Biomedical subjects

D B Wilson

Publications and source records attributed to D B Wilson.

At least 73 records · Page 4Linked to original sources

PubMed1998

Chorismate mutase-prephenate dehydratase from Escherichia coli. Study of catalytic and regulatory domains using genetically engineered proteins.

The bifunctional P-protein, which plays a central role in Escherichia coli phenylalanine biosynthesis, contains two catalytic domains (chorismate mutase and prephenate dehydratase activities) as well as one R-domain (for feedback inhibition by phenylalanine). Six genes coding for P-protein domains or subdomains were constructed and successfully expressed. Proteins containing residues 1-285 and residues 1-300 retained full mutase and dehydratase activity, but exhibited no feedback inhibition. Proteins containing residues 101-386 and residues 101-300 retained full dehydratase activity, but lacked mutase activity. Fluorescence emission spectra and binding assays indicated that residues 286-386 were crucial for phenylalanine binding. The mutase (residues 1-109), dehydratase (residues 101-285), and regulatory (residues 286-386) activities were thus shown to reside in discrete domains of the P-protein. Both the mutase domain and the native P-protein formed dimers. Deletion of the mutase domain diminished phenylalanine binding to the regulatory site as well as prephenate binding to the dehydratase domain, both through cooperative effects. Besides eliminating feedback inhibition, removal of the R-domain decreased the affinity of chorismate mutase for chorismate.

Allosteric Regulation↗

PubMed1998

Analysis of TCR beta chains in Lewis rats with experimental allergic encephalomyelitis. II. Vbeta8.2+ T cells with limited CDR3 N region additions derive from the adult thymus.

Immunization of Lewis (LEW) rats with guinea pig myelin basic protein (MBP) induces a population of encephalitogenic CD4 T cells having specificity for the dominant immunogenic peptide of MBP, 68-86. The TCR beta chains of these disease-causing T cells show three distinct features: they are almost exclusively Vbeta8.2, they use AspSer as the first two amino acid residues of the third complementarity-determining region (CDR3) and these junctional region sequences show few if any non-germline N-region nucleotide additions. This last feature raises the possibility that these autoimmune T cell precursors derive from TCR gene rearrangements occurring during early, perinatal ontogeny, a period when the enzyme terminal deoxynucleotidyl transferase (TdT), responsible for N region additions, is not expressed. An alternative possibility is that these features of the TCR of MBP 68-86-reactive T cells are dictated by considerations of antigen selection throughout ontogeny both in the thymus and in the periphery--ie., that such beta chains are conformationally the most appropriate for triggering by an epitope of 68-86 complexed to class II RT1.BI MHC molecules. We show here that active experimental allergic encephalomyelitis, while delayed in onset, occurs in heavily irradiated animals, but not in the absence of a thymus, a finding indicating that this autoimmune disease is caused by a T cell subpopulation derived from the post-irradiation adult thymus. These disease-causing T cells are heavily Vbeta8.2+, CDR3 AspSer+ and use few N region additions. We conclude that T cells with these TCR beta chain features can be generated in the adult thymus and most likely reflect requirements imposed by antigen selection.

Animals↗

PubMed1998

Acquired thymic tolerance and experimental allergic encephalomyelitis in the rat. I. Parameters and analysis of possible mechanisms.

