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Biomedical subjects

David B Wilson

Publications and source records attributed to David B Wilson.

At least 55 records · Page 3Linked to original sources
PubMed2003

Binding and reversibility of Thermobifida fusca Cel5A, Cel6B, and Cel48A and their respective catalytic domains to bacterial microcrystalline cellulose.

The binding and reversibility of Thermobifida fusca intact Cel5A, Cel5B, and Cel48A and their corresponding catalytic domains (CDs) to bacterial microcrystalline cellulose (BMCC) were studied at 5 degrees C. The binding of the intact cellulases and of corresponding CDs to BMCC was irreversible in all regions: Langmuir binding (region I), interstice penetration (region II), and interstice saturation (region III). The three cellulose binding domains (CBMs) bind reversibly in "region I" although their respective CDs do not. The irreversible binding of these enzymes in the Langmuir region does not satisfy the Langmuir assumption; however, the overall fit of the Interstice Saturation model, which includes binding in MBCC interstices as well as on the freely accessible surface (Jung et al., 2002a) is good. The main limitation of the model is that it does not explicitly address a mechanism for forming the enzyme-substrate complex within the active site of the CDs.

Actinomycetales↗
PubMed2003

An engineered chorismate mutase with allosteric regulation.

Besides playing a central role in phenylalanine biosynthesis, the bifunctional P-protein in Eschericia coli provides a unique model system for investigating whether allosteric effects can be engineered into protein catalysts using modular regulatory elements. Previous studies have established that the P-protein contains three distinct domains whose functions are preserved, even when separated: chorismate mutase (residues 1-109), prephenate dehydratase (residues 101-285), and an allosteric domain (residues 286-386) for feedback inhibition by phenylalanine. By deleting the prephenate dehydrase domain, a functional chorismate mutase linked directly to the phenylalanine binding domain has been engineered and overexpressed. This manuscript reports the catalytic properties of the mutase in the absence and presence of phenylalanine.

Allosteric Regulation↗
PubMed2003

Transcription factor GATA-6 is expressed in malignant endoderm of pediatric yolk sac tumors and in teratomas.

Transcription factors GATA-4 and GATA-6 play critical roles in mammalian yolk sac differentiation and function. Previously, we showed that GATA-4 is a potential marker for malignant yolk sac endoderm in pediatric germ cell tumors. This highly malignant tissue can cause diagnostic problems because yolk sac components may be difficult to differentiate from other, especially immature, tissue types in teratomas. In the search for new molecular markers for germ cell tumors, we have surveyed GATA-6 expression in benign and malignant pediatric germ cell tumors using mRNA in situ hybridization and immunohistochemistry. GATA-6 was expressed in most yolk sac tumors examined and also in nonmalignant tissues including gut/respiratory epithelium, sebocytes, and neuroepithelium in mature and immature teratomas. Given that GATA-6 has not been discovered in sebocytes before, this finding was confirmed by immunohistochemistry of normal mouse samples, indicating a function for this transcription factor in the mammalian skin. Taken together, GATA-6 can be used to identify yolk sac components in pediatric germ cell tumors. Furthermore, it is also expressed in specific tissues in teratomas. GATA-6, together with GATA-4, can thus be used as a novel molecular marker in characterizing of pediatric germ cell tumors.

Adolescent↗
PubMed2003

Quantitative determination of noncovalent binding interactions using automated nanoelectrospray mass spectrometry.

Electrospray ionization mass spectrometry (ESI-MS) has proven to be an extremely powerful tool for studying biomolecular structures and noncovalent interactions. Here we report a method using a fully automated, chip-based nanoESI-MS system to determine the dissociation constants (Kd) for the complexes of two different proteins with their ligands. The automated nanoelectrospray system, consisting of the NanoMate and ESI chip, serves functionally as a combination of autosampler and nanoelectrospray ionization source. This system provides all the advantages of conventional nanoelectrospray plus automated, high-throughput analyses without carryover. The automated nanoESI system was used to investigate quantitative noncovalent interactions between ribonuclease A (RNase A) and cytidylic acid ligands (2'-CMP, CTP), a well-characterized model protein-ligand complex, and between an inactive endocellulase mutant (Thermobifida fusca Cel6A D117Acd) and four oligosaccharide ligands (cellotriose, cellotetraose, cellopentaose, cellohexaose). Both titration and competitive binding approaches were performed prior to automated nanoESI-MS analysis with a Q-TOF mass spectrometer. Dissociation constants for each complex were calculated from the sum of ion peak areas of free and complexed proteins during the titration and competition experiments. The measured Kd values for the RNase A-CMP and Cel6A D117Acd-G3 complexes were found to be in excellent agreement with the available published values obtained by standard spectroscopic titration techniques. To our knowledge, this is the first report of using an ESI-MS approach to study the interactions between a cellulase and oligosaccharides. The results provide new insights for understanding the nature of cellulase-cellulose interactions.

