · h^-1, E_m varied between-153 and -129 mV and the cytosolic K⁺-activity, determined with K⁺-selective electrodes, was 83 ± 4 mM. These values yield an outwardly-directed driving force on K⁺ of at least 6.5 kJ · mol^-1. Only if external potassium is raised to the region of 1 mM does E_m become sufficient to drive net K⁺-accumulation. It is therefore concluded that at micromolar external K⁺-activities which prevail in most soils, K⁺-uptake cannot be solely energized by E_m — as exemplified by a channel-mediated mechanism. The nature of the energization mechanism is discussed in relation to processes operating in fungal and algal cells.Abbreviations and Symbols AAS atomic absorption spectrometry - E_m membrane potential - electrochemical potassium gradient - F Faraday constant (96500 C · mol^-1) We thank Peter Barraclough, Roger Leigh, David Walker and Tony Miller (Rothamsted Experimental Station, Harpenden, UK) for helpful discussions. Financial support was provided by the Agricultural and Food Research Council (Grant PG87/529).     

Sodium Uptake in Arabidopsis Roots Is Regulated by Cyclic Nucleotides

Sodium uptake from the soil is a major cause of salinity toxicity in plants, yet little is known about the mechanisms that underlie Na(+) influx. We have characterized voltage independent channels (VICs) in Arabidopsis roots that are thought to contribute to Na(+) entry. VICs showed no selectivity among monovalent cations, and their gating was found to be voltage independent. However, VIC open probability showed sensitivity to cyclic nucleotides. The presence of micromolar concentrations of cAMP or cGMP at the cytoplasmic side of the plasma membrane evoked a rapid decrease in channel open probability. In accord with predictions from electrophysiological data, our results show that short-term unidirectional Na(+) influx is also reduced in the presence of cyclic nucleotides. Moreover, addition of membrane permeable cyclic nucleotides during growth assays improved plant salinity tolerance, which corresponded with lower levels of Na(+) accumulation in plants. In summary, these data imply that Arabidopsis plants may contain a cyclic nucleotide-based signaling pathway that directly affects Na(+) transport via VICs.     

Gene Expression Analysis Reveals Inhibition of Radiation-Induced TGFβ-Signaling by Hyperbaric Oxygen Therapy in Mouse Salivary Glands

A side effect of radiation therapy in the head and neck region is injury to surrounding healthy tissues such as irreversible impaired function of the salivary glands. Hyperbaric oxygen therapy (HBOT) is clinically used to treat radiation-induced damage but its mechanism of action is largely unknown. In this study, we investigated the molecular pathways that are affected by HBOT in mouse salivary glands two weeks after radiation therapy by microarray analysis. Interestingly, HBOT led to significant attenuation of the radiation-induced expression of a set of genes and upstream regulators that are involved in processes such as fibrosis and tissue regeneration. Our data suggest that the TGFβ-pathway, which is involved in radiation-induced fibrosis and chronic loss of function after radiation therapy, is affected by HBOT. On the longer term, HBOT reduced the expression of the fibrosis-associated factor α-smooth muscle actin in irradiated salivary glands. This study highlights the potential of HBOT to inhibit the TGFβ-pathway in irradiated salivary glands and to restrain consequential radiation induced tissue injury.     

A comprehensive sensitivity analysis of microarray breast cancer classification under feature variability

Background  

Large discrepancies in signature composition and outcome concordance have been observed between different microarray breast cancer expression profiling studies. This is often ascribed to differences in array platform as well as biological variability. We conjecture that other reasons for the observed discrepancies are the measurement error associated with each feature and the choice of preprocessing method. Microarray data are known to be subject to technical variation and the confidence intervals around individual point estimates of expression levels can be wide. Furthermore, the estimated expression values also vary depending on the selected preprocessing scheme. In microarray breast cancer classification studies, however, these two forms of feature variability are almost always ignored and hence their exact role is unclear.     

