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David A Williams

Publications and source records attributed to David A Williams.

At least 91 records · Page 5Linked to original sources
PubMed2004

Purification and partial characterization of feline alpha1-proteinase inhibitor (falpha1-PI) and the development and validation of a radioimmunoassay for the measurement of falpha1-PI in serum.

Alpha(1)-proteinase inhibitor (alpha(1)-PI) of the domestic cat (Felis catus) was purified from serum and a radioimmunoassay (RIA) for the measurement of feline alpha(1)-PI concentration in serum was developed and validated. Feline alpha(1)-PI (falpha(1)-PI) was isolated using ammonium sulfate precipitation, anion-exchange, size-exclusion, ceramic hydroxyapatite, and hydrophobic interaction chromatography. The molecular weight of falpha(1)-PI was estimated at 57,000 and the relative molecular mass (M(r)) was determined to be approximately 54.5 kDa. Isoelectric focusing revealed four bands with isoelectric points (pI) between 4.3 and 4.5. The N-terminal amino acid sequence of the first 19 residues was Glu-Gly-Leu-Gln-Gly-Ala-Ala-Val-Gln-Glu-Thr-Val-Ala-Ser-Gln-His-Asp-Gln-Glu. Antiserum against feline alpha(1)-PI was raised in rabbits. Tracer was produced by iodination ((125)I) of feline alpha(1)-PI using the chloramine T method. A radioimmunoassay was established and validated by determination of sensitivity, dilutional parallelism, spiking recovery, intra-assay variability, and inter-assay variability. A control range for serum feline alpha(1)-PI concentration was established from 50 healthy cats using the central 95th percentile. The sensitivity of the assay was 0.042 mg/ml. Observed to expected ratios for serial dilutions ranged from 105% to 141.18% for four different serum samples at dilutions of 1 in 35,000, 1 in 70,000, 1 in 140,000 and 1 in 280,000. Observed to expected ratios for spiking recovery ranged from 88.14% to 152.17% for four different serum samples and five different spiking concentrations. Coefficients of variation for four different serum samples were 4.57%, 6.45%, 8.52%, and 4.27% for intra-assay variability and 6.88%, 9.57%, 7.44%, and 9.94% for inter-assay variability. The reference range was established as 0.25-0.6 mg/ml. In summary, feline alpha(1)-PI was successfully purified from serum using a rapid and efficient method. The radioimmunoassay described here is sensitive, linear, accurate, precise, and reproducible and will facilitate further studies of the physiological or potential pathological role of alpha(1)-PI in cats.

Amino Acid Sequence↗
PubMed2004

Chromosome-specific microsatellite multiplex sets for linkage studies in the domestic dog.

To expedite linkage studies and positional cloning efforts in the dog, Minimal Screening Set 2 (MSS-2) of 327 canine microsatellite markers has been multiplexed into chromosome-specific panels. MSS-2 provides 9 Mb coverage of the canine genome with no gaps larger than 17.1 Mb and is the most recent and comprehensive set of microsatellites available for whole-genome scans. Markers were labeled with fluorescent dyes based on locations and expected product sizes to facilitate the multiplexing of a maximum number of markers for each chromosome. All markers are amplified using a single thermal cycling program and PCR mix and are optimized for resolution on an ABI 3100 genetic analyzer. Sixty-nine chromosome-specific panels were created by coamplification of a maximum number of markers and subsequent coloading of the remaining markers.

Animals↗
PubMed2004

Chance or necessity? Insertional mutagenesis in gene therapy and its consequences.

Recently, unusual forms of leukemias have developed as complications following retroviral transfer of potentially therapeutic genes into hematopoietic cells. A crucial component in the pathogenesis of these complications was the upregulation of a cellular proto-oncogene by random insertion of the retroviral gene transfer vector. These findings have great implications for the genetic manipulation of somatic stem cells in medicine. This review discusses the extent to which the random oncogene activation may have required disease-specific stimuli of the transgene and the hematopoietic milieu to become leukemogenic. Based on these considerations, we propose approaches to risk prediction and prevention.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Protein-losing enteropathy in dogs is associated with decreased fecal proteolytic activity.

