Search PubMed⌕ Search

Biomedical subjects

T J Wilson

Publications and source records attributed to T J Wilson.

At least 19 recordsLinked to original sources
PubMed2005

A new Fc receptor homolog, FREB2, found in germinal center B cells.

Fc receptor homologs are a recently identified family of proteins homologous to FcgammaRI, found on human and mouse B cells. One of these, FREB/FcRX/FCRL, was found to be unique since it lacks a transmembrane domain and is expressed intracellularly within germinal center B cells. We have identified in humans and mice a new Fc receptor homolog, FREB2, that blends conserved elements of the classical Fc gamma receptors with structural motifs previously thought to be unique to FREB1. This protein is comprised of three immunoglobulin-like domains with high homology to those in FcgammaRI, and a C-terminus containing a proline-rich stalk region followed by a leucine-rich amphipathic alpha helix. Like FREB1, FREB2 is expressed as an intracellular protein. In murine splenocytes, RNA transcripts for each of the two proteins can be amplified from germinal center B cells. However, immunohistochemical analysis of human tonsils indicates that expression of FREB1 and FREB2 is mutually exclusive in non-neoplastic cells. Importantly, FREB2 expression within human tonsils appears to be limited to a small subset of nonproliferating germinal center B cells, suggesting that it may play a role in regulating clonal expansion or differentiation of B cells during the germinal center reaction.

Amino Acid Sequence↗
PubMed2005

Folding and catalysis of the hairpin ribozyme.

The active form of the hairpin ribozyme is brought about by the interaction of two formally unpaired loops. In a natural molecule, these are present on two adjacent arms of a four-way junction. Although activity can be obtained in molecules lacking this junction, the junction is important in the promotion of the folded state of the ribozyme under physiological conditions, at a rate that is faster than the chemical reaction. Single-molecule fluorescence resonance energy transfer studies show that the junction introduces a discrete intermediate into the folding process, which repeatedly juxtaposes the two loops and thus promotes their docking. Using single-molecule enzymology, the cleavage and ligation rates have been measured directly. The pH dependence of the rates is consistent with a role for nucleobases acting in general acid-base catalysis.

Catalysis↗
PubMed2004

Single-molecule studies of DNA and RNA four-way junctions.

Branched helical junctions are common in nucleic acids. In DNA, the four-way junction (Holliday junction) is an essential intermediate in homologous recombination and is a highly dynamic structure, capable of stacking conformer transitions and branch migration. Our single-molecule fluorescence studies provide unique insight into the energy landscape of Holliday junctions by visualizing these processes directly. In the hairpin ribozyme, an RNA four-way junction is an important structural element that enhances active-site formation by several orders of magnitude. Our single-molecule studies suggest a plausible mechanism for how the junction achieves this remarkable feat; the structural dynamics of the four-way junction bring about frequent contacts between the loops that are needed to form the active site. The most definitive evidence for this is the observation of three-state folding in single-hairpin ribozymes, the intermediate state of which is populated due to the intrinsic properties of the junction.

DNA↗
PubMed2003

ETS2 overexpression in transgenic models and in Down syndrome predisposes to apoptosis via the p53 pathway.

ETS2 is a transcription factor encoded by a gene on human chromosome 21 and alterations in its expression have been implicated in the pathophysiological features of Down syndrome (DS). This study demonstrates that overexpression of ETS2 results in apoptosis. This is shown in a number of circumstances, including ETS2-overexpressing transgenic mice and cell lines and in cells from subjects with DS. Indeed we report for the first time that the ETS2 overexpression transgenic mouse develops a smaller thymus and lymphocyte abnormalities similar to that observed in DS. In all circumstances of ETS2 overexpression, the increased apoptosis correlated with increased p53 and alterations in downstream factors in the p53 pathway. In the human HeLa cancer cell line, transfection with functional p53 enables ETS2 overexpression to induce apoptosis. Furthermore, crossing the ETS2 transgenic mice with p53(-/-) mice genetically rescued the thymic apoptosis phenotype. Therefore, we conclude that overexpression of human chromosome 21-encoded ETS2 induces apoptosis that is dependent on p53. These results have important consequences for understanding DS and oncogenesis and may provide new insights into therapeutic interventions.

Animals↗
PubMed2003

Transformation induced by Ewing's sarcoma associated EWS/FLI-1 is suppressed by KRAB/FLI-1.

