Anion-Sensitive Regions of L-Type CaV1.2 Calcium Channels Expressed in HEK293 Cells

L-type calcium currents (I_Ca) are influenced by changes in extracellular chloride, but sites of anion effects have not been identified. Our experiments showed that CaV1.2 currents expressed in HEK293 cells are strongly inhibited by replacing extracellular chloride with gluconate or perchlorate. Variance-mean analysis of I_Ca and cell-attached patch single channel recordings indicate that gluconate-induced inhibition is due to intracellular anion effects on Ca²⁺ channel open probability, not conductance. Inhibition of CaV1.2 currents produced by replacing chloride with gluconate was reduced from ∼75%–80% to ∼50% by omitting β subunits but unaffected by omitting α₂δ subunits. Similarly, gluconate inhibition was reduced to ∼50% by deleting an α1 subunit N-terminal region of 15 residues critical for β subunit interactions regulating open probability. Omitting β subunits with this mutant α1 subunit did not further diminish inhibition. Gluconate inhibition was unchanged with expression of different β subunits. Truncating the C terminus at AA1665 reduced gluconate inhibition from ∼75%–80% to ∼50% whereas truncating it at AA1700 had no effect. Neutralizing arginines at AA1696 and 1697 by replacement with glutamines reduced gluconate inhibition to ∼60% indicating these residues are particularly important for anion effects. Expressing CaV1.2 channels that lacked both N and C termini reduced gluconate inhibition to ∼25% consistent with additive interactions between the two tail regions. Our results suggest that modest changes in intracellular anion concentration can produce significant effects on CaV1.2 currents mediated by changes in channel open probability involving β subunit interactions with the N terminus and a short C terminal region.     

Plasmid-encoded antirestriction protein ArdA can discriminate between type I methyltransferase and complete restriction-modification system

Many promiscuous plasmids encode the antirestriction proteins ArdA (alleviation of restriction of DNA) that specifically affect the restriction activity of heterooligomeric type I restriction-modification (R-M) systems in Escherichia coli cells. In addition, a lot of the putative ardA genes encoded by plasmids and bacterial chromosomes are found as a result of sequencing of complete genomic sequences, suggesting that ArdA proteins and type I R-M systems that seem to be widespread among bacteria may be involved in the regulation of gene transfer among bacterial genomes. Here, the mechanism of antirestriction action of ArdA encoded by IncI plasmid ColIb-P9 has been investigated in comparison with that of well-studied T7 phage-encoded antirestriction protein Ocr using the mutational analysis, retardation assay and His-tag affinity chromatography. Like Ocr, ArdA protein was shown to be able to efficiently interact with EcoKI R-M complex and affect its in vivo and in vitro restriction activity by preventing its interaction with specific DNA. However, unlike Ocr, ArdA protein has a low binding affinity to EcoKI Mtase and the additional C-terminal tail region (VF-motif) is needed for ArdA to efficiently interact with the type I R-M enzymes. It seems likely that this ArdA feature is a basis for its ability to discriminate between activities of EcoKI Mtase (modification) and complete R-M system (restriction) which may interact with unmodified DNA in the cells independently. These findings suggest that ArdA may provide a very effective and delicate control for the restriction and modification activities of type I systems and its ability to discriminate against DNA restriction in favour of the specific modification of DNA may give some advantage for efficient transmission of the ardA-encoding promiscuous plasmids among different bacterial populations.     

A contribution towards resolving the nomenclature of Citharexylum (Verbenaceae). II. Remarks on implicit typifications and lectotypification of names linked to Mesoamerican taxa

During ongoing taxonomic studies in Citharexylum (Verbenaceae), sixteen names were found in need of typification or typification remarks. As a result, six names (C. brachyanthum, C. danirae, C. flabellifolium, C. lucidum, C. rugendasii and C. tristachyum f. urbanii) are here lectotypified. Furthermore, remarks on implicit typifications by Harold Moldenke for another ten names (C. altamiranum, C. berlandieri, C. hexangulare, C. macradenium, C. ovatifolium, C. pauciflorum, C. pterocladum, C. recurvatum, C. schulzii and C. stenophyllum) are provided.     

Multiple binding sites in fibrinogen for integrin alphaMbeta2 (Mac-1)

The leukocyte integrin alphaMbeta2 (Mac-1) is a multiligand receptor that mediates a range of adhesive reactions of leukocytes during the inflammatory response. This integrin binds the coagulation protein fibrinogen providing a key link between thrombosis and inflammation. However, the mechanism by which alphaMbeta2 binds fibrinogen remains unknown. Previous studies indicated that a model in which two fibrinogen gammaC domain sequences, P1 (gamma190-202) and P2 (gamma377-395), serve as the alphaMbeta2 binding sites cannot fully account for recognition of fibrinogen by integrin. Here, using surface plasmon resonance, we examined the interaction of the ligand binding alphaMI-domain of alphaMbeta2 with the D fragment of fibrinogen and showed that this ligand is capable of associating with several alphaMI-domain molecules. To localize the alternative alphaMI-domain binding sites, we screened peptide libraries covering the complete sequences of the gammaC and betaC domains, comprising the majority of the D fragment structure, for alphaMI-domain binding. In addition to the P2 and P1 peptides, the alphaMI-domain bound to many other sequences in the gammaC and betaC scans. Similar to P1 and P2, synthetic peptides derived from gammaC and betaC were efficient inhibitors of alphaMbeta2-mediated cell adhesion and were able to directly support adhesion suggesting that they contain identical recognition information. Analyses of recognition specificity using substitutional peptide libraries demonstrated that the alphaMI-domain binding depends on basic and hydrophobic residues. These findings establish a new model of alphaMbeta2 binding in which the alphaMI-domain interacts with multiple sites in fibrinogen and has the potential to recognize numerous sequences. This paradigm may have implications for mechanisms of promiscuity in ligand binding exhibited by integrin alphaMbeta2.     

