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Michael J Allen

Publications and source records attributed to Michael J Allen.

At least 19 recordsLinked to original sources
PubMed2006

Expression of a novel marine viral single-chain serine palmitoyltransferase and construction of yeast and mammalian single-chain chimera.

The genus Coccolithovirus is a recently discovered group of viruses that infect the globally important marine calcifying microalga Emiliania huxleyi. Surprisingly, the viral genome contains a cluster of putative sphingolipid biosynthetic genes not found in other viral genus. To address the role of these genes in viral pathogenesis, the ehv050 gene predicted to encode a serine palmitoyltransferase (SPT), the first and rate-limiting enzyme of sphingolipid biosynthesis, was expressed and characterized in Saccharomyces cerevisiae. We show that the encoded protein is indeed a fully functional, endoplasmic reticulum-localized, single-chain SPT. In eukaryotes SPT is a heterodimer comprised of long chain base 1 (LCB1) and LCB2 subunits. Sequence alignment and mutational analysis showed that the N-terminal domain of the viral protein most closely resembled the LCB2 subunit and the C-terminal domain most closely resembled the LCB1 subunit. Regardless of whether the viral protein was expressed as a single polypeptide or as two independent domains, it exhibited an unusual preference for myristoyl-CoA rather than palmitoyl-CoA. This preference was reflected by the increased presence of C16-sphingoid bases in yeast cells expressing the viral protein. The occurrence of a single-chain SPT suggested to us that it might be possible to create other fusion SPTs with unique properties. Remarkably, when the two subunits of the yeast SPT were thus expressed, the single-chain chimera was functional and displayed a novel substrate preference. This suggests that expression of other multisubunit membrane proteins as single-chain chimera could provide a powerful approach to the characterization of integral membrane proteins.

Amino Acid Sequence↗
PubMed2006

The coccolithovirus microarray: an array of uses.

The Coccolithoviridae is a recently discovered family of giant algal viruses. Here, we review the genomic and transcriptomic characterization of this family based on the results generated from a coccolithovirus microarray. The microarray has been used to aid the annotation of the genome, to investigate the infection process at the transcriptional level and to assess the diversity in genomic content within the family.

Genes, Viral↗
PubMed2006

2,5-diketopiperazines as potent, selective, and orally bioavailable oxytocin antagonists. 3. Synthesis, pharmacokinetics, and in vivo potency.

A short, efficient, and highly stereoselective synthesis of a series of (3R,6R,7R)-2,5-diketopiperazine oxytocin antagonists and their pharmacokinetics in rat and dog is described. Prediction of the estimated human oral absorption (EHOA) using measured lipophilicity (CHI log D) and calculated size (cMR) has allowed us to rank various 2,5-diketopiperazine templates and enabled us to focus effort on those templates with the greatest chance of high bioavailability in humans. This rapidly led to the 2',4'-difluorophenyl-dimethylamide 25 and the benzofuran 4 with high levels of potency (pK(i)) and good bioavailability in the rat and dog. Dimethylamide 25 is more potent (>20-fold) than 4 in vivo and has a high degree of selectivity toward the vasopressin receptors, >10,000 for hV1a/hV1b and approximately 500 for hV2. It has a good Cyp450 profile with no time dependent inhibition and was negative in the genotoxicity screens with a satisfactory oral safety profile in rats.

Administration, Oral↗
PubMed2006

Genome comparison of two Coccolithoviruses.

BACKGROUND: The Coccolithoviridae is a recently discovered family of viruses that infect the marine coccolithophorid Emiliania huxleyi. Following on from the sequencing of the type strain EhV-86, we have sequenced a second strain, EhV-163. RESULTS: We have sequenced approximately 80% of the EhV-163 genome, equating to more than 200 full length CDSs. Conserved and variable CDSs and a gene replacement have been identified in the EhV-86 and EhV-163 genomes. CONCLUSION: The sequencing of EhV-163 has provided a wealth of information which will aid the re-annotating of the EhV-86 genome and identified a gene insertion in EhV-163.

Base Sequence↗
PubMed2006

Annotation of environmental OMICS data: application to the transcriptomics domain.