BACKGROUND: Measurement of proteolytic activity in feces is a traditional method for the diagnosis of exocrine pancreatic insufficiency (EPI). A drawback of this method is the occurrence of falsely low results that may lead to a false-positive diagnosis of EPI. We hypothesized that intestinal loss of serum proteinase inhibitors in protein-losing enteropathy (PLE) may inhibit fecal proteolytic activity and be a potential source of false low results. OBJECTIVE: The objective of this study was to determine the effect of PLE on fecal proteolytic activity in dogs. METHODS: Fecal proteolytic activity was measured using a radial diffusion casein digestion assay in 12 samples from 4 clinically healthy control dogs and 30 samples from 16 dogs with PLE. Gastrointestinal protein loss was assessed using an ELISA to determine fecal canine alpha 1-proteinase inhibitor concentration. The relationship between the concentration of canine alpha 1-proteinase inhibitor in the feces and the diameter cleared in the casein digestion assay was determined. The mean clearing diameter was compared between control dogs and dogs with PLE. RESULTS: A significant negative correlation was observed between fecal canine alpha1-proteinase inhibitor concentration and casein clearing diameter (P <.001, Pearson r =.6317, r2 =.3999). Mean clearing diameter was significantly lower in dogs with PLE than in control dogs (12.63 vs 16.83 mm, P <.001, two-tailed Student's t-test). CONCLUSION: Increased fecal loss of alpha1-proteinase inhibitor in dogs with PLE is associated with a significant decrease in fecal proteolytic activity and may result in a false positive diagnosis of EPI.

Animals↗
PubMed2004

Application of molecular fingerprinting for qualitative assessment of small-intestinal bacterial diversity in dogs.

The aims of this study were to evaluate the use of molecular fingerprinting for assessment of bacterial diversity in canine duodenal juice and to evaluate the variation in the small intestinal microflora at repeated sampling. Two groups of dogs were used. Duodenal juice was collected from eight dogs euthanized for an unrelated project (group 1). Duodenal juice was also collected endoscopically from six dogs at weekly intervals for a total of 3 weeks (group 2). The variable V6-V8 region of bacterial 16S ribosomal DNA was amplified, and PCR amplicons separated by denaturing gradient gel electrophoresis (DGGE). The reproducibility of DGGE profiles and variations in bacterial diversity between dogs were evaluated by comparing similarity indices (Dice's coefficient; 100% represents complete identity) of DGGE profiles from group 1 dogs. Weekly variations in the flora of the small intestine were evaluated by comparison of DGGE profiles from different time points within the same individuals in group 2. The mean (+/- standard deviation) similarity of DGGE profiles of duodenal juice between the dogs in group 1 was 38.3 +/- 15.7% (range, 12.5 to 76.65%). There was a significantly higher variation in DGGE profiles between different dogs than between duplicates obtained from the same dog (P < 0.0001). DGGE profiles from samples collected at different time points varied within individuals, possibly due to variation over time or slight variation in sampling location. DGGE profiles indicate that dogs have a highly diverse microflora of the small intestine, with marked differences between individual dogs.

Animals↗
PubMed2004

Lenti in red: progress in gene therapy for human hemoglobinopathies.

Hemoglobinopathies are caused by abnormal structure or synthesis of hemoglobin chains and represent serious monogenic disorders. A new study demonstrates that lentiviral vectors can express clinically relevant levels of human transgenic beta-globin in red cells of xenografted mice. While some safety concerns must be addressed, this study is an important step toward potential clinical trials of gene therapy for hemoglobinopathies.

Animals↗
PubMed2004

Purification and partial characterization of feline pepsinogen.

OBJECTIVE: To purify and partially characterize feline pepsinogen (fPG) from the gastric mucosa and compare fPG with PGs of other species. SAMPLE POPULATION: Stomachs of 6 cats. PROCEDURE: A crude protein extract was prepared from the gastric mucosa of feline stomachs. Feline PG A was purified by ammonium sulfate precipitation, weak-anion-exchange chromatography, size-exclusion chromatography, and strong-anion exchange chromatography. Partial characterization consisted of estimation of molecular weights (MWs) and isoelectric points, N-terminal amino acid sequencing, and investigation of susceptibility to pepstatin inhibition. RESULTS: Several fPG A-group isoforms were identified. The MWs of the isoforms ranged from 37,000 to 44,820. Isoelectric points were all < pH 3.0. The proteolytic activity of the activated PGs was inhibited completely by pepstatin in a range of equimolar to 10-fold molar excess. The specific absorbance of fPG A was 1.29. The N-terminal amino acid sequence of the first 25 residues of the predominant fPG A7 had 75%, 72%, 64%, and 56% homology with PG A of dogs, rabbits, cattle, and humans, respectively. Sequences of 4 other fPG A-group isoforms were similar to fPG A7. All isoforms were immunologically cross-reactive with sheep anti-fPG A7 antiserum. CONCLUSIONS AND CLINICAL RELEVANCE: PG A is the only identified type of PG in cats and, similar to pg in other species, comprises multiple isoforms. The availability of fPG A may be used to facilitate the development of an immunoassay to quantify serum fPG A as a potential marker for gastric disorders in cats.

