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Biomedical subjects

T J Wilson

Publications and source records attributed to T J Wilson.

At least 19 recordsLinked to original sources
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↗
PubMed1999

Visualizing ion relaxation in the transport of short DNA fragments.

Ion relaxation plays an important role in a wide range of phenomena involving the transport of charged biomolecules. Ion relaxation is responsible for reducing sedimentation and diffusion constants, reducing electrophoretic mobilities, increasing intrinsic viscosities, and, for biomolecules that lack a permanent electric dipole moment, provides a mechanism for orienting them in an external electric field. Recently, a numerical boundary element method was developed to solve the coupled Navier-Stokes, Poisson, and ion transport equations for a polyion modeled as a rigid body of arbitrary size, shape, and charge distribution. This method has subsequently been used to compute the electrophoretic mobilities and intrinsic viscosities of a number of model proteins and DNA fragments. The primary purpose of the present work is to examine the effect of ion relaxation on the ion density and fluid velocity fields around short DNA fragments (20 and 40 bp). Contour density as well as vector field diagrams of the various scalar and vector fields are presented and discussed at monovalent salt concentrations of 0.03 and 0.11 M. In addition, the net charge current fluxes in the vicinity of the DNA fragments at low and high salt concentrations are briefly examined and discussed.

Biophysical Phenomena↗
PubMed1998

An apparatus for membrane-confined analytical electrophoresis.

A membrane-confined analytical electrophoresis apparatus for measuring the solution charge of macromolecules has been described previously (T. M. Laue et al., Anal. Biochem. 1989, 182, 377-382). Presented here is a design for this apparatus, which permits the on-line acquisition and display of absorbance data from up to 512 positions along an analysis chamber. Concentration distributions of macromolecules in solution can be monitored in the chamber to provide steady-state electrophoresis, electrophoretic mobility and diffusion measurements. Buffer chambers press semipermeable membranes against the open ends of a fused-silica cuvette to form the analysis chamber. This configuration permits both the flow of buffer and the establishment of an electric field across the cuvette, while retaining macromolecules in the field of view. Though a gel may be included in the analysis chamber, none is required for gradient stabilization. The volume of sample required for analysis is 8 microL, most of which is recoverable. Experimental conditions can be varied during study by simply changing the circulating buffer and/or the electric field. The analysis and buffer chambers are held in an aluminum housing that sits in an aluminum water jacket. The water jacket provides temperature control, shielding from external electrical noise and also serves as an optical mask. Plans for the cell assembly, optical system and the computer interface for data acquisition are provided. The assembly and operation of the apparatus and the analysis of data are described.

Electric Conductivity↗
PubMed1998

Two beta-tubulin genes, TUB1 and TUB8, of Arabidopsis exhibit largely nonoverlapping patterns of expression.

Chimeric reporter genes were used to investigate the patterns of expression of two beta-tubulin genes, TUB1 and TUB8, in Arabidopsis thaliana. The TUB1 chimeric gene was preferentially expressed in epidermal and cortical cells of primary roots, whereas the TUB8 chimeric gene was preferentially expressed in the endodermal and phloem cells of primary roots and in the vascular tissues of leaves, stems, and flowers of transgenic plants.

Arabidopsis↗
PubMed1998

The rpfA gene of Xanthomonas campestris pathovar campestris, which is involved in the regulation of pathogenicity factor production, encodes an aconitase.

Xanthomonas campestris pv campestris (Xcc) is a plant pathogenic bacterium that controls the production of pathogenicity factors in part by a cluster of genes designated rpf (regulation of pathogenicity factors). Sequence analysis of one of these genes (rpfA) revealed an open reading frame with amino acid sequence similarity to aconitases from other bacteria. Aconitase activity was lower in cellular extracts of an rpfA::Tn5 mutant than in those from the wild type. A zymogram of aconitase activity after native gel electrophoresis showed the presence of two distinct aconitases in Xcc; the major aconitase was absent in the rpfA::Tn5 mutant. This mutant also had reduced levels of extracellular enzymes and extracellular polysaccharide (EPS). Supplying rpfA in trans to the rpfA::Tn5 mutant restored both the major aconitase activity and the synthesis of these pathogenicity factors. The transcription of the genes for two extracellular enzymes (prtA, encoding a serine protease, and engXCA, encoding endoglucanase) was reduced in the rpfA mutant background. Because some eukaryotic aconitases are also involved in iron regulation, we explored a possible connection between rpfA and iron metabolism. Intracellular iron levels in the mutants were lower than in the wild type as assessed by sensitivity to the iron-activated antibiotic, streptonigrin. Wild-type bacteria grown in iron-deficient conditions had a similar sensitivity to streptonigrin as the aconitase mutant. Overall, these results suggest that a prokaryotic aconitase can also act as a regulator of gene expression and that the regulation is possibly related to changes in intracellular iron levels.

