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D A Williams

Publications and source records attributed to D A Williams.

At least 73 records · Page 4Linked to original sources

Conditioning across the duration of a backward conditioned stimulus.

Five conditioned suppression experiments examined the extent to which an appetitively motivated lever-press response can be punished by different components of a backward conditioned stimulus (CS). Using a 0-s unconditioned stimulus (US)-CS interval, Experiments 1 and 2 showed that the initial 3 s of a normally 30-s backward CS served as a more effective punisher than the CS as a whole. Experiment 3 found no such effect if the US-CS interval were 3 s rather than 0 s. Experiments 4A and 4B found that if the US-CS interval were 0 s, the initial part of the backward CS acquired excitatory properties although the CS as a whole passed a summation test for conditioned inhibition. By contrast, the 3-s US-CS interval supported inhibitory conditioning across the whole duration of the backward CS. Taken together, these findings support a modified version of Wagner's sometimes opponent process model, which suggests that different components of a backward CS become either excitatory or inhibitory depending on the components' temporal proximity to the US.

Animals↗

Efficient retrovirus-mediated transfer of the multidrug resistance 1 gene into autologous human long-term repopulating hematopoietic stem cells.

Pre-clinical studies indicate that efficient retrovirus-mediated gene transfer into hematopoietic stem cells and progenitor cells can be achieved by co-localizing retroviral particles and target cells on specific adhesion domains of fibronectin. In this pilot study, we used this technique to transfer the human multidrug resistance 1 gene into stem and progenitor cells of patients with germ cell tumors undergoing autologous transplantation. There was efficient gene transfer into stem and progenitor cells in the presence of recombinant fibronectin fragment CH-296. The infusion of these cells was associated with no harmful effects and led to prompt hematopoietic recovery. There was in vivo vector expression, but it may have been limited by the high rate of aberrant splicing of the multidrug resistance 1 gene in the vector. Gene marking has persisted more than a year at levels higher than previously reported in humans.

Adolescent↗

Protection of hematopoietic cells against combined O6-benzylguanine and chloroethylnitrosourea treatment by mutant forms of O6-methylguanine DNA methyltransferase.

This report demonstrates that expression of the P140A O6-methylguanine DNA methyl transferase (MGMT) mutant via retrovirus-mediated gene transfer leads to significant, but modest, resistance of cells to both 6-benzylguanine (6-BG) depletion and treatment with 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU). Expression of the P140A/G156A double mutant appeared to be associated with reduced or unstable protein in hematopoietic cells.

Animals↗

Large-scale mobilization and isolation of CD34+ cells from normal donors.

We describe collection and purification of peripheral blood CD34+ cells from volunteer, normal donors and allogeneic stem cell donors. A total of 98 aphereses were performed on 68 volunteer donors using peripheral venous access. The mean number of nucleated cells collected was 4.6 x 10(10) which included 1.9 x 10(8) CD34+ cells corresponding to 2.7 x 10(6) CD34+ cells/kg. The number of CD34+ cells collected did not differ between males and females but did correlate with the donor's weight and the total number of nucleated cells collected. The Nexell Isolex 300i cell separator was used to isolate CD34+ cells from 30 of the collections. A mean of 0.36% of the total cells was recovered and included 43 +/- 18% of the CD34+ cells. CD34+ cells represented 85 +/- 11% of the recovered cells. The total number of CD34+ cells recovered was not influenced by the number of nucleated cells placed on the Isolex 300i. The percentage of CD34+ cells recovered was not related to the number of CD34+ cells placed on the Isolex 300i. The purity of the final product was influenced by the number of CD34+ cells but not the total number of nucleated cells. An additional 38 CD34+ cell isolations were performed on normal allogeneic stem cell donors with similar results. These observations further support the safety and feasibility of peripheral blood CD34+ cell collection and purification.

Adult↗

Genetically targeted calcium sensors enhance the study of organelle function in living cells.

1. Understanding the regulation of calcium (Ca2+), the most common of the mineral ions within the human body, has always been of extreme interest to physiologists. While the importance of Ca2+ in contributing to physiological events through regulation of levels has been significantly established, seldom is consideration given to the intricacies of this ion and its mechanics in producing such diverse physiological responses in different regions of the cell. 2. The present review will summarize new methodologies used in our laboratories for the study of two major intracellular organelles, mitochondria and the nucleus. These techniques are based predominantly on the use of molecular biological approaches to both create and then target protein-based sensor molecules to specific intracellular locations. 3. The regulation of Ca2+ in the mitochondria and nucleus is of particular interest to us because of the central involvement of these organelles in: (i) cardiac cell responses during ischaemia/reperfusion; and (ii) the control of gene expression, respectively.

