Dual actions of procainamide on batrachotoxin-activated sodium channels: open channel block and prevention of inactivation.

We have investigated the action of procainamide on batrachotoxin (BTX)-activated sodium channels from bovine heart and rat skeletal muscle. When applied to the intracellular side, procainamide induced rapid, open-channel block. We estimated rate constants using amplitude distribution analysis (Yellen, G. 1984. J. Gen. Physiol. 84:157). Membrane depolarization increased the blocking rate and slowed unblock. The rate constants were similar in both magnitude and voltage dependence for cardiac and skeletal muscle channels. Qualitatively, this block resembled the fast open-channel block by lidocaine (Zamponi, G. W., D. D. Doyle, and R. J. French. 1993. Biophys. J. 65:80), but procainamide was about sevenfold less potent. Molecular modeling suggests that the difference in potency between procainamide and lidocaine might arise from the relative orientation of their aromatic rings, or from differences in the structure of the aryl-amine link. For the cardiac channels, procainamide reduced the frequency of transitions to a long-lived closed state which shows features characteristic of inactivation (Zamponi, G. W., D. D. Doyle, and R. J. French. 1993. Biophys J. 65:91). Mean durations of kinetically identified closed states were not affected. The degree of fast block and of inhibition of the slow closures were correlated. Internally applied QX-314, a lidocaine derivative and also a fast blocker, produced a similar effect. Thus, drug binding to the fast blocking site appears to inhibit inactivation in BTX-activated cardiac channels.     

Comparative analysis of the mitochondrial cytochrome c oxidase subunit I (COI) gene in ciliates (Alveolata,Ciliophora) and evaluation of its suitability as a biodiversity marker

The mitochondrial cytochrome c oxidase subunit 1 (COI) gene of ciliates was first successfully sequenced in species of the genera Tetrahymena and Paramecium (Class Oligohymenophorea). The sequence of the COI gene is extremely divergent from other eukaryotes and includes an insert, which is over 300 nucleotides long. In this study, we designed a primer pair that successfully amplified the COI gene of ciliates from five different classes: Heterotrichea, Spirotrichea, Oligohymenophorea, Nassophorea and Colpodea. These classes represent the diversity of the phylum Ciliophora very well, since they are widely distributed on the ciliate small subunit rRNA tree. The amplified region is approximately 850 nucleotides long and corresponds to the general barcoding region; it also includes the insert region. In this study, 58 new COI sequences from over 38 species in 13 orders are analysed and compared, and distance trees are constructed. While the COI gene shows high divergence within ciliates, the insert region, which is present in all classes, is even more divergent. Genetic distances calculated with and without the insert region remain in the same range at the intraspecific level, but they differ considerably at or above genus level. This suggests that the entire barcoding region is under similar selective constraints and that the evolutionary rate of the ciliate COI is extremely high and shows unequal rate variation. Although many problems still remain regarding standardization of barcoding methods in ciliates, the development of a universal or almost universal primer combination for the Phylum Ciliophora represents important progress. As shown in four examples, the resolution of COI at the intraspecific level is much greater than that of any nuclear genes and shows great potential to (1) identify species based on molecular data if a reliable database exists, and (2) resolve the relationships of closely related ciliate taxa and uncover cryptic species.     

Detailed Functional and Proteomic Characterization of Fludarabine Resistance in Mantle Cell Lymphoma Cells

Mantle cell lymphoma (MCL) is a chronically relapsing aggressive type of B-cell non-Hodgkin lymphoma considered incurable by currently used treatment approaches. Fludarabine is a purine analog clinically still widely used in the therapy of relapsed MCL. Molecular mechanisms of fludarabine resistance have not, however, been studied in the setting of MCL so far. We therefore derived fludarabine-resistant MCL cells (Mino/FR) and performed their detailed functional and proteomic characterization compared to the original fludarabine sensitive cells (Mino). We demonstrated that Mino/FR were highly cross-resistant to other antinucleosides (cytarabine, cladribine, gemcitabine) and to an inhibitor of Bruton tyrosine kinase (BTK) ibrutinib. Sensitivity to other types of anti-lymphoma agents was altered only mildly (methotrexate, doxorubicin, bortezomib) or remained unaffacted (cisplatin, bendamustine). The detailed proteomic analysis of Mino/FR compared to Mino cells unveiled over 300 differentially expressed proteins. Mino/FR were characterized by the marked downregulation of deoxycytidine kinase (dCK) and BTK (thus explaining the observed crossresistance to antinucleosides and ibrutinib), but also by the upregulation of several enzymes of de novo nucleotide synthesis, as well as the up-regulation of the numerous proteins of DNA repair and replication. The significant upregulation of the key antiapoptotic protein Bcl-2 in Mino/FR cells was associated with the markedly increased sensitivity of the fludarabine-resistant MCL cells to Bcl-2-specific inhibitor ABT199 compared to fludarabine-sensitive cells. Our data thus demonstrate that a detailed molecular analysis of drug-resistant tumor cells can indeed open a way to personalized therapy of resistant malignancies.     

