Identification and analysis of mechanisms involved in a broad-spectrum disease resistant mutant of the winter wheat cv. Guardian

M66 an X-ray induced mutant of winter wheat (Triticum aestivum) cv. Guardian exhibits broad-spectrum resistance to powdery mildew (Blumeria graminis f. sp. tritici), yellow rust (Puccinia striiformis f. sp. tritici), and leaf rust (Puccinia recondita f. sp. tritici), along with partial resistance to stagnonospora nodorum blotch (caused by the necrotroph Stagonosporum nodorum) and septoria tritici blotch (caused by the hemibiotroph Mycosphaerella graminicola) compared to the parent plant ‘Guardian’. Analysis revealed that M66 exhibited no symptoms of infection following artificial inoculation with Bgt in the glasshouse after adult growth stage (GS 45). Resistance in M66 was associated with widespread leaf flecking which developed during tillering. Flecking also occurred in M66 leaves without Bgt challenge; as a result grain yields were reduced by approximately 17% compared to ‘Guardian’ in the absence of disease. At the seedling stage, M66 exhibited partial resistance. M66, along with Tht mutants (Tht 12, Tht13), also exhibit increased tolerance to environmental stresses (abiotic), such as drought and heat stress at seedling and adult growth stages, However, adult M66 exhibited increased susceptibility to the aphid Schizaphis graminum compared to ‘Guardian’. Resistance to Bgt in M66 was characterized with increased and earlier H2O2 accumulation at the site of infection which resulted in increased papilla formation in epidermal cells, compared to ‘Guardian’. Papilla formation was associated with reduced pathogen ingress and haustorium formation, indicating that the primary cause of resistance in M66 was prevention of pathogen penetration. Heat treatment at 46º C prior to challenge with Bgt also induced partial disease resistance to Blumeria graminis f. sp. tritici in ‘Guardian’ and M66 seedlings. This was characterized by a delay in primary infection, due to increased production of ROS species, such as hydrogen peroxide, ROS-scavenging enzymes and Hsp70, resulting in cross-linking of cell wall components prior to inoculation. This actively prevented the fungus from penetrating the epidermal cell wall. Proteomics analysis using 2-D gel electrophoresis identified primary and secondary disease resistance effects in M66 including detection of ROS scavenging enzymes (4, 24 hai), such as ascorbate peroxidase and a superoxidase dismutase isoform (CuZnSOD) in M66 which were absent from ‘Guardian’. Chitinase (PR protein) was also upregulated (24 hai) in M66 compared to ‘Guardian’.Monosomic and ditelosomic analysis of M66 revealed that the mutation in M66 is located on the long arm of chromosome 2B (2BL). Chromosome 2BL is known to have key genes involved in resistance to pathogens such as those causing stripe rust and powdery mildew. The TaMloB1 gene, an orthologue of the barley Mlo gene, is also located on chromosome 2BL. Sanger sequencing of part of the coding sequence revealed no deletions in the TaMloB1 gene between ‘Guardian’ and M66.

Mutational analysis of the active centre of coronavirus 3C-like proteases.

Formation of the coronavirus replication-transcription complex involves the synthesis of large polyprotein precursors that are extensively processed by virus-encoded cysteine proteases. In this study, the coding sequence of the feline infectious peritonitis virus (FIPV) main protease, 3CL(pro), was determined. Comparative sequence analyses revealed that FIPV 3CL(pro) and other coronavirus main proteases are related most closely to the 3C-like proteases of potyviruses. The predicted active centre of the coronavirus enzymes has accepted unique replacements that were probed by extensive mutational analysis. The wild-type FIPV 3CL(pro) domain and 25 mutants were expressed in Escherichia coli and tested for proteolytic activity in a peptide-based assay. The data strongly suggest that, first, the FIPV 3CL(pro) catalytic system employs His(41) and Cys(144) as the principal catalytic residues. Second, the amino acids Tyr(160) and His(162), which are part of the conserved sequence signature Tyr(160)-Met(161)-His(162) and are believed to be involved in substrate recognition, were found to be indispensable for proteolytic activity. Third, replacements of Gly(83) and Asn(64), which were candidates to occupy the position spatially equivalent to that of the catalytic Asp residue of chymotrypsin-like proteases, resulted in proteolytically active proteins. Surprisingly, some of the Asn(64) mutants even exhibited strongly increased activities. Similar results were obtained for human coronavirus (HCoV) 3CL(pro) mutants in which the equivalent Asn residue (HCoV 3CL(pro) Asn(64)) was substituted. These data lead us to conclude that both the catalytic systems and substrate-binding pockets of coronavirus main proteases differ from those of other RNA virus 3C and 3C-like proteases.

