Stereochemical and steric control of enzymatic glucuronidation : A rational approach for the design of novel Inhibitors for the human UDP-glucuronosyltransferase 2B7

The UDP-glucuronosyltransferases (UGTs) are enzymes of the phase II metabolic system. These enzymes catalyze the transfer of α-D-glucuronic acid from UDP-glucuronic acid to aglycones bearing nucleophilic groups affording exclusively their corresponding β-D-glucuronides to render lipophilic endobiotics and xenobiotics more water soluble. This detoxification pathway aids in the urinary and biliary excretion of lipophilic compounds thus preventing their accumulation to harmful levels. The aim of this study was to investigate the effect of stereochemical and steric features of substrates on the glucuronidation catalyzed by UGTs 2B7 and 2B17. Furthermore, this study relates to the design and synthesis of novel, selective inhibitors that display high affinity for the key enzyme involved in drug glucuronidation, UGT2B7. The starting point for the development of inhibitors was to assess the influence of the stereochemistry of substrates on the UGT-catalyzed glucuronidation reaction. A set of 28 enantiomerically pure alcohols was subjected to glucuronidation assays employing the human UGT isoforms 2B7 and 2B17. Both UGT enzymes displayed high stereoselectivity, favoring the glucuronidation of the (R)-enantiomers over their respective mirror-image compounds. The spatial arrangement of the hydroxy group of the substrate determined the rate of the UGT-catalyzed reaction. However, the affinity of the enantiomeric substrates to the enzymes was not significantly influenced by the spatial orientation of the nucleophilic hydroxy group. Based on these results, a rational approach for the design of inhibitors was developed by addressing the stereochemical features of substrate molecules. Further studies showed that the rate of the enzymatic glucuronidation of substrates was also highly dependent on the steric demand in vicinity of the nucleophilic hydroxy group. These findings provided a rational approach to turn high-affinity substrates into true UGT inhibitors by addressing stereochemical and steric features of substrate molecules. The tricyclic sesquiterpenols longifolol and isolongifolol were identified as high-affinity substrates which displayed high selectivity for the UGT isoform 2B7. These compounds served therefore as lead structures for the design of potent and selective inhibitors for UGT2B7. Selective and potent inhibitors were prepared by synthetically modifying the lead compounds longifolol and isolongifolol taking stereochemical and steric features into account. The best inhibitor of UGT2B7, β-phenyllongifolol, displayed an inhibition constant of 0.91 nM.

A large-scale analysis of genetic variants within putative miRNA binding sites in prostate cancer

Prostate cancer is the second most common malignancy among men worldwide. Genome-wide association studies have identified 100 risk variants for prostate cancer, which can explain approximately 33% of the familial risk of the disease. We hypothesized that a comprehensive analysis of genetic variations found within the 3' untranslated region of genes predicted to affect miRNA binding (miRSNP) can identify additional prostate cancer risk variants. We investigated the association between 2,169 miRSNPs and prostate cancer risk in a large-scale analysis of 22,301 cases and 22,320 controls of European ancestry from 23 participating studies. Twenty-two miRSNPs were associated (P<2.3×10(-5)) with risk of prostate cancer, 10 of which were within 7 genes previously not mapped by GWAS studies. Further, using miRNA mimics and reporter gene assays, we showed that miR-3162-5p has specific affinity for the KLK3 rs1058205 miRSNP T-allele, whereas miR-370 has greater affinity for the VAMP8 rs1010 miRSNP A-allele, validating their functional role. SIGNIFICANCE Findings from this large association study suggest that a focus on miRSNPs, including functional evaluation, can identify candidate risk loci below currently accepted statistical levels of genome-wide significance. Studies of miRNAs and their interactions with SNPs could provide further insights into the mechanisms of prostate cancer risk.

