Pacemaking by HCN channels requires interaction with phosphoinositides

Hyperpolarization-activated, cyclic-nucleotide-gated (HCN) channels mediate the depolarizing cation current (termed I(h) or I(f)) that initiates spontaneous rhythmic activity in heart and brain. This function critically depends on the reliable opening of HCN channels in the subthreshold voltage-range. Here we show that activation of HCN channels at physiologically relevant voltages requires interaction with phosphoinositides such as phosphatidylinositol-4,5-bisphosphate (PIP(2)). PIP(2) acts as a ligand that allosterically opens HCN channels by shifting voltage-dependent channel activation approximately 20 mV toward depolarized potentials. Allosteric gating by PIP(2) occurs in all HCN subtypes and is independent of the action of cyclic nucleotides. In CNS neurons and cardiomyocytes, enzymatic degradation of phospholipids results in reduced channel activation and slowing of the spontaneous firing rate. These results demonstrate that gating by phospholipids is essential for the pacemaking activity of HCN channels in cardiac and neuronal rhythmogenesis.     

The yeast Arr4p ATPase binds the chloride transporter Gef1p when copper is available in the cytosol

Cellular ion homeostasis involves communication between the cytosol and the luminal compartment of organelles. This is particularly critical for metal ions because of their toxic potential. We have identified the yeast homologue of the prokaryotic ArsA protein, the homodimeric ATPase Arr4p, as a protein that binds to the yeast intracellular CLC chloride-transport protein, Gef1p. We show that binding of Arr4p to the C terminus of Gef1p requires the presence of yeast cytosol and is sensitive to a highly specific copper chelator in vitro and in vivo. Copper alone can substitute for cytosol to support the interaction of Arr4p with the C terminus of Gef1p. The migration behavior of Arr4p in nonreducing gel electrophoresis correlates with cellular copper deficiency, repletion, or stress. Our homology model of Arr4p shows that the antimony (arsenic) metal binding site of ArsA is not conserved in Arr4p. The model suggests that a pair of cysteines, Cys285 and Cys288, is located in the interface of the Arr4p dimer. These residues are required for Arr4p homodimerization and for binding to the C terminus of Gef1p. Whereas both proteins are required for normal growth under iron-limiting conditions, they play opposite roles when copper and heat stress are combined in an alkaline environment. Under these conditions, deltagef1 cells grow much better than wild type yeast, whereas deltaarr4 cells are unable to grow. Comparison of the deltaarr4 with the deltaarr4deltagef1 strain suggests that Arr4p antagonizes the function of Gef1p.     

Application of a color-shift model with heterogeneous growth to a rat hepatocarcinogenesis experiment

Several hypotheses are established to describe the formation and progression of foci of altered hepatocytes (FAH). A common model of hepatocarcinogenesis is the mutation model (MM), which is based on the assumption that cells have to undergo multiple successive changes on their way from the normal to the malignant stage. This model describes growth and phenotype change of foci on the cellular level and is based on the assumption that single cells change their phenotype through mutation into the next stage and proliferate according to a linear stochastic birth-death process. In contrast, the color-shift model (CSM) was introduced by Kopp-Schneider et al. to describe that whole colonies of altered hepatocytes simultaneously alter their phenotype. In this paper two modifications of the color-shift model are considered which allow the growth rate to vary from focus to focus. All four models are compared with respect to their ability to predict number and radii of foci in a rat hepatocarcinogenesis experiment, in which rats were treated with the carcinogens N-nitrosomorpholine, 2-acetylaminofluoren and Phenobarbital. Maximum likelihood parameter estimates are given, and predicted and empirical FAH size distributions are visualized. The Cramer-von-Mises distance is used as a measure for the discrepancy between empirical and theoretical size distributions.     

Transgenic barley plants overexpressing a 13-lipoxygenase to modify oxylipin signature