Crystallization and preliminary X-ray diffraction analysis of naphthalene dioxygenase from Rhodococcus sp strain NCIMB 12038

The three-component naphthalene dioxygenase (NDO) enzyme system carries out the first step in the aerobic degradation of naphthalene to (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene by Rhodococcus sp. strain NCIMB 12038. The terminal oxygenase component (naphthalene 1,2-dioxygenase) that catalyzes this reaction belongs to the aromatic ring hydroxylating dioxygenase family and has been crystallized. These enzymes utilize a mononuclear nonheme iron centre to catalyze the addition of dioxygen to their respective substrates. In this reaction, two electrons, two protons and a dioxygen molecule are consumed. The Rhodococcus enzyme has only 33 and 29% sequence identity to the corresponding alpha- and beta-subunits of the NDO system of Pseudomonas putida NCIMB 9816-4, for which the tertiary structure has been reported. In order to determine the three-dimensional structure of the Rhodococcus NDO, diffraction-quality crystals have been prepared by the hanging-drop method. The crystals belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 87.5, b = 144, c = 185.6 Angstrom, alpha = beta = gamma = 90degrees, and diffract to 2.3 Angstrom resolution.

Population pharmacokinetic and pharmacogenetic analysis of 6-mercaptopurine in paediatric patients with acute lymphoblastic leukaemia

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT center dot The cytotoxic effects of 6-mercaptopurine (6-MP) were found to be due to drug-derived intracellular metabolites (mainly 6-thioguanine nucleotides and to some extent 6-methylmercaptopurine nucleotides) rather than the drug itself. center dot Current empirical dosing methods for oral 6-MP result in highly variable drug and metabolite concentrations and hence variability in treatment outcome. WHAT THIS STUDY ADDS center dot The first population pharmacokinetic model has been developed for 6-MP active metabolites in paediatric patients with acute lymphoblastic leukaemia and the potential demographic and genetically controlled factors that could lead to interpatient pharmacokinetic variability among this population have been assessed. center dot The model shows a large reduction in interindividual variability of pharmacokinetic parameters when body surface area and thiopurine methyltransferase polymorphism are incorporated into the model as covariates. center dot The developed model offers a more rational dosing approach for 6-MP than the traditional empirical method (based on body surface area) through combining it with pharmacogenetically guided dosing based on thiopurine methyltransferase genotype. To investigate the population pharmacokinetics of 6-mercaptopurine (6-MP) active metabolites in paediatric patients with acute lymphoblastic leukaemia (ALL) and examine the effects of various genetic polymorphisms on the disposition of these metabolites. Data were collected prospectively from 19 paediatric patients with ALL (n = 75 samples, 150 concentrations) who received 6-MP maintenance chemotherapy (titrated to a target dose of 75 mg m(-2) day(-1)). All patients were genotyped for polymorphisms in three enzymes involved in 6-MP metabolism. Population pharmacokinetic analysis was performed with the nonlinear mixed effects modelling program (nonmem) to determine the population mean parameter estimate of clearance for the active metabolites. The developed model revealed considerable interindividual variability (IIV) in the clearance of 6-MP active metabolites [6-thioguanine nucleotides (6-TGNs) and 6-methylmercaptopurine nucleotides (6-mMPNs)]. Body surface area explained a significant part of 6-TGNs clearance IIV when incorporated in the model (IIV reduced from 69.9 to 29.3%). The most influential covariate examined, however, was thiopurine methyltransferase (TPMT) genotype, which resulted in the greatest reduction in the model's objective function (P

