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Biomedical subjects

J Errington

Publications and source records attributed to J Errington.

At least 19 recordsLinked to original sources
PubMed2007

Pharmacokinetics and metabolism of 13-cis-retinoic acid (isotretinoin) in children with high-risk neuroblastoma - a study of the United Kingdom Children's Cancer Study Group.

The administration of 13-cis-retinoic acid (13-cisRA), following myeloablative therapy improves 3-year event-free survival rates in children with high-risk neuroblastoma. This study aimed to determine the degree of inter-patient pharmacokinetic variation and extent of metabolism in children treated with 13-cisRA. 13-cis-retinoic acid (80 mg m(-2) b.d.) was administered orally and plasma concentrations of parent drug and metabolites determined on days 1 and 14 of courses 2, 4 and 6 of treatment. Twenty-eight children were studied. The pharmacokinetics of 13-cisRA were best described by a modified one-compartment, zero-order absorption model combined with lag time. Mean population pharmacokinetic parameters included an apparent clearance of 15.9 l h(-1), apparent volume of distribution of 85 l and absorption lag time of 40 min with a large inter-individual variability associated with all parameters (coefficients of variation greater than 50%). Day 1 peak 13-cisRA levels and exposure (AUC) were correlated with method of administration (P<0.02), with 2.44- and 1.95-fold higher parameter values respectively, when 13-cisRA capsules were swallowed as opposed to being opened and the contents mixed with food before administration. Extensive accumulation of 4-oxo-13-cisRA occurred during each course of treatment with plasma concentrations (mean+/-s.d. 4.67+/-3.17 microM) higher than those of 13-cisRA (2.83+/-1.44 microM) in 16 out of 23 patients on day 14 of course 2. Extensive metabolism to 4-oxo-13-cisRA may influence pharmacological activity of 13-cisRA.

Absorption↗
PubMed2006

Development of a real time PCR for the detection of Taylorella equigenitalis directly from genital swabs and discrimination from Taylorella asinigenitalis.

A discriminatory real time PCR for the detection of Taylorella equigenitalis, the causative agent of contagious equine metritis (CEM), and the related species T. asinigenitalis was developed for the direct examination of genital swabs. The 112bp amplicons produced from the two species were discriminated from each other using TaqMan probes labelled with different fluorophores. The TaqMan PCR was shown to be specific for the 16S ribosomal DNA of the two species of taylorella and did not cross-hybridise with the 16S ribosomal DNA of other bacteria tested. Direct amplification from genital swabs was shown to be equally sensitive to that of culture methods. Prevalence in a sample set from The Netherlands was shown to be equivalent to that demonstrated by culture. A companion real time PCR that amplified a fragment of the 16S rDNA gene of equine commensal bacteria was developed to ensure bacterial DNA was extracted from swab material supplied for testing. The use of a rapid and reliable real time PCR for the organism causing CEM should aid the control of this disease.

Animals↗
PubMed2006

Practical experiences of moving molecular diagnostics into routine use at the Veterinary Laboratories Agency.

The practical bench application of molecular tests (here defined as nucleic acid-based tests) in a routine testing situation is not without its challenges. This paper outlines the approaches we take at the Veterinary Laboratories Agency (VLA) and highlights some of the practical issues which we have found to be important for the successful introduction and use of molecular tests in a routine testing environment. The potential advantages of molecular tests, and factors which dictate which tests are adopted for routine testing, are discussed before giving some examples of molecular tests in routine use at the VLA. The instrumentation, reagents and assays we use are outlined, followed by sections of how we approach validation and how we manage and resource the transfer of molecular tests into routine use.

Laboratories↗
PubMed2003

Essential Bacillus subtilis genes.

To estimate the minimal gene set required to sustain bacterial life in nutritious conditions, we carried out a systematic inactivation of Bacillus subtilis genes. Among approximately 4,100 genes of the organism, only 192 were shown to be indispensable by this or previous work. Another 79 genes were predicted to be essential. The vast majority of essential genes were categorized in relatively few domains of cell metabolism, with about half involved in information processing, one-fifth involved in the synthesis of cell envelope and the determination of cell shape and division, and one-tenth related to cell energetics. Only 4% of essential genes encode unknown functions. Most essential genes are present throughout a wide range of Bacteria, and almost 70% can also be found in Archaea and Eucarya. However, essential genes related to cell envelope, shape, division, and respiration tend to be lost from bacteria with small genomes. Unexpectedly, most genes involved in the Embden-Meyerhof-Parnas pathway are essential. Identification of unknown and unexpected essential genes opens research avenues to better understanding of processes that sustain bacterial life.