Animals↗
PubMed2003

Sudden unexplained hemolysis occurring in an infant due to presumed Loxosceles envenomation.

We report the case of a 3-week-old infant referred for evaluation of sudden onset jaundice and unexplained hemolysis. After an exhaustive workup, the most likely etiology was found to be envenomation by a brown recluse spider, Loxosceles reclusa. This case underscores the fact that severe loxoscelism may occur in the absence of the classically described necrotic cutaneous lesion, and represents one of the youngest presumed cases of loxoscelism. We present the case to illustrate the importance of considering loxoscelism in the differential diagnosis of sudden massive hemolysis in children, particularly in endemic areas of the midwestern and southern United States.

Anemia, Hemolytic↗
PubMed2003

Characteristics of effective school-based substance abuse prevention.

This study summarizes, using meta-analytic techniques, results from 94 studies of school-based prevention activities that examined alcohol or other drug use outcomes. It set out to determine what features of school-based substance abuse prevention programs are related to variability in the size of program effects, It asked (1) Which populations (e.g., high risk vs. general population) should be targeted for prevention services? (2) What is the best age or developmental stage for prevention programming? (3) Does program duration matter? and (4) Does the role of the person delivering the service (e.g., teacher, law enforcement officer, peer) matter? The results suggest that targeting middle school aged children and designing programs that can be delivered primarily by peer leaders will increase the effectiveness of school-based substance use prevention programs. The results also imply that such programs need not be lengthy. The evidence related to the targeting issue is sparse, but suggests that, at least for programs teaching social competency skills, targeting higher risk youths may yield stronger effects than targeting the general population. Suggestions for future research are offered.

Adolescent↗
PubMed2003

Comparison of extracellular Escherichia coli AppA phytases expressed in Streptomyces lividans and Pichia pastoris.

A phytase gene (appA) from Escherichia coli was cloned into Streptomyces lividans and expressed as an extracellular protein which was then compared with the same enzyme expressed in Pichia pastoris. The phytase expressed in S. lividans was not glycosylated and had a molecular mass of 45 kDa. Compared with the glycosylated phytase expressed in P. pastoris, this non-glycosylated phytase was 25-50% less active (p < 0.05) at pH 2 to 3.5 or at 45 and 55 degrees C, but 50% more active (p < 0.05) at 75 degrees C. The thermo-tolerance of the non-glycosylated phytase was 26 and 48% higher (p < 0.05) than that of the glycosylated phytase at 45 and 55 degrees C, but was 80 and 94% lower (p < 0.05) at 65 degrees and 75 degrees C, respectively.

6-Phytase↗
PubMed2003

Mapping of chorismate mutase and prephenate dehydrogenase domains in the Escherichia coli T-protein.

The Escherichia coli bifunctional T-protein transforms chorismic acid to p-hydroxyphenylpyruvic acid in the l-tyrosine biosynthetic pathway. The 373 amino acid T-protein is a homodimer that exhibits chorismate mutase (CM) and prephenate dehydrogenase (PDH) activities, both of which are feedback-inhibited by tyrosine. Fifteen genes coding for the T-protein and various fragments thereof were constructed and successfully expressed in order to characterize the CM, PDH and regulatory domains. Residues 1-88 constituted a functional CM domain, which was also dimeric. Both the PDH and the feedback-inhibition activities were localized in residues 94-373, but could not be separated into discrete domains. The activities of cloned CM and PDH domains were comparatively low, suggesting some cooperative interactions in the native state. Activity data further indicate that the PDH domain, in which NAD, prephenate and tyrosine binding sites were present, was more unstable than the CM domain.

