Thermal resistance to photoreactivation of specific mutations potentiated in E. coli B/r ung by ultraviolet light

Summary Mutagenesis by ultraviolet light was studied in a strain of E. coli ung, which lacks uracil-DNA glycosylase activity. Mutation potentiated by UV in cells already induced by nalidixic acid treatment was still photoreversible suggesting that pyrimidine dimers act directly as premutational photoproducts. Secondly, irradiated cells were held in buffer at 48°C for 0 to 135 min to allow for deamination of cytosines in pyrimidine dimers. The mutation frequencies for class 2 de novo suppressor mutation, for class 2 converted suppressor mutation and for backmutation were individually determined, before and after photoreactivation, as a function of this thermal treatment. Backmutation remained sensitive to photoreactivation throughout the treatment but de novo and converted suppressor mutations rapidly developed resistance to photoreactivation. This resistance was not seen in an ung ⁺ control. A model is proposed to account for the selective resistance based on the hypothesis that class 2 de novo and converted suppressor mutations normally result from UV by GC to AT transitions at T=C dimers. The model describes deamination of the cytosine residues in these dimers to become uracil residues. In consequence, monomerization by photoreactivation in cells that can not repair uracils in DNA no longer reverses mutation and GC to AT transitions are established at the sites of uracils.     

In vitro and in vivo application of RNA interference for targeting genes involved in peritrophic matrix synthesis in a lepidopteran system

Abstract The midgut of most insects is lined with a semipermeable acellular tube, the peritrophic matrix (PM), composed of chitin and proteins. Although various genes encoding PM proteins have been characterized, our understanding of their roles in PM structure and function is very limited. One promising approach for obtaining functional information is RNA interference, which has been used to reduce the levels of specific mRNAs using double‐stranded RNAs administered to larvae by either injection or feeding. Although this method is well documented in dipterans and coleopterans, reports of its success in lepidopterans are varied. In the current study, the silencing midgut genes encoding PM proteins (insect intestinal mucin 1, insect intestinal mucin 4, PM protein 1) and the chitin biosynthetic or modifying enzymes (chitin synthase‐B and chitin deacetylase 1) in a noctuid lepidopteran, Mamestra configurata, was examined in vitro and in vivo. In vitro studies in primary midgut epithelial cell preparations revealed an acute and rapid silencing (by 24 h) for the gene encoding chitin deacetylase 1 and a slower rate of silencing (by 72 h) for the gene encoding PM protein 1. Genes encoding insect intestinal mucins were slightly silenced by 72 h, whereas no silencing was detected for the gene encoding chitin synthase‐B. In vivo experiments focused on chitin deacetylase 1, as the gene was silenced to the greatest extent in vitro. Continuous feeding of neonates and fourth instar larvae with double‐stranded RNA resulted in silencing of chitin deacetylase 1 by 24 and 36 h, respectively. Feeding a single dose to neonates also resulted in silencing by 24 h. The current study demonstrates that genes encoding PM proteins can be silenced and outlines conditions for RNA interference by per os feeding in lepidopterans.     

Structure and stability of the ankyrin domain of the Drosophila Notch receptor

The Notch receptor contains a conserved ankyrin repeat domain that is required for Notch-mediated signal transduction. The ankyrin domain of Drosophila Notch contains six ankyrin sequence repeats previously identified as closely matching the ankyrin repeat consensus sequence, and a putative seventh C-terminal sequence repeat that exhibits lower similarity to the consensus sequence. To better understand the role of the Notch ankyrin domain in Notch-mediated signaling and to examine how structure is distributed among the seven ankyrin sequence repeats, we have determined the crystal structure of this domain to 2.0 angstroms resolution. The seventh, C-terminal, ankyrin sequence repeat adopts a regular ankyrin fold, but the first, N-terminal ankyrin repeat, which contains a 15-residue insertion, appears to be largely disordered. The structure reveals a substantial interface between ankyrin polypeptides, showing a high degree of shape and charge complementarity, which may be related to homotypic interactions suggested from indirect studies. However, the Notch ankyrin domain remains largely monomeric in solution, demonstrating that this interface alone is not sufficient to promote tight association. Using the structure, we have classified reported mutations within the Notch ankyrin domain that are known to disrupt signaling into those that affect buried residues and those restricted to surface residues. We show that the buried substitutions greatly decrease protein stability, whereas the surface substitutions have only a marginal affect on stability. The surface substitutions are thus likely to interfere with Notch signaling by disrupting specific Notch-effector interactions and map the sites of these interactions.     

