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David B Wilson

Publications and source records attributed to David B Wilson.

At least 37 records · Page 2Linked to original sources
PubMed2004

Recombinant expression and enzymatic characterization of PttCel9A, a KOR homologue from Populus tremula x tremuloides.

PttCel9A is a membrane-bound, family 9 glycosyl hydrolase from Populus tremula x tremuloides that is upregulated during secondary cell wall synthesis. The catalytic domain of PttCel9A, Delta(1-105)PttCel9A, was purified, and its activity was compared to TfCel9A and TfCel9B from Thermobifida fusca. Since aromatic amino acids involved in substrate binding at subsites -4, -3, and -2 are missing in PttCel9A, the activity of TfCel9A mutant enzymes W256S, W209A, and W313G was also investigated. Delta(1-105)PttCel9A hydrolyzed a comparatively narrow range of polymeric substrates, and the preferred substrate was (carboxymethyl)cellulose 4M. Moreover, Delta(1-105)PttCel9A did not hydrolyze oligosaccharides shorter than cellopentaose, whereas TfCel9A and TfCel9B hydrolyzed cellotetraose and cellotriose, respectively. These data suggest that the preferred substrates of PttCel9A are long, low-substituted, soluble cellulosic polymers. At 30 degrees C and pH 6.0, the kcat for cellohexaose of Delta(1-105)PttCel9A, TfCel9A, and TfCel9B were 0.023 +/- 0.001, 16.9 +/- 2.0, and 1.3 +/- 0.2, respectively. The catalytic efficiency (kcat/Km) of TfCel9B was 39% of that of TfCel9A, whereas the catalytic efficiency of Delta(1-105)PttCel9A was 0.04% of that of TfCel9A. Removing tryptophan residues at subsites -4, -3, and -2 decreased the efficiency of cellohexaose hydrolysis by TfCel9A. Mutation of W313 to G had the most drastic effect, producing a mutant enzyme with 1% of the catalytic efficiency of TfCel9A. The apparent narrow substrate range and catalytic efficiency of PttCel9A are correlated with a lack of aromatic amino acids in the substrate binding cleft and may be necessary to prevent excessive hydrolysis of cell wall polysaccharides during cell wall formation.

Amino Acid Sequence↗
PubMed2004

Kinetic studies of Thermobifida fusca Cel9A active site mutant enzymes.

Thermobifida fusca Cel9A-90, an unusual family 9 enzyme, is a processive endoglucanase containing a catalytic domain closely linked to a family 3c cellulose binding domain (Cel9A-68) followed by a fibronectin III-like domain and a family 2 cellulose binding domain. To study its catalytic mechanism, 12 mutant genes with changes in five conserved residues of Cel9A-68 were constructed, cloned, and expressed in Escherichia coli. The purified mutant enzymes were assayed for their activities on (carboxymethyl)cellulose, phosphoric acid-swollen cellulose, bacterial microcrystalline cellulose, and 2,4-dinitrophenyl beta-D-cellobioside. They were also tested for ligand binding, enzyme processivity, and thermostability. The results clearly show that E424 functions as the catalytic acid, D55 and D58 are both required for catalytic base activity, and Y206 plays an important role in binding, catalysis, and processivity, while Y318 plays an important role in binding of crystalline cellulose substrates and is required for processivity. Several amino acids located in a loop at the end of the catalytic cleft (T245-L251) were deleted from Cel9A-68, and this enzyme showed slightly improved filter paper activity and binding to BMCC but otherwise behaved like the wild-type enzyme. The FnIII-like domain was deleted from Cel9A-90, reducing BMCC activity to 43% of the wild type.

Actinomycetales↗
PubMed2004

Synergistic activity of Paenibacillus sp. BP-23 cellobiohydrolase Cel48C in association with the contiguous endoglucanase Cel9B and with endo- or exo-acting glucanases from Thermobifida fusca.