Demography of an alpine population of Savannah Sparrows

ABSTRACT.   Although Savannah Sparrows ( Passerculus sandwichensis ) have been well studied across their North American range, few data are available for populations that breed in high-elevation habitats. We collected data over six years on the demography of a population of Savannah Sparrows ( P. s. anthinus ) breeding in alpine tundra and sub-alpine meadows in northern British Columbia, Canada. The mean duration of the breeding season at our site was 45.5 d, and pairs produced a maximum of one brood per season. Clutch sizes varied annually (mean = 4.37, range = 3.90 – 4.71 eggs). Nest fate also varied among years (range = 33 – 92%) due to variation in abiotic (weather) and biotic (predators) conditions. Uncorrected return rates of banded birds were 68% for adults and 17% for juveniles ( N = 22 and 102, respectively). However, when resighting probability was taken into account, apparent annual survival was 75% for adults and 34% for juveniles. Compared to populations at lower elevations, Savannah Sparrows in our study had shorter breeding seasons, fewer broods per season, larger clutches, and higher adult and juvenile return rates. Our results suggest that Savannah Sparrows that breed in high-elevation habitats have adopted a low fecundity, high survival life history strategy that enables their persistence in these challenging environments.     

Development and Validation of a New Method to Measure Walking Speed in Free-Living Environments Using the Actibelt? Platform

Walking speed is a fundamental indicator for human well-being. In a clinical setting, walking speed is typically measured by means of walking tests using different protocols. However, walking speed obtained in this way is unlikely to be representative of the conditions in a free-living environment. Recently, mobile accelerometry has opened up the possibility to extract walking speed from long-time observations in free-living individuals, but the validity of these measurements needs to be determined. In this investigation, we have developed algorithms for walking speed prediction based on 3D accelerometry data (actibelt®) and created a framework using a standardized data set with gold standard annotations to facilitate the validation and comparison of these algorithms. For this purpose 17 healthy subjects operated a newly developed mobile gold standard while walking/running on an indoor track. Subsequently, the validity of 12 candidate algorithms for walking speed prediction ranging from well-known simple approaches like combining step length with frequency to more sophisticated algorithms such as linear and non-linear models was assessed using statistical measures. As a result, a novel algorithm employing support vector regression was found to perform best with a concordance correlation coefficient of 0.93 (95%CI 0.92–0.94) and a coverage probability CP1 of 0.46 (95%CI 0.12–0.70) for a deviation of 0.1 m/s (CP2 0.78, CP3 0.94) when compared to the mobile gold standard while walking indoors. A smaller outdoor experiment confirmed those results with even better coverage probability. We conclude that walking speed thus obtained has the potential to help establish walking speed in free-living environments as a patient-oriented outcome measure.     

A novel insight into the cost–benefit model for the evolution of botanical carnivory

Background The cost–benefit model for the evolution of botanical carnivory provides a conceptual framework for interpreting a wide range of comparative and experimental studies on carnivorous plants. This model assumes that the modified leaves called traps represent a significant cost for the plant, and this cost is outweighed by the benefits from increased nutrient uptake from prey, in terms of enhancing the rate of photosynthesis per unit leaf mass or area (A_N) in the microsites inhabited by carnivorous plants.Scope This review summarizes results from the classical interpretation of the cost–benefit model for evolution of botanical carnivory and highlights the costs and benefits of active trapping mechanisms, including water pumping, electrical signalling and accumulation of jasmonates. Novel alternative sequestration strategies (utilization of leaf litter and faeces) in carnivorous plants are also discussed in the context of the cost–benefit model.Conclusions Traps of carnivorous plants have lower A_N than leaves, and the leaves have higher A_N after feeding. Prey digestion, water pumping and electrical signalling represent a significant carbon cost (as an increased rate of respiration, R_D) for carnivorous plants. On the other hand, jasmonate accumulation during the digestive period and reprogramming of gene expression from growth and photosynthesis to prey digestion optimizes enzyme production in comparison with constitutive secretion. This inducibility may have evolved as a cost-saving strategy beneficial for carnivorous plants. The similarities between plant defence mechanisms and botanical carnivory are highlighted.     

Specificity of Collybistin-Phosphoinositide Interactions: IMPACT OF THE INDIVIDUAL PROTEIN DOMAINS*

The regulatory protein collybistin (CB) recruits the receptor-scaffolding protein gephyrin to mammalian inhibitory glycinergic and GABAergic postsynaptic membranes in nerve cells. CB is tethered to the membrane via phosphoinositides. We developed an in vitro assay based on solid-supported 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine membranes doped with different phosphoinositides on silicon/silicon dioxide substrates to quantify the binding of various CB2 constructs using reflectometric interference spectroscopy. Based on adsorption isotherms, we obtained dissociation constants and binding capacities of the membranes. Our results show that full-length CB2 harboring the N-terminal Src homology 3 (SH3) domain (CB2_SH3+) adopts a closed and autoinhibited conformation that largely prevents membrane binding. This autoinhibition is relieved upon introduction of the W24A/E262A mutation, which conformationally “opens” CB2_SH3+ and allows the pleckstrin homology domain to properly bind lipids depending on the phosphoinositide species with a preference for phosphatidylinositol 3-monophosphate and phosphatidylinositol 4-monophosphate. This type of membrane tethering under the control of the release of the SH3 domain of CB is essential for regulating gephyrin clustering.     

AHNAK controls 53BP1-mediated p53 response by restraining 53BP1 oligomerization and phase separation

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