A genetic variant of MDM4 influences regulation by multiple microRNAs in prostate cancer

The oncogene MDM4, also known as MDMX or HDMX, contributes to cancer susceptibility and progression through its capacity to negatively regulate a range of genes with tumour-suppressive functions. As part of a recent genome-wide association study it was determined that the A-allele of the rs4245739 SNP (A>C), located in the 3'-UTR of MDM4, is associated with an increased risk of prostate cancer. Computational predictions revealed that the rs4245739 SNP is located within a predicted binding site for three microRNAs (miRNAs): miR-191-5p, miR-887 and miR-3669. Herein, we show using reporter gene assays and endogenous MDM4 expression analyses that miR-191-5p and miR-887 have a specific affinity for the rs4245739 SNP C-allele in prostate cancer. These miRNAs do not affect MDM4 mRNA levels, rather they inhibit its translation in C-allele-containing PC3 cells but not in LNCaP cells homozygous for the A-allele. By analysing gene expression datasets from patient cohorts, we found that MDM4 is associated with metastasis and prostate cancer progression and that targeting this gene with miR-191-5p or miR-887 decreases in PC3 cell viability. This study is the first, to our knowledge, to demonstrate regulation of the MDM4 rs4245739 SNP C-allele by two miRNAs in prostate cancer, and thereby to identify a mechanism by which the MDM4 rs4245739 SNP A-allele may be associated with an increased risk for prostate cancer.

Transcriptional regulation of a pineapple polyphenol oxidase gene and its relationship to blackheart.

Two genes encoding polyphenol oxidase (PPO) were isolated from pineapple (Ananas comosus[L.] Merr. cv. Smooth Cayenne). Sequence analyses showed that both contained a single intron and encoded typical chloroplast-localized PPO proteins, the sequences of which corresponded to two pineapple PPO cDNAs, PINPPO1 and PINPPO2, recently described by Stewart et al. (2001). Southern blot analyses suggested that pineapple contained only two PPO genes. Analysis of expression of PINPPO1 promoter GUS fusion constructs showed this promoter had a low basal activity and was cold- and wound-inducible, consistent with known mRNA expression profiles. Striking homologies to gibberellin response complexes (GARC) were observed in sequences of both the PINPPO1 and PINPPO2 promoters. Transient assays in mature pineapple fruit and stable expression in transgenic tobacco showed that PINPPO1 promoter-GUS fusions were indeed gibberellin (GA) responsive. A role for the element within the putative GARCs in mediating GA-responsiveness of the PINPPO1 promoter was confirmed by mutational analysis. PINPPO2 was also shown to be GA-responsive by RT-PCR analysis. Mutant PINPPO1 promoter-GUS fusion constructs, which were no longer GA-inducible, showed a delayed response to cold induction in pineapple fruit in transient assays, suggesting a role for GA in blackheart development. This was supported by observations that exogenous GA3 treatment induced blackheart in the absence of chilling. Sequences showing homology to GARCs are also present in some PPO promoters in tomato, suggesting that GA regulates PPO expression in diverse species.

Discrepancy Between Antennal and Behavioral Responses for Enantiomers of a-Pinene: Electrophysiology and Behavior of Helicoverpa Armigera (Lepidoptera)

The ability of adult cotton bollworm, Helicoverpa armigera (Hubner), to distinguish and respond to enantiomers of α-pinene was investigated with electrophysiological and behavioral methods. Electroantennogram recordings using mixtures of the enantiomers at saturating dose levels, and single unit electrophysiology, indicated that the two forms were detected by the same receptor neurons. The relative size of the electroantennogram response was higher for the (−) compared to the (+) form, indicating greater affinity for the (−) form at the level of the dendrites. Behavioral assays investigated the ability of moths to discriminate between, and respond to the (+) and (−) forms of α pinene. Moths with no odor conditioning showed an innate preference for (+)-α-pinene. This preference displayed by naıve moths was not significantly different from the preferences of moths conditioned on (+)-α-pinene. However, we found a significant difference in preference between moths conditioned on the (−) enantiomer compared to naıve moths and moths conditioned on (+)-α-pinene, showing that learning plays an important role in the behavioral response. Moths are less able to distinguish between enantiomers of α-pinene than different odors (e.g., phenylacetaldehyde versus (−)-α-pinene) in learning experiments. The relevance of receptor discrimination of enantiomers and learning ability of the moths in host plant choice is discussed.

