Biomedical subjects

Patrick J Collins

Publications and source records attributed to Patrick J Collins.

16 recordsLinked to original sources

PubMed2006

Disruption of iron homeostasis increases phosphine toxicity in Caenorhabditis elegans.

The aim of this study is to identify the biochemical mechanism of phosphine toxicity and resistance, using Caenorhabditis elegans as a model organism. To date, the precise mode of phosphine action is unclear. In this report, we demonstrate the following dose-dependent actions of phosphine, in vitro: (1) reduction of ferric iron (Fe3+) to ferrous iron (Fe2+), (2) release of iron from horse ferritin, (3) and the peroxidation of lipid as a result of iron release from ferritin. Using in situ hybridization, we show that the ferritin genes of C. elegans, both ferritin-1 and ferritin-2, are expressed along the digestive tract with greatest expression at the proximal and distal ends. Basal expression of the ferritin-2 gene, as determined by quantitative PCR, is approximately 80 times that of ferritin-1. However, transcript levels of ferritin-1 are induced at least 20-fold in response to phosphine, whereas there is no change in the level of ferritin-2. This resembles the reported pattern of ferritin gene regulation by iron, suggesting that phosphine toxicity may be related to an increase in the level of free iron. Indeed, iron overload increases phosphine toxicity in C. elegans at least threefold. Moreover, we demonstrate that suppression of ferritin-2 gene expression by RNAi, significantly increases sensitivity to phosphine. This study identifies similarities between phosphine toxicity and iron overload and demonstrates that phosphine can trigger iron release from storage proteins, increasing lipid peroxidation, leading to cell injury and/or cell death.

Animals↗

PubMed2006

The structure of H5N1 avian influenza neuraminidase suggests new opportunities for drug design.

The worldwide spread of H5N1 avian influenza has raised concerns that this virus might acquire the ability to pass readily among humans and cause a pandemic. Two anti-influenza drugs currently being used to treat infected patients are oseltamivir (Tamiflu) and zanamivir (Relenza), both of which target the neuraminidase enzyme of the virus. Reports of the emergence of drug resistance make the development of new anti-influenza molecules a priority. Neuraminidases from influenza type A viruses form two genetically distinct groups: group-1 contains the N1 neuraminidase of the H5N1 avian virus and group-2 contains the N2 and N9 enzymes used for the structure-based design of current drugs. Here we show by X-ray crystallography that these two groups are structurally distinct. Group-1 neuraminidases contain a cavity adjacent to their active sites that closes on ligand binding. Our analysis suggests that it may be possible to exploit the size and location of the group-1 cavity to develop new anti-influenza drugs.

Acetamides↗

PubMed2006

The Agilent in situ-synthesized microarray platform.

Microarray technology has become a standard tool in many laboratories. Agilent Technologies manufactures a variety of catalog and custom long-oligonucleotide (60-mer) microarrays that can be used in multiple two-color microarray applications. Optimized methods and techniques have been developed for two such applications: gene expression profiling and comparative genomic hybridization. Methods for a third technique, location analysis, are evolving rapidly. This chapter outlines current best methods for using Agilent microarrays, provides detailed instructions for the most recently developed techniques, and discusses solutions to common problems encountered with two-color microarrays.

Animals↗

PubMed2006

Evaluation of external RNA controls for the assessment of microarray performance.

External RNA controls (ERCs), although important for microarray assay performance assessment, have yet to be fully implemented in the research community. As part of the MicroArray Quality Control (MAQC) study, two types of ERCs were implemented and evaluated; one was added to the total RNA in the samples before amplification and labeling; the other was added to the copyRNAs (cRNAs) before hybridization. ERC concentration-response curves were used across multiple commercial microarray platforms to identify problematic assays and potential sources of variation in the analytical process. In addition, the behavior of different ERC types was investigated, resulting in several important observations, such as the sample-dependent attributes of performance and the potential of using these control RNAs in a combinatorial fashion. This multiplatform investigation of the behavior and utility of ERCs provides a basis for articulating specific recommendations for their future use in evaluating assay performance across multiple platforms.

