Coassembly of big conductance Ca2+-activated K+ channels and L-type voltage-gated Ca2+ channels in rat brain

Based on electrophysiological studies, Ca(2+)-activated K(+) channels and voltage-gated Ca(2+) channels appear to be located in close proximity in neurons. Such colocalization would ensure selective and rapid activation of K(+) channels by local increases in the cytosolic calcium concentration. The nature of the apparent coupling is not known. In the present study we report a direct coassembly of big conductance Ca(2+)-activated K(+) channels (BK) and L-type voltage-gated Ca(2+) channels in rat brain. Saturation immunoprecipitation studies were performed on membranes labeled for BK channels and precipitated with antibodies against alpha(1C) and alpha(1D) L-type Ca(2+) channels. To confirm the specificity of the interaction, precipitation experiments were carried out also in reverse order. Also, additive precipitation was performed because alpha(1C) and alpha(1D) L-type Ca(2+) channels always refer to separate ion channel complexes. Finally, immunochemical studies showed a distinct but overlapping expression pattern of the two types of ion channels investigated. BK and L-type Ca(2+) channels were colocalized in various compartments throughout the rat brain. Taken together, these results demonstrate a direct coassembly of BK channels and L-type Ca(2+) channels in certain areas of the brain.     

On the maximal cliques in c-max-tolerance graphs and their application in clustering molecular sequences

Given a set S of n locally aligned sequences, it is a needed prerequisite to partition it into groups of very similar sequences to facilitate subsequent computations, such as the generation of a phylogenetic tree. This article introduces a new method of clustering which partitions S into subsets such that the overlap of each pair of sequences within a subset is at least a given percentage c of the lengths of the two sequences. We show that this problem can be reduced to finding all maximal cliques in a special kind of max-tolerance graph which we call a c-max-tolerance graph. Previously we have shown that finding all maximal cliques in general max-tolerance graphs can be done efficiently in O(n ³ + out). Here, using a new kind of sweep-line algorithm, we show that the restriction to c-max-tolerance graphs yields a better runtime of O(n ² log n + out). Furthermore, we present another algorithm which is much easier to implement, and though theoretically slower than the first one, is still running in polynomial time. We then experimentally analyze the number and structure of all maximal cliques in a c-max-tolerance graph, depending on the chosen c-value. We apply our simple algorithm to artificial and biological data and we show that this implementation is much faster than the well-known application Cliquer. By introducing a new heuristic that uses the set of all maximal cliques to partition S, we finally show that the computed partition gives a reasonable clustering for biological data sets.     

Selecting effective siRNAs based on guide RNA structure

In RNA interference, guide RNAs direct RNA-induced silencing complexes to mRNA targets, mediating cleavage and ultimately leading to gene silencing. We have observed that unstructured guide strands, which either completely lack complementary bases or in which internal base pairing is thermodynamically unlikely, confer strongest silencing, whereas structures with base-paired ends are inactive. Thus, the structure of the guide strand represents a major determinant of small interfering RNA activity. Here we describe a detailed computational protocol for identification of unstructured guide strands for a given mRNA target sequence. Sequentially, all guide sequences with target complementarity are simulated, their corresponding structures are folded and unstructured guide strands are selected and rated according to thermodynamic parameters. Although this procedure is new and remains to be validated by the community, it allows reliable identification of highly active siRNAs that can be used for functional target validation or drug development.     

ATM requirement in gene expression responses to ionizing radiation in human lymphoblasts and fibroblasts

The heritable disorder ataxia telangiectasia (AT) is caused by mutations in the AT-mutated (ATM) gene with manifestations that include predisposition to lymphoproliferative cancers and hypersensitivity to ionizing radiation (IR). We investigated gene expression changes in response to IR in human lymphoblasts and fibroblasts from seven normal and seven AT-affected individuals. Both cell types displayed ATM-dependent gene expression changes after IR, with some responses shared and some responses varying with cell type and dose. Interestingly, after 5 Gy IR, lymphoblasts displayed ATM-independent responses not seen in the fibroblasts at this dose, which likely reflect signaling through ATM-related kinases, e.g., ATR, in the absence of ATM function.     