Intrathymic injection of guinea pig myelin basic protein (MBP) or the immunodominant, encephalitogenic fragment of MPB, 68-86, without otherwise compromising the peripheral lymphocyte pool in adult LEW rats, dramatically inhibits onset of experimental allergic encephalomyelitis (EAE) caused by the usual peripheral inoculation with MBP in complete Freund's adjuvant. This surprising finding demonstrates that interaction of antigen and one or more components of an intact thymus can down-regulate systemic responses by mature T cells already existing in the peripheral lymphocyte pool. How this happens is not known. In studies designed to explore possible mechanisms: (a) adult thymectomized animals remain susceptible to active EAE, thus EAE cannot be attributed solely to recent thymic emigrants that might be inactivated by antigen deposited in the thymus; (b) heterotopic isografts of injected thymic lobes transfer thymic tolerance to secondary recipients, thus the tolerance effect is dominant over an intact, non-treated thymus; (c) T cells from made thymic tolerant but not immunized donors are less effective in causing EAE following adoptive transfer into, and active immunization of, secondary, irradiated recipients; and (d) animals resistant to active EAE as a consequence of thymic tolerance are fully vulnerable to adoptive EAE caused by already activated MBP-specific T cell subpopulations. These results rule out a possible mechanism previously proposed for acquired thymic tolerance, i. e., that potentially pathogenic T cells traffic to the antigen-injected thymus where they are inactivated or eliminated.

Adoptive Transfer↗

PubMed1998

The role of regulatory T cells in Lewis rats resistant to EAE.

Adult Lewis (LEW) rats are highly susceptible to experimental autoimmune encephalomyelitis (EAE), induced actively by immunization with guinea pig (GP) myelin basic protein (MBP) in complete Freund's adjuvant or adoptively transferred with activated T lymphocytes reactive to GP MBP peptide 68-88. Once LEW rats recover from active EAE or when given MBP in incomplete Freund's adjuvant (IFA), they become resistant to further attempts to induce active or passive EAE. In this study, we examined whether such EAE-resistant rats after MBP-IFA immunization have reduced frequencies of MBP-reactive T cells, whether these T cells are anergized, and whether the activity of regulatory T cells is increased to the event that they prevent activation of MBP-specific T cell subpopulations. By limiting dilution analyses (LDA) of unfractionated splenic T cells, the levels of MBP-reactive T cells in EAE-resistant rats appeared to be approximately 5% of the levels in EAE-susceptible rats. However, a subsequent analysis of CD4+ enriched T cell populations, depleted of the CD8 subset, showed similar frequencies of MBP-reactive cells in susceptible and resistant LEW rats. Not only were the frequencies on LDA altered by suppressor cells, but also LDA comparisons based on cell proliferation and cytokine production as indicators of MBP reactive cell frequencies gave markedly different results. We conclude that MBP-reactive T cells in this model of EAE-resistant LEW rats are hyporeactive to MBP as the result of an increased activity of a regulatory subset of CD8+ T cells. These results also demonstrate that the quantitation of MBP-reactive CD4+ T cells by LDA is strongly influenced by the presence of functionally antagonistic CD8+ T cells, which cause an underestimation of responder T cell frequencies, and by the method of detecting T cell reactivity.

Animals↗

PubMed1998

Regulation of biosynthesis of individual cellulases in Thermomonospora fusca.

Regulation of the biosynthesis of the six cellulases comprising the cellulolytic system of the thermophilic soil bacterium Thermomonospora fusca ER1 was studied. The levels of the individual enzymes produced on different noninducing and inducing carbon sources were determined. The lowest level of cellulase synthesis (3 nM) was observed with xylose as a carbon source, and the highest level (247 to 1,670 nM for different enzymes) was found in cultures grown on microcrystalline cellulose. Endocellulases and exocellulases showed distinctly different regulation patterns. Differences in the regulation of individual enzymes appear to be determined by the specific structural organization of the upstream regulatory sequences of their genes.

Actinomycetaceae↗

PubMed1998

Roles of the catalytic domain and two cellulose binding domains of Thermomonospora fusca E4 in cellulose hydrolysis.