Cellobiohydrolase Cel48C from Paenibacillus sp. BP-23, an enzyme displaying limited activity on most cellulosic substrates, was assayed for activity in the presence of other bacterial endo- or exocellulases. Significant enhanced activity was observed when Cel48C was incubated in the presence of Paenibacillus sp. BP-23 endoglucanase Cel9B or Thermobifida fusca cellulases Cel6A and Cel6B, indicating that Cel48C acts synergistically with them. Maximum synergism rates on bacterial microcrystalline cellulose or filter paper were obtained with a mixture of Paenibacillus cellulases Cel9B and Cel48C, accompanied by T. fusca exocellulase Cel6B. Synergism was also observed in cell extracts from recombinant clone E. coli pUCel9-Cel48 expressing the two contiguous Paenibacillus cellulases Cel9B and Cel48C. The enhanced cellulolytic activity displayed by the cellulase mixtures assayed could be used as an efficient tool for biotechnological applications like pulp and paper manufacturing.

Actinomycetales↗
PubMed2004

Adrenocortical tumorigenesis in transgenic mice expressing the inhibin alpha-subunit promoter/simian virus 40 T-antigen transgene: relationship between ectopic expression of luteinizing hormone receptor and transcription factor GATA-4.

We have analyzed the ontogeny and putative mechanisms of transregulation of LH receptor (LHR) and transcription factor GATA-4, coexpressed during the adrenocortical tumorigenesis of prepubertally gonadectomized transgenic (TG) mice expressing the inhibin alpha-subunit promoter/simian virus 40 T-antigen (inhalpha/Tag) transgene. The onset of adrenal LHR mRNA and protein expression coincided with that of GATA-4 at the age of 4 months and preceded the appearance of discernible adrenal tumors at about 6 months. In situ hybridization and double-immunohistochemistry demonstrated colocalization of the LHR and GATA-4 messages and proteins in the adrenal cortex. A GATA-4 expression plasmid cotransfected with a murine LHR promoter-driven luciferase reporter plasmid, containing a consensus GATA-binding site, induced a dose-dependent significant transactivation of the LHR promoter in nonsteroidogenic human embryonic kidney 293, steroidogenic murine mLTC-1 Leydig cells and in murine adrenal Y-1 cells. The Calpha1 cells derived from an Inhalpha/Tag adrenal tumor did not show this response, apparently due to their high endogenous GATA-4 expression. However, an additional link between GATA-4 and LHR in Calpha1 cells was provided upon the LH/human chorionic gonadotropin stimulation of LHR promoter activity; mutations or deletion of the consensus GATA-4 binding site of the LHR promoter abolished this transactivation. EMSAs further proved GATA-4 binding to the putative consensus GATA recognition site. Our results demonstrate direct interrelationship between LHR and GATA-4 expression during adrenocortical tumorigenesis of the inhalpha/Tag mice. There is apparently a positive and reciprocal feed-forward amplification link between LHR and GATA-4 expression. This mechanism gradually and in synergy with Tag expression leads to formation of the LH-dependent adrenocortical tumors.

Adrenal Cortex Neoplasms↗
PubMed2004

Red-green-blue model.

We experimentally study the red-green-blue model, which is a system of loops obtained by superimposing three dimer coverings on offset hexagonal lattices. We find that when the boundary conditions are "flat," the red-green-blue loops are closely related to stochastic Loewner evolution with parameter kappa=4 (SLE4) and double-dimer loops, which are the loops formed by superimposing two dimer coverings of the Cartesian lattice. But we also find that the red-green-blue loops are more tightly nested than the double-dimer loops. We also investigate the two-dimensional minimum spanning tree, and find that it is not conformally invariant.

Journal Article↗
PubMed2004

GATA-4, GATA-5, and GATA-6 activate the rat liver fatty acid binding protein gene in concert with HNF-1alpha.