Transmission genetics of Nicotiana hybrids produced by protoplast fusion

Somatic cell hybrids were produced by fusing protoplasts isolated from callus cells of a tobacco line transformed by Agrobacterium tumefaciens (octopine synthesizing strain B6S3), and mesophyll protoplasts from haploid plants of Nicotiana plumbaginifolia. Hybrids were selected by using differential medium (hormone-independent growth plus greening capacity), or by mechanical isolation and cloning of individual heterokaryocytes. The analysis of hybrid cell lines included the determination of lysopine dehydrogenase activity (encoded by the T-region of Agrobacterium tumefaciens plasmid), examination of isozymes of esterase, and study of chromosome number and morphology. All eight cell lines selected on the screening medium were identified as nuclear hybrids, while only three of the eight evaluated clones obtained by mechanical isolation and cloning were found to be nuclear hydrids; the rest of them were nuclear segregants of tobacco [1] or N. plumbaginifolia [4] type. These data give independent evidence for the occurrence of non-fusion and segregation of nuclei in fusion products, that can be revealed only by using nonselective methods for hybrid screening. In this paper we demonstrate the value of microisolation for the recovery of cytoplasmic hybrids.     

Probable mechanism of sexual dimorphism in insulin control of Drosophila heat stress resistance

Adult males of Drosophila species (Drosophila melanogaster L. and Drosophila virilis) show a lower tolerance to heat stress compared with females. The present study investigates the effects of RNA interference (RNAi) knockdown of the insulin‐like receptor in the corpus allatum of D. melanogaster males on dopamine metabolism and content, heat stress resistance and juvenile hormone metabolism. In male flies, the knockdown of insulin‐like receptor in the corpus allatum is shown to change metabolism of juvenile hormone but not dopamine. It is also shown that knockdown of the insulin‐like receptor in the corpus allatum results in a decrease of heat stress resistance in females but not males, and that juvenile hormone rescues this decrease. The results suggest that at least one of the ways in which insulin‐like growth factors affect heat stress resistance is by successive mediation through juvenile hormone and dopamine, which could lead to sexual dimorphism in the trait.     

miR‐200/375 control epithelial plasticity‐associated alternative splicing by repressing the RNA‐binding protein Quaking

Members of the miR‐200 family are critical gatekeepers of the epithelial state, restraining expression of pro‐mesenchymal genes that drive epithelial–mesenchymal transition (EMT) and contribute to metastatic cancer progression. Here, we show that miR‐200c and another epithelial‐enriched miRNA, miR‐375, exert widespread control of alternative splicing in cancer cells by suppressing the RNA‐binding protein Quaking (QKI). During EMT, QKI‐5 directly binds to and regulates hundreds of alternative splicing targets and exerts pleiotropic effects, such as increasing cell migration and invasion and restraining tumour growth, without appreciably affecting mRNA levels. QKI‐5 is both necessary and sufficient to direct EMT‐associated alternative splicing changes, and this splicing signature is broadly conserved across many epithelial‐derived cancer types. Importantly, several actin cytoskeleton‐associated genes are directly targeted by both QKI and miR‐200c, revealing coordinated control of alternative splicing and mRNA abundance during EMT. These findings demonstrate the existence of a miR‐200/miR‐375/QKI axis that impacts cancer‐associated epithelial cell plasticity through widespread control of alternative splicing.     

Mitochondrial binding of hexokinase II inhibits Bax-induced cytochrome c release and apoptosis.

Proapoptotic proteins such as Bax, undergo translocation to the mitochondria during apoptosis, where they mediate the release of intermembrane space proteins including cytochrome c. Bax binds to the voltage-dependent anion channel (VDAC). VDAC is a beta-barrel protein located in the outer mitochondrial membrane. In planar lipid bilayers, Bax and VDAC form a channel through which cytochrome c can pass. Hexokinase II (HXK II) also binds to VDAC. HXK II catalyzes the first step of glycolysis and is highly expressed in transformed cells, where over 70% of it is bound to the mitochondria. The present study demonstrates that HXK II interferes with the ability of Bax to bind to mitochondria and release cytochrome c. Detachment of HXK II from the mitochondria-enriched fraction isolated from HeLa cells promoted the binding of recombinant Bax-Delta19 and subsequent cytochrome c release. Similarly, the addition of recombinant HXK II to the mitochondria-enriched fraction isolated from hepatocytes, cells that do not express HXK II endogenously, prevented the ability of recombinant Bax-Delta19 to bind to the mitochondria and promote cytochrome c release. Similar results were found in intact cells, in which the detachment of mitochondrial bound HXK II or its overexpression potentiated and inhibited, respectively, Bax-induced mitochondrial dysfunction and cell death.