Binding Sites↗
PubMed2003

Cloning, expression and characterization of a family-74 xyloglucanase from Thermobifida fusca.

Thermobifida fusca xyloglucan-specific endo-beta-1,4-glucanase (Xeg)74 and the Xeg74 catalytic domain (CD) were cloned, expressed in Escherichia coli, purified and characterized. This enzyme has a glycohydrolase family-74 CD that is a specific xyloglucanase followed by a family-2 carbohydrate binding module at the C terminus. The Michaelis constant (Km) and maximal rate (Vmax) values for hydrolysis of tamarind seed xyloglucan (tamXG) are 2.4 micro m and 966 micro mol xyloglucan oligosaccharides (XGOs) min-1. micro mol protein-1. More than 75% of the activity was retained after a 16-h incubation at temperatures up to 60 degrees C. The enzyme was most active at pH 6.0-9.4. NMR analysis showed that its catalytic mechanism is inverting. The oligosaccharide products from hydrolysis of tamXG were determined by MS analysis. Cel9B, an active carboxymethylcellulose (CMC)ase from T. fusca, was also found to have activity on xyloglucan (XG) at 49 micro mol.min-1. micro mol protein-1, but it could not hydrolyze XG units containing galactose. An XG/cellulose composite was prepared by growing Gluconacetobacterxylinus on glucose with tamXG in the medium. Although a mixture of purified cellulases was unable to degrade this material, the composite material was fully hydrolyzed when Xeg74 was added. T. fusca was not able to grow on tamXG, but Xeg74 was found in the culture supernatant at the same level as was found in cultures grown on Solka Floc. The function of this enzyme appears to be to break down the XG surrounding cellulose fibrils found in biomass so that T. fusca can utilize the cellulose as a carbon source.

Acetobacter↗
PubMed2003

Isolated CNS relapse following stem cell transplantation for juvenile myelomonocytic leukemia.

A 1-year-old girl with juvenile myelomonocytic leukemia (JMML) underwent allogeneic bone marrow transplantation (BMT) from her HLA-matched brother. A few months after BMT she experienced a bone marrow relapse that did not respond to withdrawal of immunosuppression. To enhance the graft-versus-leukemia (GVL) effect, she underwent peripheral stem cell transplantation (PSCT) from the same donor, using a nonmyeloablative conditioning regimen. She achieved clinical remission and developed chronic graft-versus-host disease (GVHD), which was treated with prednisone and cyclosporine A. One year after PSCT she experienced an isolated central nervous system (CNS) relapse. She was treated with intrathecal Ara-C followed by craniospinal irradiation and achieved a third clinical remission. While extramedullary relapses have been described in JMML, this is the first report of a CNS relapse. Based on this case and others in the literature, the authors suggest that newer therapies are changing the natural history of JMML. By manipulating the GVL effect it is possible to achieve a prolonged bone marrow remission, but only at the expense of unmasking the risk of late extramedullary relapse.

Central Nervous System Neoplasms↗
PubMed2003

Mouse strain susceptibility to gonadectomy-induced adrenocortical tumor formation correlates with the expression of GATA-4 and luteinizing hormone receptor.

Certain inbred strains of mice, including DBA/2J, develop adrenocortical tumors in response to gonadectomy. Spindle-shaped cells with limited steroidogenic capacity, termed A cells, appear in the subcapsular region of the adrenal gland, followed by sex steroid-producing cells known as B cells. These changes result from unopposed gonadotropin production by the pituitary, but the adrenocortical factors involved in tumorigenesis have not been characterized. GATA-4, a transcription factor normally expressed in fetal, but not adult, adrenocortical cells, was found in neoplastic cells that proliferate in the adrenal cortex of gonadectomized DBA/2J mice. GATA-4 mRNA was detected in the adrenal glands of female mice 0.5 months after ovariectomy and reached a maximum by 4 months. Castrated male mice developed adrenocortical tumors more slowly than gonadectomized females, and the onset of GATA-4 expression in the adrenal was delayed. In situ hybridization and immunohistochemistry revealed GATA-4 mRNA and protein in A and B cells, but not in normal adrenocortical cells. mRNA encoding another factor associated with adrenocortical tumorigenesis, LH receptor (LHR), was detected in A and B cells. In addition, transcripts for P450 17 alpha-hydroxylase/C17-C20 lyase, an enzyme essential for the production of sex steroids, and inhibin-alpha were found in B cells. Unilateral ovarian regeneration, a phenomenon known to occur in gonadectomized mice, was observed in a subset of DBA/2J mice undergoing complete ovariectomy. In these animals, adrenocortical tumor progression was arrested; A cells and GATA-4 expression were evident, but there was no expression of LHR or P450 17 alpha-hydroxylase/C17-C20 lyase. Strain susceptibility to adrenocortical tumorigenesis (DBA/2J >> FVB/N) correlated with the expression of GATA-4 and LHR, implicating these factors in the process of adrenocortical neoplasia in response to continuous gonadotropin stimulation.