Thermomonospora fusca E4 is an unusual 90.4-kDa endocellulase comprised of a catalytic domain (CD), an internal family IIIc cellulose binding domain (CBD), a fibronectinlike domain, and a family II CBD. Constructs containing the CD alone (E4-51), the CD plus the family IIIc CBD (E4-68), and the CD plus the fibronectinlike domain plus the family II CBD (E4-74) were made by using recombinant DNA techniques. The activities of each purified protein on bacterial microcrystalline cellulose (BMCC), filter paper, swollen cellulose, and carboxymethyl cellulose were measured. Only the whole enzyme, E4-90, could reach the target digestion of 4.5% on filter paper. Removal of the internal family IIIc CBD (E4-51 and E4-74) decreased activity markedly on every substrate. E4-74 did bind to BMCC but had almost no hydrolytic activity, while E4-68 retained 32% of the activity on BMCC even though it did not bind. A low-activity mutant of one of the catalytic bases, E4-68 (Asp55Cys), did bind to BMCC, although E4-51 (Asp55Cys) did not. The ratios of soluble to insoluble reducing sugar produced after filter paper hydrolysis by E4-90, E4-68, E4-74, and E4-51 were 6.9, 3.5, 1.3, and 0.6, respectively, indicating that the family IIIc CBD is important for E4 processivity.

Amino Acid Sequence↗

PubMed1998

Thyroid reoperations: indications and risks.

Thyroid reoperations can be technically difficult and associated with an increased risk of complications. To determine the indications for reoperations and the risk of postoperative complications with these procedures, records of 362 patients undergoing thyroidectomy by a single surgeon (R.A.P.) were reviewed. Thirty-two patients had a reoperation. The group consists of 21 women and 11 men with an average age of 55 years (range, 31-79). Twenty-four patients had 1 prior operation, and 8 patients had 2 or more. We performed 4 of the initial operations, and 28 were done by surgeons at other centers. Fourteen reoperations were done for symptomatic multinodular goiter (MNG), and 5 because of a change in the histologic diagnosis from benign to cancerous. The remainder were for further treatment of malignancy. The most common operation was completion thyroidectomy (31). In 3 patients, either unilateral or bilateral modified radical neck exploration was performed. One patient required median sternotomy. One subtotal thyroidectomy was also performed. Recurrent laryngeal nerve injury occurred in 2 patients. It resolved in 1 patient but was permanent in another, who had 3 operations for MNG. One of the 2 patients with preoperative unilateral vocal cord paralysis had return of function after removal of a substernal goiter. The other had a permanent nerve injury from the original surgery. Three patients had postoperative hypocalcemia (calcium <8.0 mg/dL). This resolved in all patients within 1 to 6 months. One patient who had a third operation for MNG had postoperative hemorrhage necessitating tracheostomy for airway control. Another patient developed a seroma that resolved within 2 months. We conclude that reoperations are indicated for both benign and malignant thyroid disease. Because they carry a higher risk of complications, every effort should be made to avoid them by performing definitive initial treatment.

Female↗

PubMed1997

Surface residue mutations which change the substrate specificity of Thermomonospora fusca endoglucanase E2.

The three dimensional structure of a T. fusca endoglucanase catalytic domain (E2cd) has been determined by X-ray crystallography at 1.0 A resolution (Wilson et al., 1995). The availability of a high resolution structure for E2cd allows us to initiate structure-based efforts to engineer cellulases with a high activity on native cellulose. The low activity on crystalline cellulose suggests that the entry of a cellulose molecule into the active site rather than catalysis may be the rate limiting step for hydrolysis of crystalline cellulose. Movement of a loop upon substrate binding has been proposed to play a crucial role in catalysis. A total of 15 surface mutants and 5 loop mutants were created by site-directed mutagenesis and their effects on activity and substrate specificity were determined. Circular dichroism spectra were used to monitor structural changes, and no major changes were found. The binding constants for two methyl umbelliferyl oligosaccharides and cellotriose were measured for some of the mutants and all of them showed binding similar to wild type E2. These results provide the first direct link between loop movement and catalysis by E2 and show that surface residues can affect its substrate specificity.

Cellulase↗

PubMed1997

Wild-type endoderm abrogates the ventral developmental defects associated with GATA-4 deficiency in the mouse.