Algorithms↗

PubMed2006

The MicroArray Quality Control (MAQC) project shows inter- and intraplatform reproducibility of gene expression measurements.

Over the last decade, the introduction of microarray technology has had a profound impact on gene expression research. The publication of studies with dissimilar or altogether contradictory results, obtained using different microarray platforms to analyze identical RNA samples, has raised concerns about the reliability of this technology. The MicroArray Quality Control (MAQC) project was initiated to address these concerns, as well as other performance and data analysis issues. Expression data on four titration pools from two distinct reference RNA samples were generated at multiple test sites using a variety of microarray-based and alternative technology platforms. Here we describe the experimental design and probe mapping efforts behind the MAQC project. We show intraplatform consistency across test sites as well as a high level of interplatform concordance in terms of genes identified as differentially expressed. This study provides a resource that represents an important first step toward establishing a framework for the use of microarrays in clinical and regulatory settings.

Equipment Design↗

PubMed2006

Performance comparison of one-color and two-color platforms within the MicroArray Quality Control (MAQC) project.

Microarray-based expression profiling experiments typically use either a one-color or a two-color design to measure mRNA abundance. The validity of each approach has been amply demonstrated. Here we provide a simultaneous comparison of results from one- and two-color labeling designs, using two independent RNA samples from the Microarray Quality Control (MAQC) project, tested on each of three different microarray platforms. The data were evaluated in terms of reproducibility, specificity, sensitivity and accuracy to determine if the two approaches provide comparable results. For each of the three microarray platforms tested, the results show good agreement with high correlation coefficients and high concordance of differentially expressed gene lists within each platform. Cumulatively, these comparisons indicate that data quality is essentially equivalent between the one- and two-color approaches and strongly suggest that this variable need not be a primary factor in decisions regarding experimental microarray design.

Equipment Design↗

PubMed2005

Use of a mixed tissue RNA design for performance assessments on multiple microarray formats.

The comparability and reliability of data generated using microarray technology would be enhanced by use of a common set of standards that allow accuracy, reproducibility and dynamic range assessments on multiple formats. We designed and tested a complex biological reagent for performance measurements on three commercial oligonucleotide array formats that differ in probe design and signal measurement methodology. The reagent is a set of two mixtures with different proportions of RNA for each of four rat tissues (brain, liver, kidney and testes). The design provides four known ratio measurements of >200 reference probes, which were chosen for their tissue-selectivity, dynamic range coverage and alignment to the same exemplar transcript sequence across all three platforms. The data generated from testing three biological replicates of the reagent at eight laboratories on three array formats provides a benchmark set for both laboratory and data processing performance assessments. Close agreement with target ratios adjusted for sample complexity was achieved on all platforms and low variance was observed among platforms, replicates and sites. The mixed tissue design produces a reagent with known gene expression changes within a complex sample and can serve as a paradigm for performance standards for microarrays that target other species.

Animals↗

PubMed2004

The purification and characterisation of novel dipeptidyl peptidase IV-like activity from bovine serum.

The discovery of a potentially novel proline-specific peptidase from bovine serum is presented which is capable of cleaving the dipeptidyl peptidase IV (DPIV) substrate Gly-Pro-MCA. The enzyme was isolated and purified with the use of Phenyl Sepharose Hydrophobic Interaction, Sephacryl S-300 Gel Filtration, and Q-Sephacryl Anion Exchange, producing an overall purification factor of 257. SDS PAGE resulted in a monomeric molecular mass of 158kDa while size exclusion chromatography generated a native molecular mass of 328kDa. The enzyme remained active over a broad pH range with a distinct preference for a neutral pH range of 7-8.5. Chromatofocusing and isoelectric focusing (IEF) revealed the enzyme's isoelectric point to be 4.74. DPIV-like activity was not inhibited by serine protease inhibitors but was by the metallo-protease inhibitors, the phenanthrolines. The enzyme was also partially inhibited by bestatin. Substrate specificity studies proved that the enzyme is capable of sequential cleavage of bovine beta-Casomorphin and Substance P. The peptidase cleaved the standard DPIV substrate, Gly-Pro-MCA with a K(M) of 38.4 microM, while Lys-Pro-MCA was hydrolysed with a K(M) of 103 microM. The DPIV-like activity was specifically inhibited by both Diprotin A and B, non-competitively, generating a K(i) of 1.4 x 10(-4) M for both inhibitors. Ile-Thiazolidide and Ile-Pyrrolidide both inhibited competitively with an inhibition constant of 3.7 x 10(-7) and 7.5 x 10(-7) M, respectively. It is concluded that bovine serum DPIV-like activity share many biochemical properties with DPIV and DPIV-like enzymes but not exclusively, suggesting that the purified peptidase may play an important novel role in bioactive oligopeptide degradation.