Amino Acid Sequence↗
PubMed2004

Effects of exchange of dietary medium chain triglycerides for long-chain triglycerides on serum biochemical variables and subjectively assessed well-being of dogs with exocrine pancreatic insufficiency.

OBJECTIVE: To test the hypothesis that exchange of medium-chain triglycerides (MCTs) for long-chain triglycerides (LCTs) in the diet of dogs with well-managed exocrine pancreatic insufficiency (EPI) changes serum biochemical variables and to subjectively assess the well-being of dogs with EPI in response to experimental diets. ANIMALS: 21 dogs with EPI and 6 healthy control dogs. PROCEDURE: The effects of 3 diets containing 0%, 16%, or 35% of the total fat content as MCTs were examined in a randomized controlled double-blind crossover trial. The 3 diets were fed for 12 weeks each. Dietary effects were evaluated by both subjective and objective variables. RESULTS: Analysis of subjective data revealed no significant difference in appetite, attitude, drinking behavior, volume of feces, defecation frequency, color of feces, consistency of feces, flatulence, or borborygmus among dogs fed the 3 experimental diets. A high MCT content in the diet was associated with significantly higher serum vitamin E, cholesterol, triglyceride, retinyl stearate, retinyl palmitate, and total vitamin A concentrations in dogs with EPI and significantly higher serum vitamin E concentrations in control dogs, compared with low MCT content. High MCT content in the diet was also associated with significantly lower concentrations of serum linoleic acid (C18:2[n-6]) in dogs with EPI and in control dogs, compared with low MCT content. CONCLUSIONS AND CLINICAL RELEVANCE: A high MCT content in the diet leads to increases in serum concentrations of cholesterol and certain fat-soluble vitamins. However, no effect was found on the subjective well-being of the dogs as evaluated by their owners.

Animals↗
PubMed2004

Kinetic analysis of demethylation of 13C-aminopyrine in healthy dogs.

OBJECTIVE: To describe the kinetics of demethylation of 13C-aminopyrine in healthy dogs for use in determining the most appropriate time for collection of blood samples for a 13C-aminopyrine demethylation blood test for evaluation of hepatic function. ANIMALS: 9 healthy dogs. PROCEDURES: A 2-mL baseline blood sample was collected into an evacuated heparinized tube, and 13C-aminopyrine was administered to each dog (2 mg/kg, IV). Additional 2-mL blood samples were collected 15, 30, 45, 60, 75, 90, 105, 120, 135, 150, 180, 240, 300, and 360 minutes after 13C-aminopyrine administration. The CO2 was extracted from blood samples by addition of a strong acid, and the percentage dose of 13CO2 (PCD) in the extracted gas was determined by fractional mass spectrometry. RESULTS: No dogs had gross evidence of adverse effects, and all had an increase in PCD after IV administration of 13C-aminopyrine. The PCD had the least variability among 5 variables used to evaluate hepatic demethylating capacity. Peak PCD was detected at 30 minutes in 1 dog, 45 minutes in 5 dogs, 60 minutes in 2 dogs, and 75 minutes in 1 dog. The mean PCD for the 9 dogs peaked at 45 minutes after 13C-aminopyrine administration. CONCLUSIONS AND CLINICAL RELEVANCE: PCD appears to be the preferable variable for evaluation of hepatic demethylating capacity. Intravenous administration of 13C-aminopyrine leads to a consistent increase in PCD. Mean PCD peaked 45 minutes after administration, suggesting that blood sample collection 45 minutes after 13C-aminopyrine administration may be appropriate for use in estimating hepatic demethylating capacity.

Aminopyrine↗
PubMed2004

Evaluation of urine sucrose concentration for detection of gastric ulcers in horses.

