Impact of angiotensin II type 2 receptor blockade on experimental radiation nephropathy

In the rat, blockade of angiotensin II type 1 receptors diminishes the functional changes that occur after kidney irradiation. It has been hypothesized that some of the beneficial effects of angiotensin II type 1 blockers in renal disease are caused by a rise in angiotensin II that stimulates the angiotensin II type 2 receptor. If this hypothesis applied in this model, blockade of the type 2 receptor should exacerbate radiation nephropathy and/or counteract the beneficial effects of type 1 receptor blockade. To assess this hypothesis, rats were given total-body irradiation plus bone marrow transplantation and then treated for 12 weeks with a type 1 receptor blocker (L158,809), a type 2 blocker (PD123319), both blockers, or no blockers. Rats were assessed for renal function (proteinuria, hypertension, azotemia) and renal failure for up to 62 weeks. Contrary to the hypothesis, the type 2 blocker alone produced a temporary delay in the development of radiation nephropathy, and it substantially enhanced the efficacy of the type 1 blocker. This implies that both type 1 and type 2 angiotensin receptors need to be blocked to achieve the maximum level of prophylaxis of radiation nephropathy. We speculate that the beneficial effect of the angiotensin II type 2 receptor blocker is due to a reduction in radiation-induced renal cell proliferation or fibrosis.     

Re-infestation of houses by Triatoma dimidiata after intra-domicile insecticide application in the Yucatán peninsula, Mexico

In most countries, Chagas disease transmission control remains based on domestic insecticide application. We thus evaluated the efficacy of intra-domicile cyfluthrin spraying for the control of Triatoma dimidiata, the only Chagas disease vector in the Yucatán peninsula, Mexico, and monitored potential re-infestation every 15 days for up to 9 months. We found that there was a re-infestation of houses by adult bugs starting 4 months after insecticide application, possibly from sylvatic/peridomicile areas. This points out the need to take into account the potential dispersal of sylvatic/peridomestic adult bugs into the domiciles as well as continuity action for an effective vector control.     

Cross talk between 2,4-diacetylphloroglucinol-producing biocontrol pseudomonads on wheat roots

The performance of Pseudomonas biocontrol agents may be improved by applying mixtures of strains which are complementary in their capacity to suppress plant diseases. Here, we have chosen the combination of Pseudomonas fluorescens CHA0 with another well-characterized biocontrol agent, P. fluorescens Q2-87, as a model to study how these strains affect each other's expression of a biocontrol trait. In both strains, production of the antimicrobial compound 2,4-diacetylphloroglucinol (DAPG) is a crucial factor contributing to the suppression of root diseases. DAPG acts as a signaling compound inducing the expression of its own biosynthetic genes. Experimental setups were developed to investigate whether, when combining strains CHA0 and Q2-87, DAPG excreted by one strain may influence expression of DAPG-biosynthetic genes in the other strain in vitro and on the roots of wheat. DAPG production was monitored by observing the expression of lacZ fused to the biosynthetic gene phlA of the respective strain. Dual-culture assays in which the two strains were grown in liquid medium physically separated by a membrane revealed that Q2-87 but not its DAPG-negative mutant Q2-87::Tn5-1 strongly induced phlA expression in a DeltaphlA mutant of strain CHA0. In the same way, phlA expression in a Q2-87 background was induced by DAPG produced by CHA0. When coinoculated onto the roots of wheat seedlings grown under gnotobiotic conditions, strains Q2-87 and CHA0, but not their respective DAPG-negative mutants, were able to enhance phlA expression in each other. In summary, we have established that two nonrelated pseudomonads may stimulate each other in the expression of an antimicrobial compound important for biocontrol. This interpopulation communication occurs in the rhizosphere, i.e., at the site of pathogen inhibition, and is mediated by the antimicrobial compound itself acting as a signal exchanged between the two pseudomonads.     