Bacillus subtilis↗
PubMed2001

Two essential DNA polymerases at the bacterial replication fork.

DNA replication in bacteria is carried out by a multiprotein complex, which is thought to contain only one essential DNA polymerase, specified by the dnaE gene in Escherichia coli and the polC gene in Bacillus subtilis. Bacillus subtilis genome analysis has revealed another DNA polymerase gene, dnaE(BS), which is homologous to dnaE. We show that, in B. subtilis, dnaE(BS) is essential for cell viability and for the elongation step of DNA replication, as is polC, and we conclude that there are two different essential DNA polymerases at the replication fork of B. subtilis, as was previously observed in eukaryotes. dnaE(BS) appears to be involved in the synthesis of the lagging DNA strand and to be associated with the replication factory, which suggests that two different polymerases carry out synthesis of the two DNA strands in B. subtilis and in many other bacteria that contain both polC and dnaE genes.

Bacillus subtilis↗
PubMed2001

Division site selection protein DivIVA of Bacillus subtilis has a second distinct function in chromosome segregation during sporulation.

DivIVA is a coiled-coil, tropomyosin-like protein of Gram-positive bacteria. Previous work showed that this protein is targeted to division sites and retained at the cell poles after division. In vegetative cells, DivIVA sequesters the MinCD division inhibitor to the cell poles, thereby helping to direct cell division to the correct midcell site. We now show that DivIVA has a second, quite separate role in sporulating cells of Bacillus subtilis. It again acts at the cell pole but in this case interacts with the chromosome segregation machinery to help position the oriC region of the chromosome at the cell pole, in preparation for polar division. We isolated mutations in divIVA that separate the protein's role in sporulation from its vegetative function in cell division. DivIVA therefore appears to be a bifunctional protein with distinct roles in division-site selection and chromosome segregation.

Adenosine Triphosphatases↗
PubMed2001

Control of cell shape in bacteria: helical, actin-like filaments in Bacillus subtilis.

In the absence of an overt cytoskeleton, the external cell wall of bacteria has traditionally been assumed to be the primary determinant of cell shape. In the Gram-positive bacterium Bacillus subtilis, two related genes, mreB and mbl, were shown to be required for different aspects of cell morphogenesis. Subcellular localization of the MreB and Mbl proteins revealed that each forms a distinct kind of filamentous helical structure lying close to the cell surface. The distribution of the proteins in different species of bacteria, and the similarity of their sequence to eukaryotic actins, suggest that the MreB-like proteins have a cytoskeletal, actin-like role in bacterial cell morphogenesis.

Actins↗
PubMed2001

Septation and chromosome segregation during sporulation in Bacillus subtilis.

The early stages of sporulation in Bacillus subtilis incorporate a modified, highly asymmetric cell division. It is now clear that most, if not all, of the components of the vegetative division machinery are used also for asymmetric division. However, the machinery for chromosome segregation may differ significantly between vegetative growth and sporulation. Several interesting checkpoint mechanisms couple cell cycle events to gene expression early in sporulation. This review summarises important advances in the understanding of chromosome segregation and cell division at the onset of sporulation in B.subtilis in the past three years.

Bacillus subtilis↗
PubMed2001

Dynamic proteins in bacteria.

The MinCDE system regulates the position of the division plane in rod-shaped bacteria. New results from Escherichia coli provide insight into how this operates by showing that MinE stimulates the ATPase activity of MinD.

Adenosine Triphosphatases↗
PubMed2001

DNA transport in bacteria.

DNA transport is important in various biological contexts--particularly chromosome segregation and intercellular gene transfer. Recently, progress has been made in understanding the function of a family of bacterial proteins involved in DNA transfer, and we focus here on one of the best-understood members, SpoIIIE. Studies of SpoIIIE-like proteins show that they might couple DNA transport to processes such as cell division, conjugation (mating) and the resolution of chromosome dimers.

Bacteria↗
PubMed2001

Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli.

The twin-arginine translocation (Tat) system targets cofactor-containing proteins across the Escherichia coli cytoplasmic membrane via distinct signal peptides bearing a twin-arginine motif. In this study, we have analysed the mechanism and capabilities of the E. coli Tat system using green fluorescent protein (GFP) fused to the twin-arginine signal peptide of TMAO reductase (TorA). Fractionation studies and fluorescence measurements demonstrate that GFP is exported to the periplasm where it is fully active. Export is almost totally blocked in tat deletion mutants, indicating that the observed export in wild-type cells occurs predominantly, if not exclusively, by the Tat pathway. Imaging studies reveal a halo of fluorescence in wild-type cells corresponding to the exported periplasmic form; the GFP is distributed uniformly throughout the cytoplasm in a tat mutant. Because previous work has shown GFP to be incapable of folding in the periplasm, we propose that GFP is exported in a fully folded, active state. These data also show for the first time that heterologous proteins can be exported in an active form by the Tat pathway.