OBJECTIVE: To evaluate the use of sucrose permeability testing to detect ulcers in the gastric squamous mucosa of horses. ANIMALS: 13 adult horses ranging from 5 to 19 years of age. PROCEDURE: Following induction of gastric ulcers by intermittent feed deprivation, horses underwent sucrose permeability testing (administration of sucrose by nasogastric intubation followed by collection of urine at 2 and 4 hours after intubation) and gastric endoscopy. Squamous ulcers were assigned a severity score (range, 0 to 3) by use of an established scoring system. Horses were subsequently administered omeprazole for 21 days, and sucrose testing and endoscopy were repeated. Pair-wise comparisons of urine sucrose concentration were made between horses with induced ulcers before and after omeprazole treatment. Urine sucrose concentrations also were compared on the basis of ulcer severity score. RESULTS: Urine sucrose concentrations and ulcer severity scores were significantly higher in horses with induced ulcers before omeprazole treatment than after treatment. Urine sucrose concentrations were significantly higher for horses with ulcer severity scores > 1. Use of a cut-point value of 0.7 mg/mL revealed that the apparent sensitivity and specificity of sucrose permeability testing to detect ulcers with severity scores > 1 was 83% and 90%, respectively. Results were similar after adjusting sucrose concentrations for urine osmolality. CONCLUSIONS AND CLINICAL RELEVANCE: Urine sucrose concentration appears to be a reliable but imperfect indicator of gastric squamous ulcers in horses. Sucrose permeability testing may provide a simple, noninvasive test to detect and monitor gastric ulcers in horses.

Animals↗
PubMed2004

Development and analytical validation of an enzyme linked immunosorbent assay for the measurement of canine gastric lipase immunoreactivity in serum.

The objective of this study was to develop and analytically validate an enzyme linked immunosorbent assay (ELISA) for measurement of canine gastric lipase immunoreactivity (cGLI). A sandwich ELISA was developed using canine gastric lipase (cGL) purified from canine stomachs and polyclonal antibodies directed against cGL, raised in rabbits and purified by affinity chromatography. The assay was validated by determination of sensitivity, working range, linearity, accuracy, precision, reproducibility, and the upper limit of the control range by determining the 97.5th percentile of serum cGLI concentration in 74 healthy canines. Sensitivity and working range in serum were 200 ng/L and 200 to 39 160 ng/L, respectively. Observed to expected ratios for dilutional parallelism for 3 serum samples and 3 dilutions ranged from 86.1% to 244.2% (mean +/- standard deviation [s]; 125.4% +/- 48.2%). Observed to expected ratios for spiking recoveries for 3 serum samples and 6 spiking concentrations ranged from 66.4% to 152.5% (mean +/- s; 104.5% +/- 22.9%). Intra-assay and interassay variabilities for 3 different serum samples were 25.5%, 9.4%, and 13.4% and 26.0%, 17.2%, and 14.4%, respectively. The upper limit of the control range for serum cGLI was 662 ng/L. We concluded that the ELISA for cGLI described here is highly sensitive and shows a wide working range. However, the validation characteristics for this assay are suboptimal and below values of approximately 2.000 ng/L the assay is more semiquantitative in nature. Despite its limitations, whether this assay is useful for the diagnosis of canine gastric disorders remains to be determined.

Animals↗
PubMed2004

Development and analytical validation of a radioimmunoassay for the measurement of feline pancreatic lipase immunoreactivity in serum.

Pancreatitis is recognized as an important cause for morbidity and mortality in cats, but diagnosis remains difficult in many cases. As a first step in trying to identify a better diagnostic tool for feline pancreatitis the objective of this project was to develop and analytically validate a radioimmunoassay for the measurement of feline pancreatic lipase immunoreactivity (fPLI). Feline pancreatic lipase (fPL) was purified from pancreatic tissue and antiserum against fPL was raised in rabbits. Tracer was produced by iodination of fPL using the chloramine T method. A radioimmunoassay was established and analytically validated by determination of sensitivity, dilutional parallelism, spiking recovery, intra-assay variability, and interassay variability. A control range for fPLI in cat serum was established from 30 healthy cats using the central 95th percentile. The sensitivity of the assay was 1.2 microg/L. Observed to expected ratios for serial dilutions ranged from 98.8% to 164.3% for 3 different serum samples. Observed to expected ratios for spiking recovery ranged from 76.9% to 147.6% for 3 different serum samples. Coefficients of variation for intra- and interassay variability for 4 different serum samples were 10.1%, 4.5%, 2.2%, and 3.9% and 24.4%, 15.8%, 16.6%, and 21.3%, respectively. A reference range for fPLI was established as 1.2 to 3.8 microg/L. We conclude that the assay described is sensitive, accurate, and precise with limited linearity in the lower and limited reproducibility in the lower and higher end of the working range. Further studies to evaluate the clinical usefulness of this assay are needed and in progress.

Animals↗
PubMed2004

Evaluation of the cationic trypsinogen gene for potential mutations in miniature schnauzers with pancreatitis.