Laboratory evaluation of flight performance of the plum curculio (Coleoptera: Curculionidae)

Flight performance of laboratory-reared adults of the plum curculio, Conotrachelus nenuphar (Herbst) (Coleoptera: Curculionidae), was investigated under controlled conditions by using a flight mill system. Across all insects tested (n=198), median values of total distance traveled, total flight time, and maximum uninterrupted flight time were 122.7 m day(-1), 23.5 min day(-1), and 2.0 min, respectively. The latter result indicates that flight occurred primarily in short bursts. Although females had a significantly higher body mass than males, there were no significant differences in flight performance between the two sexes. Flight during the first 24-h test period (especially the first 6 h) was dominated by escape behavior, i.e., elevated levels of activity presumably associated with attempts by the insects to regain freedom of movement; during the second 24 h, flight activity was very limited throughout the late morning and afternoon, increased around sunset, and remained high during the night. All flight performance variables decreased linearly and significantly with insect age over the age range tested (2-16 d after emergence). Nutritional status also had a significant effect, whereby insects that had been provided with apples as a food source for 2 d after emergence showed considerably improved flight performance compared with those that had been given no food or only water during the same period. There was no significant effect of mating status on flight performance of male or female insects.     

CD1 antigen presentation by human dendritic cells as a target for herpes simplex virus immune evasion

In contrast to MHC molecules, which present peptides, the CD1 molecules have been discovered to present lipid Ags to T cells. CD1-restricted T lymphocytes have been recently associated with resistance to virus infection. The mechanisms underlying activation of CD1-restricted T cells in the course of virus infection are not defined. In this study, we wanted to investigate the interaction of HSV with the antiviral CD1 Ag presentation system in human dendritic cells (DC). In response to low titers of HSV, the surface expression of CD1b and CD1d on human DC was up-regulated. These phenotypic changes enhanced the capacity of infected DC to stimulate proliferation of CD1-restricted T lymphocytes. High titers of HSV, however, lead to strong down-regulation of all surface CD1 molecules. This modulation of surface expression was associated with intracellular accumulation, colocalization with viral proteins, and disruption of the CD1 recycling machinery. Finally, even at low titers HSV interfered with the capacity of infected DC to stimulate the release of important cytokines by CD1d-restricted NKT cells. Thus, we demonstrate both the existence of a CD1 pathway allowing human DC to react to viral infection, as well as its blockage by a human herpesvirus.     

Adaphostin and other anticancer drugs quench the fluorescence of mitochondrial potential probes

Fluorescent dyes are widely used to monitor changes in mitochondrial transmembrane potential (DeltaPsim). When MitoTracker Red CMXRos, tetramethylrhodamine methyl ester (TMRM), and 3,3'dihexyloxacarbocyanine iodide (DiOC6(3)) were utilized to examine the effects of the experimental anticancer drug adaphostin on intact cells or isolated mitochondria, decreased fluorescence was observed. In contrast, measurement of tetraphenylphosphonium uptake by the mitochondria using an ion-selective microelectrode failed to show any effect of adaphostin on DeltaPsim. Instead, further experiments demonstrated that adaphostin quenches the fluorescence of the mitochondrial dyes. Structure-activity analysis revealed that the adamantyl and p-aminobenzoic acid moieties of adaphostin are critical for this quenching. Anticancer drugs containing comparable structural motifs, including mitoxantrone, aminoflavone, and amsacrine, also quenched the mitochondrial probes. These results indicate the need for caution when mitochondrial dyes are utilized to examine the effects of xenobiotics on DeltaPsim and suggest that some previously reported direct effects of anticancer drugs on mitochondria might need re-evaluation.     

Cell orientation by a microgrooved substrate can be predicted by automatic control theory

Cells have the ability to measure and respond to extracellular signals like chemical molecules and topographical surface features by changing their orientation. Here, we examined the orientation of cultured human melanocytes exposed to grooved topographies. To predict the cells' orientation response, we describe the cell behavior with an automatic controller model. The predicted dependence of the cell response to height and spatial frequency of the grooves is obtained by considering the symmetry of the system (cell + substrate). One basic result is that the automatic controller responds to the square of the product of groove height and spatial frequency or to the aspect ratio for symmetric grooves. This theoretical prediction was verified by the experiments, in which melanocytes were exposed to microfabricated poly(dimethylsiloxane) substrates having parallel rectangular grooves of heights (h) between 25 and 200 nm and spatial frequencies (L) between 100 and 500 mm(-1). In addition, the model of the cellular automatic controller is extended to include the case of different guiding signals acting simultaneously.