Transcriptional regulation by GATA-4, GATA-5, and GATA-6 in intestine and liver was explored using a transgene constructed from the proximal promoter of the rat liver fatty acid binding protein gene (Fabpl). An immunohistochemical survey detected GATA-4 and GATA-6 in enterocytes, GATA-6 in hepatocytes, and GATA-5 in neither cell type in adult animals. In cell transfection assays, GATA-4 or GATA-5 but not GATA-6 activated the Fabpl transgene solely through the most proximal of three GATA binding sites in the Fabpl promoter. However, all three factors activated transgenes constructed from each Fabpl site upstream of a minimal viral promoter. GATA factors interact with hepatic nuclear factor (HNF)-1alpha, and the proximal Fabpl GATA site adjoins an HNF-1 site. GATA-4, GATA-5, or GATA-6 bounded to HNF-1alpha in solution, and all cooperated with HNF-1alpha to activate the Fabpl transgene. Mutagenizing all Fabpl GATA sites abrogated transgene activation by GATA factors, but GATA-4 activated the mutagenized transgene in the presence of HNF-1alpha. These in vitro results suggested GATA/HNF-1alpha interactions function in Fabpl regulation, and in vivo relevance was determined with subsequent experiments. In mice, the Fabpl transgene was active in enterocytes and hepatocytes, a transgene with mutagenized HNF-1 site was silent, and a transgene with mutagenized GATA sites had identical expression as the native transgene. Mice mosaic for biallelic Gata4 inactivation lost intestinal but not hepatic Fabpl expression in Gata4-deficient cells but not wild-type cells. These results demonstrate GATA-4 is critical for intestinal gene expression in vivo and suggest a specific GATA-4/HNF-1alpha physical and functional interaction in Fabpl activation.

Animals↗
PubMed2004

Cloning, characterization and phylogenetic relationships of cel5B, a new endoglucanase encoding gene from Thermobifida fusca.

Thermobifida fusca, a thermophilic, aerobic, cellulolytic bacterium has a highly complex cellulase system comprising three endoglucanases, two exoglucanases and one processive endoglucanase. Zymogram analysis indicated that additional cellulases may exist in T. fusca strain TM51, therefore a TM51 expression library was prepared in Streptomyces lividans TK24 and screened for hydrolases. A new endoglucanase gene, named Tf cel5B, was identified. Heterologous Cel5B, produced in S. lividans, had temperature and pH optima of 77 degrees C and 8.2, respectively and retained more than 60% of its activity after 24 h incubation at 60 degrees C. Domain analysis revealed an N-terminal catalytic domain with homology to known endoglucanases in family GH5 and a C-terminal cellulose binding module III domain (CBD). Comparing the domain structures of all seven known T. fusca cellulases showed, that the cellulase system of this organism consists of pairs of enzymes from the same GH family, including Cel5A--Cel5B, Cel6A--Cel6B and Cel9A--Cel9B plus a single family GH48 enzyme (Cel48A). Furthermore, the catalytic and substrate binding domains of enzymes, belonging to the same GH family were arranged in opposite orientations. Phylogenetic comparisons of the catalytic domain sequences of the T. fusca cellulases to other family GH5, GH6, GH9 and GH48 cellulases of bacterial origin revealed that the enzyme pairs in the same GH family are not closely related to each other, instead they showed significant similarities to various cellulase enzymes from taxonomically distinct organisms. Therefore, the complex and highly efficient cellulase system of T. fusca seems to be evolved as a result of horizontal gene transfers rather than gene duplication events.

Actinomycetales↗
PubMed2004

Studies of Thermobifida fusca plant cell wall degrading enzymes.

I have been studying the Thermobifida fusca cellulose degrading proteins for the past 25 years. In this period, we have purified and characterized the six extracellular cellulases and an intracellular beta- glucosidase used by T. fusca for cellulose degradation, cloned and sequenced the structural genes encoding these enzymes, and helped to determine the 3-dimensional structures of two of the cellulase catalytic domains. This research determined the mechanism of a novel class of cellulase, family 9 processive endoglucanases, and helped to show that there were two types of exocellulases, ones that attacked the non-reducing ends of cellulose and ones that attacked the reducing ends. It also led to the sequencing of the T. fusca genome by the DOE Joint Genome Institute. We have studied the mechanisms that regulate T. fusca cellulases and have shown that cellobiose is the inducer and that cellulase synthesis is repressed by any good carbon source. A regulatory protein (CelR) that functions in the induction control has been purified, characterized, and its structural gene cloned and expressed in E. coli. I have also carried out research on two rumen bacteria, Prevotella ruminicola and Fibrobacter succinogenes, in collaboration with Professor James Russell, helping to arrange for the genomes of these two organisms to be sequenced by TIGR, funded by a USDA grant to the North American Consortium for Genomics of Fibrolytic Ruminal Biology.