The purpose of this study was to evaluate the cationic trypsinogen gene in miniature schnauzers for possible mutations. Genetic mutations have been linked with hereditary pancreatitis in humans. Four miniature schnauzers were selected on the basis of a clinical history of pancreatitis. One healthy miniature schnauzer and 1 healthy mixed breed canine were enrolled as controls. DNA was extracted from these canines using a commercial kit. Primers were designed to amplify the entire canine cationic trypsinogen cDNA sequence. A polymerase chain reaction (PCR) was performed and products were purified and sequenced. All sequences were then compared. The healthy control canine, a healthy miniature schnauzer, and the 4 miniature schnauzers with pancreatitis showed identical sequences of the cationic trypsinogen gene to the published sequence. We conclude that, in contrast to humans with hereditary pancreatitis, mutations of the cationic trypsinogen gene do not play a major role in the genesis of pancreatitis in the miniature schnauzer.

Amino Acid Sequence↗
PubMed2003

In vivo selection of human hematopoietic cells in a xenograft model using combined pharmacologic and genetic manipulations.

Strategies that increase the ability of human hematopoietic stem and progenitor cells to repair alkylator-induced DNA damage may prevent the severe hematopoietic toxicity in patients with cancer undergoing high-dose alkylator therapy. In the context of genetic diseases, this approach may allow for selection of small numbers of cells that would not otherwise have a favorable growth advantage. No studies have tested this approach in vivo using human hematopoietic stem and progenitor cells. Human CD34(+) cells were transduced with a bicistronic oncoretrovirus vector that coexpresses a mutant form of O(6)-methylguanine DNA methyltransferase (MGMT(P140K)) and the enhanced green fluorescent protein (EGFP) and transplanted into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Mice were either not treated or treated with O(6)-benzylguanine (6BG) and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). At 8-weeks postinjection, a 2- to 8-fold increase in the percentage of human CD45(+)EGFP(+) cells in 6BG/BCNU-treated versus nontreated mice was observed in the bone marrow and was associated with increased MGMT(P140K)-repair activity. Functionally, 6BG/BCNU-treated mice demonstrated multilineage differentiation in vivo, although some skewing in the maturation of myeloid and B cells was observed in mice transplanted with granulocyte-colony stimulating factor (G-CSF)-mobilized peripheral blood compared to umbilical cord blood. Expansion of human cells in 6BG/BCNU-treated mice was observed in the majority of mice previously transplanted with transduced umbilical cord blood cells. In addition, a significant increase in the number of EGFP(+) progenitor colonies in treated versus nontreated mice were observed in highly engrafted mice indicating that selection and maintenance of human progenitor cells can be accomplished by expression of MGMT(P140K) and treatment with 6BG/BCNU.

Animals↗
PubMed2003

Hematopoietic cell regulation by Rac1 and Rac2 guanosine triphosphatases.

The Rho guanosine triphosphatases (GTPases) Rac1 and Rac2 are critical signaling regulators in mammalian cells. The deletion of both Rac1 and Rac2 murine alleles leads to a massive egress of hematopoietic stem/progenitor cells (HSC/Ps) into the blood from the marrow, whereas Rac1-/- but not Rac2-/- HSC/Ps fail to engraft in the bone marrow of irradiated recipient mice. In contrast, Rac2, but not Rac1, regulates superoxide production and directed migration in neutrophils, and in each cell type, the two GTPases play distinct roles in actin organization, cell survival, and proliferation. Thus, Rac1 and Rac2 regulate unique aspects of hematopoietic development and function.

Actins↗
PubMed2003

Critical roles for Rac1 and Rac2 GTPases in B cell development and signaling.

The Rac1 guanosine triphosphatase (GTPase) has been implicated in multiple cellular functions, including actin dynamics, proliferation, apoptosis, adhesion, and migration resulting from signaling by multiple receptors, including the B cell antigen receptor (BCR). We used conditional gene targeting to generate mice with specific Rac1 deficiency in the B cell lineage. In the absence of both Rac1 and the highly related Rac2, B cell development was almost completely blocked. Both GTPases were required to transduce BCR signals leading to proliferation, survival and up-regulation of BAFF-R, a receptor for BAFF, a key survival molecule required for B cell development and maintenance.

Animals↗
PubMed2003

Medicine. Gene therapy--new challenges ahead.

The successful use of retroviral gene transfer to treat 10 patients with X-linked severe combined immunodeficiency (SCID-X1) has been welcomed as evidence of the therapeutic potential of gene therapy. However, as Williams and Baum suggest in their Perspective, the discovery that 2 of the 10 patients developed leukemia within 3 years of gene therapy (Hacein-Bey-Abina et al.) reinforces the need to develop even more specific gene therapy interventions.

Adaptor Proteins, Signal Transducing↗