Researchers working on environmentally relevant organisms, populations, and communities are increasingly turning to the application of OMICS technologies to answer fundamental questions about the natural world, how it changes over time, and how it is influenced by anthropogenic factors. In doing so, the need to capture meta-data that accurately describes the biological "source" material used in such experiments is growing in importance. Here, we provide an overview of the formation of the "Env" community of environmental OMICS researchers and its efforts at considering the meta-data capture needs of those working in environmental OMICS. Specifically, we discuss the development to date of the Env specification, an informal specification including descriptors related to geographic location, environment, organism relationship, and phenotype. We then describe its application to the description of environmental transcriptomic experiments and how we have used it to extend the Minimum Information About a Microarray Experiment (MIAME) data standard to create a domain-specific extension that we have termed MIAME/Env. Finally, we make an open call to the community for participation in the Env Community and its future activities.

Ecology↗
PubMed2006

The response of Escherichia coli to exposure to the biocide polyhexamethylene biguanide.

The global response of Escherichia coli to the broad-spectrum biocide polyhexamethylene biguanide (PHMB) was investigated using transcriptional profiling. The transcriptional analyses were validated by direct determination of the PHMB-tolerance phenotypes of derivatives of E. coli MG1655 carrying either insertionally inactivated genes and/or plasmids expressing the cognate open reading frames from a heterologous promoter in the corresponding chromosomally inactivated strains. The results showed that a wide range of genes was altered in transcriptional activity and that all of the corresponding knockout strains subsequently challenged with biocide were altered in tolerance. Of particular interest was the induction of the rhs genes and the implication of enzymes involved in the repair/binding of nucleic acids in the generation of tolerance, suggesting a novel dimension in the mechanism of action of PHMB based on its interaction with nucleic acids.

Bacterial Outer Membrane Proteins↗
PubMed2006

Locus-specific gene expression pattern suggests a unique propagation strategy for a giant algal virus.

Emiliania huxleyi virus strain 86 is the largest algal virus sequenced to date and is unique among the Phycodnaviridae since its genome is predicted to contain six RNA polymerase subunit genes. We have used a virus microarray to profile the temporal transcription strategy of this unusual virus during infection. There are two distinct transcription phases to the infection process. The primary phase is dominated by a group of coding sequences (CDSs) expressed by 1 h postinfection that are localized to a subregion of the genome. The CDS of the primary group have no database homologues, and each is associated with a unique promoter element. The remainder of the CDSs are expressed in a secondary phase between 2 and 4 hours postinfection. Compartmentalized transcription of the two distinctive phases is discussed. We hypothesize that immediately after infection the nucleic acid of the virus targets the host nucleus, where primary-phase genes are transcribed by host RNA polymerase which recognizes the viral promoter. Secondary-phase transcription may then be conducted in the cytoplasm.

Cell Nucleus↗
PubMed2005

2,5-diketopiperazines as potent, selective, and orally bioavailable oxytocin antagonists. 2. Synthesis, chirality, and pharmacokinetics.

A short stereoselective synthesis of a series of chiral 7-aryl-2,5-diketopiperazines oxytocin antagonists is described. Varying the functionality and substitution pattern of substituents in the 7-aryl ring and varying the chirality of this exocyclic ring have produced potent oxytocin antagonists (pK(i) > 8.5). SAR and pharmacokinetic profiling of this series of (3R,6R,7R)-2,5-diketopiperazines together with the introduction of an ortho F group in the 7-aryl ring to improve rat pK has culminated in the 2',4'-difluorophenyldiketopiperazine derivative 37, a highly potent oxytocin antagonist against the human oxytocin receptor (pK(i) = 8.9) that has >1000-fold selectivity over all three vasopressin receptors V1a, V2, and V1b. It has good bioavailability (46%) in the rat and moderate bioavailability (13-31%) in the dog and is more active in vivo in the rat than atosiban (rat DR(10) = 0.44 mg/kg iv).

Administration, Oral↗
PubMed2005

Evolutionary history of the Coccolithoviridae.

We recently determined the genome sequence of the Coccolithoviridae strain Emiliania huxleyi virus 86 (EhV-86), a giant double-stranded DNA (dsDNA) algal virus from the family Phycodnaviridae that infects the marine coccolithophorid E. huxleyi. Here, we determine the phylogenetic relationship between EhV-86 and other large dsDNA viruses. Twenty-five core genes common to nuclear-cytoplasmic large dsDNA virus genomes were identified in the EhV-86 genome; sequence from eight of these genes were used to create a phylogenetic tree in which EhV-86 was placed firmly with the two other members of the Phycodnaviridae. We have also identified a 100-kb region of the EhV-86 genome which appears to have transferred into this genome from an unknown source. Furthermore, the presence of six RNA polymerase subunits (unique among the Phycodnaviridae) suggests both a unique evolutionary history and a unique lifestyle for this intriguing virus.