Aconitate Hydratase↗
PubMed1997

A novel regulatory system required for pathogenicity of Xanthomonas campestris is mediated by a small diffusible signal molecule.

Mutations in the seven clustered rpf genes cause downregulated synthesis of extracellular enzymes and reduced virulence of Xanthomonas campestris pathovar campestris (Xcc). The phenotype of mutants in one of the genes, rpfF, can be restored by a diffusible extracellular factor (DSF) produced by all Xcc strains tested, apart from rpfF and rpfB mutants. DSF accumulates in early stationary phase (when synthesis of enzymes is maximal), but levels decline subsequently. Addition of DSF to exponentially-growing wild-type bacteria does not cause precocious enzyme synthesis. rpfB and rpfF are expressed throughout growth, but the rate increases in early stationary phase. RpfB is predicted to be a long-chain fatty acyl CoA ligase, and RpfF shows some relatedness to enoyl CoA hydratases. The properties of DSF suggest that it may be a fatty-acid derivative, and certain lipid preparations possess DSF activity at higher concentrations. These include lipid extracts and acid-hydrolysed lipoplysaccharide and lipid A from Xcc, and purified dodecanoic and hydroxydodecanoic acid. DSF production is confined to certain xanthomonads. We propose a model for the DSF system, which represents a novel mechanism for regulating virulence factor synthesis in response to physiological or environmental changes.

Diffusion↗
PubMed1996

Down's syndrome-like skeletal abnormalities in Ets2 transgenic mice.

Expression of Ets2, a proto-oncogene and transcription factor, occurs in a variety of cell types. During murine development it is highly expressed in newly forming cartilage, including in the skull precursor cells and vertebral primordia. Ets2 is located on human chromosome 21 (ref. 8) and is overexpressed in Down's syndrome (trisomy 21). Here we generate transgenic mice to investigate the consequences of overexpression of Ets2. We find that mice with less than 2-fold Ets2 overexpression in particular organs develop neurocranial, viscerocranial and cervical skeletal abnormalities. These abnormalities have similarities with the skeletal anomalies found in trisomy-16 mice and humans with Down's syndrome, in which the gene dosage of Ets2 is increased. Our results indicate that Ets2 has a role in skeletal development and implicate the overexpression of Ets2 in the genesis of some skeletal abnormalities that occur in Down's syndrome.

Abnormalities, Multiple↗
PubMed1996

Insights from a new analytical electrophoresis apparatus.

Charge is a fundamental property of macromolecules that is inextricably linked to their structure, solubility, stability, and interactions. Progress has been made on the theoretical and structural aspects of these relationships. However, for several reasons, charge is difficult to measure in solution. Consequently, there is a lack of experimental data that, independent of other macro-ion properties, determines the effective charge. To overcome this problem, novel instrumentation and methods are being developed in our laboratory. Described here is an analytical electrophoresis apparatus that permits both the measurement of electrophoretic mobilities and the determination of steady-state electrophoresis concentration distributions. The latter provides a different-perspective on the processes that influence macro-ion behavior in an electric field. In addition, the apparatus permits the determination of diffusion coefficients either from boundary spreading during transport or from the decay of a concentration gradient. All of these determinations can be made with a single, 8-microL sample in a variety of solvents, thus providing unique insights into the charge properties of a macro-ion. Presented here is a progress report about this emerging technology, including the description of a prototype apparatus and examples of its use with a DNA oligonucleotide.

Diffusion↗
PubMed1996

Effector-induced self-association and conformational changes in the enhancer-binding protein NTRC.