GATA-4 knockout mice die by 9.5 days postcoitum and exhibit profound defects in ventral morphogenesis, including abnormal foregut formation and a failure of fusion of the bilateral myocardial primordia. During early mouse development, GATA-4 is expressed in cardiogenic splanchnic mesoderm and associated endoderm, suggesting that the presence of this transcription factor in one or both of these tissue types is essential for ventral development. To distinguish whether GATA-4 expression in mesoderm or endoderm accounts for the phenotype of the knockout mouse, we prepared chimeric mice by injecting Gata4-/- ES cells into 8-cell stage ROSA26(Gata4+/+) embryos. We identified a series of high percentage null chimeras (8-10 days postcoitum) in which Gata4+/+ cells were restricted to visceral yolk sac endoderm and small portions of the foregut/hindgut endoderm. Despite an absence of GATA-4 in all other cells of these embryos, there was normal development of the heart, foregut, and surrounding tissues. We conclude that expression of GATA-4 in endoderm rather than cardiogenic mesoderm is required for ventral morphogenesis.

Animals↗

PubMed1997

Is there a causal relationship between alcohol use and violence? A synthesis of evidence.

This chapter reviews the evidence bearing on the question of whether those individuals who consume alcohol have an increased probability of subsequent violent behavior. Four bodies of relevant research are examined: experimental studies with animals, experimental studies with humans, individual-level correlational studies, and macro-level correlational studies. All these research approaches provide some evidence of an association between alcohol consumption and violent behavior, but no firm conclusion can be drawn about whether alcohol plays a causal role in such behavior. Various limitations, deficiencies, and ambiguities of available research that contribute to this state of affairs are discussed.

Alcohol Drinking↗

PubMed1997

A Prevotella ruminicola B(1)4 operon encoding extracellular polysaccharide hydrolases.

When Escherichia coli XL1-Blue MRA (P2) was infected with lambda DNA containing Prevotella ruminicola B(1)4 chromosomal DNA, only a few plaques produced beta-1,4-endoglucanase activity, and all of these had mannanase activity. Positive phage contained a 17-kb SacI DNA fragment that gave six bands after EcoRI digestion. The EcoRI fragments were ligated into pBluescript and sequenced. The order of the fragments was verified by PCR and by restriction mapping. The DNA sequence contained 6 open reading frames (ORFs). The 4th and 5th ORFs encoded two related beta-1,4-endoglucanases. E. coli clones carrying ORF5 and ORF6 had beta-1,4-endoglucanase and mannanase activities, while a clone carrying only ORF6 hydrolyzed mannan but not carboxymethylcellulose. The 6th ORF had three regions of homology to mannanase A from Pseudomonas fluorescens. Based on these results, ORF6 encoded the mannanase gene. The 3rd ORF had 10 regions of homology with cellulose-binding protein A from Clostridium cellulovorans. The 1st and 2nd ORFs had no significant homology to genes or amino acid sequences in GeneBank or SwissProt. All of the ORFs except 1 encoded a potential signal peptide sequence. The upstream region of ORF1 contained four direct repeats and four inverted repeat elements, but no apparent sigma70 sequence-like promoter was present. The segment of DNA containing the 6 ORFs was preceded and followed by potential transcription termination signals suggesting a single transcriptional unit.

Amino Acid Sequence↗

PubMed1997

Results of a phase I clinical trial of a T-cell receptor peptide vaccine in patients with multiple sclerosis. I. Analysis of T-cell receptor utilization in CSF cell populations.