A proteomic analysis of thioacetamide-induced hepatotoxicity and cirrhosis in rat livers

Thioacetamide (TAA) administration is an established technique for generating rat models of liver fibrosis and cirrhosis. Oxidative stress is believed to be involved as TAA-induced liver fibrosis is initiated by thioacetamide S-oxide, which is derived from the biotransformation of TAA by the microsomal flavine-adenine dinucleotide (FAD)-containing monooxygense (FMO) and cytochrome P450 systems. A two-dimensional gel electrophoresis-mass spectrometry approach was applied to analyze the protein profiles of livers of rats administered with sublethal doses of TAA for 3, 6 and 10 weeks respectively. With this approach, 59 protein spots whose expression levels changed significantly upon TAA administration were identified, including three novel proteins. These proteins were then sorted according to their common biochemical properties and functions, so that pathways involved in the pathogenesis of rat liver fibrosis due to TAA-induced toxicity could be elucidated. As a result, it was found that TAA-administration down-regulated the enzymes of the primary metabolic pathways such as fatty acid beta-oxidation, branched chain amino acids and methionine breakdown. This phenomenon is suggestive of the depletion of succinyl-CoA which affects heme and iron metabolism. Up-regulated proteins, on the other hand, are related to oxidative stress and lipid peroxidation. Finally, these proteomics data and the data obtained from the scientific literature were integrated into an

Genetic analysis of the dTDP-rhamnose biosynthesis region of the Escherichia coli VW187 (O7:K1) rfb gene cluster:identification of functional homologs of rfbB and rfbA in the rff cluster and correct location of the rffE gene

The O-repeating unit of the Escherichia coli O7-specific lipopolysaccharide is made of galactose, mannose, rhamnose, 4-acetamido-4,6-dideoxyglucose, and N-acetyglucosamine. We have recently characterized the genes involved in the biosynthesis of the sugar precursor GDP-mannose occurring in the E. coli O7:K1 strain VW187 (C. L. Marolda and M. A. Valvano, J. Bacteriol. 175:148-158, 1993). In the present study, we identified and sequenced the rfbBDAC genes encoding the enzymes for the biosynthesis of another precursor, dTDP-rhamnose. These genes are localized on the upstream end of the rfbEcO7 region, and they are strongly conserved compared with similar genes found in various enteric and nonenteric bacteria. Upstream of rfbB we identified a DNA segment containing the rfb promoter and a highly conserved untranslated leader sequence also present in the promoter regions of other surface polysaccharide gene clusters. Also, we have determined that rfbB and rfbA have homologs, rffG (o355) and rffH (o292), respectively, located on the rff cluster, which is involved in the synthesis of enterobacterial common antigen. We provide biochemical evidence that rffG and rffH encode dTDP-glucose dehydratase and glucose-1-phosphate thymidylyltransferase activities, respectively, and we also show that rffG complemented the rfbB defect in the O7+ cosmid pJHCV32. We also demonstrate that rffG is distinct from rffE and map the rffE gene to the second gene of the rff cluster.

An integrated transcriptomics and proteomics analysis of the secretome of the helminth pathogen Fasciola hepatica: Proteins associated with invasion and infection of the mammalian host

To infect their mammalian hosts, Fasciola hepatica larvae must penetrate and traverse the intestinal wall of the duodenum, move through the peritoneum, and penetrate the liver. After migrating through and feeding on the liver, causing extensive tissue damage, the parasites move to their final niche in the bile ducts where they mature and produce eggs. Here we integrated a transcriptomics and proteomics approach to profile Fasciola secretory proteins that are involved in host-pathogen interactions and to correlate changes in their expression with the migration of the parasite. Prediction of F. hepatica secretory proteins from 14,031 expressed sequence tags (ESTs) available from the Wellcome Trust Sanger Centre using the semiautomated EST2Secretome pipeline showed that the major components of adult parasite secretions are proteolytic enzymes including cathepsin L, cathepsin B, and asparaginyl endopeptidase cysteine proteases as well as novel trypsin-like serine proteases and carboxypeptidases. Proteomics analysis of proteins secreted by infective larvae, immature flukes, and adult F. hepatica showed that these proteases are developmentally regulated and correlate with the passage of the parasite through host tissues and its encounters with different host macromolecules. Proteases such as FhCL3 and cathepsin B have specific functions in larvae activation and intestinal wall penetration, whereas FhCL1, FhCL2, and FhCL5 are required for liver penetration and tissue and blood feeding. Besides proteases, the parasites secrete an array of antioxidants that are also highly regulated according to their migration through host tissues. However, whereas the proteases of F. hepatica are secreted into the parasite gut via a classical endoplasmic reticulum/Golgi pathway, we speculate that the antioxidants, which all lack a signal sequence, are released via a non-classical trans-tegumental pathway.