The Klebsiella pneumoniae nitrogen regulatory protein NTRC is a response regulator which activates transcription in response to nitrogen limitation, and is a member of the family of sigma N-dependent enhancer-binding proteins. Using limited trypsin digestion, two domains of NTRC were detected and conformational changes within the protein in response to the binding of ligands were also observed. In the absence of ligands, the major digestion products were 42, 36 and 12.5 kDa bands corresponding to the central plus C-terminal domain, the central domain, and the N-terminal domains, respectively. Upon binding of purine but not pyrimidine nucleotides, the 36 kDa band was insensitive to further proteolysis, indicative of a conformational change in the central domain. Analysis of the dependence of this insensitivity on ATP gamma S concentration suggested an apparent dissociation constant (Kd) for ATP gamma S of 150 microM. In the presence of DNA, both the central and C-terminal domains of NTRC were insensitive to proteolytic cleavage, indicative of a further conformational change. NTRC S160F, a mutant form of NTRC that is active in the absence of phosphorylation, was more stable to proteolysis than the wild-type protein. This mutant protein is apparently locked in a conformation resembling the DNA-bound form of wild-type NTRC. The involvement of ligands in self-association was studied using sedimentation equilibrium analysis. In the absence of ligand, wild-type NTRC displayed a monomer-dimer equilibrium with a Kd of 6 microM. In the presence of ATP gamma S the equilibrium was shifted towards the dimer form (Kd = 0.8 microM). A similar dissociation constant for the monomer-dimer interaction was observed with NTRC S160F in the absence of ATP gamma S (Kd = 0.5 microM). The addition of ATP gamma S induced a significant association of NTRC S160F to higher-order states with a dimer-octamer model producing a slightly, but not significantly better fit to the data than a dimer-hexamer model. We propose that ligand-mediated self-association provides a common mechanism for activation of this class of transcriptional regulatory proteins.

Bacterial Proteins↗
PubMed1996

A gene for superoxide dismutase from Xanthomonas campestris pv. campestris and its expression during bacterial-plant interactions.

A recombinant plasmid selected from a library of Xanthomonas campestris pv. campestris genomic DNA by functional complementation of a superoxide dismutase (SOD)-deficient strain of Escherichia coli contained a gene encoding the major SOD activity of X. campestris pv. campestris. Inhibition and renaturation studies suggested that manganese was the metal cofactor for this SOD. Examination of the nucleotide sequence of an active subclone revealed a 612-bp open reading frame that encodes a protein with high amino acid sequence homology to a range of SOD enzymes. The sod gene was mutagenized with Tn5-lacZ. None of the insertions that abolished SOD-conferring activity were in the correct orientation for lacZ expression. Repeated attempts to introduce these insertions into the chromosome of X. campestris pv. campestris were unsuccessful and it was concluded that the sod gene may be essential for viability. In order to monitor the expression of the sod gene, a sod-gus transcriptional fusion was constructed. Expression of the sod gene varied according to the growth stage of the organism in culture. In planta, the sod gene was induced within 3 to 4 h of inoculation, with similar kinetics during compatible and incompatible interactions with turnip and pepper, respectively.

Amino Acid Sequence↗
PubMed1996

Cloning and characterization of the rpfC gene of Xanthomonas oryzae pv. oryzae: involvement in exopolysaccharide production and virulence to rice.

rpfC is one of a cluster of genes which coordinately regulate the synthesis of extracellular enzymes and exopolysaccharide and pathogenicity in Xanthomonas campestris pv. campestris, the black rot pathogen of brassicas. An rpfC homolog which could functionally complement an rpfC mutant of X. campestris pv. campestris was identified in Xanthomonas oryzae pv. oryzae and the gene was characterized. Mutation of this gene in X. oryzae pv. oryzae had no effect on extracellular enzymes, but exopolysaccharide synthesis and virulence to rice were substantially reduced.

Bacterial Proteins↗
PubMed1995

Phytochrome A and phytochrome B mediate the hypocotyl-specific downregulation of TUB1 by light in arabidopsis.

