Crystallization and preliminary X-ray analysis of the sporulation factor SpoIIAA in its native and phosphorylated forms

Sporulation in Bacillus begins with an asymmetric cell division producing two progeny with identical chromosomes but different developmental fates. As such, it is a simple example of cellular differentiation. The establishment of cell type is controlled by a series of alternate RNA polymerase sigma subunits. The first compartment-specific sigma factor is sigma (F), whose activity is controlled by SpoIIAB, an anti-sigma factor, and SpoIIAA, an anti-sigma factor antagonist which is phosphorylated by the kinase activity of SpoIIAB. Here, the preliminary crystallographic analysis of SpoIIAA and phosphorylated SpoIIAA from B. sphaericus in forms suitable for high-resolution structure determination are reported

Transcription activator structure reveals redox control of a replication initiation reaction†

Redox changes are one of the factors that influence cell-cycle progression and that control the processes of cellular proliferation, differentiation, senescence and apoptosis. Proteins regulated through redox-sensitive cysteines have been characterized but specific ‘sulphydryl switches’ in replication proteins remain to be identified. In bovine papillomavirus type-1, DNA replication begins when the viral transcription factor E2 recruits the viral initiator protein E1 to the origin of DNA replication (ori). Here we show that a novel dimerization interface in the E2 transcription activation domain is stabilized by a disulphide bond. Oxidative cross-linking via Cys57 sequesters the interaction surface between E1 and E2, preventing pre-initiation and replication initiation complex formation. Our data demonstrate that as well as a mechanism for regulating DNA binding, redox reactions can control replication by modulating the tertiary structure of critical protein factors using a specific redox sensor

Pharmacological and genetic characterisation of the canine P2X4 receptor

Background and Purpose: P2X4 receptors are emerging therapeutic targets for treating chronic pain and cardiovascular disease. Dogs are well-recognised natural models of human disease, but information regarding P2X4 receptors in dogs is lacking. To aid the development and validation of P2X4 receptor ligands, we have characterised and compared canine and human P2X4 receptors. Experimental Approach: Genomic DNA was extracted from whole blood samples from 101 randomly selected dogs and sequenced across the P2RX4 gene to identify potential missense variants. Recombinant canine and human P2X4 receptors tagged with Emerald GFP were expressed in 1321N1 and HEK293 cells and analysed by immunoblotting and confocal microscopy. In these cells, receptor pharmacology was characterised using nucleotide-induced Fura-2 AM measurements of intracellular Ca 2+ and known P2X4 receptor antagonists. P2X4 receptor-mediated inward currents in HEK293 cells were assessed by automated patch clamp. Key Results: No P2RX4 missense variants were identified in any canine samples. Canine and human P2X4 receptors were localised primarily to lysosomal compartments. ATP was the primary agonist of canine P2X4 receptors with near identical efficacy and potency at human receptors. 2′(3′)-O-(4-benzoylbenzoyl)-ATP, but not ADP, was a partial agonist with reduced potency for canine P2X4 receptors compared to the human orthologues. Five antagonists inhibited canine P2X4 receptors, with 1-(2,6-dibromo-4-isopropyl-phenyl)-3-(3-pyridyl)urea displaying reduced sensitivity and potency at canine P2X4 receptors. Conclusion and Implications: P2X4 receptors are highly conserved across dog pedigrees and display expression patterns and pharmacological profiles similar to human receptors, supporting validation and use of therapeutic agents for P2X4 receptor-related disease onset and management in dogs and humans

Dynamic thyroid testing, thyroid histology and thyroxine replacement therapy in Basenji dogs

Objective: To monitor the long-term health status of a cohort of client-owned Basenji dogs over several years; to compare the accuracy of various laboratory methods to assess thyroid status; and to document any adverse effects of routinely recommended doses of thyroxine for confirmed or presumptive hypothyroidism. Methods: Health outcomes were followed by 1-8 years of clinical surveillance using serum total thyroid hormone (TT4) and canine thyroid-stimulating hormone (cTSH) concentrations, and either dynamic thyroid testing using recombinant human (rh) TSH, and/or thyroid histopathology at necropsy. The long-term accuracy of the various diagnostic interpretative criteria for rhTSH stimulation was analysed. The clinical history and serial concentrations of TT4 and cTSH in dogs suspected of exhibiting adverse clinical responses to standard thyroxine supplementation were reviewed. Results: When rhTSH interpretative criteria were compared with thyroid histology at necropsy, Larsson's equation correctly identified normal Basenji dogs from dogs with hypothyroidism and extensive thyroid pathology, yet provided an equivocal result for dogs that were not hypothyroid. Despite sequential low TT4 concentration measurements over several years of clinical surveillance, these 'equivocal' dogs had unremarkable thyroid histology (consistent with breed and age) at necropsy. Thyroxine supplementation at recommended dose rates resulted in clinical signs of thyrotoxicosis in six elderly Basenji dogs. Conclusion: Age and breed may account for dose-related adverse responses in Basenji dogs dosed with thyroxine at recommended doses. We recommend that when conducting therapeutic thyroid supplementation trials in this breed, the drug be administered in gradually increasing doses to avoid dose-related side effects. In a breed with a lower than 'normal' reference interval for serum TT4 concentration, attempts to achieve the post-pill TT4 concentration achieved in normal dogs may induce signs of thyrotoxicosis. Serial cTSH concentration measurements are helpful when adjusting thyroxine supplementation in Basenji dogs. Larsson's equation can be of assistance in interpreting dynamic thyroid testing using rhTSH in this breed. The overall prevalence of hypothyroidism in the Basenji breed appears to be low.9 page(s