Three chimeric gene constructs were designed comprising the full length cDNA of a lipoxygenase (LOX) from barley (LOX2:Hv:1) including its chloroplast targeting sequence (cTP) under control of either (1) CaMV35S- or (2) polyubiquitin-1-promoter, whereas the third plasmid contains 35S promoter and the cDNA without cTP. Transgenic barley plants overexpressing LOX2:Hv:1 were generated by biolistics of scutella from immature embryos. Transformation frequency for 35S::LOX with or without cTP was in a range known for barley particle bombardment, whereas for Ubi::cTP-LOX no transgenic plants were detected. In general, a high number of green plantlets selected on bialaphos became yellow and finally died either in vitro or after potting. All transgenic plants obtained were phenotypically indistinguishable from wild type plants and all of them set seeds. The corresponding protein (LOX-100) in transgenic T0 and T1 plants accumulated constitutively to similar levels as in the jasmonic acid methyl ester (JAME)-treated wild type plants. Moreover, LOX-100 was clearly detectable immunocytochemically within the chloroplasts of untreated T0 plants containing the LOX-100-cDNA with the chloroplast target sequence. In contrast, an exclusive localization of LOX-100 in the cytoplasm was detectable when the target sequence was removed. In comparison to sorbitol-treated wild type leaves, analysis of oxylipin profiles in T2 progenies showed higher levels of jasmonic acid (JA) for those lines that displayed elevated levels of LOX-100 in the chloroplasts and for those lines that harboured LOX-100 in the cytoplasm, respectively. The studies demonstrate for the first time the constitutive overexpression of a cDNA coding for a 13-LOX in a monocotyledonous species and indicate a link between the occurrence of LOX-100 and senescence.     

Sugar concentrations along and across the Ricinus communis L. hypocotyl measured by single cell sampling analysis

Single cell sap sampling and analysis were used to measure the longitudinal and radial distribution of sucrose, glucose and fructose in the apical cell division zone and in the basal, elongated zone of the Ricinus hypocotyl. Sucrose and hexose increased in concentration from the apex to the base of the seedling axis. In the cell division zone low hexose and sucrose concentrations prevailed in cortex and pith, with a slightly higher hexose concentration in pith cells. The sucrose concentrations in sieve tubes and in phloem were much higher than in the cortex and pith cells. In the basal zone of the hypocotyl high levels of sucrose in phloem, cortex and pith were found, therefore radial, diffusional sucrose flow away from the phloem was considered unlikely. It is proposed that radial flow of growth-water to the hypocotyl periphery together with the down-regulation of a sucrose transporter at the phloem leads to a preferential sucrose flow to the expanding cortex. The pith cells, which do not experience flow of growth-water, are probably insufficiently supplied with sucrose from the phloem resulting eventually in cell death as the plant grows. Shortage of sucrose supply, experimentally achieved by removal of the endosperm, led to sucrose hydrolysis in the pith. The sucrose levels in the other tissues decreased less. It appears that the hydrolysis to hexose was initiated to maintain the osmotic value in the pith cell sap. It is speculated that high hexose levels in the cells are indicative of insufficient sucrose supply via the phloem and that the pith cells are confronted with that situation during early seedling development.     

A dominant complement fixation pathway for pneumococcal polysaccharides initiated by SIGN-R1 interacting with C1q

The intricate system of serum complement proteins provides resistance to infection. A pivotal step in the complement pathway is the assembly of a C3 convertase, which digests the C3 complement component to form microbial binding C3 fragments recognized by leukocytes. The spleen and C3 provide resistance against blood-borne S. pneumoniae infection. To better understand the mechanisms involved, we studied SIGN-R1, a lectin that captures microbial polysaccharides in spleen. Surprisingly, conditional SIGN-R1 knockout mice developed deficits in C3 catabolism when given S. pneumoniae or its capsular polysaccharide intravenously. There were marked reductions in proteolysis of serum C3, deposition of C3 on organisms within SIGN-R1(+) spleen macrophages, and formation of C3 ligands. We found that SIGN-R1 directly bound the complement C1 subcomponent, C1q, and assembled a C3 convertase, but without the traditional requirement for either antibody or factor B. The transmembrane lectin SIGN-R1 therefore contributes to innate resistance by an unusual C3 activation pathway.     

Readout technologies for highly miniaturized kinase assays applicable to high-throughput screening in a 1536-well format

This article discusses the development of homogeneous, miniaturized assays for the identification of novel kinase inhibitors from very large compound collections. In particular, the suitability of time-resolved fluorescence resonance energy transfer (TR-RET) based on phospho-specific antibodies, an antibody-independent fluorescence polarization (FP) approach using metal-coated beads (IMAP technology), and the determination of adenosine triphosphate consumption through chemiluminescence is evaluated. These readouts are compared with regard to assay sensitivity, compound interference, reagent consumption, and performance in a 1536-well format, and practical considerations for their application in primary screening or in the identification of kinase substrates are discussed. All of the tested technologies were found to be suitable for miniaturized high-throughput screening (HTS) in principle, but each of them has distinct limitations and advantages. Therefore, the target-specific selection of the most appropriate readout technology is recommended to ensure maximal relevance of HTS campaigns.     

