Cholesterol-rich membrane microdomains mediate cell cycle arrest induced by Actinobacillus actinomycetemcomitans cytolethal-distending toxin

We have previously shown that Actinobacillus actinomycetemcomitans cytolethal-distending toxin (Cdt) is a potent immunosuppressive agent that induces G2/M arrest in human lymphocytes. In this study, we explored the possibility that Cdt-mediated immunotoxicity involves lipid membrane microdomains. We first determined that following treatment of Jurkat cells with Cdt holotoxin all three Cdt subunits localize to these microdomains. Laser confocal microscopy was employed to colocalize the subunits with GM1-enriched membrane regions which are characteristic of membrane rafts. Western blot analysis of isolated lipid rafts also demonstrated the presence of Cdt peptides. Cholesterol depletion, using methyl beta-cyclodextrin, protected cells from the ability of the Cdt holotoxin to induce G2 arrest. Moreover, cholesterol depletion reduced the ability of the toxin to associate with Jurkat cells. Thus, lipid raft integrity is vital to the action of Cdt on host cells. The implications of our observations with respect to Cdt mode of action are discussed.     

A Deviant Genetic Code in the Reduced Mitochondrial Genome of the Picoplanktonic Green Alga Pycnococcus provasolii

Reduction in size of flagellated chlorophytes occurred multiple times during evolution, providing the opportunity to study the consequences of cell reduction on genome architecture. Recent investigations on the chloroplast genomes of the tiny prasinophyceans Ostreococcus tauri (Mamiellales), Micromonas sp. RCC299 (Mamiellales), and Pycnococcus provasolii (Pseudocourfieldiales) highlighted their extreme compaction and reduced gene repertoires. Genome compaction is also exemplified by the Ostreococcus and Micromonas mitochondrial DNAs (mtDNAs) although they have retained almost all of the about 65 genes presumably present in the mitochondria of ancestral prasinophyceans. In this study, the mitochondrial genome of Pycnococcus was sequenced and compared to those of previously examined chlorophytes. Our results document the first case where cellular reduction of a free-living alga was accompanied by marked reduction in gene content of both the mitochondrial and chloroplast genomes. At 24,321 bp, the intronless Pycnococcus mitochondrial genome falls within the lower size range displayed by green algal mtDNAs. The 36 conserved genes, specifying two rRNAs with conventional structures, 16 tRNAs and 18 proteins, are all encoded on the same DNA strand and represent 88% of the genome. Besides a pronounced codon bias, the protein-coding genes feature a variant genetic code characterized by the use of TGA (normally a stop codon) to code for tryptophan, and the unprecedented use of TTA and TTG (normally leucine codons) as stop codons. We conclude that substantial reduction of the mitochondrial genome occurred in at least three independent chlorophyte lineages and that this process entailed a number of convergent changes in these lineages.     

The complete chloroplast DNA sequences of the charophycean green algae <Emphasis Type="Italic">Staurastrum</Emphasis> and <Emphasis Type="Italic">Zygnema</Emphasis> reveal that the chloroplast genome underwent extensive changes during the evolution of the Zygnematales

Background  

The Streptophyta comprise all land plants and six monophyletic groups of charophycean green algae. Phylogenetic analyses of four genes from three cellular compartments support the following branching order for these algal lineages: Mesostigmatales, Chlorokybales, Klebsormidiales, Zygnematales, Coleochaetales and Charales, with the last lineage being sister to land plants. Comparative analyses of the Mesostigma viride (Mesostigmatales) and land plant chloroplast genome sequences revealed that this genome experienced many gene losses, intron insertions and gene rearrangements during the evolution of charophyceans. On the other hand, the chloroplast genome of Chaetosphaeridium globosum (Coleochaetales) is highly similar to its land plant counterparts in terms of gene content, intron composition and gene order, indicating that most of the features characteristic of land plant chloroplast DNA (cpDNA) were acquired from charophycean green algae. To gain further insight into when the highly conservative pattern displayed by land plant cpDNAs originated in the Streptophyta, we have determined the cpDNA sequences of the distantly related zygnematalean algae Staurastrum punctulatum and Zygnema circumcarinatum.     

A Reinforcing Circuit Action of Extrasynaptic GABAA Receptor Modulators on Cerebellar Granule Cell Inhibition

GABA_A receptors (GABARs) are the targets of a wide variety of modulatory drugs which enhance chloride flux through GABAR ion channels. Certain GABAR modulators appear to acutely enhance the function of δ subunit-containing GABAR subtypes responsible for tonic forms of inhibition. Here we identify a reinforcing circuit mechanism by which these drugs, in addition to directly enhancing GABAR function, also increase GABA release. Electrophysiological recordings in cerebellar slices from rats homozygous for the ethanol-hypersensitive (α6100Q) allele show that modulators and agonists selective for δ-containing GABARs such as THDOC, ethanol and THIP (gaboxadol) increased the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) in granule cells. Ethanol fails to augment granule cell sIPSC frequency in the presence of glutamate receptor antagonists, indicating that circuit mechanisms involving granule cell output contribute to ethanol-enhancement of synaptic inhibition. Additionally, GABAR antagonists decrease ethanol-induced enhancement of Golgi cell firing. Consistent with a role for glutamatergic inputs, THIP-induced increases in Golgi cell firing are abolished by glutamate receptor antagonists. Moreover, THIP enhances the frequency of spontaneous excitatory postsynaptic currents in Golgi cells. Analyses of knockout mice indicate that δ subunit-containing GABARs are required for enhancing GABA release in the presence of ethanol and THIP. The limited expression of the GABAR δ subunit protein within the cerebellar cortex suggests that an indirect, circuit mechanism is responsible for stimulating Golgi cell GABA release by drugs selective for extrasynaptic isoforms of GABARs. Such circuit effects reinforce direct actions of these positive modulators on tonic GABAergic inhibition and are likely to contribute to the potent effect of these compounds as nervous system depressants.     