Adrenal Cortex↗
PubMed2003

Evaluation of an automated, latex-enhanced turbidimetric D-dimer test (advanced D-dimer) and usefulness in the exclusion of acute thromboembolic disease.

Advanced D-dimer (Dade Behring, Marburg, Germany) is a latex-enhanced turbidimetric test for the quantitative determination of D-dimers. To evaluate this assay, examine test performance on 2 instruments (BCS Analyzer and Sysmex CA-1500, Dade Behring), establish negative predictive value cutoffs (NPVCs), and compare the test with the qualitative SimpliRed test (Agen Biomedical, Brisbane, Australia), we evaluated 117 patients for possible acute thromboembolic disease (pulmonary embolus or deep venous thrombosis). Patients selected for the study were those with confirmatory tests (ultrasound, spiral computed tomography, ventilation-perfusion scan, or angiography) and those without certain medical conditions that might be associated with increased D-dimers. An NPVC of 1.8 mg/L for the BCS Analyzer resulted in 41.0% negative results (48/117) and sensitivity, specificity, and negative and positive predictive values of 100%, 48%, 100%, and 21%, respectively. An NPVC of 1.1 mg/L for the Sysmex CA-1500 resulted in 31.6% negative results (37/117) and sensitivity, specificity, and negative and positive predictive values of 100%, 36%, 100%, and 18%, respectively. The SimpliRed had 31.6% negative results (37/117) and sensitivity, specificity, and negative and positive predictive values of 86%, 34%, 96%, and 15%, respectively.

Acute Disease↗
PubMed2002

Retinoid X receptor alpha represses GATA-4-mediated transcription via a retinoid-dependent interaction with the cardiac-enriched repressor FOG-2.

Dietary vitamin A and its derivatives, retinoids, regulate cardiac growth and development. To delineate mechanisms involved in retinoid-mediated control of cardiac gene expression, the regulatory effects of the retinoid X receptor alpha (RXR alpha) on atrial naturietic factor (ANF) gene transcription was investigated. The transcriptional activity of an ANF promoter-reporter in rat neonatal ventricular myocytes was repressed by RXR alpha in the presence of 9-cis-RA and by the constitutively active mutant RXR alpha F318A indicating that liganded RXR confers the regulatory effect. The RXR alpha-mediated repression mapped to the proximal 147 bp of the rat ANF promoter, a region lacking a consensus retinoid response element but containing several known cardiogenic cis elements including a well characterized GATA response element. Glutathione S-transferase "pull-down" assays revealed that RXR alpha interacts directly with GATA-4, in a ligand-independent manner, via the DNA binding domain of RXR alpha and the second zinc finger of GATA-4. Liganded RXR alpha repressed the activity of a heterologous promoter-reporter construct containing GATA-response element recognition sites in cardiac myocytes but not in several other cell types, suggesting that additional cardiac-enriched factors participate in the repression complex. Co-transfection of liganded RXR alpha and the known cardiac-enriched GATA-4 repressor, FOG-2, resulted in additive repression of GATA-4 activity in ventricular myocytes. In addition, RXR alpha was found to bind FOG-2, in a 9-cis-RA-dependent manner. These data reveal a novel mechanism by which retinoids regulate cardiogenic gene expression through direct interaction with GATA-4 and its co-repressor, FOG-2.