Actinomycetales↗
PubMed2004

Neutropenic enterocolitis as a presenting complication of acute lymphoblastic leukemia: an unusual case marked by delayed perforation of the descending colon.

Neutropenic enterocolitis (NE) is a life-threatening complication most commonly seen in patients receiving intensive chemotherapy for acute leukemia. The condition usually affects the terminal ileum, cecum, or ascending colon. In rare instances, NE may occur before the initiation of chemotherapy or involve more distal bowel. The authors report the case of a 2-year-old girl who had NE affecting the descending colon as a presenting complication of acute lymphoblastic leukemia. Despite aggressive medical interventions, including granulocyte infusions, she had a delayed bowel perforation that was managed successfully with surgery. This case highlights the challenges of treating patients who have NE as an initial manifestation of acute leukemia.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed2004

Mesenteric artery disease in the elderly.

PURPOSE: The purpose of this study was to estimate the population-based prevalence of mesenteric artery stenosis (MAS) and occlusion among independent elderly Americans. METHOD: As part of an ancillary investigation to the Cardiovascular Health Study (CHS), participants in the Forsyth County, NC cohort had visceral duplex sonography of the celiac arteries and superior mesenteric arteries (SMAs). Critical MAS was defined by celiac peak systolic velocity >or=2.0 m/s and/or SMA peak systolic velocity >or=2.7 m/s. Occlusion of either vessel was defined by lack of a Doppler-shifted signal within the imaged artery. Demographic data, blood pressures, and blood lipid levels were collected as part of the baseline CHS examination. Participants' weights were measured at baseline and before the duplex exam. Univariate tests of association were performed with two-way contingency tables, Student t tests, and Fisher exact tests. Multivariate associations were examined with logistic regression analysis. RESULTS: A total of 553 CHS participants had visceral duplex sonography technically adequate to define the presence or absence of MAS. The study group had a mean age of 77.2 +/- 4.9 years and comprised 63% women and 37% men. Participant race was 76% white and 23% African-American. Ninety-seven participants (17.5%) had MAS. There was no significant difference in age, race, gender, body mass index, blood pressure, cholesterol, or low-density lipoproteins for participants with or without MAS. Forward stepwise variable selection found renal artery stenosis (P =.008; odds ratio [OR], 2.85; 95% confidence interval [CI], 1.31, 6.21) and high-density lipoprotein >40 (P =.02; OR, 3.03; 95% CI, 1.17, 7.81) significantly associated with MAS in a multivariate logistic regression model. Eighty-three of the 97 participants with MAS (15.0% of the cohort) had isolated celiac stenosis. Seven participants (1.3% of the cohort) had combined celiac and SMA stenosis. Five participants (0.9% of the cohort) had isolated SMA stenosis. Two participants (0.4% of the cohort) had celiac occlusion. Considering all participants with MAS, there was no association with weight change. However, SMA stenosis and celiac occlusion demonstrated an independent association with annualized weight loss (P =.028; OR, 1.54; 95% CI, 1.05, 2.26) and with renal artery stenosis (P =.001; OR, 9.48; 95% CI, 2.62, 34.47). CONCLUSION: This investigation provides the first population-based estimate of the prevalence of MAS among independent elderly Americans. MAS existed in 17.5% of the study cohort. The majority had isolated celiac disease. SMA stenosis and celiac artery occlusion demonstrated a significant and independent association with weight loss and concurrent renal artery disease.

Age Factors↗
PubMed2004

Surgical management of renal artery aneurysms.