Ewing's sarcoma is a childhood bone tumour with poor prognosis, most commonly associated with a t(11;22)(q24;q12) reciprocal translocation that fuses the EWS and FLI-1 genes, resulting in the production of an aberrant chimeric transcription factor EWS/FLI-1. To elucidate the mechanisms by which EWS/FLI-1 mediates transformation in mouse models, we have generated a murine Ews/Fli-1 fusion protein. We demonstrate that this protein transforms fibroblast cells in vitro similar to human EWS/FLI-1 as demonstrated by serum and anchorage-independent growth, the formation of tumours in nude mice and elevation of the oncogenic marker c-myc. Furthermore, transformation of these cells was inhibited by a specific repressor, KRAB/FLI-1. The KRAB/FLI-1 repressor also suppressed the tumorigenic phenotype of a human Ewing's sarcoma cell line. These findings suggest that the transformed phenotype of Ewing's sarcoma cells can be reversed by using the sequence-specific FLI-1-DNA-binding domain to target a gene repressor domain. The inhibition of EWS/FLI-1 is the first demonstration of the KRAB domain suppressing the action of an ETS factor. This approach provides potential avenues for the elucidation of the biological mechanisms of EWS/FLI-1 oncogenesis and the development of novel therapeutic strategies.

3T3 Cells↗
PubMed2001

The A730 loop is an important component of the active site of the VS ribozyme.

The core of the VS ribozyme comprises five helices, that act either in cis or in trans on a stem-loop substrate to catalyse site-specific cleavage. The structure of the 2-3-6 helical junction indicates that a cleft is formed between helices II and VI that is likely to serve as a receptor for the substrate. Detailed analysis of sequence variants suggests that the base bulges of helices II and VI play an architectural role. By contrast, the identity of the nucleotides in the A730 loop is very important for ribozyme activity. The base of A756 is particularly vital, and substitution by any other nucleotide or ablation of the base leads to a major reduction in cleavage rate. However, variants of A756 bind substrate efficiently, and are not defective in global folding. These results suggest that the A730 loop is an important component of the active site of the ribozyme, and that A756 could play a key role in catalysis.

Adenine↗
PubMed2001

Lewis antigen expression by Helicobacter pylori.

Although Helicobacter pylori express Lewis antigens as a component in the lipopolysaccharide, their role in the infectious process is not well understood. Lewis antigen expression with growth phase was investigated, as well as the distribution of Lewis antigens among isolates from asymptomatic and symptomatic individuals. Lewis antigens are expressed by H. pylori in a growth phase-dependent manner, with the greatest expression occurring in the logarithmic phase of growth. As growth proceeds, an increasing amount of Lewis antigens are shed into the culture supernatant. Lewis antigen expression among H. pylori isolates from asymptomatic individuals is characterized by an absence of type I Lewis antigens, a decrease in Le(x) expression, and an increase in nontypeable H. pylori, as compared with that among H. pylori isolates from symptomatic patients. The data support a role for Lewis antigens in the pathogenesis associated with symptomatic H. pylori infection in colonized individuals.

Helicobacter Infections↗
PubMed2001

Importance of specific nucleotides in the folding of the natural form of the hairpin ribozyme.

The hairpin ribozyme in its natural context consists of two loops in RNA duplexes that are connected as arms of a four-way helical junction. Magnesium ions induce folding into the active conformation in which the two loops are in proximity. In this study, we have investigated nucleotides that are important to this folding process. We have analyzed the folding in terms of the cooperativity and apparent affinity for magnesium ions as a function of changes in base sequence and functional groups, using fluorescence resonance energy transfer. Our results suggest that the interaction between the loops is the sum of a number of component interactions. Some sequence variants such as A10U, G+1A, and C25U exhibit loss of cooperativity and reduced affinity of apparent magnesium ion binding. These variants are also very impaired in ribozyme cleavage activity. Nucleotides A10, G+1, and C25 thus appear to be essential in creating the conformational environment necessary for ion binding. The double variant G+1A/C25U exhibits a marked recovery of both folding and catalytic activity compared to either individual variant, consistent with the proposal of a triple-base interaction among A9, G+1, and C25 [Pinard, R., Lambert, D., Walter, N. G., Heckman, J. E., Major, F., and Burke, J. M. (1999) Biochemistry 38, 16035-16039]. However, substitution of A9 leads to relatively small changes in folding properties and cleavage activity, and the double variant G+1DAP/C25U (DAP is 2,6-diaminopurine), which could form an isosteric triple-base interaction, exhibits folding and cleavage activities that are both very impaired compared to those of the natural sequence. Our results indicate an important role for a Watson--Crick base pair between G+1 and C25; this may be buttressed by an interaction with A9, but the loss of this has less significant consequences for folding. 2'-Deoxyribose substitution leads to folding with reduced magnesium ion affinity in the following order: unmodified RNA > dA9 > dA10 > dC25 approximately dA10 plus dC25. The results are interpreted in terms of an interaction between the ribose ring of C25 and the ribose and base of A10, in agreement with the proposal of Ryder and Strobel [Ryder, S. P., and Strobel, S. A. (1999) J. Mol. Biol. 291, 295-311]. In general, there is a correlation between the ability to undergo ion-induced folding and the rate of ribozyme cleavage. An exception to this is provided by G8, for which substitution with uridine leads to severe impairment of cleavage but folding characteristics that are virtually unaltered from those of the natural species. This is consistent with a direct role for the nucleobase of G8 in the chemistry of cleavage.