Cannabidiol inhibits lung cancer cell invasion and metastasis via intercellular adhesion molecule-1

Cannabinoids inhibit cancer cell invasion via increasing tissue inhibitor of matrix metalloproteinases-1 (TIMP-1). This study investigates the role of intercellular adhesion molecule-1 (ICAM-1) within this action. In the lung cancer cell lines A549, H358, and H460, cannabidiol (CBD; 0.001-3 μM) elicited concentration-dependent ICAM-1 up-regulation compared to vehicle via cannabinoid receptors, transient receptor potential vanilloid 1, and p42/44 mitogen-activated protein kinase. Up-regulation of ICAM-1 mRNA by CBD in A549 was 4-fold at 3 μM, with significant effects already evident at 0.01 μM. ICAM-1 induction became significant after 2 h, whereas significant TIMP-1 mRNA increases were observed only after 48 h. Inhibition of ICAM-1 by antibody or siRNA approaches reversed the anti-invasive and TIMP-1-upregulating action of CBD and the likewise ICAM-1-inducing cannabinoids Δ(9)-tetrahydrocannabinol and R(+)-methanandamide when compared to isotype or nonsilencing siRNA controls. ICAM-1-dependent anti-invasive cannabinoid effects were confirmed in primary tumor cells from a lung cancer patient. In athymic nude mice, CBD elicited a 2.6- and 3.0-fold increase of ICAM-1 and TIMP-1 protein in A549 xenografts, as compared to vehicle-treated animals, and an antimetastatic effect that was fully reversed by a neutralizing antibody against ICAM-1 [% metastatic lung nodules vs. isotype control (100%): 47.7% for CBD + isotype antibody and 106.6% for CBD + ICAM-1 antibody]. Overall, our data indicate that cannabinoids induce ICAM-1, thereby conferring TIMP-1 induction and subsequent decreased cancer cell invasiveness.     

Structure and activity of the only human RNase T2

Mutations in the gene of human RNase T2 are associated with white matter disease of the human brain. Although brain abnormalities (bilateral temporal lobe cysts and multifocal white matter lesions) and clinical symptoms (psychomotor impairments, spasticity and epilepsy) are well characterized, the pathomechanism of RNase T2 deficiency remains unclear. RNase T2 is the only member of the Rh/T2/S family of acidic hydrolases in humans. In recent years, new functions such as tumor suppressing properties of RNase T2 have been reported that are independent of its catalytic activity. We determined the X-ray structure of human RNase T2 at 1.6 Å resolution. The α+β core fold shows high similarity to those of known T2 RNase structures from plants, while, in contrast, the external loop regions show distinct structural differences. The catalytic features of RNase T2 in presence of bivalent cations were analyzed and the structural consequences of known clinical mutations were investigated. Our data provide further insight into the function of human RNase T2 and may prove useful in understanding its mode of action independent of its enzymatic activity.     

An Archaeal Immune System Can Detect Multiple Protospacer Adjacent Motifs (PAMs) to Target Invader DNA

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system provides adaptive and heritable immunity against foreign genetic elements in most archaea and many bacteria. Although this system is widespread and diverse with many subtypes, only a few species have been investigated to elucidate the precise mechanisms for the defense of viruses or plasmids. Approximately 90% of all sequenced archaea encode CRISPR/Cas systems, but their molecular details have so far only been examined in three archaeal species: Sulfolobus solfataricus, Sulfolobus islandicus, and Pyrococcus furiosus. Here, we analyzed the CRISPR/Cas system of Haloferax volcanii using a plasmid-based invader assay. Haloferax encodes a type I-B CRISPR/Cas system with eight Cas proteins and three CRISPR loci for which the identity of protospacer adjacent motifs (PAMs) was unknown until now. We identified six different PAM sequences that are required upstream of the protospacer to permit target DNA recognition. This is only the second archaeon for which PAM sequences have been determined, and the first CRISPR group with such a high number of PAM sequences. Cells could survive the plasmid challenge if their CRISPR/Cas system was altered or defective, e.g. by deletion of the cas gene cassette. Experimental PAM data were supplemented with bioinformatics data on Haloferax and Haloquadratum.