Proteomics and phylogenetic analysis of the cathepsin L protease family of the helminth pathogen Fasciola hepatica:Expansion of a repertoire of virulence-associated factors

Cathepsin L proteases secreted by the helminth pathogen Fasciola hepatica have functions in parasite virulence including tissue invasion and suppression of host immune responses. Using proteomics methods alongside phylogenetic studies we characterized the profile of cathepsin L proteases secreted by adult F. hepatica and hence identified those involved in host-pathogen interaction. Phylogenetic analyses showed that the Fasciola cathepsin L gene family expanded by a series of gene duplications followed by divergence that gave rise to three clades associated with mature adult worms (Clades 1, 2, and 5) and two clades specific to infective juvenile stages (Clades 3 and 4). Consistent with these observations our proteomics studies identified representatives from Clades 1, 2, and 5 but not from Clades 3 and 4 in adult F. hepatica secretory products. Clades 1 and 2 account for 67.39 and 27.63% of total secreted cathepsin Ls, respectively, suggesting that their expansion was positively driven and that these proteases are most critical for parasite survival and adaptation. Sequence comparison studies revealed that the expansion of cathepsin Ls by gene duplication was followed by residue changes in the S2 pocket of the active site. Our biochemical studies showed that these changes result in alterations in substrate binding and suggested that the divergence of the cathepsin L family produced a repertoire of enzymes with overlapping and complementary substrate specificities that could cleave host macromolecules more efficiently. Although the cathepsin Ls are produced as zymogens containing a prosegment and mature domain, all secreted enzymes identified by MS were processed to mature active enzymes. The prosegment region was highly conserved between the clades except at the boundary of prosegment and mature enzyme. Despite the lack of conservation at this section, sites for exogenous cleavage by asparaginyl endopeptidases and a Leu-Ser[downward arrow]His motif for autocatalytic cleavage by cathepsin Ls were preserved.

REMA: A computer-based mapping tool for analysis of restriction sites in multiple DNA sequences

REMA is an interactive web-based program which predicts endonuclease cut sites in DNA sequences. It analyses Multiple sequences simultaneously and predicts the number and size of fragments as well as provides restriction maps. The users can select single or paired combinations of all commercially available enzymes. Additionally, REMA permits prediction of multiple sequence terminal fragment sizes and suggests suitable restriction enzymes for maximally discriminatory results. REMA is an easy to use, web based program which will have a wide application in molecular biology research. Availability: REMA is written in Perl and is freely available for non-commercial use. Detailed information on installation can be obtained from Jan Szubert (jan.szubert@gmail.com) and the web based application is accessible on the internet at the URL http://www.macaulay.ac.uk/rema. Contact: b.singh@macaulay.ac.uk. (C) 2007 Elsevier B.V. All rights reserved.

Hepatocellular carcinoma and polymorphisms in carcinogen-metabolizing and DNA repair enzymes in a population with aflatoxin exposure and hepatitis B virus endemicity