Arabidopsis contains six alpha-tubulin and nine beta-tubulin genes that are expressed in a tissue-specific and developmentally regulated manner. We analyzed the effects of light on tubulin mRNA abundance in Arabidopsis seedlings using RNA gel blot hybridizations and gene-specific probes. Transcript levels of all 15 tubulin genes were decreased by continuous white light, although to different degrees. Detailed analysis was performed with the beta-tubulin TUB1 gene. The transcript level of TUB1 was high in etiolated seedlings and decreased to approximately 20% of the dark mRNA level after 2 to 6 hr of white light treatment. We showed that this downregulation requires high-irradiance light treatment and that multiple photoreceptors are involved. In particular, using phytochrome mutants and narrow wave band light, we demonstrated that both the phytochrome A (phyA)-mediated far-red light high-irradiance response and the phytochrome B (phyB)-mediated red light high-irradiance response are involved in the downregulation of TUB1 expression by white light. Histochemical analysis of transgenic plants expressing a TUB1-beta-glucuronidase chimeric transgene indicated that the downregulation observed only in hypocotyls and not in roots is controlled transcriptionally.

Arabidopsis↗
PubMed1995

Analysis of an Escherichia coli mutant TyrR protein with impaired capacity for tyrosine-mediated repression, but still able to activate at sigma 70 promoters.

In Escherichia coli, TyrR represses and activates transcription of operons required for tyrosine, phenylalanine and tryptophan biosynthesis and uptake. The TyrR central domain is homologous with NtrC and some other bacterial regulatory proteins, although TyrR regulates sigma 70, not sigma 54, promoters. We isolated a central domain TyrR mutant (TyrR E274Q) by substitution of a normally conserved amino acid. The mutant was unable to bring about tyrosine-mediated repression of aroF, aroL, tyrB, and tyrP and had diminished capability for tyrosine- and phenylalanine-mediated repression of aroP. In contrast, it was able to effect wild-type levels of phenylalanine-mediated repression of aroG, tryptophan-mediated repression of aroP and transcriptional activation of mtr and tyrP. The binding of purified TyrR E274Q to ATP (a requirement for tyrosine binding) and to the strong TyrR box of tyrP operator DNA were normal, but tyrosine binding and tyrosine-dependent hexamerization were significantly impaired. These properties are consistent with the proposal that self association is essential for tyrosine-mediated repression by TyrR but not for tyrosine- or phenylalanine-mediated activation. E274 of TyrR must participate in either the binding of tyrosine, or the coupling of ATP binding with a conformational change that alters the affinity of the ATP-dependent aromatic amino acid-binding site.

Adenosine Triphosphatases↗
PubMed1995

Evidence for two aromatic amino acid-binding sites, one ATP-dependent and the other ATP-independent, in the Escherichia coli regulatory protein TyrR.

In Escherichia coli, genetic regulation of aromatic amino acid biosynthesis and uptake is effected by the protein TyrR, which acts via ligand-mediated repression and activation. Characterization of the interactions of tyrosine, phenylalanine and tryptophan with TyrR revealed the presence of two separate aromatic amino acid-binding sites, one ATP-dependent, the other ATP-independent. Binding to the ATP-dependent site induces the self-association of TyrR. Using sedimentation equilibrium analyses, dissociation constants for this site in the dimeric and hexameric forms of TyrR were determined to be 330 microM and 24 microM, respectively, for tyrosine, and 55 mM and 3.7 mM, respectively, for phenylalanine. Tryptophan bound with a strength similar to that of phenylalanine, and both phenylalanine and tryptophan competed with the binding of tyrosine. The ATP-independent site, which has not been observed previously, was characterized by ultraviolet (u.v.) difference spectroscopy and a sedimentation-velocity meniscus-depletion method. Phenylalanine bound co-operatively to this site, exhibiting half-saturation at 260 microM. Tryptophan competed weakly with phenylalanine, half-saturation occurring at 1.2 mM. No binding of tyrosine to this site could be detected. We propose that the binding of phenylalanine or tryptophan to this ATP-independent site is responsible for phenylalanine- and tryptophan-mediated regulation by TyrR.

Adenosine Triphosphate↗
PubMed1994

Ligand-induced self-association of the Escherichia coli regulatory protein TyrR.