Animals↗

PubMed2004

Purification, identification and characterisation of seprase from bovine serum.

The study and identification for the first time of a soluble form of a seprase activity from bovine serum is presented. To date, this activity has only been reported to be an integral membrane protease but has been known to shed from its membrane. The activity was purified 30,197-fold to homogeneity, using a combination of column chromatographies, from bovine serum. Inhibition by DFP, resulting in an IC(50) of 100:nM confirms classification as a serine protease. The protease after separation and visualisation by native PAGE was subjected to tryptic digestion and the subsequent peptides sequenced. Each peptide sequenced was found to be present in the primary structure of seprase/fibroblast activation protein (FAP), a serine gelatinase specific for proline-containing peptides and macromolecules. Substrate specificity studies using kinetic, RP-HPLC and LC-MS analysis of synthetic peptides suggest that this peptidase has an extended substrate-binding region in addition to the primary specificity site S(1). This analysis revealed at least five subsites to be involved in enzyme-substrate binding, with the smallest peptide cleaved being a tetrapeptide. A proline residue in position P(1) was absolutely necessary therefore showing high primary substrate specificity for the Pro-X bond, while a preference for a hydrophobic residue at the C-terminal end of the scissile bond (P'(1)) was evident. The enzyme also showed complete insensitivity to the prolyl oligopeptidase specific inhibitors, JTP-4819, Fmoc-Ala-pyrrCN and Z-Phe-Pro-BT. To date, no physiological substrate has clearly been defined for this protease but its ability to effectively degrade gelatin suggests a candidate protein substrate in vivo and a possible role in extracellular matrix protein degradation.

Amino Acid Sequence↗

PubMed2004

Developmental validation of a single-tube amplification of the 13 CODIS STR loci, D2S1338, D19S433, and amelogenin: the AmpFlSTR Identifiler PCR Amplification Kit.

Analysis of length polymorphism at short tandem repeat (STR) loci utilizing the polymerase chain reaction (PCR) process has proven to be an ideal assay for human identification purposes. The short length of STR loci coupled with the amplification of target sequence through PCR allows for a robust, sensitive, and specific assay for highly polymorphic markers. A multiplex containing fifteen STR loci plus the gender-determining locus Amelogenin was developed to provide a single amplification/detection of all CODIS (Combined DNA Index System) STR loci (CSF1PO, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, FGA, TH01, TPOX, and vWA) as well as two internationally-accepted STRs (D2S1338 and D19S433). By incorporating five-dye fragment analysis technology and non-nucleotide linkers, previously optimized AmpFlSTR kit primer sequences have been maintained. This kit has been developed in accordance with the standards of the forensic community as defined by the DNA Advisory Board. Validation studies were performed to include developmental validation, and the results support the use of the AmpFlSTR Identifiler PCR Amplification Kit for human identity and parentage testing.

Animals↗

PubMed2003

Caenorhabditis elegans mutants resistant to phosphine toxicity show increased longevity and cross-resistance to the synergistic action of oxygen.