PURPOSE: This retrospective review describes the surgical management and clinical outcome for renal artery aneurysms (RAAs) in 62 consecutive patients. METHODS: From January 1987 through July 2003, 804 patients had operative renal artery (RA) repair involving 1206 kidneys at our center. A subgroup of 62 patients (42 women, 20 men; mean age 46 +/- 18 years) received repair of 72 RAAs. Demographic data, comorbidity, and surgical technique were examined. Blood pressure and renal function response were determined. Patency of repair was evaluated by renal duplex sonography. Primary patency and patient survival were estimated by life-table methods. Tests of association were performed using chi(2) and the Student t tests. RESULTS: Seventy-two RAs were repaired for RAA with a mean diameter of 2.6 cm (range, 1.3 to 5.5 cm). Bilateral RAAs were present in 21 patients. Associated conditions included fibromuscular dysplasia, atherosclerosis, and arteritis in 54%, 35%, and 7%, respectively. Hypertension was present in 89% (mean blood pressure, 171 +/- 35/95 +/- 19 mm Hg; mean medications, 2.2 +/- 1.2 drugs) and renal insufficiency was present in 8% (mean serum creatinine, 1.9 +/- 0.6 mg/dL). RAA repair included bypass (67%), aneurysmorrhaphy (15%), or a combination (17%). One planned nephrectomy (1%) was performed for un-reconstructable disease. Branch RA reconstruction in 78% used ex vivo cold perfusion in 50%, in situ cold perfusion in 29%, and warm in situ repair in 21%. Of 9 bilateral RAA repairs, 7 (78%) were staged and 2 (22%) were simultaneous. Combined aortic reconstruction was required in 6 (10%) patients. Perioperative death occurred in 1 patient (1.6%), and significant morbidity was observed in 8 patients (12%). Hypertension was considered improved in 54%, cured in 21%, and unchanged in 25% at mean follow-up of 48 months (range, 1-156 months). Among patients with renal insufficiency, renal function was improved in 3 (60%), unchanged in 1 (20%), and declined in 1 (20%). Follow-up patency (mean, 33 months; range, 1-118 months) was determined for 64 (91%) RA reconstructions. Product-limit estimate of primary patency at 48 months was 96%. Product-limit estimate of survival was 91% at 120 months. CONCLUSION: RAAs were repaired with low morbidity and mortality. Complex branch RAA repair using cold perfusion preservation and ex vivo techniques resulted in no unplanned nephrectomy, with an estimated primary patency of 96% at 48 months. Beneficial blood pressure response was observed in the majority of hypertensive patients. These results support selective surgical management of RAA.

Adult↗
PubMed2004

Dyskeratosis congenita and telomerase.

PURPOSE OF REVIEW: Dyskeratosis congenita, a rare condition characterized by mucocutaneous abnormalities and bone marrow failure, is caused by inherited defects in the telomerase complex. Autosomal dominant dyskeratosis congenita is associated with mutations in the RNA component of telomerase, hTERC, while X-linked dyskeratosis congenita is due to mutations in the gene encoding dyskerin, a protein implicated in both telomerase function and ribosomal RNA processing. This review highlights recent research on dyskeratosis congenita and its relevance to other fields, including cancer and aging. RECENT FINDINGS: Newly developed animal models suggest that defects in ribosomal RNA processing contribute to the phenotype of X-linked dyskeratosis congenita. Bone marrow dysfunction may be the first manifestation of dyskeratosis congenita in children, and hTERC mutations have been detected in a subset of patients presumed to have idiopathic aplastic anemia or myelodysplastic syndrome. In vitro studies suggest that hTERC mutations associated with dyskeratosis congenita or aplastic anemia either impair the specific activity of telomerase, decrease hTERC stability, or disrupt assembly of the telomerase complex. Recent clinical reports suggest that nonmyeloablative conditioning regimens afford better outcomes in patients with dyskeratosis congenita who require hematopoietic stem cell transplantation. SUMMARY: Studies of dyskeratosis congenita have shed light on the pathobiology of aplastic anemia and other forms of bone marrow dysfunction. It seems likely that mutations in other genes involved in telomere maintenance will be linked to bone marrow failure or other human diseases. Genetic testing for occult dyskeratosis congenita may be warranted in selected patients with aplastic anemia or myelodysplastic syndrome, as this may impact the choice of therapies.

Animals↗
PubMed2004

Purification and characterization of Thermobifida fusca xylanase 10B.

