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The convergent synthesis of geometrically degradable dendrimers based on the 2,4-bis(hydroxymethyl)phenol subunit is presented. The key step of the synthetic scheme involves the CuI/3,4,7,8-tetramethyl-1,10-phenanthroline-catalyzed coupling of aryl iodides and alcohols. The synthesis and disassembly of these compounds is discussed.

The biosynthesis of many natural products of clinical interest involves large, multidomain enzymes called nonribosomal peptide synthetases (NRPSs). In bacteria, many of the gene clusters coding for NRPSs also code for a member of the MbtH-like protein superfamily, which are small proteins of unknown function. Using MbtH-like proteins from three separate NRPS systems, we show that these proteins copurify with the NRPSs and influence amino acid activation. As a consequence, MbtH-like proteins are integral components of NRPSs.

The structural behaviour of CuInSe(2) under high pressure has been studied up to 53 GPa using angle-dispersive x-ray powder diffraction techniques. The previously reported structural phase transition from its ambient pressure tetragonal structure to a high pressure phase with a NaCl-like cubic structure at 7.6 GPa has been confirmed. On further compression, another structural phase transition is observed at 39 GPa. A full structural study of this high pressure phase has been carried out and the high pressure structure has been identified as orthorhombic with space group Cmcm and lattice parameters a = 4.867(8) Å, b = 5.023(8) Å and c = 4.980(3) Å at 53.2(2) GPa. This phase transition behaviour is similar to those of analogous binary and trinary semiconductors, where the orthorhombic Cmcm structure can also be viewed as a distortion of the cubic NaCl-type structure.

Nucleic acid cytidine deaminases of the activation-induced deaminase (AID)/APOBEC family are critical players in active and innate immune responses, playing roles as target-directed, purposeful mutators. AID specifically deaminates the host immunoglobulin (Ig) locus to evolve antibody specificity, whereas its close relative, APOBEC3G (A3G), lethally mutates the genomes of retroviral pathogens such as HIV. Understanding the basis for the target-specific action of these enzymes is essential, as mistargeting poses significant risks, potentially promoting oncogenesis (AID) or fostering drug resistance (A3G). AID prefers to deaminate cytosine in WRC (W = A/T, R = A/G) motifs, whereas A3G favors deamination of CCC motifs. This specificity is largely dictated by a single, divergent protein loop in the enzyme family that recognizes the DNA sequence. Through grafting of this substrate-recognition loop, we have created enzyme variants of A3G and AID with altered local targeting to directly evaluate the role of sequence specificity on immune function. We find that grafted loops placed in the A3G scaffold all produced efficient restriction of HIV but that foreign loops in the AID scaffold compromised hypermutation and class switch recombination. Local targeting, therefore, appears alterable for innate defense against retroviruses by A3G but important for adaptive antibody maturation catalyzed by AID. Notably, AID targeting within the Ig locus is proportionally correlated to its in vitro ability to target WRC sequences rather than non-WRC sequences. Although other mechanisms may also contribute, our results suggest that local sequence targeting by AID/APOBEC3 enzymes represents an elegant example of co-evolution of enzyme specificity with its target DNA sequence.

We have determined the full crystal structure of the high-pressure phase methane A. X-ray single-crystal diffraction data were used to determine the carbon-atom arrangement, and neutron powder diffraction data from a deuterated sample allowed the deuterium atoms to be located. It was then possible to refine all the hydrogen positions from the single-crystal x-ray data. The structure has 21 molecules in a rhombohedral unit cell, and is quite strongly distorted from the cubic close-packed structure of methane I, although some structural similarities remain. Full knowledge of this structure is important for modeling of methane at higher pressures, including in relation to the mineralogy of the outer solar system. We discuss interesting structural parallels with the carbon tetrahalides.