Abacá mosaic virus: a distinct strain of Sugarcane mosaic virus

Abacá mosaic virus (AbaMV) is related to members of the sugarcane mosaic virus subgroup of the genus Potyvirus. The ~2 kb 3′ terminal region of the viral genome was sequenced and, in all areas analysed, found to be most similar to Sugarcane mosaic virus (SCMV) and distinct from Johnsongrass mosaic virus (JGMV), Maize dwarf mosaic virus (MDMV) and Sorghum mosaic virus (SrMV). Cladograms of the 3′ terminal region of the NIb protein, the coat protein core and the 3′ untranslated region showed that AbaMV clustered with SCMV, which was a distinct clade and separate from JGMV, MDMV and SrMV. The N-terminal region of the AbaMV coat protein had a unique amino acid repeat motif different from those previously published for other strains of SCMV. The first experimental transmission of AbaMV from abacá (Musa textilis) to banana (Musa sp.), using the aphid vectors Rhopalosiphum maidis and Aphis gossypii, is reported. Polyclonal antisera for the detection of AbaMV in western blot assays and ELISA were prepared from recombinant coat protein expressed in E. coli. A reverse transcriptase PCR diagnostic assay, with microtitre plate colourimetric detection, was developed to discriminate between AbaMV and Banana bract mosaic virus, another Musa-infecting potyvirus. Sequence data, host reactions and serological relationships indicate that AbaMV should be considered a distinct strain of SCMV, and the strain designation SCMV-Ab is suggested.

miR-514a regulates the tumour suppressor NF1 and modulates BRAFi sensitivity in melanoma

To identify ‘melanoma-specific’ microRNAs (miRNAs) we used an unbiased microRNA profiling approach to comprehensively study cutaneous melanoma in relation to other solid malignancies, which revealed 233 differentially expressed (≥ 2 fold, p < 0.05) miRNAs. Among the top 20 most significantly different miRNAs was hsa-miR-514a-3p. miR-514a is a member of a cluster of miRNAs (miR-506-514) involved in initiating melanocyte transformation and promotion of melanoma growth. We found miR-514a was expressed in 38/55 (69%) melanoma cell lines but in only 1/34 (3%) other solid cancers. To identify miR-514a regulated targets we conducted a miR-514a-mRNA ‘pull-down’ experiment, which revealed hundreds of genes, including: CTNNB1, CDK2, MC1R, and NF1, previously associated with melanoma. NF1 was selected for functional validation because of its recent implication inacquired resistance to BRAFV600E-targeted therapy. Luciferase-reporter assays confirmed NF1 as a direct target of miR-514a and over-expression of miR-514a in melanoma cell lines inhibited NF1 expression, which correlated with increased survival of BRAFV600E cells treated with PLX4032. These data provide another mechanism for the dysregulation of the MAPK pathway which may contribute to the profound resistance associated with current RAF-targeted therapies.

Phenotypic and genotypic characterisation of Pasteurella multocida isolated from pigs at slaughter in New Zealand

AIMS: To examine pigs at slaughter in New Zealand for the presence of Pasteurella multocida, and to determine for isolates, their biochemical profi les, somatic and capsular types, and the presence or absence of the HSB and toxA genes, associated with haemorrhagic septicaemia (HS) and progressive atrophic rhinitis (PAR), respectively. METHODS: Swabs from 173 lungs, 158 palatine tonsils and 82 nasal passages of pigs at two abattoirs in New Zealand were cultured for P. multocida using conventional techniques, and isolated colonies were subjected to biochemical tests for identi- fi cation of biovars. Somatic serotyping was conducted using an agar gel immunodiffusion (AGID) test. Polymerase chain reaction (PCR) assays were used to confi rm phenotypic identifi cation of colonies using species-specifi c primers, capsule type using serogroup-specifi c primers and multiplex PCR, and to test for the presence of HSB and toxA genes. RESULTS: Pasteurella multocida was isolated from 11/173 (6.4%) lung, 32/158 (20.2%) palatine tonsil and 5/82 (6.1 %) nasal swab samples, a total of 48 isolates from 413 samples (11.6%). Isolation rates per farm ranged from 1–53% of tissue samples collected from pigs 5–6 months of age. On phenotypic characterisation, isolates were allocated to seven main biovars, viz 1, 2, 3, 5, 9, 12, and a dulcitol-negative variant of Biovar 8, the majority (30/48) being Biovar 3. Of the 42 isolates for which somatic serotyping was conducted, 10% were Serovar 1, 79% were Serovar 3, 2% were Serovar 6,1, 2% were Serovar 12, and 7% could not be typed. All 48 isolates were confi rmed as P. multocida using a species-specifi c PCR. In the capsular multiplex PCR, 92% of isolates were Capsular (Cap) type A, 2% were Cap D, and 6% could not be typed. None of the samples were positive for the HSB or toxA genes. CONCLUSION: Serovars or capsular types of P. multocida associated with HS or PAR in pigs were not detected. Establishment of species-specifi c, capsular and toxin PCR assays allowed the rapid screening of isolates of P. multocida, while serotyping provided an additional tool for epidemiological and tracing purposes.