Cluster Analysis↗
PubMed2005

Complete genome sequence and lytic phase transcription profile of a Coccolithovirus.

The genus Coccolithovirus is a recently discovered group of viruses that infect the globally important marine calcifying microalga Emiliania huxleyi. Among the 472 predicted genes of the 407,339-base pair genome are a variety of unexpected genes, most notably those involved in biosynthesis of ceramide, a sphingolipid known to induce apoptosis. Uniquely for algal viruses, it also contains six RNA polymerase subunits and a novel promoter, suggesting this virus encodes its own transcription machinery. Microarray transcriptomic analysis reveals that 65% of the predicted virus-encoded genes are expressed during lytic infection of E. huxleyi.

Apoptosis↗
PubMed2005

2,5-Diketopiperazines as potent and selective oxytocin antagonists 1: Identification, stereochemistry and initial SAR.

This paper covers efforts to discover orally active potent and selective oxytocin antagonists. Screening pooled libraries identified a novel series of 2,5-diketopiperazine derivatives with antagonist activity at the human oxytocin receptor. We report the initial structure-activity relationship investigations and the determination of the stereochemistry of the most potent compounds.

Humans↗
PubMed2004

Physical characterization of controlled release of paclitaxel from the TAXUS Express2 drug-eluting stent.

The polymer carrier technology in the TAXUS drug-eluting stent consists of a thermoplastic elastomer poly(styrene-b-isobutylene-b-styrene) (SIBS) with microphase-separated morphology resulting in optimal properties for a drug-delivery stent coating. Comprehensive physical characterization of the stent coatings and cast film formulations showed that paclitaxel (PTx) exists primarily as discrete nanoparticles embedded in the SIBS matrix. Thermal and chemical analysis did not show any evidence of solubility of PTx in SIBS or of any molecular miscibility between PTx and SIBS. Atomic force microscope data images revealed for the first time three-dimensional stent coating surfaces at high spatial resolutions in air and in situ under phosphate-buffered saline as drug was released. PTx release involves the initial dissolution of drug particles from the PTx/SIBS coating surface. Morphological examination of the stent coatings in vitro supported an early burst release in most formulations because of surface PTx followed by a sustained slower release of PTx from the bulk coating. The in vitro PTx release kinetics were dependent on the formulation and correlated to the drug-to-polymer ratio. Atomic force microscopy analysis confirmed this correlation and further supported the concept of a matrix-based drug-release coating.

Antineoplastic Agents, Phytogenic↗
PubMed2004

Cooperativity in the binding of the cationic biocide polyhexamethylene biguanide to nucleic acids.

The interaction between the broad-spectrum antimicrobial agent, polyhexamethylene biguanide (PHMB), and various nucleic acids was investigated. Titration of either single- or double-stranded 100-bp DNA, or mixed-molecular weight marker DNA, or tRNA with PHMB caused precipitation of a complex between nucleic acid and PHMB in which the nucleotide/biguanide ratio was always close to unity. Binding of PHMB was highly cooperative, with apparent Hill coefficients 10.3-14.6. When a fluorescent derivative of PHMB was titrated with increasing amounts of nucleic acid, all four forms of nucleic acid caused strong polarisation of fluorescence, demonstrating the association with PHMB. The intensity and broad-spectrum binding of PHMB to all forms of nucleic acid has significant implications for the mechanism of action of this biocide.

Anti-Infective Agents↗
PubMed2004

Force spectroscopy of the double-tethered concanavalin-A mannose bond.