Animals↗
PubMed2002

Binding mechanisms for Thermobifida fusca Cel5A, Cel6B, and Cel48A cellulose-binding modules on bacterial microcrystalline cellulose.

The family II cellulose-binding modules (CBM) from Thermobifida fusca Cel5A and Cel48A were cloned in the Escherichia coli/Streptomyces shuttle vector pD730, and the plasmids were transformed into Streptomyces lividans TKM31. CBM(Cel5A), and CBM(Cel48A), CBM(Cel6B) were expressed and purified from S. lividans. The molecular masses were determined by mass spectrometry, and the values were 10595 +/- 2, 10915 +/- 2, and 11291 +/- 2 Da for CBM(Cel5A), CBM(Cel6B), and CBM(Cel48A), respectively. Three different binding models (Langmuir, Interstice Penetration, and Interstice Saturation) were tested to describe the binding isotherms of these CBMs on bacterial microcrystalline cellulose (BMCC). The experimental binding isotherms of T. fusca family II CBMs on BMCC are best modeled by the Interstice Saturation model, which includes binding to the constrained interstice surface of BMCC as well as traditional Langmuir binding on the freely accessible surface. The Interstice Saturation model consists of three different steps (Langmuir binding, interstice binding, and interstice saturation). Full reversibility only occurred in the Langmuir region. The irreversibility in the interstice binding and saturation regions probably was caused by interstice entrapment. Temperature shift experiments in different binding regions support the interstice entrapment assumption. There was no systematic difference in binding between the two types of exocellulase CBMs--one that hydrolyzes cellulose from the nonreducing (CBM(Cel6B)) end and one that hydrolyzes cellulose from the reducing end (CBM(Cel48A)).

Actinomycetales↗
PubMed2002

Genetic mosaic analysis reveals that GATA-4 is required for proper differentiation of mouse gastric epithelium.

During mouse embryogenesis GATA-4 is expressed first in primitive endoderm and then in definitive endoderm derivatives, including glandular stomach and intestine. To explore the role of GATA-4 in specification of definitive gastric endoderm, we generated chimeric mice by introducing Gata4(-/-) ES cells into ROSA26 morulae or blastocysts. In E14.5 chimeras, Gata4(-/-) cells were represented in endoderm lining the proximal and distal stomach. These cells expressed early cytodifferentiation markers, including GATA-6 and ApoJ. However, by E18.5, only rare patches of Gata4(-/-) epithelium were evident in the distal stomach. This heterotypic epithelium had a squamous morphology and did not express markers associated with differentiation of gastric epithelial cell lineages. Sonic Hedgehog, an endoderm-derived signaling molecule normally down-regulated in the distal stomach, was overexpressed in Gata4(-/-) cells. We conclude that GATA-4-deficient cells have an intrinsic defect in their ability to differentiate. Similarities in the phenotypes of Gata4(-/-) chimeras and mice with other genetically engineered mutations that affect gut development suggest that GATA-4 may be involved in the gastric epithelial response to members of the TGF-beta superfamily.

Animals↗
PubMed2002

Differential expression of GATA-4 and GATA-6 in fetal and adult mouse and human adrenal tissue.

Earlier work implicates transcription factors GATA-4 and GATA-6 in murine adrenal function. We have now studied their expression during mouse and human adrenal development in detail. GATA-4 and GATA-6 mRNAs and protein are readily detectable from embryonic d 14 and gestational wk 19 onwards in the mouse and human adrenal cortex, respectively. In the postnatal adrenal, GATA-4 expression is down-regulated, whereas GATA-6 mRNA and protein continue to be expressed. To clarify the significance of GATA-4 for early adrenocortical development, Gata4-/- ES cells were injected into eight-cell-stage embryos derived from ROSA26 mice, a transgenic line expressing beta-galactosidase in all cell types, including the adrenocortical cells. The resultant chimeric embryos were stained with X-gal to discriminate ES cell- and host-derived tissue. Gata4-/- cells contributed to adrenocortical cells in these chimeras, and these cells also expressed GATA-6. Taken together, our findings suggest that GATA-6 expression is needed throughout adrenal development from fetal to adult age. GATA-4, on the other hand, may serve a role in the fetal adrenal gene regulation, although it is not essential for early adrenocortical differentiation.

Adrenal Glands↗