Predicting survival of western rock lobsters Panulirus cygnus using discriminant analysis of hemolymph parameters taken immediately following simulated handling treatments

Instances of morbidity amongst rock lobsters (Panulirus cygnus) arriving at factories in Western Australia (WA) have been attributed to stress during post-harvest handling. This study used discriminant analysis to determine whether physiological correlates of stress following a period of simulated post-harvest handling had any validity as predictors of future rejection or morbidity of western rock lobsters. Groups of 230 western rock lobsters were stored for 6 h in five environments (submerged/flowing sea water, submerged/re-circulating sea water, humid air, flowing sea water spray, and re-circulated sea water spray). The experiment was conducted in late spring (ambient sea water 22°C), and repeated again in early autumn (ambient sea water 26°C). After 6 h treatment, each lobster was graded for acceptability for live export, numbered, and its hemolymph was sampled. The samples were analysed for a number of physiological and health status parameters. The lobsters were then stored for a week in tanks in the live lobster factory to record mortality. The mortality of lobsters in the factory was associated with earlier deviations in hemolymph parameters as they emerged from the storage treatments. Discriminant analysis (DA) of the hemolymph assays enabled the fate of 80-90% of the lobsters to be correctly categorised within each experiment. However, functions derived from one experiment were less accurate at predicting mortality when applied to the other experiments. One of the reasons for this was the higher mortality and the more severe patho-physiological changes observed in lobsters stored in humid air or sprays at the higher temperature. The analysis identified lactate accumulation during emersion and associated physiological and hemocyte-related effects as a major correlate of mortality. Reducing these deviations, for example by submerged transport, is expected to ensure high levels of survival. None of the indicators tested predicted mortality with total accuracy. The simplest and most accurate means of comparing emersed treatments was to count the mortality afterwards.

Genetic variability of Tomato spotted wilt virus in Australia and validation of real time RT-PCR for its detection in single and bulked leaf samples

The potential for large-scale use of a sensitive real time reverse transcription polymerase chain reaction (RT-PCR) assay was evaluated for the detection of Tomato spotted wilt virus (TSWV) in single and bulked leaf samples by comparing its sensitivity with that of DAS-ELISA. Using total RNA extracted with RNeasy® or leaf soak methods, real time RT-PCR detected TSWV in all infected samples collected from 16 horticultural crop species (including flowers, herbs and vegetables), two arable crop species, and four weed species by both assays. In samples in which DAS-ELISA had previously detected TSWV, real time RT-PCR was effective at detecting it in leaf tissues of all 22 plant species tested at a wide range of concentrations. Bulk samples required more robust and extensive extraction methods with real time RT-PCR, but it generally detected one infected sample in 1000 uninfected ones. By contrast, ELISA was less sensitive when used to test bulked samples, once detecting up to 1 infected in 800 samples with pepper but never detecting more than 1 infected in 200 samples in tomato and lettuce. It was also less reliable than real time RT-PCR when used to test samples from parts of the leaf where the virus concentration was low. The genetic variability among Australian isolates of TSWV was small. Direct sequencing of a 587 bp region of the nucleoprotein gene (S RNA) of 29 isolates from diverse crops and geographical locations yielded a maximum of only 4.3% nucleotide sequence difference. Phylogenetic analysis revealed no obvious groupings of isolates according to geographic origin or host species. TSWV isolates, that break TSWV resistance genes in tomato or pepper did not differ significantly in the N gene region studied, indicating that a different region of the virus genome is responsible for this trait.