Phakopsora pachyrhizi is an obligate pathogen that causes Asian soybean rust. Asian soybean rust has an unusually broad host range and infects by direct penetration through the leaf cuticle. In order to understand the early events in the infection process, it is important to identify and characterize proteins in P. pachyrhizi. Germination of the urediniospore is the first stage in the infection process and represents a critical life stage applicable to studies with this obligate pathogen. We have applied a 2-DE and MS approach to identify 117 proteins from the National Center of Biotechnology Information nonredundant protein database and a custom database of Basidiomycota EST sequences. Proteins with roles in primary metabolism, energy transduction, stress, cellular regulation and signaling were identified in this study. This data set is accessible at http://world-2dpage.expasy.org/repository/database=0018.

No abstract given.

Recognition and repair of cellular damage is crucial if organisms are to survive harmful environmental conditions. In mammals, the Keap1 protein orchestrates this response, but how it perceives adverse circumstances is not fully understood. Herein, we implicate NO, Zn(2+), and alkenals, endogenously occurring chemicals whose concentrations increase during stress, in this process. By combining molecular modeling with phylogenetic, chemical, and functional analyses, we show that Keap1 directly recognizes NO, Zn(2+), and alkenals through three distinct sensors. The C288 alkenal sensor is of ancient origin, having evolved in a common ancestor of bilaterans. The Zn(2+) sensor minimally comprises H225, C226, and C613. The most recent sensor, the NO sensor, emerged coincident with an expansion of the NOS gene family in vertebrates. It comprises a cluster of basic amino acids (H129, K131, R135, K150, and H154) that facilitate S-nitrosation of C151. Taken together, our data suggest that Keap1 is a specialized sensor that quantifies stress by monitoring the intracellular concentrations of NO, Zn(2+), and alkenals, which collectively serve as second messengers that may signify danger and/or damage.

A series of 2,2-dimethyl-3,3-diphenyl-propanamides as novel glucocorticoid receptor modulators is reported. SAR exploration led to the identification of 4-hydroxyphenyl propanamide derivatives displaying good agonist activity in GR-mediated transrepression assays and reduced agonist activity in GR-mediated transactivation assays. Compounds 17 and 30 showed anti-inflammatory activity comparable to prednisolone in the rat carrageenan-induced paw edema model, with markedly decreased side effects with regard to increases in blood glucose and expression of hepatic tyrosine aminotransferase. A hypothetical binding mode accounting for the induction of the functional activity by a 4-hydroxyl group is proposed.

BACKGROUND:
Allotetraploids carry pairs of diverged homoeologs for most genes. With the genome doubled in size, the number of putative interactions is enormous. This poses challenges on how to coordinate the two disparate genomes, and creates opportunities by enhancing the phenotypic variation. New combinations of alleles co-adapt and respond to new environmental pressures. Three stages of the allopolyploidization process--parental species divergence, hybridization, and genome duplication--have been well analyzed. The last stage of evolutionary adjustments remains mysterious.

RESULTS:
Homoeolog-specific retention and use were analyzed in Arabidopsis suecica (As), a species derived from A. thaliana (At) and A. arenosa (Aa) in a single event 12,000 to 300,000 years ago. We used 405,466 diagnostic features on tiling microarrays to recognize At and Aa contributions to the As genome and transcriptome: 324 genes lacked Aa contributions and 614 genes lacked At contributions within As. In leaf tissues, 3,458 genes preferentially expressed At homoeologs while 4,150 favored Aa homoeologs. These patterns were validated with resequencing. Genes with preferential use of Aa homoeologs were enriched for expression functions, consistent with the dominance of Aa transcription. Heterologous networks--mixed from At and Aa transcripts--were underrepresented.

CONCLUSIONS:
Thousands of deleted and silenced homoeologs in the genome of As were identified. Since heterologous networks may be compromised by interspecies incompatibilities, these networks evolve co-biases, expressing either only Aa or only At homoeologs. This progressive change towards predominantly pure parental networks might contribute to phenotypic variability and plasticity, and enable the species to exploit a larger range of environments.