Phosphine (hydrogen phosphide, PH3) is the fumigant most widely used to protect stored products from pest infestation. Despite the importance of this chemical, little is known about its mode of action. We have created three phosphine-resistant lines (pre-1, pre-7, pre-33) in the model organism C. elegans, with LC50 values 2, 5, and 9 times greater than the fully susceptible parental strain. Molecular oxygen was shown to be an extremely effective synergist with phosphine as, under hyperoxic conditions, 100% mortality was observed in wild-type nematodes exposed to 0.1 mg/l phosphine, a nonlethal concentration in air. All three mutants were resistant to the synergistic effects of oxygen in proportion to their resistance to phosphine with one mutant, pre-33, showing complete resistance to this synergism. We take the proportionality of cross-resistance between phosphine and the synergistic effect of oxygen to imply that all three mutants circumvent a mechanism of phosphine toxicity that is directly coupled to oxygen metabolism. Compared with the wild-type strain, all three mutants have an extended average life expectancy of from 12.5 to 25.3%. This is consistent with the proposed involvement of oxidative stress in both phosphine toxicity and ageing. Because the wild-type and mutant nematodes develop at the same rate, the longevity is unlikely to be caused by a clk-type reduction in oxidative metabolism, a potential alternative mechanism of phosphine resistance.

Aging↗

PubMed2003

Inhibition of egg development by phosphine in the cosmopolitan pest of stored products Liposcelis bostrychophila (Psocoptera: Liposcelididae).

Phosphine-induced delay in development of eggs was investigated as a mechanism of resistance to this fumigant in Liposcelis bostrychophila Badonnel. One-day-old eggs of a susceptible and a strongly resistant strain of L bostrychophila were exposed to a range of phosphine concentrations for 6days at 30 (+/- 1) degrees C and 70 (+/- 2)% RH. Delay in mean hatching period occurred in both susceptible and resistant eggs, although it was more pronounced in the latter. A maximum delay of 2.65 days was recorded for eggs of the susceptible strain at 0.01 mg litre(-1) (the highest concentration at which eggs survived) and 13.39 days for the resistant strain at 1 mg litre(-1) (the highest concentration tested). Delay in egg development time was positively correlated with increasing phosphine concentration. Our results reveal that the most successful strategy to control resistant L bostrychophila is to apply relatively low concentrations of phosphine for extended exposure times (eg 0.05 mg litre(-1) for 16 days) that allow all eggs to hatch to the much less tolerant nymph stage.

Animals↗

PubMed2002

Effects of time and concentration on mortality of phosphine-resistant Sitophilus oryzae (L) fumigated with phosphine.

The effects of exposure period and phosphine concentration on mortality of susceptible and resistant Sitophilus oryzae (L) were investigated. Although S oryzae is one of the world's most serious pests of stored grain there are few data on the practical significance of phosphine resistance in this species. The strains investigated were an Australian susceptible strain, a homozygous resistant strain exhibiting a level of resistance common in Australia and an unselected field strain from China with a much stronger resistance. Fumigations were carried out at 25 degrees C on adults and mixed-age cultures. For adults of all three strains and mixed-age cultures of the susceptible and resistant Australian strains, the relationship between concentration and time could be described by equations of the form Cnt = k. In all cases n < 1, indicating that time was a more important variable than concentration. In all fumigations of adults the resistant strains were harder to kill than the susceptible strain. However, in fumigations of mixed-age cultures, which contained the tolerant pupal stage, the difference between susceptible and resistant strains was more pronounced at lower concentrations than higher concentrations. For example, at 0.02 mg litre-1 the estimated LT99.9 for mixed-age cultures of the Australian resistant strain (27 days) is 3.4 times that of the susceptible strain (8 days), but at 1 mg litre-1 there is no difference between the two strains (4 days). Limited data on the Chinese resistant strain supported this finding. Twenty-three days exposure at 0.02 mg litre-1 had no effect on mixed-age cultures of this strain, but there were no survivors after 5 days exposure to 1 mg litre-1.

Algorithms↗

PubMed2002

Long-term effectiveness of grain protectants and structural treatments against Liposcelis decolor (Pearman) (Psocoptera: Liposcelididae), a pest of stored products.