Association of a P2RX7 gene missense variant with brachycephalic dog breeds

Missense variants are associated with various phenotypic traits and disorders in dogs. The canine P2RX7 gene, coding the ATP-gated P2X7 receptor ion channel, contains four known missense variants. The current study aimed to examine the presence of these variants in a random sample of pedigree and mixed-pedigree dogs. Exons 3, 8, 11 and 13 of the P2RX7 gene, encoding these four respective variants, in 65 dogs were assessed by Sanger sequencing and combined with existing sequencing data from another 69 dogs. The distribution of these variants was then evaluated in all 134 dogs combined and separately within individual breeds including 35 different pure breeds. The rs23314713 (p.Phe103Leu) and rs23315462 (p.Pro452Ser) variants were present in 47 and 40% of all dogs studied respectively, with the rs23314713 variant associated with brachycephalic breeds. Among pedigree dogs, the rs23314713 and rs23315462 variants were associated with brachycephalic and non-brachycephalic breeds respectively. The rs851148233 (p.Arg270Cys) and rs850760787 (p.Arg365Gln) variants were present only in dogs of Cocker Spaniel and Labrador Retriever pedigrees respectively. No other missense variants were found in exons 3, 8, 11 and 13 of the P2RX7 gene within the dogs. In conclusion, the rs23314713 and rs23315462 missense variants of the P2RX7 gene are present in a large proportion of dogs, with the rs23314713 variant associated with a number of brachycephalic breeds. However, the association of this variant with dogs of bulldog ancestry, not brachycephaly per se, cannot be excluded

Structure of the Bacillus cell fate determinant SpoIIAA in phosphorylated and unphosphorylated forms

Background: The asymmetric cell division during sporulation in Bacillus subtilis gives rise to two compartments: the mother cell and the forespore. Each follow different programs of gene expression coordinated by a succession of alternate RNA polymerase σ factors. The activity of the first of these σ factors, σF, is restricted to the forespore although σF is present in the predivisional cell and partitions into both compartments following the asymmetric septation. For σF to become active, it must escape from a complex with its cognate anti-σ factor, SpoIIAB. This relief from SpoIIAB inhibition requires the dephosphorylation of the anti-σ factor antagonist, SpoIIAA. The phosphorylation state of SpoIIAA is thus a key determinant of σF activity and cell fate. Results: We have solved the crystal structures of SpoIIAA from Bacillus sphaericus in its phosphorylated and unphosphorylated forms. The overall structure consists of a central β-pleated sheet, one face of which is buried by a pair of α helices, while the other is largely exposed to solvent. The site of phosphorylation, Ser57, is located at the N terminus of helix α2. The phosphoserine is exceptionally well defined in the 1.2 Å electron density maps, revealing that the structural changes accompanying phosphorylation are slight. Conclusions: Comparison of unphosphorylated and phosphorylated SpoIIAA shows that covalent modification has no significant effect on the global structure of the protein. The phosphoryl group has a passive role as a negatively charged flag rather than the active role it plays as a nucleus of structural reorganization in many eukaryotic signaling systems

Características de plantas de cultivares de arroz irrigado relacionadas à habilidade competitiva com plantas concorrentes Characteristics of flooded rice cultivar plants related to competitive ability against weeds concurrent plants