Epidemic clone I-specific genetic markers in strains of Listeria monocytogenes serotype 4b from foods

Listeria monocytogenes contamination of ready-to-eat foods has been implicated in numerous outbreaks of food-borne listeriosis. However, the health hazards posed by L. monocytogenes detected in foods may vary, and speculations exist that strains actually implicated in illness may constitute only a fraction of those that contaminate foods. In this study, examination of 34 serogroup 4 (putative or confirmed serotype 4b) isolates of L. monocytogenes obtained from various foods and food-processing environments, without known implication in illness, revealed that many of these strains had methylation of cytosines at GATC sites in the genome, rendering their DNA resistant to digestion by the restriction endonuclease Sau3AI. These strains also harbored a gene cassette with putative restriction-modification system genes as well as other, genomically unlinked genetic markers characteristic of the major epidemic-associated lineage of L. monocytogenes (epidemic clone I), implicated in numerous outbreaks in Europe and North America. This may reflect a relatively high fitness of strains with these genetic markers in foods and food-related environments relative to other serotype 4b strains and may partially account for the repeated involvement of such strains in human food-borne listeriosis.     

Highly efficient production of laccase by the basidiomycete Pycnoporus cinnabarinus

An efficient transformation and expression system was developed for the industrially relevant basidiomycete Pycnoporus cinnabarinus. This was used to transform a laccase-deficient monokaryotic strain with the homologous lac1 laccase gene placed under the regulation of its own promoter or that of the SC3 hydrophobin gene or the glyceraldehyde-3-phosphate dehydrogenase (GPD) gene of Schizophyllum commune. SC3-driven expression resulted in a maximal laccase activity of 107 nkat ml(-1) in liquid shaken cultures. This value was about 1.4 and 1.6 times higher in the cases of the GPD and lac1 promoters, respectively. lac1-driven expression strongly increased when 25 g of ethanol liter(-1) was added to the medium. Accordingly, laccase activity increased to 1,223 nkat ml(-1). These findings agree with the fact that ethanol induces laccase gene expression in some fungi. Remarkably, lac1 mRNA accumulation and laccase activity also strongly increased in the presence of 25 g of ethanol liter(-1) when lac1 was expressed behind the SC3 or GPD promoter. In the latter case, a maximal laccase activity of 1,393 nkat ml(-1) (i.e., 360 mg liter(-1)) was obtained. Laccase production was further increased in transformants expressing lac1 behind its own promoter or that of GPD by growth in the presence of 40 g of ethanol liter(-1). In this case, maximal activities were 3,900 and 4,660 nkat ml(-1), respectively, corresponding to 1 and 1.2 g of laccase per liter and thus representing the highest laccase activities reported for recombinant fungal strains. These results suggest that P. cinnabarinus may be a host of choice for the production of other proteins as well.     

PCR-denaturing gradient gel electrophoresis profiling of inter- and intraspecies 18S rRNA gene sequence heterogeneity is an accurate and sensitive method to assess species diversity of arbuscular mycorrhizal fungi of the genus Gigaspora

Despite the importance of arbuscular mycorrhizal fungi in the majority of terrestrial ecosystems, their ecology, genetics, and evolution are poorly understood, partly due to difficulties associated with detecting and identifying species. We explored the inter- and intraspecies variations of the 18S rRNA genes of the genus Gigaspora to assess the use of this marker for the discrimination of Gigaspora isolates and of Gigasporaceae populations from environmental samples. Screening of 48 Gigaspora isolates by PCR-denaturing gradient gel electrophoresis (DGGE) revealed that the V3-V4 region of the 18S rRNA gene contained insufficient variation to discriminate between different Gigaspora species. In contrast, the patterns of 18S ribosomal DNA (rDNA) heterogeneity within the V9 region of this marker could be used for reliable identification of all recognized species within this genus. PCR-DGGE patterns provided insight into some putative misidentifications and could be used to differentiate geographic isolates of G. albida, G. gigantea, and G. margarita but not G. rosea. Two major clusters were apparent based upon PCR-DGGE ribotype patterns, one containing G. albida, G. candida, G. ramisporophora, and G. rosea and the other containing G. decipiens and G. margarita. Dissection of the DGGE patterns by cloning, DGGE screening, and sequencing confirmed these groupings and revealed that some ribotypes were shared across species boundaries. Of the 48 isolates examined, only two displayed any spore-to-spore variation, and these exceptions may be indicative of coisolation of more than one species or subspecies within these cultures. Two Brazilian agricultural soils were also analyzed with a Gigasporaceae-specific nested PCR approach, revealing a dominance of G. margarita within this family.     

