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James Thomas

Publications and source records attributed to James Thomas.

27 records · Page 2Linked to original sources
PubMed2003

Formation of disulfide bond in p53 correlates with inhibition of DNA binding and tetramerization.

The p53 tumor suppressor protein is susceptible to oxidation, which prevents it from binding to its DNA response element. The goal of the current research was to determine the nature of the cysteine residue thiol oxidation that prevents p53 from binding its DNA target and its effect on p53 structure. Recombinant p53, purified in the presence of the reducing agent dithiothreitol (DTT), contains five free thiol groups on the surface of the protein. In the absence of DTT, p53 contains only four thiol groups, indicating that an average of one surface thiol group is readily susceptible to oxidation. Sulfite-mediated disulfide bond cleavage followed by reaction with 2-nitro-5-thiosulfobenzoate showed that oxidized p53 contains a single disulfide bond per monomer. By atomic force microscopy, we determined that reduced p53 binds to a double-stranded DNA containing the p53 promoter element of the MDM2 gene. The DNA-bound reduced p53 has an average cross-sectional diameter of 8.61 nm and a height of 4.12 nm. The amount of oxidized p53 that bound to the promoter element was ninefold lower, and it has an 18% larger average cross-sectional diameter. Electromobility shift assays showed that binding of oxidized p53 to DNA was enhanced upon addition of DTT, indicating that oxidation is reversible. The possibility that oxidized p53 contained significant amounts of sulfenic (-SOH), sulfinic (-SO2H), or sulfonic acid (-SO3H) was ruled out. Gel filtration chromatography indicated that oxidation increases the percentage of p53 monomers and high-molecular-weight oligomers (>1,000 kDa) relative to tetrameric p53. Protein modeling studies suggest that a mixed disulfide glutathione adduct on Cys182 could account for the observed stoichiometry of oxidized thiols and structural changes. The glutathione adduct may prevent proper helix-helix interaction within the DNA binding domain and contribute to tetramer dissociation.

Binding Sites↗
PubMed2003

The second murine autoantibody workshop: remarkable interlaboratory concordance for radiobinding assays to identify insulin autoantibodies in nonobese diabetic mice.

In October 2000, the First Murine Autoantibody Workshop was held as part of an International Workshop on Lessons from Animal Models for Human Type 1 Diabetes. This first workshop identified insulin, but not glutamic acid decarboxylase (GAD) or IA-2, as specific autoantigens of humoral immunity in nonobese diabetic (NOD) mice. The goals of the Second Murine Autoantibody Workshop, part of the Sixth Annual Meeting of the IDS, were to increase the number of participating investigators, attempt standardization of insulin autoantibody (IAA) results across laboratories, identify serologic evidence of humoral immunity to other beta cell antigens, and allow for validation of ELISA assays for autoantibody detection in NOD mice. Sixty-three coded samples (26 pooled NOD sera, 23 pooled C57BL/6 sera, and 14 diluted samples of an anti-insulin monoclonal antibody) were distributed to 12 participating laboratories. This second workshop demonstrated that, for nearly all laboratories, IAA measured by radioimmunoassay (RIA) provided a sensitive and specific assay capable of distinguishing diabetes-prone from nondiabetes-prone mice. Analyses involving the serially diluted anti-insulin monoclonal antibody offered hope that a standard reference unit for reactivity could be established. Surprisingly, two ELISA assays for IAA detection proved remarkably sensitive (i.e., 65% and 92%). However, subsequent absorption studies performed after the workshop (presented at the IDS meeting) brought into question whether ELISA assays for IAA do, in reality, detect anti-insulin immunities and whether assays for GAD and IA-2 autoantibodies distinguish diabetes-prone from nondiabetes-prone mice. In sum, this workshop continued to support the notion that IAA, as determined by RIA, could provide a sensitive and specific marker of anti-beta cell immunity in NOD mice.

Animals↗
PubMed2002

Genetic and physical maps of jerker (Espn(je)) on mouse chromosome 4.

The jerker mutation causes degeneration of cochlea and vestibular sensory hair cells in mice. A frame-shift mutation in the actin bundling gene Espin (Espn) leads to hair bundle defects by disrupting the actin filament assembly in stereocilia. Previously, jerker was mapped to distal mouse chromosome 4. Here, analyzing 2536 informative meioses derived from two intersubspecific intercrosses, we localize jerker to a 0.51+/-0.14cM interval on chromosome 4. The following order and distances of genes and markers were determined: D4Mit180-0.44+/-0.13cM-Hes2, Espn(je)-0.08+/-0.06cM-D4Mit356-0.28+/-0.1cM-D4Mit208. A 300kb physical bacterial artificial chromosome (BAC) contig was generated containing the Espn(je) locus. The human homologous region maps to 1p36.31. We present a detailed high-resolution genetic and physical map of markers located at distal chromosome 4 and demonstrate concordance of Espn with jerker.

Animals↗
PubMed2002

Meiotic arrest and aneuploidy in MLH3-deficient mice.

