Electrostatic interactions during acidic phospholipid reactivation of DnaA protein, the Escherichia coli initiator of chromosomal replication.

The initiation of Escherichia coli chromosomal replication by DnaA protein is strongly influenced by the tight binding of the nucleotides ATP and ADP. Anionic phospholipids in a fluid bilayer promote the conversion of inactive ADP-DnaA protein to replicatively active ATP-DnaA protein in vitro, and thus likely play a key role in regulating DnaA activity. Previous studies have revealed that, during this reactivation, a specific region of DnaA protein inserts into the hydrophobic portion of the lipid bilayer in an acidic phospholipid-dependent manner. To elucidate the requirement for acidic phospholipids in the reactivation process, the contribution of electrostatic forces in the interaction of DnaA and lipid was examined. DnaA-lipid binding required anionic phospholipids, and DnaA-lipid binding as well as lipid-mediated release of DnaA-bound nucleotide were inhibited by increased ionic strength, suggesting the involvement of electrostatic interactions in these processes. As the vesicular content of acidic phospholipids was increased, both nucleotide release and DnaA-lipid binding increased in a linear, parallel manner. Given that DnaA-membrane binding, the insertion of DnaA into the membrane, and the consequent nucleotide release all require anionic phospholipids, the acidic headgroup may be necessary to recruit DnaA protein to the membrane for insertion and subsequent reactivation for replication.     

Reconstitution of a GTPase Activity a 50S Ribosomal Protein from <Emphasis Type="Italic">E. coli</Emphasis>

DURING each step of peptide chain elongation the ribosome shifts up one triplet along the messenger RNA with concomitant movement of the peptidyl-transfer RNA from the donor to the acceptor site. This process, commonly known as translocation, is triggered by a supernatant protein, factor G, which in association with the ribosome cleaves GTP into GDP and inorganic phosphate^1,2 and it has been argued that the energy liberated in this reaction is used “to carry the complex one triplet forward”³.     

Single-Stranded RNAs Use RNAi to Potently and Allele-Selectively Inhibit Mutant Huntingtin Expression

Mutant huntingtin (HTT) protein causes Huntington disease (HD), an incurable neurological disorder. Silencing mutant HTT using nucleic acids would eliminate the root cause of HD. Developing nucleic acid drugs is challenging, and an ideal clinical approach to gene silencing would combine the simplicity of single-stranded antisense oligonucleotides with the efficiency of RNAi. Here, we describe RNAi by single-stranded siRNAs (ss-siRNAs). ss-siRNAs are potent (>100-fold more than unmodified RNA) and allele-selective (>30-fold) inhibitors of mutant HTT expression in cells derived from HD patients. Strategic placement of mismatched bases mimics micro-RNA recognition and optimizes discrimination between mutant and wild-type alleles. ss-siRNAs require Argonaute protein and function through the RNAi pathway. Intraventricular infusion of ss-siRNA produced selective silencing of the mutant HTT allele throughout the brain in a mouse HD model. These data demonstrate that chemically modified ss-siRNAs function through the RNAi pathway and provide allele-selective compounds for clinical development.     

Single-Stranded siRNAs Activate RNAi in Animals

The therapeutic utility of siRNAs is limited by the requirement for complex formulations to deliver them to tissues. If potent single-stranded RNAs could be identified, they would provide a simpler path to pharmacological agents. Here, we describe single-stranded siRNAs (ss-siRNAs) that silence gene expression in animals absent lipid formulation. Effective ss-siRNAs were identified by iterative design by determining structure-activity relationships correlating chemically modified single strands and Argonaute 2 (AGO2) activities, potency in cells, nuclease stability, and pharmacokinetics. We find that the passenger strand is not necessary for potent gene silencing. The guide-strand activity requires AGO2, demonstrating action through the RNAi pathway. ss-siRNA action requires a 5' phosphate to achieve activity in?vivo, and we developed a metabolically stable 5'-(E)-vinylphosphonate (5'-VP) with conformation and sterioelectronic properties similar to the natural phosphate. Identification of potent ss-siRNAs offers an additional option for RNAi therapeutics and an alternate perspective on RNAi mechanism.     