Molecular population genetics of the alcohol dehydrogenase locus in the Hawaiian drosophilid D. mimica

Sequence variation among 10 alleles of the alcohol dehydrogenase (Adh) gene of the Hawaiian drosophilid D. mimica was analyzed with reference to the evolutionary history of the Hawaiian subgroup as well as to levels and patterns of polymorphism of the Adh gene in continental drosophilid species. The Adh gene of D. mimica is less polymorphic than that of other drosophilid species, and no replacement substitutions were found. Statistical analyses of the Adh alleles suggested the action of balancing selection and revealed significant linkage disequilibrium among three of the variable sites. The effective population size was estimated to be only slightly smaller than that of continental species and, surprisingly, on the same order of magnitude as the actual size.      

Contrasting roles in ion transport of two K+-channel types in root cells of Arabidopsis thaliana

Plant roots accumulate K⁺ over a range of external concentrations. Root cells have evolved at least two parallel plasma-membrane K⁺ transporters which operate at millimolar and micromolar external [K⁺]: high-affinity K⁺ uptake is energised by symport with H⁺, while low-affinity uptake is assumed to occur via ion channels. To determine the role of ion channels in low-affinity K⁺ uptake, a characterisation of the principal K⁺-selective ion channels in the plasma membrane of Arabidopsis thaliana (L.) Heynh. cv. Columbia roots was undertaken. Two classes of K⁺-selective channels were frequently observed: one inward (IRC) and one outward (ORC) rectifying with unitary conductances of 5 pS, 20 pS (IRCs) and 15 pS (ORC), measured in symmetrical 10 mM KCl. The dominant IRC (5 pS) and ORC (15 pS) were highly cation-selective (P_Cl P_K < 0.025) but less selective amongst monovalent cations (P_NaP_K0.17–0.3). Both the IRC and the ORC were blocked by Ba²⁺, Cs⁺ and tetra-ethyl-ammonium, whereas 4-aminopyridine and quinidine selectively inhibited the ORC. The ORC open probability was steeply voltage-dependent and ORC activation potentials were close to the potassium equilibrium potential (E_K+), enabling ORCs to conduct mainly outward, but occasionally inward, K⁺ current. By contrast, gating of the 5-pS IRC was weakly voltageependent and IRC gating was invariably restricted to membrane potentials more negative than E_K+, ensuring K⁺ transport was always inwardly directed. Studies on channel activity were conducted for a large number of root cells grown at two levels of external [K⁺], one where K⁺ uptake is likely to be principally through channels (6 mM K⁺) and one where it must be energised (100 M K⁺). Shifting growth conditions from high to low K⁺ did not affect single-channel properties such as conductance and selectivity, nor the manifestation of the ORC and 20-pS IRC, but led to enhanced activity of the 5-pS IRC. The enhanced activity of the 5-pS IRC was mirrored by a parallel increase in unidirectional ⁸⁶Rb⁺ influx after low-K⁺ growth, clearly indicating a dominant role of this particular channel in K⁺ uptake at supra millimolar external [K⁺].Abbreviations E_K+ potassium equilibrium potential - E_m membrane potential - HK high [K⁺] - IRC inward rectifying channel - LK low [K⁺] - ORC outward rectifying channel - TEA tetra-ethyl-ammonium Financial support was provided by the Biotechnology and Biological Sciences Research Council (Grant PG87/529) and by the European Union (Framework III, Biotechnology Programme).     

Nonparametric inference for competing risks current status data with continuous, discrete or grouped observation times

New methods and theory have recently been developed to nonparametrically estimate cumulative incidence functions for competing risks survival data subject to current status censoring. In particular, the limiting distribution of the nonparametric maximum likelihood estimator and a simplified naive estimator have been established under certain smoothness conditions. In this paper, we establish the large-sample behaviour of these estimators in two additional models, namely when the observation time distribution has discrete support and when the observation times are grouped. These asymptotic results are applied to the construction of confidence intervals in the three different models. The methods are illustrated on two datasets regarding the cumulative incidence of different types of menopause from a cross-sectional sample of women in the United States and subtype-specific HIV infection from a sero-prevalence study in injecting drug users in Thailand.