n-3 PUFA attenuate lipopolysaccharide-induced down-regulation of toll-like receptor 4 expression in porcine adipose tissue but does not alter the expression of other immune modulators

The objective of this study was to test the hypothesis that the inflammatory response to lipopolysaccharide (LPS) in vivo is accompanied by down-regulation of toll-like receptor (TLR) 4 in adipose tissue, and a source of protected n-3 polyunsaturated fatty acid (PUFA) attenuates this response. Seventy-two castrated male pigs were individually fed either a control (CONT) diet, or the CONT diet containing 1.87% (LF) or 7.50% (HF) protected n-3 PUFA on a weight basis for 7 weeks. Adipose and muscle tissue biopsy samples were taken at Weeks 1, 2, 3, 4 and 7 to assess gene expression and/or confirm tissue enrichment with eicosapentaenoic acid and docosahexaenoic acid and reflected the n-3 PUFA contained in the diet. The LPS challenge was performed at week 7 and consisted of sequential injections of 10 and 2.5 mug LPS per kilogram of body weight 23 h apart. The LPS challenge resulted in a marked down-regulation (P=.004) of TLR4 at the protein level in the adipose tissue of challenged vs. control pigs, but LF and HF clearly blocked this response at the mRNA level. Although LF and HF also attenuated (P<.001) the LPS-induced acute febrile response and lowered (P<.002) serum concentrations of tumour necrosis factor alpha. Cyclooxygenase 2 and 12-lipoxygenase were readily expressed in porcine adipose tissue, but there was no effect of LF, HF or LPS on expression levels of these inflammatory mediators, or that of TNF and interleukin 6, at the conclusion of the challenge period. These findings indicate that adipose tissue responds to LPS administration in vivo by reducing TLR4 mRNA and protein abundance and that the anti-inflammatory effects of n-3 PUFA do not include down-regulation of TLR4 in adipose tissue.     

Compensatory substitutions in the HCV NS3/4A protease cleavage sites are not observed in patients treated unsuccessfully with telaprevir combination treatment

ABSTRACT: BACKGROUND: Development of compensatory mutations within the HIV p7/p1 and p1/p6 protease cleavage site region has been observed in HIV-infected patients treated with protease inhibitors. Mechanisms of fitness compensation may occur in HCV populations upon treatment of HCV protease inhibitors as well. FINDINGS: In this study, we investigated whether substitutions in protease cleavage site regions of HCV occur in response to a treatment regimen containing the NS3/4A protease inhibitor telaprevir (TVR). Evaluation of viral populations from 569 patients prior to treatment showed that the four NS3/4A cleavage sites were well conserved. Few changes in the cleavage site regions were observed in the 159 patients who failed TVR combination treatment, and no residues displayed evidence of directional selection after the acquisition of TVR-resistance. CONCLUSIONS: Cleavage site mutations did not occur after treatment with the HCV protease inhibitor telaprevir.     

Dance Education Matters: Rebuilding Postsecondary Dance Education for Twenty-First Century Relevance and Resonance

Postsecondary dance education is at a crucial juncture in its history in academe. Emerging from women's physical education programs in the 1930s, the profession's realignment with the arts broadly and arts-based education specifically has been characterized by ambitious goals and steady growth through the 1990s. However, a number of critical developments over the past decade have displaced many previous gains and undermined the overall stability and integrity of the field. Four primary challenges are investigated in this article: curricular equity, expansive dance education programs, graduate study opportunities, and national leadership. I urge dance educators and administrators to re-envision and expand P–12 dance education in the liberal arts tradition to include private studio, commercial-sector dance in community and related teaching professions. This article provides recommendations and strategies for developing relevant and resonant twenty-first-century dance education programs beyond current confines.     