We present the measurement of the force required to rupture a single protein-sugar bond using a methodology that provides selective discrimination between specific and nonspecific binding events and helps verify the presence of a single functional molecule on the atomic force microscopy tip. In particular, the interaction force between a polymer-tethered concanavalin-A protein (ConA) and a similarly tethered mannose carbohydrate was measured as 47 +/- 9 pN at a bond loading rate of approximately 10 nN/s. Computer simulations of the polymer molecular configurations were used to determine the angles that the polymers could sweep out during binding and, in conjunction with mass spectrometry, used to separate the angular effects from the effects due to a distribution of tether lengths. We find that when using commercially available polymer tethers that vary in length from 19 to 29 nm, the angular effects are relatively small and the rupture distributions are dominated by the 10-nm width of the tether length distribution. In all, we show that tethering both a protein and its ligand allows for the determination of the single-molecule bond rupture force with high sensitivity and includes some validation for the presence of a single-tethered functional molecule on the atomic force microscopy tip.

Computer Simulation↗
PubMed2004

The effect of deinsuring chlorpropamide on the prescribing of oral antihyperglycemics for Nova Scotia Seniors' Pharmacare beneficiaries.

On behalf of the Nova Scotia Seniors' Pharmacare Program, the Drug Evaluation Alliance of Nova Scotia developed a multicomponent intervention plan to facilitate the removal of chlorpropamide as an insured benefit. Chlorpropamide has caused serious hypoglycemia in seniors to a greater extent than some other agents. Pharmacy administrative claims were used to compute monthly use rates for insulin and each oral antihyperglycemic drug from January 1, 2000-December 30, 2002, in an intervention cohort (patients receiving chlorpropamide) and a control cohort (patients receiving an antihyperglycemic agent other than chlorpropamide). Initially, 630 patients were receiving chlorpropamide therapy. By the time chlorpropamide was deinsured, only 10% of the treatment cohort continued receiving chlorpropamide; shortly after deinsurance, no beneficiaries continued receiving the drug. The antihyperglycemics with the greatest increase in prescription were glyburide and gliclazide. The deinsuring of chlorpropamide and the educational strategies that accompanied it resulted in the selection of more appropriate antihyperglycemics for Nova Scotia seniors.

Aged↗
PubMed2003

Voriconazole, a novel wide-spectrum triazole: oral pharmacokinetics and safety.

AIMS: Voriconazole is a potent new triazole with broad-spectrum antifungal activity against clinically significant and emerging pathogens. The present study evaluated the safety, toleration, and pharmacokinetics of oral voriconazole after single and multiple dosing. METHODS: Sixty-four healthy subjects were randomized to receive treatment and 56 completed the study. Groups of eight subjects each received voriconazole doses of 2 mg kg-1 twice daily, 4 mg kg-1 once daily, 2 mg kg-1 three times daily, or 3 mg kg-1 twice daily. Eleven subjects received 1.5 mg kg-1 three times daily, and 21 subjects were administered placebo. RESULTS: Voriconazole exhibited nonlinear (dose- and time-dependent) pharmacokinetics. This deviation from linear pharmacokinetics was confirmed by linearity ratios of > 1 and decreasing kel values on multiple dosing, with a consequent increase in the terminal phase t1/2. There was also notable intersubject variability in Cmax and AUCtau. The absorption of voriconazole was rapid (mean tmax= 0.9-1.7 h) after single and multiple dosing and the decline in plasma concentration-time curves after tmax was generally biphasic. By day 12, the Cmax, AUCtau, tmax, and t1/2 values for the 3 mg kg-1 twice-daily group were 2356 ng ml-1, 11 170 ng.h ml-1, 1.1 h, and 6.4 h, respectively. The observed accumulation of voriconazole after multiple dosing was greater than predicted from single-dose data. Accumulation ratios for Cmax and AUCtau, which were 1.97 and 3.55, respectively, for the group given voriconazole 3 mg kg-1 twice daily, varied between treatment groups and appeared to be influenced by total daily dose and the frequency and duration of dosing. Visual inspection of Cmin values together with statistical analyses of Cmax and AUCtau values suggest that steady-state levels were achieved by the fifth to sixth day of multiple dosing. Plasma concentrations of voriconazole were well above the minimum inhibitory concentrations (MICs) for Aspergillus spp., Candida spp., and for most emerging fungal pathogens (Cmin > 0.8 micro g ml-1). Voriconazole was well tolerated: most treatment-related adverse events (abnormal vision, headache, dizziness) were mild and resolved within an hour of dosing. CONCLUSIONS: The oral dosing regimen selected for subsequent Phase II/III clinical trials on the basis of these results was 200 mg twice daily, equivalent to 3 mg kg-1 twice daily.

Administration, Oral↗