Plantas que apresentam rápido crescimento tendem a ocupar precocemente nichos disponíveis, utilizando o espaço de suas vizinhas. Em geral, esses vegetais adquirem prioridade na utilização dos recursos do meio. O objetivo deste trabalho foi avaliar variações em características de crescimento de cultivares de arroz irrigado. Para isso, conduziu-se um experimento em campo na estação de crescimento de 2000/2001, em Cachoeirinha-RS. Investigou-se o comportamento de oito cultivares de arroz, cultivados em presença e ausência de plantas de arroz do cultivar EEA 406, simulando infestação de arroz-vermelho, estabelecida com densidade média de 30 plantas m-2. Entre 15 e 60 dias após a semeadura (DAS), avaliou-se a evolução de área foliar, estatura e massa aérea das plantas de arroz. Com esses dados, calcularam-se razão de área foliar (RAF), taxa de crescimento relativo (TCR) e taxa de assimilação líquida (TAL) dos cultivares. Os cultivares Ligeirinho e XL 6 apresentaram elevadas velocidades de ganho em área foliar, estatura e massa aérea; com isso, alcançaram as maiores coberturas do solo, juntamente com o cultivar IR 841. Por outro lado, os cultivares Bluebelle e Formosa mostraram lento crescimento absoluto no período avaliado, mas, em geral, apresentaram os maiores valores para TCR e TAL.<br>Fast-growing plants tend to occupy available niches utilizing these spaces ahead of their neighbors. In general, these plants have priority over others in the use of environmental resources. The objective of this research was to evaluate variations in growth characteristics of flooded rice cultivars. A field experiment was carried out during the 2000/01 summer season, in Cachoeirinha, RS, Brazil. The behavior of eight rice cultivars, grown in the absence or presence of the EEA 406 rice genotype was investigated, simulating a red rice infestation, established at an average density of 30 plants m-2. From 15 to 60 days after seeding (DAS), variations in leaf area, plant height, and dry weight of rice plants were evaluated. The data were used to calculate leaf area rate (LAR), relative growth rate (RGR), and net assimilation rate (NAR) of the cultivars. The cultivars Ligeirinho and XL 6 presented the fastest increments in leaf area, plant height, and dry weight, reaching the highest soil cover values, together with the cultivar IR 841. On the other hand, cultivars Bluebelle and Formosa showed slow absolute growth during the evaluated period, even though presenting the highest values for RGR and NAR, overall

Competitividade de cultivares de arroz irrigado com cultivar simuladora de arroz-vermelho Competitiveness of flooded rice cultivars with a red rice simulating cultivar

Características morfológicas e fisiológicas de plantas cultivadas podem afetar sua habilidade competitiva com plantas daninhas. Este trabalho objetivou investigar a competitividade de cultivares de arroz irrigado (Oryza sativa L.) com cultivar simuladora de arroz-vermelho. Investigou-se na safra 2000/2001 o comportamento de oito genótipos de arroz, cultivados na presença ou ausência da cultivar de arroz EEA 406, que simulou infestação de arroz-vermelho. Aos 45 e aos 60 dias após a semeadura, avaliou-se a resposta da simuladora em relação às cultivares de arroz. Na colheita, foram determinados estatura de planta, componentes do rendimento e produtividade das plantas. A cultivar tardia IR 841 suprimiu o crescimento da simuladora, apresentando com a cultivar superprecoce Ligeirinho as menores reduções de produtividade quando em competição. Por sua vez, as cultivares IAS 12-9 Formosa e Bluebelle permitiram maior crescimento e produção de sementes pela concorrente, também sofrendo as maiores reduções de produtividade de grãos na condição de competição.<br>Morphologic and physiologic characteristics of crop plants can affect their competitive ability with weeds. This research aimed to investigate competitiveness of flooded rice (Oryza sativa L.) cultivars with a red rice simulating genotype. It was investigated, during the 2000/2001 warm season, the behavior of eight rice genotypes, grown in presence or absence of the rice cultivar EEA 406, simulating a red rice infestation. At 45 and 60 days after rice seeding, it was evaluated the response of red rice mimicker genotype in relation to rice cultivars. At harvest, it was determined plant height, yield components, and grain yield of the cultivars. The late season genotype IR 841 suppressed growth of the simulative cultivar, presenting, together with the veryearly cultivar Ligeirinho, the lowest reductions in grain yield under competition. In opposite, IAS 12-9 Formosa and Bluebelle cultivars allowed the greatest growth and seed production by the concurrent rice, also presenting the largest decreases of grain yield under the competition condition

AT7867 Is a Potent and Oral Inhibitor of AKT and p70 S6 Kinase That Induces Pharmacodynamic Changes and Inhibits Human Tumor Xenograft Growth

The serine/threonine kinase AKT plays a pivotal role in signal transduction events involved in malignant transformation and chemoresistance and is an attractive target for the development of cancer therapeutics. Fragment-based lead discovery, combined with structure-based drug design, has recently identified AT7867 as a novel and potent inhibitor of both AKT and the downstream kinase p70 S6 kinase (p70S6K) and also of protein kinase A. This ATP-competitive small molecule potently inhibits both AKT and p70S6K activity at the cellular level, as measured by inhibition of GSK3? and S6 ribosomal protein phosphorylation, and also causes growth inhibition in a range of human cancer cell lines as a single agent. Induction of apoptosis was detected by multiple methods in tumor cells following AT7867 treatment. Administration of AT7867 (90 mg/kg p.o. or 20 mg/kg i.p.) to athymic mice implanted with the PTEN-deficient U87MG human glioblastoma xenograft model caused inhibition of phosphorylation of downstream substrates of both AKT and p70S6K and induction of apoptosis, confirming the observations made in vitro. These doses of AT7867 also resulted in inhibition of human tumor growth in PTEN-deficient xenograft models. These data suggest that the novel strategy of AKT and p70S6K blockade may have therapeutic value and supports further evaluation of AT7867 as a single-agent anticancer strategy