Analyses of the sedimentation properties of the Escherichia coli regulatory protein TyrR indicated that it undergoes a ligand-induced hexamerization. This phenomenon was observed at protein concentrations approximating to those found in vivo. In the absence of added ligands, TyrR sedimented as a single molecular species with a sedimentation coefficient of 5.3 S and a relative molecular mass of 113,000. Given a subunit relative molecular mass of 57,640 for TyrR, it was concluded that this species is a dimer. Similar sedimentation properties were observed when TyrR was sedimented in the presence of either tyrosine, phenylalanine, ATP or ATP gamma S, a non-hydrolysable analogue of ATP. However, in the presence of saturating ATP gamma S and 500 microM tyrosine or 25 mM phenylalanine the sedimentation behaviour of TyrR yielded relative molecular masses of 340,000 and 310,000, respectively, indicative of hexamer formation. The sedimentation data obtained across a range of TyrR concentrations fitted equally well to dimer-hexamer and dimer-tetramer-hexamer models. For the latter model, the predicted overall association constant was 3.2 x 10(13) M-2 at saturating tyrosine, while the relative values of the association constants for the two individual steps indicated a concerted mechanism with the tetramer a minor component. There was no indication of dimer dissociation when dilute TyrR solutions (100 nM) were sedimented. A model to explain the role of hexamerization in tyrosine-mediated repression of transcription by TyrR is proposed. It is suggested that the hexameric form of TyrR is the active repressing species, interacting with two or three specific sequences (TyrR boxes) in the targeted regulatory DNA. The hexamerization reaction that takes place when the tyrosine concentration rises is envisaged as occurring in situ on the DNA, with a TyrR dimer that permanently occupies one of the TyrR boxes acting as a nucleation site for the development of the hexamer-DNA complex.

Adenosine Triphosphate↗
PubMed1994

Purification of the Escherichia coli regulatory protein TyrR and analysis of its interactions with ATP, tyrosine, phenylalanine, and tryptophan.

A plasmid that directs the overexpression of the Escherichia coli regulatory protein TyrR was constructed. Cell extracts of an E. coli strain harboring the plasmid were used to develop a two-step procedure for purifying homogeneous TyrR. The weight-average molecular weight of the pure protein was determined by sedimentation equilibrium analyses to be 110,000 +/- 5,000, indicating that native TyrR is a homodimer. The binding of ligands to TyrR was investigated by the techniques of sedimentation velocity meniscus depletion and steady state dialysis. One mol of ATP bound per mol of TyrR subunit with half-maximal saturation at 5-7 microM ATP. ATP binding curves exhibited positive cooperativity, with a value of 1.3 for the Hill constant, nH. The binding was not significantly affected by the presence of either 500 microM tyrosine or 2 mM phenylalanine. Binding of tyrosine to TyrR (40 microM subunit) could not be detected in the absence of ATP, indicating that the TyrR-tyrosine complex has a dissociation constant (Kd) in excess of 180 microM. However, binding was observed in the presence of saturating ATP (200 microM), where 1 mol of tyrosine bound per mol of TyrR subunit with half-maximal saturation at 50 microM tyrosine. The binding exhibited positive cooperativity (nH of 1.2). There was no detectable binding of either phenylalanine or tryptophan to TyrR (40 microM) in the absence or presence of 200 microM ATP, indicating that any binding of these amino acids to TyrR or TyrR.ATP also has a Kd in excess of 180 microM. Each of these amino acids was found to inhibit the binding of tyrosine by TyrR.ATP when present in large molar excess (20 microM tyrosine and 2 or 10 mM phenylalanine or tryptophan), indicating that TyrR binds each of these amino acids, albeit more weakly than it binds tyrosine.

Adenosine Triphosphate↗
PubMed1994

Decreased natural killer cell activity in endometriosis patients: relationship to disease pathogenesis.

A number of reports have measured NK cell activity in patients with endometriosis with varied results. Therefore we have examined the NK activity of PBL from 44 gynecological patients undergoing laparoscopy. This analysis has demonstrated a significant reduction in NK activity only in more severe stages of endometriosis (stages III and IV) relative to patients with milder disease and controls. These data indicate that decreased NK activity is unlikely to be a primary etiological factor in the development of endometriosis but may indicate that decreased NK activity is related to the development of the more frequent and/or larger lesions characteristic of severe endometriosis. These data could indicate potential for immunotherapy of patients with advanced endometriosis by the upregulation of NK activity in vivo.

Cytotoxicity Tests, Immunologic↗