Detection and discovery of RNA modifications using microarrays

Using a microarray that tiles all known yeast non-coding RNAs, we compared RNA from wild-type cells with RNA from mutants encoding known and putative RNA modifying enzymes. We show that at least five types of RNA modification (dihydrouridine, m1G, m2(2)G, m1A and m6(2)A) catalyzed by 10 different enzymes (Trm1p, Trm5, Trm10p, Dus1p-Dus4p, Dim1p, Gcd10p and Gcd14p) can be detected by virtue of differential hybridization to oligonucleotides on the array that are complementary to the modified sites. Using this approach, we identified a previously undetected m1A modification in GlnCTG tRNA, the formation of which is catalyzed by the Gcd10/Gcd14 complex. complex.     

Effects of parathyroid hormone on Wnt signaling pathway in bone

The Wnt signaling pathway has recently been demonstrated to play an important role in bone cell function. In previous studies using DNA microarray analyses, we observed a change in some of the molecular components of the canonical Wnt pathway namely, frizzled-1 (FZD-1) and axil, in response to continuous parathyroid hormone (PTH) treatment in rats. In the present study, we further explored other components of the Wnt signaling pathway in rat distal metaphyseal bone in vivo, and rat osteoblastic osteosarcoma cells (UMR 106) in culture. Several Wnt pathway components, including low-density lipoprotein-receptor-related protein 5 (LRP5), LRP6, FZD-1, Dickkopf-1 (Dkk-1), and Kremen-1 (KRM-1), were expressed in bone in vivo and in osteoblasts in vitro. Continuous exposure to PTH (1-38) both in vivo and in vitro upregulated the mRNA expression of LRP6 and FZD-1 and decreased LRP5 and Dkk-1. These effects in UMR 106 cells were associated with an increase in beta-catenin as measured by Western blots and resulted in functional activation (three to six-fold) of a downstream Wnt responsive TBE6-luciferase (TCF/LEF-binding element) reporter gene. Activation of the TBE6-luciferase reporter gene by PTH (1-38) in UMR 106 cells was inhibited by the protein kinase A (PKA) inhibitor, H89. Activation was mimicked by PTH (1-31), PTH-related protein (1-34), and forskolin, but both PTH (3-34) and (7-34) had no effect. These findings suggest that the effect of PTH on the canonical Wnt signaling pathway occurs at least in part via the cAMP-PKA pathway through the differential regulation of the receptor complex proteins (FZD-1/LRP5 or LRP6) and the antagonist (Dkk-1). Taken together, these results reveal a possible role for the Wnt signaling pathway in PTH actions in bone.     

Patterns of diversifying selection in the phytotoxin-like scr74 gene family of Phytophthora infestans

Phytophthora infestans, the organism responsible for the Irish famine, causes late blight, a re-emerging disease of potato and tomato. Little is known about the molecular evolution of P. infestans genes. To identify candidate effector genes (virulence or avirulence genes) that may have co-evolved with the host, we mined expressed sequence tag (EST) data from infection stages of P. infestans for secreted and potentially polymorphic genes. This led to the identification of scr74, a gene that encodes a predicted 74-amino acid secreted cysteine-rich protein with similarity to the Phytophthora cactorum phytotoxin PcF. The expression of scr74 was upregulated approximately 60-fold 2 to 4 days after inoculation of tomato and was also significantly induced during early stages of colonization of potato. The scr74 gene was found to belong to a highly polymorphic gene family within P. infestans with 21 different sequences identified. Using the approximate and maximum likelihood (ML) methods, we found that diversifying selection likely caused the extensive polymorphism observed within the scr74 gene family. Pairwise comparisons of 17 scr74 sequences revealed elevated ratios of nonsynonymous to synonymous nucleotide-substitution rates, particularly in the mature region of the proteins. Using ML, all 21 polymorphic amino acid sites were identified to be under diversifying selection. Of these 21 amino acids, 19 are located in the mature protein region, suggesting that selection may have acted on the functional portions of the proteins. Further investigation of gene copy number and organization revealed that the scr74 gene family comprises at least three copies located in a region of no more than 300 kb of the P. infestans genome. We found evidence that recombination contributed to sequence divergence within at least one gene locus. These results led us to propose an evolutionary model that involves gene duplication and recombination, followed by functional divergence of scr74 genes. This study provides support for using diversifying selection as a criterion for identifying candidate effector genes from sequence databases.     