2-Aminopurine↗
PubMed2001

Spontaneous mutations that confer antibiotic resistance in Helicobacter pylori.

In this study, we systematically examined in vitro frequencies and spectra of the spontaneous mutations in Helicobacter pylori that confer resistance to clarithromycin (Cla(r)), metronidazole (Mtz(r)), amoxicillin (Amx(r)), ciprofloxacin (Cip(r)), and rifampin (Rif(r)). The mutation rate of Rif(r) or Cip(r) determined in a fluctuation assay is 1 x 10(-8) to 2 x 10(-8) per cell per division. In contrast, the mutation rates of Cla(r), Mtz(r), and Amx(r) are much lower (<10(-9)). However, Mtz(r) mutants could be readily selected in vitro by using the serial passage method, suggesting that the mutagenic effect and selective effect of a sublethal dose of metronidazole contribute to the rapid development of Mtz(r). Analysis of spontaneous Rif(r), Cla(r), and Cip(r) mutants confirmed previous results indicating that mutations within the rpoB gene, the 23S rRNA gene, and the gyrA gene, respectively, are responsible; also, several new mutant alleles were identified. Mtz(r) mutants resulted most frequently, but not always, from mutations in the rdxA gene. DNA fragments containing each mutant allele could readily transform susceptible H. pylori strains to resistance, confirming that each mutant allele is responsible for the resistance phenotype.

Anti-Bacterial Agents↗
PubMed2000

Early growth response (Egr)-1 gene induction in the thymus in response to TCR ligation during early steps in positive selection is not required for CD8 lineage commitment.

The early growth response gene 1 (Egr-1) is induced during positive selection in the thymus and has been implicated in the differentiation of CD4+ thymocytes. Here, we show that signals that specifically direct CD8 lineage commitment also induce Egr-1 DNA-binding activity in the nucleus. However, we find that pharmacological inhibition of mitogen-activated protein kinase/extracellular signal-related kinase kinase activity potently inhibits Egr-1 DNA-binding function at concentrations that promote differentiation of CD8+ thymocytes, suggesting Egr-1 activity is not essential for CD8 commitment. To further determine the role of Egr-1 in thymocyte development, we compare steady-state Egr-1 DNA-binding activity in thymocytes from mice with defined defects in positive selection. The data indicate that the appearance of functional Egr-1 is downstream of signals induced by TCR/MHC engagement, whereas it is less sensitive to alterations in Lck-mediated signals, and does not correlate directly with proficient positive selection. Egr-1 is one of the earliest transcription factors induced upon TCR ligation on immature thymocytes, and plays a potential role in the transcription of genes involved in thymocyte selection.

Animals↗
PubMed2000

Fluorescence resonance energy transfer as a structural tool for nucleic acids.

Fluorescence resonance energy transfer is a spectroscopic method that provides distance information on macromolecules in solution in the range 20-80 A. It is particularly suited to the analysis of the global structure of nucleic acids because the long-range distance information provides constraints when modelling these important structures. The application of fluorescence resonance energy transfer to nucleic acid structure has seen a resurgence of interest in the past decade, which continues to increase. An especially exciting development is the recent extension to single-molecule studies.

Nucleic Acid Conformation↗
PubMed2000

The folding of the hairpin ribozyme: dependence on the loops and the junction.