To identify a panel of multiple sclerosis patients (MS) for a phase I clinical trial of a T-cell receptor (TCR) peptide vaccine we characterized the T-cell populations present in the cerebrospinal fluids (CSF) of a large group of patients with respect to surface phenotype and state of activation, TCR beta chain utilization, features of the CDR3 junctional region, the extent of clonality and persistence of selected clonotypes over time. These CSF cell populations consist of approximately 60% CD4+ T-cells, half of which bear IL-2 receptors, indicating these activated T-cells may be part of the pathogenic process in MS. When these activated CD4+ T-cells were selectively expanded in IL-2/IL-4 supplemented cultures, an over-representation of several TCRV beta families was noted in 39/47 patients, the most frequent being V beta 6.5, V beta 6.7, V beta 2, V beta 5 and V beta 4. Biased expression of various members of the V beta 6 family was seen in 21 of this group of 39 patients. Clonal analysis of TCR beta 6 CDR3 sequences, revealed two notable features: clonal dominance and clonal persistence. CSF cells from two-thirds of MS patients contained a dominant clone comprising 50% or more of sequences and the same patient-specific clone could be shown to persist for up to 18 months. This clonal prevalence and over representation of V beta 6+TCR raises the possibility that immunization with a V beta 6 peptide vaccine may produce a regulatory immune response leading to a clinical benefit.

Adult↗

PubMed1997

Results of a phase I clinical trial of a T-cell receptor vaccine in patients with multiple sclerosis. II. Comparative analysis of TCR utilization in CSF T-cell populations before and after vaccination with a TCRV beta 6 CDR2 peptide.

We report here the results of a phase I trial of a T-cell receptor (TCR) V beta 6 CDR2 region peptide vaccine in 10 patients with multiple sclerosis who showed biased over-representations of V beta 6 mRNA among T-cells in their cerebrospinal fluids (CSF). One group of 5 patients was immunized twice during a four week period with 100 micrograms of the TCRV beta 6 peptide 39-LGQGPEF LTYFQNEAQLEKS-58 emulsified in incomplete Freund's adjuvant (IFA); the second group of 5 MS patients received 300 micrograms of the same peptide in IFA over a similar time period. Patients were monitored for adverse events, immunogenicity of the peptide and changes in their CSF T-cell populations. The results indicate that this peptide was immunogenic (T-cell proliferation assays and recall DTH responses) in some of the patients, although none of the immunized patients produced detectable anti-peptide antibodies. More importantly, we show that the 5 patients treated with higher doses of the vaccine displayed a slight decrease in CSF cellularity, a lack of growth of CSF cells in cytokine supplemented expansion cultures that implies a significant absence of a subset of activated CD4 T-cells and a marked diminution in V beta 6 mRNA levels among T-cells in these cultures. By comparison, in 5 patients receiving the lower dosage of the vaccine, CSF cellularity was the same or slightly increased over pre-vaccination levels, CSF cells from 1 patient failed to grow in expansion cultures and cultured CSF cells from 2 patients underwent a change from an oligoclonal V beta 6 pattern to one that was more polyclonal. These results justify a more through exploration of the use of TCR peptide vaccines as a possible therapeutic treatment for MS.

Adult↗

PubMed1997

Induction of yolk sac endoderm in GATA-4-deficient embryoid bodies by retinoic acid.

GATA-4, a transcription factor implicated in lineage determination, is expressed in both parietal and visceral endoderm of the early mouse embryo. In embryonic stem cell-derived embryoid bodies, GATA-4 mRNA is first detectable at 4-5 days of differentiation and is confined to visceral endoderm cells on the surface of the bodies. Previously we reported that targeted mutagenesis of the Gata4 gene in embryonic stem cells results in a block in visceral endoderm differentiation in vitro. In an attempt to elucidate the role of GATA-4 in the formation of visceral endoderm, we have now differentiated Gata4 -/- and wild type embryoid bodies in the presence of retinoic acid +/- dbcAMP, known inducers of endoderm formation. We show that differentiation of Gata4 -/- embryoid bodies in the presence of retinoic acid results in formation of visceral endoderm, while differentiation of Gata4 -/- embryoid bodies in the presence of retinoic acid plus dbcAMP causes parietal endoderm formation. The presence of these yolk sac endoderm layers was confirmed by light microscopy and analysis of biochemical markers including alpha-fetoprotein, type IV collagen, laminin, and binding sites for Dolichos biflorus agglutinin. Treatment of Gata4 -/- embryoid bodies with retinoic acid induces expression of another GATA-binding protein, GATA-6, in both visceral and parietal endoderm cells. That another GATA-binding protein is induced in the absence of GATA-4 suggests that this family of transcription factors plays an important role in yolk sac differentiation.