Antibiotic resistance in Campylobacter jejuni and Campylobacter coli isolated from poultry in the South-East Queensland region

Objectives: The aim of this study was to determine the antimicrobial resistance patterns of 125 Campylobacter jejuni and 27 Campylobacter coli isolates from 39 Queensland broiler farms. Methods: Two methods, a disc diffusion assay and an agar-based MIC assay, were used. The disc diffusion was performed and interpreted as previously described (Huysmans MB, Turnidge JD. Disc susceptibility testing for thermophilic campylobacters. Pathology 1997; 29: 209–16), whereas the MIC assay was performed according to CLSI (formerly NCCLS) methods and interpreted using DANMAP criteria. Results: In both assays, no C. jejuni or C. coli isolates were resistant to ciprofloxacin or chloramphenicol, no C. coli were resistant to nalidixic acid, and no C. jejuni were resistant to erythromycin. In the MIC assay, no C. jejuni isolate was resistant to nalidixic acid, whereas three isolates (2.4%) were resistant in the disc assay. The highest levels of resistance of the C. jejuni isolates were recorded for tetracycline (19.2% by MIC and 18.4% by disc) and ampicillin (19.2% by MIC and 17.6% by disc). The C. coli isolates gave very similar results (tetracycline resistance 14.8% by both MIC and disc; ampicillin resistance 7.4% by MIC and 14.8% by disc). Conclusions: This work has shown that the majority of C. jejuni and C. coli isolates were susceptible to the six antibiotics tested by both disc diffusion and MIC methods. Disc diffusion represents a suitable alternative methodology to agar-based MIC methods for poultry Campylobacter isolates.

Assessing for genetic and environmental effects on ruminant feed quality in barley ( Hordeum vulgare )

Grain samples from a combined intermediate and advanced stage barley breeding trial series, grown at two sites in two consecutive years were assessed for detailed grain quality and ruminant feed quality. The results indicated that there were significant genetic and environmental effects for “feed” traits as measured using grain hardness, acid detergent fibre (ADF), starch and in-sacco dry matter digestibility (ISDMD) assays. In addition, there was strong genotypic discrimination for the regressed feed performance traits, namely Net Energy (NE) and Average Daily Gain (ADG). There was considerable variation in genetic correlations for all traits based on variance from the cultivars used, sites or laboratory processing effects. There was a high level of heritability ranging from 89% to 88% for retention, 60% to 80% for protein and 56% to 68% for ADF. However, there were only low to moderate levels of heritability for the feed traits, with starch 30–39%, ISDMD 55–63%, ADF 56–68%, particle size 47–73%, 31–48% NE and ADG 44–51%. These results suggest that there were real differences in the feed performance of barleys and that selection for cattle feed quality is potentially a viable option for breeding programs.

Heterologous signals allow efficient targeting of a nuclear-encoded fusion protein to plastids and endoplasmic reticulum in diverse plant species

Approximately 30% of plant nuclear genes appear to encode proteins targeted to the plastids or endoplasmic reticulum (ER). The signals that direct proteins into these compartments are diverse in sequence, but, on the basis of a limited number of tests in heterologous systems, they appear to be functionally conserved across species. To further test the generality of this conclusion, we tested the ability of two plastid transit peptides and an ER signal peptide to target green fluorescent protein (GFP) in 12 crops, including three monocots (barley, sugarcane, wheat) and nine dicots (Arabidopsis, broccoli, cabbage, carrot, cauliflower, lettuce, radish, tobacco, turnip). In all species, transient assays following microprojectile bombardment or vacuum infiltration using Agrobacterium showed that the plastid transit peptides from tomato DCL (defective chloroplast and leaves) and tobacco RbcS [ribulose bisphosphate carboxylase (Rubisco) small subunit] genes were effective in targeting GFP to the leaf plastids. GFP engineered as a fusion to the N-terminal ER signal peptide from Arabidopsis basic chitinase and a C-terminal HDEL signal for protein retention in the ER was accumulated in the ER of all species. The results in tobacco were confirmed in stably transformed cells. These signal sequences should be useful to direct proteins to the plastid stroma or ER lumen in diverse plant species of biotechnological interest for the accumulation of particular recombinant proteins or for the modification of particular metabolic streams.