A small study was carried out in order to examine the molecular presence of bla CTX-M gene phylogenetic groups in E. coli (n=263) isolated from food (n=54), water (n=7), animal sources (n=69), using consensus bla CTX-M primers and PCR, in addition to human faecal isolates (n=69) and VTEC O157:H7 (n=64). None of the clinically significant faecal VTEC O157:H7 isolates were shown to carry blaCTX-M type phylogenetic groups, nor were such phylogenetic groups observed in any of the food, water and animal isolates. One community faecal isolate (1/69; 1.4%), dating from 1997, carried this phylogenetic group. As recent work has indicated that a significant proportion of such phylogenetic groups are carried in community isolates of E. coli with little or no hospital contact, it is important that surveillance is increased to identify potential source(s) and reservoirs of such resistance in the community. Further prospective surveillance is thus required to help elucidate the origins of such phylogenetic group in the community. The significance of this study is that the ESBL-producing E. coli associated with local hospital outbreaks is not commonly found in local food, water or animal sources. In addition, given that ESBL-producing E. coli is now a significant organism, both in hospitals and nursing homes in Northern Ireland, this report demonstrates that such organisms were present in the community, as early as 1997.

In order to understand at the tissue level how Aedes aegypti copes with toxic ammonia concentrations that result from the rapid metabolism of blood meal proteins, we investigated the incorporation of (15)N from (15)NH(4)Cl into amino acids using an in vitro tissue culture system. Fat body or midgut tissues from female mosquitoes were incubated in an Aedes saline solution supplemented with glucose and (15)NH(4)Cl for 10-40min. The media were then mixed with deuterium-labeled amino acids, dried and derivatized. The (15)N-labeled and unlabeled amino acids in each sample were quantified by mass spectrometry techniques. The results demonstrate that both tissues efficiently incorporate ammonia into amino acids, however, the specific metabolic pathways are distinct. In the fat body, the (15)N from (15)NH(4)Cl is first incorporated into the amide side chain of Gln and then into the amino group of Gln, Glu, Ala and Pro. This process mainly occurs via the glutamine synthetase (GS) and glutamate synthase (GltS) pathway. In contrast, (15)N in midgut is first incorporated into the amino group of Glu and Ala, and then into the amide side chain of Gln. Interestingly, our data show that the GS/GltS pathway is not functional in the midgut. Instead, midgut cells detoxify ammonia by glutamate dehydrogenase, alanine aminotransferase and GS. These data provide new insights into ammonia metabolism in A. aegypti mosquitoes.

Thioredoxins reduce disulfide bonds and other thiol modifications in all cells using a CXXC motif. Human thioredoxin 1 is unusual in that it codes for an additional three cysteines in its 105 amino acid sequence, each of which have been implicated in other reductive activities. Cys 62 and Cys 69 are buried in the protein interior and lie at either end of a short helix (helix 3), and yet can disulfide link under oxidizing conditions. Cys 62 is readily S-nitrosated, giving rise to a SNO modification, which is also buried. Here, we present two crystal structures of the C69S/C73S mutant protein under oxidizing (1.5 A) and reducing (1.1 A) conditions. In the oxidized structure, helix 3 is unraveled and displays a new conformation that is stabilized by a series of new hydrogen bonds and a disulfide link with Cys 62 in a neighboring molecule. The new conformation provides an explanation for how a completely buried residue can participate in SNO exchange reactions.

We have sequenced the genomes of 18 isolates of the closely related human pathogenic fungi Coccidioides immitis and Coccidioides posadasii to more clearly elucidate population genomic structure, bringing the total number of sequenced genomes for each species to 10. Our data confirm earlier microsatellite-based findings that these species are genetically differentiated, but our population genomics approach reveals that hybridization and genetic introgression have recently occurred between the two species. The directionality of introgression is primarily from C. posadasii to C. immitis, and we find more than 800 genes exhibiting strong evidence of introgression in one or more sequenced isolates. We performed PCR-based sequencing of one region exhibiting introgression in 40 C. immitis isolates to confirm and better define the extent of gene flow between the species. We find more coding sequence than expected by chance in the introgressed regions, suggesting that natural selection may play a role in the observed genetic exchange. We find notable heterogeneity in repetitive sequence composition among the sequenced genomes and present the first detailed genome-wide profile of a repeat-induced point mutation (RIP) process distinctly different from what has been observed in Neurospora. We identify promiscuous HLA-I and HLA-II epitopes in both proteomes and discuss the possible implications of introgression and population genomic data for public health and vaccine candidate prioritization. This study highlights the importance of population genomic data for detecting subtle but potentially important phenomena such as introgression.