Amino Acid Sequence↗
PubMed2001

Cytological and biochemical characterization of the FtsA cell division protein of Bacillus subtilis.

The actin-like protein FtsA is present in many eubacteria, and genetic experiments have shown that it plays an important, sometimes essential, role in cell division. Here, we show that Bacillus subtilis FtsA is targeted to division sites in both vegetative and sporulating cells. As in other organisms FtsA is probably recruited immediately after FtsZ. In sporulating cells of B. subtilis FtsZ is recruited to potential division sites at both poles of the cell, but asymmetric division occurs at only one pole. We have now found that FtsA is recruited to only one cell pole, suggesting that it may play an important role in the generation of asymmetry in this system. FtsA is present in much higher quantities in B. subtilis than in Escherichia coli, with approximately one molecule of FtsA for five of FtsZ. This means that there is sufficient FtsA to form a complete circumferential ring at the division site. Therefore, FtsA may have a direct structural role in cell division. We have purified FtsA and shown that it behaves as a dimer and that it has both ATP-binding and ATP-hydrolysis activities. This suggests that ATP hydrolysis by FtsA is required, together with GTP hydrolysis by FtsZ, for cell division in B. subtilis (and possibly in most eubacteria).

Adenosine Triphosphatases↗
PubMed2001

Genetic analysis of the chromosome segregation protein Spo0J of Bacillus subtilis: evidence for separate domains involved in DNA binding and interactions with Soj protein.

Spo0J and Soj belong to the ParB/ParA family of proteins involved in chromosome and plasmid segregation in bacteria. In Bacillus subtilis, Spo0J protein binds to several specific sites, parS, located on both sides of the origin of DNA replication, oriC, and apparently self-associates to form large discrete foci visible by fluorescence microscopy. Soj protein forms large 'patches' probably associated with the nucleoid, which can undergo dynamic, co-operative jumping from nucleoid to nucleoid in the presence of Spo0J. Patches of Soj protein somehow help to bring about the condensation of Spo0J foci. Soj is also a negative regulator of transcription. In the absence of Spo0J, Soj is statically distributed on each of the nucleoids in the cell and blocks the transcription of several sporulation genes. To analyse the functional interaction between Spo0J and Soj further, we have constructed and studied a collection of spo0J mutants. Most of the mutants completely prevent Spo0J from interacting with DNA. One mutation impairs the formation of compact Spo0J foci and simultaneously results in loss of Soj movement. We also isolated one spo0J mutant, in which the frequency of Soj internucleoid oscillation is highly increased. Both mutations affecting the interaction with Soj lie in the N-terminal coding part of spo0J, whereas the substitutions affecting DNA binding lie in the mid- to C-terminal coding region.

Alleles↗
PubMed2001

Isolation and characterization of topological specificity mutants of minD in Bacillus subtilis.

In rod-shaped bacteria such as Bacillus subtilis, division site selection is mediated by MinC and MinD, which together function as a division inhibitor. Topological specificity is imposed by DivIVA, which ensures that MinCD specifically inhibits division close to the cell poles, while allowing division at mid-cell. MinD plays a central role in this process, as it positions and activates MinC and is dependent on DivIVA for its own positioning at the poles. To investigate MinD activities further, we have constructed and analysed a collection of minD mutants. Mutations in the conserved ATPase motifs lead to an inactive protein, possibly unable to oligomerize, but which nevertheless retains some affinity for the cell membrane. Several mutations affecting the mid- to C-terminal parts of MinD led to a protein probably unable to interact with DivIVA, but that could still stimulate division inhibition by MinC. These findings suggest that the ATPase activity of MinD is necessary for all its functions (possibly in part by controlling the oligomerization state of the protein). The other mutations may identify a surface of MinD involved in its interactions with DivIVA and a possible mechanism for control of MinD by DivIVA.

Adenosine Triphosphatases↗
PubMed2001

The impact of p53 status on cellular sensitivity to antifolate drugs.