Predicted Effect of Landscape Position on Wildlife Habitat Value of Conservation Reserve Enhancement Program Wetlands in a Tile‐drained Agricultural Region

Justification for investment in restored or constructed wetland projects are often based on presumed net increases in ecosystem services. However, quantitative assessment of performance metrics is often difficult and restricted to a single objective. More comprehensive performance assessments could help inform decision‐makers about trade‐offs in services provided by alternative restoration program design attributes. The primary goal of the Iowa Conservation Reserve Enhancement Program is to establish wetlands that efficiently remove nitrates from tile‐drained agricultural landscapes. A secondary objective is provision of wildlife habitat. We used existing wildlife habitat models to compare relative net change in potential wildlife habitat value for four alternative landscape positions of wetlands within the watershed. Predicted species richness and habitat value for birds, mammals, amphibians, and reptiles generally increased as the wetland position moved lower in the watershed. However, predicted average net increase between pre‐ and post‐project value was dependent on taxonomic group. The increased average wetland area and changes in surrounding upland habitat composition among landscape positions were responsible for these differences. Net change in predicted densities of several grassland bird species at the four landscape positions was variable and species‐dependent. Predicted waterfowl breeding activity was greater for lower drainage position wetlands. Although our models are simplistic and provide only a predictive index of potential habitat value, we believe such assessment exercises can provide a tool for coarse‐level comparisons of alternative proposed project attributes and a basis for constructing informed hypotheses in auxiliary empirical field studies.     

Mourning dove reporting probabilities for web-address versus toll-free bands

Federal migratory bird bands have been inscribed with a postal address and toll-free telephone number (toll-free bands) since 1995 for their reporting, but in 2007 the postal mail address was replaced with an internet address (web-address bands). The reporting rate of web-address bands is unknown, and knowledge of band reporting probabilities is an integral part of bird band-recovery studies designed to obtain unbiased estimates of harvest rates and recovery rates if use of band types differs across space and time. We estimated web-address band reporting probabilities for mourning doves (Zenaida macroura) in the United States by comparing the relative recovery rate (ratio) of web-address to toll-free bands and applying this as an adjustment factor to the known reporting probabilities of toll-free bands. Cooperators banded 132,386 doves representing 57,982 (44%) web-address and 74,404 (56%) toll-free bands in July and August during 2008–2010 across 34 states, 12 regions, and 3 management units. Of the birds banded, 4,642 (3.5%) were direct recoveries, as a result of being shot, and all but 3 were reported via telephone or internet. Nationally, internet reporting accounted for 16.3% of toll-free bands and 42.9% of web-address bands recovered. Web-address bands had a greater recovery rate than toll-free bands (ratio = 1.081, SE = 0.027). We found some evidence of spatial variation in ratio estimates at management unit and regional scales, but the variation was primarily at the regional level where it was not great (range = 1.015–1.213) and statistical support for this variation was not particularly strong (SE = 0.129). Nationally, the web-address band reporting probability was 0.535 (SE = 0.021), an absolute increase of 0.040 compared to the reporting probability of toll-free bands. The additional reporting probability of web-address bands relative to toll-free bands must be accounted for in band-recovery studies used to obtain estimates of harvest rates and recovery rates if use of band types differs across space and time for mourning doves and possibly other species. © 2011 The Wildlife Society.     

The site-specific DNA endonuclease encoded by a group I intron in the Chlamydomonas pallidostigmatica chloroplast small subunit rRNA gene introduces a single-strand break at low concentrations of Mg2+.

Two group I introns (CpSSU.1 and CpSSU.2) that each potentially encode a protein with two copies of the LAGLI-DADG motif were identified in the Chlamydomonas pallidostigmatica chloroplast small subunit rRNA gene. They both belong to subgroup IA3 and represent novel insertion positions in this gene (sites 508 and 793 in the Escherichia coli 16S rRNA). The proteins encoded by the two introns were synthesized in vitro and tested for their ability to cleave the homing site of their respective introns. Only the CpSSU.1-encoded protein (I-CpaII) was found to display specific DNA endonuclease activity. At 0.1 mM MgCl2, I-CpaII nicks only the bottom (transcribed) DNA strand, but at concentrations ranging from 0.5 to 5.0 mM, it cleaves both DNA strands (leaving a 4 nucleotide single-stranded extension with 3'-OH overhangs) while preferentially nicking the bottom strand. The rate of cleavage of the top strand increases with increasing concentration of MgCl2. The preliminary data derived from these endonuclease assays suggest that the mode of DNA cleavage by I-CpaII is directed by the availability of Mg2+ and the affinity of different binding sites for this cation.