MutL homolog 3 (Mlh3) is a member of a family of proteins conserved during evolution and having dual roles in DNA mismatch repair and meiosis. The pathway in eukaryotes consists of the DNA-binding components, which are the homologs of the bacterial MutS protein (MSH 2 6), and the MutL homologs, which bind to the MutS homologs and are essential for the repair process. Three of the six homologs of MutS that function in these processes, Msh2, Msh3 and Msh6, are involved in the mismatch repair of mutations, frameshifts and replication errors, and two others, Msh4 and Msh5, have specific roles in meiosis. Of the four MutL homologs, Mlh1, Mlh3, Pms1 and Pms2, three are involved in mismatch repair and at least two, Pms2 and Mlh1, are essential for meiotic progression in both yeast and mice. To assess the role of Mlh3 in mammalian meiosis, we have generated and characterized Mlh3(-/-) mice. Here we show that Mlh3(-/-) mice are viable but sterile. Mlh3 is required for Mlh1 binding to meiotic chromosomes and localizes to meiotic chromosomes from the mid pachynema stage of prophase I. Mlh3(-/-) spermatocytes reach metaphase before succumbing to apoptosis, but oocytes fail to complete meiosis I after fertilization. Our results show that Mlh3 has an essential and distinct role in mammalian meiosis.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Phase I clinical and pharmacokinetic study of NSC 655649, a rebeccamycin analogue, given in both single-dose and multiple-dose formats.

PURPOSE: NSC 655649 was given in both single- and multiple-dose formats, to characterize maximum tolerated dose (MTD), toxicity, and pharmacokinetic profile. EXPERIMENTAL DESIGN: Patients with advanced malignancies were treated with escalating doses of NSC 655649 in either a single-dose format (step 1) or a multiple-dose format (step 2). In step 1, NSC 655649 was given as a 30-60 min infusion. In step 2, the NSC 655649 dose was divided into three consecutive daily doses. Plasma and urine were sampled to assess the pharmacokinetic and excretory characteristics of NSC 655649. A total of 12 patients were enrolled at the MTD for the purpose of gender equity. RESULTS: Forty-three patients were treated with NSC 655649 for a total of 108 cycles in step 1, and 26 patients were treated for a total of 41 cycles in step 2. The MTD for both steps 1 and 2 was determined to be 572 mg/m(2). Myelosuppression was the dose-limiting toxicity. Local venous irritation was generally grade 1-2 in severity but could only be adequately prevented by administration of study drug through central i.v. access. One patient with adenocarcinoma of unknown primary experienced a partial response on step 1. Four patients experienced stable disease of >100 days duration. CONCLUSIONS: NSC 655649 may be safely given at an MTD of 572 mg/m(2) in both single-dose and multiple-dose formats. Optimally, this drug should be administered through central i.v. access.

Adult↗
PubMedDate not supplied

Chondroblastoma with associated aneurysmal bone cyst of the cuboid.

The authors describe a young adult patient with a chondroblastoma and associated aneurysmal bone cyst of the cuboid. Although chondroblastoma has been reported to occur in tarsal bones, the cuboid is a very rare location. The association of chondroblastoma with an aneurysmal bone cyst in long bones has been well documented. However, this association in the cuboid has not been reported in the English literature. A 20-year-old man with a 4-month history of foot pain localized to the lateral border of the foot and ankle presented with an expansile lesion in the cuboid bone with a nondisplaced pathologic fracture. A computed tomography scan showed an expansile lesion with discontinuity of the cortex, and magnetic resonance imaging showed marrow replacement within the cuboid with surrounding periosteal edema. An open biopsy was performed, followed by curettage and cementation of the lesion. The histology showed a chondroblastoma with an associated aneurysmal bone cyst of the cuboid.

Adult↗
PubMedDate not supplied

Systematic reviews and their application to research in speech and language therapy: a response to T. R. Pring's 'Ask a silly question: two decades of troublesome trials' (2004).

BACKGROUND: The advent of evidence-based healthcare has seen a rise in the use of systematic reviews to bring together the findings from research studies. The use of systematic reviews in speech and language therapy (SLT) was criticized in this journal in 2004 by T. R. Pring. It was claimed that their findings are misleading due to the potential inclusion of biased data, and uninformative due to a lack of detail in the reporting of interventions. It is argued that outcome research should be carried out in a series of phases in which small-scale research precedes large-scale research. This, it is argued, is most likely to demonstrate statistically significant effects and also help to ensure that therapies become sufficiently defined so that clinicians can apply them in practice. AIMS: This paper argues that the above criticism of systematic reviews is based on a narrow conception of their capabilities: on the popular misapprehension that all systematic reviews answer effectiveness questions using only experimental studies and contain meta-analyses. Different methods for systematic reviews are described and their application within clinical outcome research is discussed with reference to a phased structure for empirical enquiry. MAIN CONTRIBUTION: Systematic reviews seek to identify and synthesize information within a given topic area. They are used to answer a wide range of research questions and the studies they include are not limited exclusively to experimental designs. Methods of synthesis can include both statistical approaches, such as meta-analysis, and 'qualitative' approaches, such as meta-ethnography and thematic analysis. Knowledge of the current state of research is essential for a sequentially phased approach within outcome research to operate. Since systematic reviews are summaries of research activity, they can provide this knowledge and should therefore be considered a valuable tool within outcome research. CONCLUSIONS: A systematic review using 'qualitative' and/or statistical methods for combining studies can be carried out within or across any of the phases within outcome research. Far from being uninformative, this can help bring together what is, and what is not, known and indicate the kinds of therapies that may be beneficial in the clinical setting and therapies which would benefit from further research and development.

Evidence-Based Medicine↗