Summer and winter sensitivity of leaves and xylem to minimum freezing temperatures: a comparison of co-occurring Mediterranean oaks that differ in leaf lifespan

Freezing sensitivity of leaves and xylem was examined in four co-occurring Mediterranean oaks (Quercus spp.) grown in a common garden to determine whether freezing responses of leaves and xylem were coordinated and could be predicted by leaf lifespan. Freezing-induced embolism and loss of photosynthetic function were measured after overnight exposure to a range of subzero temperatures in both summer and winter. Both measures were found to be dependent on minimum freezing temperature and were correlated with leaf lifespan and vessel diameter. The dependence of xylem embolism on minimum freezing temperature may result from the decline in water potential with ice temperature that influences the redistribution of water during freezing and leads to an increase in xylem tension. Winter acclimatization had a relatively small effect on the vulnerability to freezing-induced embolism, although leaf photosynthetic function showed a strong acclimatization response, particularly in the two evergreen species. Quercus ilex, the species with the longest leaf lifespan and narrowest vessel diameters, showed the highest freezing tolerance. This helps explain its ability to inhabit a broad range throughout the Mediterranean region. By contrast, the inability of the deciduous oaks to maintain photosynthetic and vascular function throughout the winter indicates a competitive disadvantage that may prevent them from expanding their ranges.     

Field studies to determine mancozeb based spray programmes with minimal impact on predatory mites in European vine cultivation

Mancozeb is an ethylene bisdithiocarbamate (EBDC) fungicide with contact activity against a wide range of economically important fungal diseases. Its multi-site mode of action means that to date there have been no recorded incidences of resistance developing despite many years of use on high risk diseases. One such disease, Grape downy mildew (Plasmopara viticola) has developed resistance to a number of important oomycete specific fungicides following their introduction onto the market. The role of Mancozeb either as a mixing or alternation partner in helping to manage these resistance situations remains critically important. Historical use patterns for mancozeb in tree and vine crops involved many applications of product at high use rates. Although this gave excellent disease control, a negative impact on predatory mites has been reported by researchers. This has lead to the development of mancozeb spray programmes in vines and other crops with a much reduced impact on predatory mites. A range of field studies was conducted in France, Germany, Italy, Portugal and Spain where either 2 or 4 applications of mancozeb containing products were made per season at different spray timings. These trials covered the representative range of uses, agronomic practices, mite species and geographical locations in Europe. In this paper findings from ten field studies in five different vine growing regions in Europe indicated that two to four applications of mancozeb at 1.6 kg a.i./ha as part of a spray programme caused minimal impact on naturally occurring populations of predatory mites which in turn was compatible with Integrated Pest Management programmes and the conservation of predatory mites.     

Arrestin times for compartmentalised cAMP signalling and phosphodiesterase-4 enzymes

Various methods reveal that cyclic AMP (cAMP) signalling in cells is compartmentalised. These methods use FRET probes based upon either protein kinase A (PKA) or EPAC, cAMP-gated ion channels, or the selective activation of AKAP-anchored PKA isoforms. The basis of compartmentalisation involves point sources of cAMP generation within sub-domains of the plasma membrane coupled to degradation by spatially segregated, anchored forms of cAMP phosphodiesterases. cAMP-specific phosphodiesterase-4 (PDE4) isoforms play a central role in determining compartmentalisation, as exemplified in cardiac myocytes and T cells. The PKA phosphorylation status of the beta2-adrenoreceptor, and hence its ability to switch its signalling from G(s) to G(i) and thus to activate ERK, is regulated dynamically by the agonist-stimulated recruitment of PDE4 to the receptor in complex with beta-arrestin. The co-receptor CD28 enhances signalling through the T-cell receptor by recruiting a PDE4/beta-arrestin complex, which then attenuates PKA phosphorylation of Csk.     