Two direct repeats cause most human mtDNA deletions

Mitochondrial DNA (mtDNA) deletions are a common cause of human mitochondrial disease and also occur as part of normal aging. However, it is unknown how the deletions actually occur. To gain further insight, we studied the sequences that flank 263 different human mtDNA deletions. The distribution of deletion breakpoints did not correspond to the basic parameters of wild-type mtDNA that are thought to predispose to deletion formation. But there was a striking correspondence to the position of two 13-bp direct repeats beginning at nucleotides 8470 and 13 447. The vast majority of different mtDNA deletions appear to be related to these two repeats, suggesting a common mechanism related to mtDNA replication.     

RIP: the evolutionary cost of genome defense

Repeat-induced point mutation (RIP) is a homology-based process that mutates repetitive DNA and frequently leads to epigenetic silencing of the mutated sequences through DNA methylation. Consistent with the hypothesis that RIP serves to control selfish DNA, an analysis of the Neurospora crassa genome sequence reveals a complete absence of intact mobile elements. As in most eukaryotes, the centromeric regions of N. crassa are rich in sequences that are related to transposable elements; however, in N crassa these sequences have been heavily mutated. The analysis of the N. crassa genome sequence also reveals that RIP has impacted genome evolution significantly through gene duplication, which is considered to be crucial for the evolution of new functions. Most if not all paralogs in N. crassa duplicated and diverged before the emergence of RIP. Thus, RIP illustrates the extraordinary extent to which genomes will go to defend themselves against mobile genetic elements.     

Logos: a modular bayesian model for de novo motif detection

The complexity of the global organization and internal structure of motifs in higher eukaryotic organisms raises significant challenges for motif detection techniques. To achieve successful de novo motif detection, it is necessary to model the complex dependencies within and among motifs and to incorporate biological prior knowledge. In this paper, we present LOGOS, an integrated LOcal and GlObal motif Sequence model for biopolymer sequences, which provides a principled framework for developing, modularizing, extending and computing expressive motif models for complex biopolymer sequence analysis. LOGOS consists of two interacting submodels: HMDM, a local alignment model capturing biological prior knowledge and positional dependency within the motif local structure; and HMM, a global motif distribution model modeling frequencies and dependencies of motif occurrences. Model parameters can be fit using training motifs within an empirical Bayesian framework. A variational EM algorithm is developed for de novo motif detection. LOGOS improves over existing models that ignore biological priors and dependencies in motif structures and motif occurrences, and demonstrates superior performance on both semi-realistic test data and cis-regulatory sequences from yeast and Drosophila genomes with regard to sensitivity, specificity, flexibility and extensibility.     

Synthesis of chiral phosphoantigens and their activity in gamma delta T cell stimulation

Gammadelta T cells expressing Vgamma2Vdelta2 T cell receptors are activated by a broad range of phosphorus-containing small molecules, termed phosphoantigens, and are of interest in the context of the chemotherapy of B cell malignancies. Here, we report the synthesis of four pairs of chiral phosphoantigens: the bromohydrins of isopentenyl diphosphate (Phosphostim), the epoxides of isopentenyl diphosphate (EIPP); and the corresponding bromohydrin and epoxide analogs of but-3-enyl diphosphate. The ability of each compound to stimulate human Vgamma2Vdelta2 T cells was determined by TNF-alpha release and cell proliferation. In these assays, the (R)-bromohydrin diphosphates were, on average, about twice as active as the (S)-bromohydrin diphosphates. In contrast, the (S)-form of EIPP was about twice as active as (R)-EIPP. The activities of the epoxy but-3-enyl diphosphates were both very low. These results suggest that chiral phosphoantigens, as opposed to racemic mixtures, may have utility in immunotherapy.