Animals↗

PubMed1997

Low dose epidural bupivacaine/fentanyl infusion does not mask uterine rupture.

A patient is described in whom the symptoms and signs of uterine rupture were not masked by combined spinal epidural analgesia with an epidural infusion of 0.1% bupivacaine and 1.5 microg ml(-l) fentanyl. Early recognition of the dehiscence of a previous caesarean section scar resulted in an excellent neonatal and maternal outcome.

Journal Article↗

PubMed1997

Genetic engineering of bacteria and their potential for Hg2+ bioremediation.

Ion exchange or biosorptive processes for metal removal generally lack specificity in metal binding and are sensitive to ambient conditions, e.g. pH, ionic strength and the presence of metal chelators. In this study, cells of a genetically engineered Escherichia coli strain, JM109, which expresses metallothionein and a Hg2+ transport system after induction were evaluated for their selectivity for Hg2+ accumulation in the presence of sodium, magnesium, or cadmium ions and their sensitivity to pH or the presence of metal chelators during Hg2+ bioaccumulation. The genetically engineered E. coli cells in suspension accumulated Hg2+ effectively at low concentrations (0-20 microM) over a broad range of pH (3 to 11). The presence of 400 mM sodium chloride, 200 mM magnesium chloride, or 100 microM cadmium ions did not have a significant effect on the bioaccumulation of 5 microM Hg2+, indicating that this process is not sensitive to high ionic strength and is highly selective against sodium, magnesium, or cadmium ions. Metal chelators usually interfere with ion exchange or biosorptive processes. However, two common metal chelators, EDTA and citrate, had no significant effect on Hg2+ bioaccumulation by the genetically engineered strain. These results suggest that this E. coli strain could be used for selective removal of Hg2+ from waste water or from contaminated solutions which are resistant to common treatments. A second potential application would be to remove Hg2+ from Hg(2+)-contaminated soil, sediment, or particulates by washing them with a Hg2+ chelator and regenerating the chelator by passing the solution through a reactor containing the strain.

Cadmium↗

PubMed1997

Structure and mechanism of endo/exocellulase E4 from Thermomonospora fusca.

Cellulase E4 from Thermomonospora fusca is unusual in that it has characteristics of both exo- and endo-cellulases. Here we report the crystal structure of a 68K M(r) fragment of E4 (E4-68) at 1.9 A resolution. E4-68 contains both a family 9 catalytic domain, exhibiting an (alpha/alpha)6 barrel fold, and a family III cellulose binding domain, having an antiparallel beta-sandwich fold. While neither of these folds is novel, E4-68 provides the first cellulase structure having interacting catalytic and cellulose binding domains. The complexes of E4-68 with cellopentaose, cellotriose and cellobiose reveal conformational changes associated with ligand binding and allow us to propose a catalytic mechanism for family 9 enzymes. We also provide evidence that E4 has two novel characteristics: first it combines exo- and endo-activities and second, when it functions as an exo-cellulase, it cleaves off cellotetraose units.

Actinomycetales↗

PubMed1997

The Medical Skills Centre at the University of Calgary Medical School.

The aim of the Medical Skills Centre at the University of Calgary is to provide a predictable learning environment and standardized teaching methods in the area of clinical skills teaching. The centre uses volunteer patients and trained actors for all levels of medical education and evaluation. A computer database enables easy access to groups of patients and varying medical issues. This article describes the organization and operation of the Medical Skills Centre and notes a variety of courses which use the unique facilities of the centre.

Academic Medical Centers↗