In plants, double fertilization requires successful sperm cell delivery into the female gametophyte followed by migration, recognition and fusion of the two sperm cells with two female gametes. We isolated a null allele (lre-5) of LORELEI, which encodes a putative glycosylphosphatidylinositol (GPI)-anchored protein implicated in reception of the pollen tube by the female gametophyte. Although most lre-5 female gametophytes do not allow pollen tube reception, in those that do, early seed development is delayed. A fraction of lre-5/lre-5 seeds underwent abortion due to defect(s) in the female gametophyte. The aborted seeds contained endosperm but no zygote/embryo, reminiscent of autonomous endosperm development in the pollen tube reception mutants scylla and sirene. However, unpollinated lre-5/lre-5 ovules did not initiate autonomous endosperm development and endosperm development in aborted seeds began after central cell fertilization. Thus, the egg cell probably remained unfertilized in aborted lre-5/lre-5 seeds. The lre-5/lre-5 ovules that remain undeveloped due to defective pollen tube reception did not induce synergid degeneration and repulsion of supernumerary pollen tubes. In ovules, LORELEI is expressed during pollen tube reception, double fertilization and early seed development. Null mutants of LORELEI-like-GPI-anchored protein 1 (LLG1), the closest relative of LORELEI among three Arabidopsis LLG genes, are fully fertile and did not enhance reproductive defects in lre-5/lre-5 pistils, suggesting that LLG1 function is not redundant with that of LORELEI in the female gametophyte. Our results show that, besides pollen tube reception, LORELEI also functions during double fertilization and early seed development.

Double fertilization, uniquely observed in plants, requires successful sperm cell delivery by the pollen tube to the female gametophyte, followed by migration, recognition and fusion of the two sperm cells with two female gametic cells. The female gametophyte not only regulates these steps but also controls the subsequent initiation of seed development. Previously, we reported that loss of LORELEI, which encodes a putative glycosylphosphatidylinositol (GPI)-anchored protein, in the female reproductive tissues causes a delay in initiation of seed development. From these studies, however, it was unclear if embryos derived from fertilization of lre-5 gametophytes continued to lag behind wild type during seed development. Additionally, it was not determined if the delay in initiation of seed development had any lingering effects during seed germination. Finally, it was not known if loss of LORELEI function affects seedling development given that LORELEI is expressed in eight-day-old seedlings. Here, we showed that despite a delay in initiation, lre-5/lre-5 embryos recover, becoming equivalent to the developing wild-type embryos beginning at 72 hours after pollination. Additionally, lre-5/lre-5 seed germination, and seedling and root development are indistinguishable from wild type indicating that loss of LORELEI is tolerated, at least under standard growth conditions, in vegetative tissues.

Translational development - in the sense of translating a mature methodology from one area of application to another, evolving area - is discussed for the use of benchmark doses in quantitative risk assessment. Illustrations are presented with traditional applications of the benchmark paradigm in biology and toxicology, and also with risk endpoints that differ from traditional toxicological archetypes. It is seen that the benchmark approach can apply to a diverse spectrum of risk management settings. This suggests a promising future for this important risk-analytic tool. Extensions of the method to a wider variety of applications represent a significant opportunity for enhancing environmental, biomedical, industrial, and socio-economic risk assessments.

Many inferential procedures for generalized linear models rely on the asymptotic normality of the maximum likelihood estimator (MLE). Fahrmeir & Kaufmann (1985, Ann. Stat., 13, 1) present mild conditions under which the MLEs in GLiMs are asymptotically normal. Unfortunately, limited study has appeared for the special case of binomial response models beyond the familiar logit and probit links, and for more general links such as the complementary log-log link, and the less well-known complementary log link. We verify the asymptotic normality conditions of the MLEs for these models under the assumption of a fixed number of experimental groups and present a simple set of conditions for any twice differentiable monotone link function. We also study the quality of the approximation for constructing asymptotic Wald confidence regions. Our results show that for small sample sizes with certain link functions the approximation can be problematic, especially for cases where the parameters are close to the boundary of the parameter space.