SAR and biological evaluation of novel trans-3,4-dimethyl-4-arylpiperidine derivatives as opioid antagonists

The phenolic hydroxy group of opiate-derived ligands is of known importance for biological activity. We have developed a SAR study around LY255582 by comparing the effect of the hydroxy group in the 2- and 4-position of the phenyl ring. Also, we have proved that the 3-position of the phenyl ring is optimal for opioid activity. Furthermore, we have successfully replaced the hydroxy group in LY255582 by carbamate and carboxamide groups. The new analogs have high affinity for the opioid receptors comparable to the corresponding phenol. Carboxamide analog 12 has an improved metabolism profile and proved to be efficacious in in vivo studies.     

Differential effects of estrogen and raloxifene on messenger RNA and matrix metalloproteinase 2 activity in the rat uterus

A detailed analysis of the differential effects of estrogen (E) compared to raloxifene (Ral), a selective estrogen receptor modulator (SERM), following estrogen receptor (ER) binding in gynecological tissues was conducted using gene microarrays, Northern blot analysis, and matrix metalloproteinase (MMP) 2 activity studies. We profiled gene expression in the uterus following acute (1 day) and prolonged daily (5 wk) treatment of E and Ral in ovariectomized rats. Estrogen regulated twice as many genes as Ral, largely those associated with catalysis and metabolism, whereas Ral induced genes associated with cell death and negative cell regulation. Follow-up studies confirmed that genes associated with matrix integrity were differentially regulated by Ral and E at various time points in uterine and vaginal tissues. Additional experiments were conducted to determine the levels of MMP2 activity in uterus explants from ovariectomized rats following 2 wk of treatment with E, Ral, or one of two additional SERMs: lasofoxifene, and levormeloxifene. Both E and lasofoxifene stimulated uterine MMP2 activity to a level twofold that of Ral, whereas levormeloxifene elevated MMP2 activity to a level 12-fold that of Ral. These data show that one of the significant differences between E and Ral signaling in the uterus is the regulation of genes and proteins associated with matrix integrity. This may be a potential key difference between the action of SERMs in the uterus of postmenopausal women.     

Molecular and functional characterization of a novel splice variant of ANKHD1 that lacks the KH domain and its role in cell survival and apoptosis

Multiple ankyrin repeat motif-containing proteins play an important role in protein-protein interactions. ANKHD1 proteins are known to possess multiple ankyrin repeat domains and a single KH domain with no known function. Using yeast two-hybrid system analysis, we identified a novel splice variant of ANKHD1. This splice variant of ANKHD1, which we designated as HIV-1 Vpr-binding ankyrin repeat protein (VBARP), does not contain the signature KH domain, and codes for only a single ankyrin repeat motif. We characterized VBARP by molecular and functional analysis, revealing that VBARP is ubiquitously expressed in different tissues as well as cell lines of different lineage. In addition, blast searches indicated that orthologs and homologs to VBARP exist in different phyla, suggesting that VBARP might be evolutionarily conserved, and thus may be involved in basic cellular function(s). Furthermore, biochemical analysis revealed the presence of two VBARP isoforms coding for 69 and 49 kDa polypeptides, respectively, that are primarily localized in the cytoplasm. Functional analysis using short interfering RNA approaches indicate that this gene product is essential for cell survival through its regulation of caspases. Taken together, these results indicate that VBARP is a novel splice variant of ANKHD1 and may play a role in cellular apoptosis (antiapoptotic) and cell survival pathway(s).