Nuclear envelope phosphatase 1-regulatory subunit 1 (formerly TMEM188) is the metazoan Spo7p ortholog and functions in the lipin activation pathway

Lipin-1 catalyzes the formation of diacylglycerol from phosphatidic acid. Lipin-1 mutations cause lipodystrophy in mice and acute myopathy in humans. It is heavily phosphorylated, and the yeast ortholog Pah1p becomes membrane-associated and active upon dephosphorylation by the Nem1p-Spo7p membrane complex. A mammalian ortholog of Nem1p is the C-terminal domain nuclear envelope phosphatase 1 (CTDNEP1, formerly "dullard"), but its Spo7p-like partner is unknown, and the need for its existence is debated. Here, we identify the metazoan ortholog of Spo7p, TMEM188, renamed nuclear envelope phosphatase 1-regulatory subunit 1 (NEP1-R1). CTDNEP1 and NEP1-R1 together complement a nem1Δspo7Δ strain to block endoplasmic reticulum proliferation and restore triacylglycerol levels and lipid droplet number. The two human orthologs are in a complex in cells, and the amount of CTDNEP1 is increased in the presence of NEP1-R1. In the Caenorhabditis elegans embryo, expression of nematode CTDNEP1 and NEP1-R1, as well as lipin-1, is required for normal nuclear membrane breakdown after zygote formation. The expression pattern of NEP1-R1 and CTDNEP1 in human and mouse tissues closely mirrors that of lipin-1. CTDNEP1 can dephosphorylate lipins-1a, -1b, and -2 in human cells only in the presence of NEP1-R1. The nuclear fraction of lipin-1b is increased when CTDNEP1 and NEP1-R1 are co-expressed. Therefore, NEP1-R1 is functionally conserved from yeast to humans and functions in the lipin activation pathway.     

Genome size rather than content might affect call properties in toads of three ploidy levels (Anura: Bufonidae: Bufo viridis subgroup)

In vertebrates, genome size has been shown to correlate with nuclear and cell sizes, and influences phenotypic features, such as brain complexity. In three different anuran families, advertisement calls of polyploids exhibit longer notes and intervals than diploids, and difference in cellular dimensions have been hypothesized to cause these modifications. We investigated this phenomenon in green toads (Bufo viridis subgroup) of three ploidy levels, in a different call type (release calls) that may evolve independently from advertisement calls, examining 1205 calls, from ten species, subspecies, and hybrid forms. Significant differences between pulse rates of six diploid and four polyploid (3n, 4n) green toad forms across a range of temperatures from 7 to 27 °C were found. Laboratory data supported differences in pulse rates of triploids vs. tetraploids, but failed to reach significance when including field recordings. This study supports the idea that genome size, irrespective of call type, phylogenetic context, and geographical background, might affect call properties in anurans and suggests a common principle governing this relationship. The nuclear‐cell size ratio, affected by genome size, seems the most plausible explanation. However, we cannot rule out hypotheses under which call‐influencing genes from an unexamined diploid ancestral species might also affect call properties in the hybrid‐origin polyploids. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 105 , 584–590.     

THE EFFECT OF SOME BEVERAGES ON THE PERCEPTION OF SUCCEEDING BASIC TASTES

The objective of this study was to investigate the effect beverages have on subsequently perceived basic tastes. Two types of milk, Cola and water were used to rinse the mouth prior to conducting a Time Intensity study with basic taste solutions. From this some effects of the prior beverage onto the TI parameters was found. Especially Cola showed an effect on the intensity of the subsequently tasted sweet taste, and whole milk showed a stronger effect than skim milk. The effects showed both in size and timing of the basic taste intensities.     

CspD, a novel DNA replication inhibitor induced during the stationary phase in Escherichia coli

CspD is a stationary phase-induced, stress response protein in the CspA family of Escherichia coli. Here, we demonstrate that overproduction of CspD is lethal, with the cells displaying a morphology typical of cells with impaired DNA replication. CspD consists mainly of beta-strands, and the purified protein exists exclusively as a dimer and binds to single-stranded (ss)DNA and RNA in a dose-dependent manner without apparent sequence specificity. CsdD effectively inhibits both the initiation and the elongation steps of minichromosome replication in vitro. Electron microscopic studies revealed that CspD tightly packs ssDNA, resulting in structures distinctly different from those of SSB-coated DNA. We propose that CspD dimers, with two independent beta-sheets interacting with ssDNA, function as a novel inhibitor of DNA replication and play a regulatory role in chromosomal replication in nutrient-depleted cells.