Numerous strains of the psocid pest, Liposcelis decolor (Pearman) were collected from farms and central storages and interbred to form three representative strains from three major grain-growing states of Australia: Queensland, South Australia and New South Wales. These were tested against the grain protectants and structural treatments currently registered for use in Australia. Recently, L decolor has become an important pest of stored grain in Australia, particularly in the eastern and southern parts. There is no published information available on management of this pest and the current pest-management strategy, based predominantly on phosphine fumigation, has failed to control infestations of this pest in numerous grain storages in Australia. Alternative methods of control such as use of contact insecticides were explored in the present work to supplement phosphine fumigation to manage this new pest. From eight grain protectants tested as admixtures, only chlorpyrifos-methyl, bioresmethrin plus piperonyl butoxide, and fenitrothion were found to provide long term (3-9 months) protection against all three strains of L decolor. Chlorpyrifos-methyl gave the best protection, providing a minimum of 7.5 to a maximum of 9 months protection, depending on the strains tested. Three structural treatments, azamethiphos, azamethiphos plus carbaryl and permethrin provided long-term control (8-9 months) of all three strains of L decolor on galvanised steel surfaces, with permethrin delivering 9 months protection against all strains. However, all of these treatments failed to provide long-term control of any strains on concrete surfaces. We conclude that chlorpyrifos-methyl as a grain admixture can be incorporated into fumigation strategies to optimise the control of L decolor infestations. Structural treatments, such as permethrin can be used to support a fumigation strategy in storages made of galvanised steel.

Animals↗

PubMed2002

Genetic linkage analysis of the lesser grain borer Rhyzopertha dominica identifies two loci that confer high-level resistance to the fumigant phosphine.

High levels of inheritable resistance to phosphine in Rhyzopertha dominica have recently been detected in Australia and in an effort to isolate the genes responsible for resistance we have used random amplified DNA fingerprinting (RAF) to produce a genetic linkage map of R. dominica. The map consists of 94 dominant DNA markers with an average distance between markers of 4.6 cM and defines nine linkage groups with a total recombination distance of 390.1 cM. We have identified two loci that are responsible for high-level resistance. One provides approximately 50x resistance to phosphine while the other provides 12.5x resistance and in combination, the two genes act synergistically to provide a resistance level 250x greater than that of fully susceptible beetles. The haploid genome size has been determined to be 4.76 x 10(8) bp, resulting in an average physical distance of 1.2 Mbp per map unit. No recombination has been observed between either of the two resistance loci and their adjacent DNA markers in a population of 44 fully resistant F5 individuals, which indicates that the genes are likely to reside within 0.91 cM (1.1 Mbp) of the DNA markers.

Animals↗

PubMed2002

Genetics of resistance to phosphine in Rhyzopertha dominica (Coleoptera: Bostrichidae).

The inheritance of resistance to phosphine was studied in two strains of the lesser grain borer, Rhyzopertha dominica (F.), labeled 'Weak-R' and 'Strong-R'. These strains were purified versions of field-selected populations collected in Queensland, Australia. Weak-R and Strong-R were, respectively, 23.4 times (20-h exposure) and 600 times (48-h exposure) resistant to phosphine compared with a reference susceptible strain (S-strain). Each -R strain was crossed with the S-strain and the response to phosphine was measured in their respective F1, F2, and F1-backcross (F1-BC) progenies. Data from testing of reciprocal F1 progeny indicated that resistance in Weak-R was autosomal and incompletely recessive with a degree of dominance -0.96. Modified chi-square analysis and contingency analysis of the observed response to phosphine of F1-BC and F2 progenies rejected the hypothesis of single gene inheritance of resistance. Analysis of the response of the F1, F2, and F1-BC progeny from the Strong-R x S-strain cross also rejected the null hypothesis for single gene resistance. Resistance in the Strong-R strain was autosomal and incompletely recessive with a degree of dominance of -0.64. The Weak-R and Strong-R strains were then crossed. Analysis ofthe F1 and F2 progenies of this reciprocal cross revealed that the strong resistance phenotype was coded by a combination of the genes already present in the Weak-R genotype plus an extra major, incompletely recessive gene. There was also evidence of a minor dominant gene present in approximately 5% of Strong-R individuals.

Animals↗