High rates of hepatocellular carcinoma (HCC) in The Gambia, West Africa, are primarily due to a high prevalence of chronic hepatitis B virus infection and heavy aflatoxin exposure via groundnut consumption. We investigated genetic polymorphisms in carcinogen-metabolizing (GSTM1, GSTT1, HYL1*2) and DNA repair (XRCC1) enzymes in a hospital-based case-control study. Incident HCC cases (n = 216) were compared with frequency-matched controls (n = 408) with no clinically apparent liver disease. Although the prevalence of variant genotypes was generally low, in multivariable analysis (adjusting for demographic factors, hepatitis B virus, hepatitis C virus, and TP53 status), the GSTM1-null genotype [odds ratio (OR), 2.45; 95% confidence interval (95% CI), 1.21-4.95] and the heterozygote XRCC1-399 AG genotype (OR, 3.18; 95% CI, 1.35-7.51) were significantly associated with HCC. A weak association of the HYL1*2 polymorphism with HCC was observed but did not reach statistical significance. GSTT1 was not associated with HCC. The risk for HCC with null GSTM1 was most prominent among those with the highest groundnut consumption (OR, 4.67; 95% CI, 1.45-15.1) and was not evident among those with less than the mean groundnut intake (OR, 0.64; 95% Cl, 0.20-2.02). Among participants who had all three suspected aflatoxin-related high-risk genotypes [GSTM1 null, HLY1*2 (HY/HH), and XRCC1 (AG/GG)], a significant 15-fold increased risk of HCC was observed albeit with imprecise estimates (OR, 14.7; 95% CI, 1.27-169). Our findings suggest that genetic modulation of carcinogen metabolism and DNA repair can alter susceptibility to HCC and that these effects may be modified by environmental factors.

Phase I and II biotransformation enzymes and polycyclic aromatic hydrocarbons in the Mediterranean mussel (Mytilus galloprovincialis, Lamarck, 1819) collected in front of an oil refinery

The aim of the present study was to investigate the responses of phase I and II biotransformation enzymes and levels of PAHs in the Mediterranean mussel (Mytilus galloprovincialis, Lamarck, 1819) collected from three sites at different distance from an oil refinery. Phase I enzyme activities as NAD(P)H-cyt c red, NADH ferry red, B(a)PMO and phase II as UDPGT. GST were measured in digestive gland while 16 PAHs (US-EPA) in whole soft tissue. An added value to the data obtained in the present study rely on the RDA analysis which showed close correlations between PAHs levels and phase I enzyme activities in mussels collected in front of the refinery. And again a significant spatial correlation between B(a)P levels and NADPH-cyt c red activities was observed using linear models. No differences among sites for B(a) PMO and phase II GST activities were observed, while the application of UDPGT as biomarkers requires further investigation. (C) 2012 Elsevier Ltd. All rights reserved.

Transcriptome analysis of a parasitic clade V nematode:comparative analysis of potential molecular anthelmintic targets in Cylicostephanus goldi

Clade V nematodes comprise several parasitic species that include the cyathostomins, primary helminth pathogens of horses. Next generation transcriptome datasets are available for eight parasitic clade V nematodes, although no equine parasites are included in this group. Here, we report next generation transcriptome sequencing analysis for the common cyathostomin species, Cylicostephanus goldi. A cDNA library was generated from RNA extracted from 17 C. goldi male and female adult parasites. Following sequencing using a 454 GS FLX pyrosequencer, a total of 475,215 sequencing reads were generated, which were assembled into 26,910 contigs. Using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases, 27% of the transcriptome was annotated. Further in-depth analysis was carried out by comparing the C. goldi dataset with the next generation transcriptomes and genomes of other clade V nematodes, with the Oesophagostomum dentatum transcriptome and the Haemonchus contortus genome showing the highest levels of sequence identity with the cyathostomin dataset (45%). The C. goldi transcriptome was mined for genes associated with anthelmintic mode of action and/or resistance. Sequences encoding proteins previously associated with the three major anthelmintic classes used in horses were identified, with the exception of the P-glycoprotein group. Targeted resequencing of the glutamate gated chloride channel α4 subunit (glc-3), one of the primary targets of the macrocyclic lactone anthelmintics, was performed for several cyathostomin species. We believe this study reports the first transcriptome dataset for an equine helminth parasite, providing the opportunity for in-depth analysis of these important parasites at the molecular level. Sequences encoding enzymes involved in key processes and genes associated with levamisole/pyrantel and macrocyclic lactone resistance, in particular the glutamate gated chloride channels, were identified. This novel data will inform cyathostomin biology and anthelmintic resistance studies in future.