The impact of p53 status on cellular sensitivity to antifolate drugs has been examined in seven human cell lines (A549, MCF7, T-47D, CCRF-CEM, COR-L23, A2780, and HCT-116) and p53 nonfunctional counterparts of two of the cell lines (HCT-116/N7 and A2780/CP70). p53 status was determined by sequencing and functional assays. The sensitivities of the cell lines to growth inhibition (sulphorhodamine B assay) produced by four antifolate drugs (Alimta, methotrexate, raltitrexed, and lometrexol) were studied. There was no clear relationship between functional p53 status and sensitivity to methotrexate or lometrexol, whereas a functional p53 status was possibly associated with resistance to Alimta- and raltitrexed-induced growth inhibition. In contrast, in the two pairs of related human tumor cell lines (HCT-116 and HCT-116/N7 and A2780 and A2780/CP70) cells with functional p53 were more sensitive to Alimta- and raltitrexed-induced growth inhibition (P = 0.002). Detailed studies were performed with the A2780 cell lines, and in the parental cells sensitivity to Alimta- and raltitrexed-induced cytotoxicity (clonogenic assay) was similar to the sensitivity determined in the sulphorhodamine B assay. However, in A2780/CP70 cells, 1 microM of drug resulted in only 40-60% growth inhibition yet > or = 85% cytotoxicity. After Alimta and raltitrexed exposure for < or = 72 h, there were no differences between the A2780 and A278/CP70 cell lines in cell cycle phase distribution, absolute cell number, or the induction of apoptosis. However, the cellular protein content of the A2780/CP70 cells was 3-6-fold higher than in A2780 cells after Alimta and raltitrexed treatment, suggesting that cells without functional p53 can maintain protein synthesis in the absence of cell division (unbalanced cell growth). In conclusion, the apparent impact of functional p53 status on sensitivity to antifolate drugs may depend upon the phenotypic/genotypic background as well as the assay used to measure cellular sensitivity.

Apoptosis↗
PubMed2001

Influence of cellular factors and pharmacokinetics on the formation of platinum-DNA adducts in leukocytes of children receiving cisplatin therapy.

The formation of platinum (Pt)-DNA adducts is thought to be crucial to the antitumor activity of cisplatin, and relationships between adduct formation in peripheral blood leukocytes (PBLs) and response to cisplatin therapy have been reported. The current study directly tests, for the first time, whether pharmacokinetic or other factors predominantly determine the drug-target interaction of cisplatin in a pediatric patient population. Cisplatin pharmacokinetics and Pt-DNA adduct formation in PBLs were determined in 10 children in parallel with measurement of adduct levels after incubation of pretreatment blood samples with cisplatin in vitro. Total and unbound plasma Pt concentrations were determined by atomic absorption spectrophotometry and adduct measurements performed by competitive ELISA. Pt-DNA adduct levels determined after cisplatin treatment showed considerable interindividual variation (peak levels at 24 h ranged from 0.15 to 1.31 nmol/g DNA) and correlated strongly with adduct levels determined after incubation of pretreatment whole blood with cisplatin (r = 0.92; P = 0.0002). No significant correlation was observed between in vivo adduct formation and either unbound or total cisplatin plasma concentrations (r = 0.14 and 0.18, respectively). A correlation was also observed between the degree of myelosuppression, as determined by WBC nadirs measured over a 14-day period after cisplatin treatment, and the extent of adduct formation, with greater WBC toxicity observed in patients with higher levels of Pt-DNA adducts (P = 0.010). These preliminary results provide evidence that interpatient variation in formation of Pt-DNA adducts in PBLs of children is determined by host-specific factors other than cisplatin pharmacokinetics. These results imply that analysis of adducts in PBLs after incubation of pretreatment blood samples with cisplatin may be used to predict in vivo adduct levels, leukopenia, and, potentially, response to cisplatin therapy.

Adolescent↗
PubMed2000

Role of Bacillus subtilis SpoIIIE in DNA transport across the mother cell-prespore division septum.

The SpoIIIE protein of Bacillus subtilis is required for chromosome segregation during spore formation. The COOH-terminal cytoplasmic part of SpoIIIE was shown to be a DNA-dependent adenosine triphosphatase (ATPase) capable of tracking along DNA in the presence of ATP, and the NH(2)-terminal part of the protein was found to mediate its localization to the division septum. Thus, during sporulation, SpoIIIE appears to act as a DNA pump that actively moves one of the replicated pair of chromosomes into the prespore. The presence of SpoIIIE homologs in a broad range of bacteria suggests that this mechanism for active transport of DNA may be widespread.

Adenosine Triphosphatases↗