In its natural context, the hairpin ribozyme is constructed around a four-way helical junction. This presents the two loops that interact to form the active site on adjacent arms, requiring rotation into an antiparallel structure to bring them into proximity. In the present study we have compared the folding of this form of the ribozyme and subspecies lacking either the loops or the helical junction using fluorescence resonance energy transfer. The complete ribozyme as a four-way junction folds into an antiparallel structure by the cooperative binding of magnesium ions, requiring 20-40 microM for half-maximal extent of folding ([Mg2+]1/2) and a Hill coefficient n = 2. The isolated junction (lacking the loops) also folds into a corresponding antiparallel structure, but does so noncooperatively (n = 1) at a higher magnesium ion concentration ([Mg2+]1/2 = 3 mM). Introduction of a G + 1A mutation into loop A of the ribozyme results in a species with very similar folding to the simple junction, and complete loss of ribozyme activity. Removal of the junction from the ribozyme, replacing it either with a strand break (serving as a hinge) or a GC5 bulge, results in greatly impaired folding, with [Mg2+]1/2 > 20 mM. The results indicate that the natural form of the ribozyme undergoes ion-induced folding by the cooperative formation of an antiparallel junction and loop-loop interaction to generate the active form of the ribozyme. The four-way junction thus provides a scaffold in the natural RNA that facilitates the folding of the ribozyme into the active form.

Base Sequence↗
PubMed2000

Lewis antigen expression and stability in Helicobacter pylori isolated from serial gastric biopsies.

The expression of Lewis antigens by the gastric pathogen Helicobacter pylori in serial biopsy isolates was investigated to assess antigen distribution and stability. A total of 26 asymptomatic subjects were given various doses of 3' sialyllactose for up to 56 days. Gastric biopsies were performed during the dosing period, as well as 30 days after dosing, which provided 127 H. pylori isolates that were examined by use of ELISA and immunoblot. A large proportion of subjects (14/26) yielded sequential H. pylori isolates, which appeared genetically identical but had variable Lewis antigen expression. The proportion of subjects with H. pylori isolates not expressing any Lewis antigens was greater than that previously reported (10/26). Thus, the expression of the Lewis antigens by H. pylori does not appear to be a requirement for colonization, whereas the antigen expression after human infection is more variable than the previously reported rate observed with in vitro cultures.

Adult↗
PubMed1999

Analysis of a ptsH homologue from Streptomyces coelicolor A3(2).

A ptsH homologue of Streptomyces coelicolor A3(2) was identified in the emerging genome sequence, cloned in Escherichia coli and the S. coelicolor HPr over-produced and purified. The protein was phosphorylated in vitro in a phosphoenolpyruvate (PEP)-dependent manner by purified enzyme I (EI) from Bacillus subtilis, and much less efficiently in an ATP-dependent manner by purified HPr kinase, also from B. subtilis. There was no indication of ATP-dependent phosphorylation of the purified protein by cell extracts of either S. coelicolor or Streptomyces lividans. Deletion of the ptsH homologue from the S. coelicolor and S. lividans chromosomes had no effect on growth when fructose was supplied as sole carbon source, and in S. coelicolor it had no effect on glucose repression of agarase and galactokinase synthesis, suggesting that the HPr encoded by this gene does not play an essential role in fructose transport nor a general role in carbon catabolite repression.

Adenosine Triphosphate↗
PubMed1999

Elevated immunoglobulin E (IgE) levels in children with exposure to environmental lead.

Lead has been reported to be an immunosuppressive agent in animal systems at levels far below those recognized as overtly toxic. Little data exist on lead's effects on the human immune system, especially in young children who are at greatest risk for exposure to this environmental hazard. The effects of environmental lead exposure on the human immune system were examined in a population of young children, age 9 months-6 years, from the urban population of Springfield-Greene County, Missouri. Reported here are data from 279 children with blood lead levels ranging from 1 to 45 microg/dl. White blood cell populations have been enumerated and examined for cell surface expression of activation markers. Serum has been analyzed for IgE, specific titers to Rubella vaccine, sCD25 (the soluble form of the IL2 receptor), sCD27 (the soluble form of the lymphocyte specific member of the tumor necrosis factor receptor family), and IL4 (the cytokine interleukin 4). Variation of these assays with age of the child was considered in statistical analysis of data. A statistically significant relationship of IgE and blood lead level was found in this population; as blood lead (PbB) level increases, IgE level increases. No other statistically significant differences between risk categories or other associations with blood lead level were found. The exact mechanism for this apparent stimulus of IgE-producing B cells remains to be elucidated. The development of allergic symptoms is often preceded by an increase in IgE. These data indicate that ingested lead could play a role in this process by stimulating IgE production.

Age Factors↗