Genome analysis of a simultaneously predatory and prey-independent, novel Bdellovibrio bacteriovorus from the River Tiber, supports in silico predictions of both ancient and recent lateral gene transfer from diverse bacteria

BACKGROUND: Evolution equipped Bdellovibrio bacteriovorus predatory bacteria to invade other bacteria, digesting and replicating, sealed within them thus preventing nutrient-sharing with organisms in the surrounding environment. Bdellovibrio were previously described as "obligate predators" because only by mutations, often in gene bd0108, are 1 in ~1x10(7) of predatory lab strains of Bdellovibrio converted to prey-independent growth. A previous genomic analysis of B. bacteriovorus strain HD100 suggested that predatory consumption of prey DNA by lytic enzymes made Bdellovibrio less likely than other bacteria to acquire DNA by lateral gene transfer (LGT). However the Doolittle and Pan groups predicted, in silico, both ancient and recent lateral gene transfer into the B. bacteriovorus HD100 genome.

RESULTS: To test these predictions, we isolated a predatory bacterium from the River Tiber- a good potential source of LGT as it is rich in diverse bacteria and organic pollutants- by enrichment culturing with E. coli prey cells. The isolate was identified as B. bacteriovorus and named as strain Tiberius. Unusually, this Tiberius strain showed simultaneous prey-independent growth on organic nutrients and predatory growth on live prey. Despite the prey-independent growth, the homolog of bd0108 did not have typical prey-independent-type mutations. The dual growth mode may reflect the high carbon content of the river, and gives B. bacteriovorus Tiberius extended non-predatory contact with the other bacteria present. The HD100 and Tiberius genomes were extensively syntenic despite their different cultured-terrestrial/freshly-isolated aquatic histories; but there were significant differences in gene content indicative of genomic flux and LGT. Gene content comparisons support previously published in silico predictions for LGT in strain HD100 with substantial conservation of genes predicted to have ancient LGT origins but little conservation of AT-rich genes predicted to be recently acquired.

CONCLUSIONS: The natural niche and dual predatory, and prey-independent growth of the B. bacteriovorus Tiberius strain afforded it extensive non-predatory contact with other marine and freshwater bacteria from which LGT is evident in its genome. Thus despite their arsenal of DNA-lytic enzymes; Bdellovibrio are not always predatory in natural niches and their genomes are shaped by acquiring whole genes from other bacteria.

Low alanine aminotransferase levels and higher number of cardiovascular events in people with Type 2 diabetes: analysis of the Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) study.

AIMS: To determine whether alanine aminotransferase or gamma-glutamyltransferase levels, as markers of liver health and non-alcoholic fatty liver disease, might predict cardiovascular events in people with Type 2 diabetes.

METHODS: Data from the Fenofibrate Intervention and Event Lowering in Diabetes study were analysed to examine the relationship between liver enzymes and incident cardiovascular events (non-fatal myocardial infarction, stroke, coronary and other cardiovascular death, coronary or carotid revascularization) over 5 years.

RESULTS: Alanine aminotransferase level had a linear inverse relationship with the first cardiovascular event occurring in participants during the study period. After adjustment, for every 1 sd higher baseline alanine aminotransferase value (13.2 U/l), the risk of a cardiovascular event was 7% lower (95% CI 4-13; P=0.02). Participants with alanine aminotransferase levels below and above the reference range 8-41 U/l for women and 9-59 U/l for men, had hazard ratios for a cardiovascular event of 1.86 (95% CI 1.12-3.09) and 0.65 (95% CI 0.49-0.87), respectively (P=0.001). No relationship was found for gamma-glutamyltransferase.

CONCLUSIONS: The data may indicate that in people with Type 2 diabetes, which is associated with higher alanine aminotransferase levels because of prevalent non-alcoholic fatty liver disease, a low alanine aminotransferase level is a marker of hepatic or systemic frailty rather than health. This article is protected by copyright. All rights reserved.

Bacteriophage lytic enzymes and their engineering towards improved antibacterial efficacy

Tese de doutoramento, Farmácia (Microbiologia), Universidade de Lisboa, Faculdade de Farmácia, 2015