Borate protection of softwood from Coptotermes acinaciformis (Isoptera: Rhinotermitidae) damage: Variation in protection thresholds explained

Laboratory and field data reported in the literature are confusing with regard to “adequate” protection thresholds for borate timber preservatives. The confusion is compounded by differences in termite species, timber species and test methodology. Laboratory data indicate a borate retention of 0.5% mass/mass (m/m) boric acid equivalent (BAE) would cause >90% termite mortality and restrict mass loss in test specimens to ≤5%. Field data generally suggest that borate retentions appreciably >0.5% m/m BAE are required. We report two field experiments with varying amounts of untreated feeder material in which Coptotermes acinaciformis (Froggatt) (Isoptera: Rhinotermitidae) responses to borate-treated radiata (Monterey) pine, Pinus radiata D. Don, were measured. The apparently conflicting results between laboratory and field data are explained by the presence or absence of untreated feeder material in the test environment. In the absence of untreated feeder material, wood containing 0.5% BAE provided adequate protection from Coptotermes sp., whereas in the presence of untreated feeder material, increased retentions were required. Furthermore, the retentions required increased with increased amounts of susceptible material present. Some termites, Nasutitermes sp. and Mastotermes darwiniensis Froggatt, for example, are borate-tolerant and borate timber preservatives are not a viable management option with these species. The lack of uniform standards for termite test methodology and assessment criteria for efficacy across the world is recognized as a difficulty with research into the performance of timber preservatives with termites. The many variables in laboratory and field assays make “prescriptive” standards difficult to recommend. The use of “performance” standards to define efficacy criteria (“adequate” protection) is discussed.

A high-density consensus map of barley linking DArT markers to SSR, RFLP and STS loci and agricultural traits

Background: Molecular marker technologies are undergoing a transition from largely serial assays measuring DNA fragment sizes to hybridization-based technologies with high multiplexing levels. Diversity Arrays Technology (DArT) is a hybridization-based technology that is increasingly being adopted by barley researchers. There is a need to integrate the information generated by DArT with previous data produced with gel-based marker technologies. The goal of this study was to build a high-density consensus linkage map from the combined datasets of ten populations, most of which were simultaneously typed with DArT and Simple Sequence Repeat (SSR), Restriction Enzyme Fragment Polymorphism (RFLP) and/or Sequence Tagged Site (STS) markers. Results: The consensus map, built using a combination of JoinMap 3.0 software and several purpose-built perl scripts, comprised 2,935 loci (2,085 DArT, 850 other loci) and spanned 1,161 cM. It contained a total of 1,629 'bins' (unique loci), with an average inter-bin distance of 0.7 ± 1.0 cM (median = 0.3 cM). More than 98% of the map could be covered with a single DArT assay. The arrangement of loci was very similar to, and almost as optimal as, the arrangement of loci in component maps built for individual populations. The locus order of a synthetic map derived from merging the component maps without considering the segregation data was only slightly inferior. The distribution of loci along chromosomes indicated centromeric suppression of recombination in all chromosomes except 5H. DArT markers appeared to have a moderate tendency toward hypomethylated, gene-rich regions in distal chromosome areas. On the average, 14 ± 9 DArT loci were identified within 5 cM on either side of SSR, RFLP or STS loci previously identified as linked to agricultural traits. Conclusion: Our barley consensus map provides a framework for transferring genetic information between different marker systems and for deploying DArT markers in molecular breeding schemes. The study also highlights the need for improved software for building consensus maps from high-density segregation data of multiple populations.