Detection and identification of Lactobacillus species in crops of broilers of different ages by using PCR-denaturing gradient gel electrophoresis and amplified ribosomal DNA restriction analysis

The microflora of the crop was investigated throughout the broiler production period (0 to 42 days) using PCR combined with denaturing gradient gel electrophoresis (PCR-DGGE) and selective bacteriological culture of lactobacilli followed by amplified ribosomal DNA restriction analysis (ARDRA). The birds were raised under conditions similar to those used in commercial broiler production. Lactobacilli predominated and attained populations of 10(8) to 10(9) CFU per gram of crop contents. Many of the lactobacilli present in the crop (61.9% of isolates) belonged to species of the Lactobacillus acidophilus group and could not be differentiated by PCR-DGGE. A rapid and simple ARDRA method was developed to distinguish between the members of the L. acidophilus group. HaeIII-ARDRA was used for preliminary identification of isolates in the L. acidophilus group and to identify Lactobacillus reuteri and Lactobacillus salivarius. MseI-ARDRA generated unique patterns for all species of the L. acidophilus group, identifying Lactobacillus crispatus, Lactobacillus johnsonii, and Lactobacillus gallinarum among crop isolates. The results of our study provide comprehensive knowledge of the Lactobacillus microflora in the crops of birds of different ages using nucleic acid-based methods of detection and identification based on current taxonomic criteria.     

Sgt1 acts via an LKB1/AMPK pathway to establish cortical polarity in larval neuroblasts

Drosophila neuroblasts are a model system for studying stem cell self-renewal and the establishment of cortical polarity. Larval neuroblasts generate a large apical self-renewing neuroblast, and a small basal cell that differentiates. We performed a genetic screen to identify regulators of neuroblast self-renewal, and identified a mutation in sgt1 (suppressor-of-G2-allele-of-skp1) that had fewer neuroblasts. We found that sgt1 neuroblasts have two polarity phenotypes: failure to establish apical cortical polarity at prophase, and lack of cortical Scribble localization throughout the cell cycle. Apical cortical polarity was partially restored at metaphase by a microtubule-induced cortical polarity pathway. Double mutants lacking Sgt1 and Pins (a microtubule-induced polarity pathway component) resulted in neuroblasts without detectable cortical polarity and formation of "neuroblast tumors." Mutants in hsp83 (encoding the predicted Sgt1-binding protein Hsp90), LKB1, or AMPKα all show similar prophase apical cortical polarity defects (but no Scribble phenotype), and activated AMPKα rescued the sgt1 mutant phenotype. We propose that an Sgt1/Hsp90-LKB1-AMPK pathway acts redundantly with a microtubule-induced polarity pathway to generate neuroblast cortical polarity, and the absence of neuroblast cortical polarity can produce neuroblast tumors.     

Interaction of the trp RNA-binding attenuation protein (TRAP) with anti-TRAP

The trp RNA-binding attenuation protein (TRAP) negatively regulates expression of the tryptophan biosynthesis genes of Bacillus subtilis. In the presence of tryptophan, TRAP is activated to bind to the 5'-leader region of the trp mRNA resulting in termination prior to the structural genes. In addition, accumulation of uncharged tRNA(Trp) induces synthesis of anti-TRAP (AT), which binds to TRAP and inhibits its function. Both of these proteins consist of oligomers of identical subunits. Here, we characterize the self-association of each of these proteins and the TRAP-AT interaction in free solution using equilibrium and velocity analytical ultracentrifugation. TRAP exists as a stable 11-mer in the absence and in the presence of tryptophan. Tryptophan binding induces a conformational change in TRAP. AT exists in a reversible equilibrium between trimer and dodecamer with an equilibrium constant of approximately 3 x 10(14)M(-3). About 20% of the trimer is incompetent to form dodecamer. The AT equilibrium is slow on the time-scale of the velocity experiment. Formation of TRAP-AT complexes occurs only in the presence of tryptophan. A complex containing one TRAP 11-mer and one AT 12-mer forms with high affinity. At higher ratios of TRAP:AT complexes containing two TRAP 11-mers and one AT 12-mer are detected. A model for the structure of the complex is proposed.