Lymphodepletion before adoptive cell transfer (ACT)-based immunotherapies can enhance anti-tumor responses by augmenting innate immunity, by increasing access to homeostatic cytokines, and by depressing the numbers of regulatory T cells and myeloid-derived suppressor cells. Although it is clear that high-dose total body irradiation given together with hematopoietic stem cell (HSC) transplantation effectively enhances ACT, the relationship between the intensity of lymphodepletion and tumor treatment efficacy has not been systematically studied. Using the pmel-1 mouse model of self/tumor-reactive CD8 T cells, we observed a strong correlation between the intensity of the conditioning regimen and the efficacy of ACT-based treatments using linear regression analysis. This was the case for preparative total body irradiation administered either as a single dose (R=0.97, P<0.001) or in fractionated doses (R=0.94, P<0.001). Increased amounts of preparative total body irradiation were directly correlated with progressively more favorable ratios of transferred tumor-reactive CD8 T cells toward endogenous cells with the potential for inhibitory activity including: CD4 cells (potentially T regulatory cells); Gr1 cells (which are capable of functioning as myeloid-derived suppressor cells); and endogenous CD8 and natural killer 1.1 cells (that can act as "sinks" for homeostatic cytokines in the postablative setting). With increasing ablation, we also observed elevated lipopolysaccharide levels in the sera and heightened levels of systemic inflammatory cytokines. Thus, increased intensity lymphodepletion triggers enhanced tumor treatment efficacy and the benefits of high-dose total body irradiation must be titrated against its risks.

The ideal experimental system would be cheap and easy to maintain, amenable to a variety of techniques, and would be supported by an extensive literature and genome sequence database. Cultured Drosophila S2 cells, the product of disassociated 20-24 hour old embryos, possess all these properties. Consequently, S2 cells are extremely well-suited for the analysis of cellular processes, including the discovery of the genes encoding the molecular components of the process or mechanism of interest. The features of S2 cells that are most responsible for their utility are the ease with which they are maintained, their exquisite sensitivity to double-stranded (ds)RNA-mediated interference (RNAi), and their tractability to fluorescence microscopy as either live or fixed cells. S2 cells can be grown in a variety of media, including a number of inexpensive, commercially-available, fully-defined, serum-free media. In addition, they grow optimally and quickly at 21-24 degrees C and can be cultured in a variety of containers. Unlike mammalian cells, S2 cells do not require a regulated atmosphere, but instead do well with normal air and can even be maintained in sealed flasks. Complementing the ease of RNAi in S2 cells is the ability to readily analyze experimentally-induced phenotypes by phase or fluorescence microscopy of fixed or live cells. S2 cells grow in culture as a single monolayer but do not display contact inhibition. Instead, cells tend to grow in colonies in dense cultures. At low density, S2 cultures grown on glass or tissue culture-treated plastic are round and loosely-attached. However, the cytology of S2 cells can be greatly improved by inducing them to flatten extensively by briefly culturing them on a surface coated with the lectin, concanavalin A (ConA). S2 cells can also be stably transfected with fluorescently-tagged markers to label structures or organelles of interest in live or fixed cells. Therefore, the usual scenario for the microscopic analysis of cells is this: first, S2 cells (which can possess transgenes to express tagged markers) are treated by RNAi to eliminate a target protein(s). RNAi treatment time can be adjusted to allow for differences in protein turn-over kinetics and to minimize cell trauma/death if the target protein is important for viability. Next, the treated cells are transferred to a dish containing a coverslip pre-coated with conA to induce cells to spread and tightly adhere to the glass. Finally, cells are imaged with the researcher's choice of microscopy modes. S2 cells are particularly good for studies requiring extended visualization of live cells since these cells stay healthy at room temperature and normal atmosphere.

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