Thermobifida fusca grows well on cellulose and xylan, and produces a number of cellulases and xylanases. The gene encoding a previously unstudied endoxylanase, xyl10B, was overexpressed in E. coli, and the protein was purified and characterized. Mature Xyl10B is a 43-kDa glycohydrolase with a short basic domain at the C-terminus. It has moderate thermostability, maintaining 50% of its activity after incubation for 16 h at 62 degrees C, and is most active between pH 5 and 8. Xyl10B is produced by growth of T. fusca on xylan or Solka Floc but not on pure cellulose. Mass spectroscopic analysis showed that Xyl10B produces xylobiose as the major product from birchwood and oat spelts xylan and that its hydrolysis products differ from those of T. fusca Xyl11A. Xyl10B hydrolyzes various p-nitrophenyl-sugars, including p-nitrophenyl alpha-D-arabinofuranoside, p-nitrophenyl-beta-D-xylobioside, p-nitrophenyl-beta-D-xyloside, and p-nitrophenyl-beta-D-cellobioside. Xyl11A has higher activity on xylan substrates, but Xyl10B produced more reducing sugars from corn fiber than did Xyl11A.

Actinomycetales↗
PubMed2004

Characterization and comparison of metal accumulation in two Escherichia coli strains expressing either CopA or MntA, heavy metal-transporting bacterial P-type adenosine triphosphatases.

MntA from Lactobacillus plantarum and copA from Enterococcus hirae both encode membrane proteins that are members of the P-type family of adenosine triphosphatases (ATPases). Both transporters act as metal importers to take up nutritionally required substrates; MntA translocates Mn(II) and CopA translocates Cu(I). Both ATPases can also translocate secondary substrates, Cd(II) and Ag(I), respectively. Although functionally and sequentially similar, these ATPases differ in several key residues and in their membrane topologies. The bioaccumulation properties of these two proteins were examined by coexpressing the transporters with overexpressed metallothionein in Escherichia coli cells, a system that has previously shown high levels of substrate-specific uptake. Both strains exhibited rapid metal accumulation, both saturated at around 50 microM metal, and both displayed temperature-sensitive uptake. However, the transporters responded differently when external conditions were varied; MntA displayed increased sensitivity to ionic strength, while CopA was more pH sensitive and more inhibited by chelating agents. The differences in accumulation are likely owing to structural differences in the transmembrane region of these two ATPases.

Adenosine Triphosphatases↗
PubMed2004

Amifostine-induced fever: case report and review of the literature.

Amifostine, also known as WR-2721 (S-2-(3-aminopropylamino)-ethylphosphorothioic acid), is an organic thiophosphate cytoprotective agent used to reduce the frequency of severe xerostomia in patients with cancer undergoing postoperative radiation of the head and neck. A 56-year-old Caucasian man who received concomitant chemotherapy and radiation for head and neck cancer developed fever concurrent with the administration of amifostine. To our knowledge, this is the first case report that demonstrates the occurrence of fever with low-dose amifostine therapy without the manifestation of accompanying rash or hypotension. Patients receiving amifostine who develop only fever should be evaluated for an adverse drug reaction, as well as for sepsis and fevers of neutropenia, and it may be necessary to discontinue the drug. Recognition of amifostine as the cause of this adverse event may prevent the cost and inconvenience of a hospital admission.

Amifostine↗
PubMed2004

3 steps to profitable managed care contracts.

An effective managed care contract negotiation strategy should be founded on: Internal analysis that compares performance of your current managed care contracts in terms of volume versus discount rates and volume versus profit. External analysis that compares your current contracts with those of competitors and assesses prevailing market rates, contract language and provisions, and premium trends Payment performance analysis that identifies payment promptness and accuracy and associated penalty dollars for each payer.

Contracts↗
PubMed2003

Winding angle variance of Fortuin-Kasteleyn contours.

The variance in the winding number of various random fractal curves, including the self-avoiding walk, the loop-erased random walk, contours of Fortuin-Kastelyn clusters, and stochastic Loewner evolution, has been studied by numerous researchers. Usually the focus has been on the winding at the end points. We measure the variance in winding number at typical points along the curve. More generally, we study the winding at points where k strands come together, and some adjacent strands may be conditioned not to hit each other. The measured values are consistent with an interesting conjecture.

Journal Article↗