Amino Acid Sequence↗

Osmometric and permeability characteristics of human placental/umbilical cord blood CD34+ cells and their application to cryopreservation.

The transplantation of placental/cord blood-derived HPC (e.g., CD34+ cells) has become a useful treatment for a broad spectrum of malignant and nonmalignant diseases. The ability to cryopreserve this cell type with high efficiency adds considerable flexibility to cord blood transplantation. The purpose of this study was to develop an understanding of the fundamental cryobiologic factors of these cells, including the osmotic/permeability characteristics, and to use a theoretical approach to optimize freezing procedures. To that end, biophysical parameters, including the osmotically inactive cell volume (Vb), hydraulic conductivity (Lp), and cryoprotectant permeability coefficient (P(CPA)) for DMSO and propylene glycol were measured using a modified Coulter Counter (Coulter Electronics, Inc., Hialeah, FL) at 22 degrees C. In addition, the osmotic tolerance of PCB CD34+ cells was assessed using a colony-forming assay. These experimentally determined parameters were used in a mathematical model to predict optimal cryoprotectant addition and removal procedures. The results demonstrate a Vb of 0.32 x V(iso), an average Lp of 0.17 +/- 0.03 (microm/min/atm +/- SD), and a PCPA of 0.94 +/- 0.004 or 1.0 +/- 0.004 cm/min (x10(-3)) for DMSO or propylene glycol, respectively. No significant difference was determined between the two cryoprotectants used. The osmotic tolerance limits were determined to be 200 and 600 mOsm/kg (1.29 and 0.62 x V(iso), respectively). These results indicate potential benefits of modifications to the widely used method of Rubinstein et al. Proc Natl Acad Sci USA 92:10119-10122, 1995) for cord blood CD34+ cell cryopreservation. As opposed to Rubinstein's method in which DMSO is added to cooled cell suspensions over a 15-min interval, our data indicate that better results may be obtained by introducing and removing the cryoprotectant at ambient temperature over 5 min both to increase viability by avoiding unnecessary risks from osmotic shock and to simplify the protocol. In addition, substitution of propylene glycol for DMSO may be of benefit during the actual freezing and thawing process.

Antigens, CD34↗

Role of a glycocalyx on coronary arteriole permeability to proteins: evidence from enzyme treatments.

Whereas the glycocalyx of endothelial cells has been shown to influence solute flux from capillary microvessels, little is known about its contribution to the movement of macromolecules across the walls of other microvessels. We evaluated the hypothesis that a glycocalyx contributes resistance to protein flux measured in coronary arterioles. Apparent solute permeability (P(s)) to two proteins of different size and similar charge, alpha-lactalbumin (alpha-lactalb) and porcine serum albumin (PSA), was determined in arterioles isolated from the hearts of 43 female Yucatan miniature swine. P(s) was assessed in arterioles with an "intact" glycocalyx under control conditions and again after suffusion with adenosine (Ado, 10(-5) M, n = 42 arterioles, N = 29 pigs). In a second set of experiments (n = 21 arterioles, N = 21 pigs) arteriolar P(s) was determined before and after perfusion with enzyme (pronase or heparinase), which was used to digest the glycocalyx. P(s) was assessed a third time on those microvessels after exposure to Ado. Consistent with the hypothesis, P(s) for PSA (P(PSA)(s)) and P(s) for alpha-lactalb (P(alpha-lactalb)(s)) increased from basal levels following enzyme treatment. Subsequent suffusion with Ado, a significant metabolite known to alter coronary vascular smooth muscle tone and permeability, resulted in a significant reduction of basal P(alpha-lactalb)(s) in both untreated and enzyme-treated arterioles. Furthermore, in untreated arterioles, P(PSA)(s) was unchanged by Ado suffusion, whereas Ado induced a pronounced reduction in P(PSA)(s) of enzyme-treated vessels. These data demonstrate that in intact coronary arterioles an enzyme-sensitive layer, most likely at the endothelial cell surface, contributes significantly to net barrier resistance to solute flux.

Animals↗

Hyperbaric oxygen improves contractile function of regenerating rat skeletal muscle after myotoxic injury.

There is growing interest in hyperbaric oxygen (HBO) as an adjunctive treatment for muscle injuries. This experiment tested the hypothesis that periodic inhalation of HBO hastens the functional recovery and myofiber regeneration of skeletal muscle after myotoxic injury. Injection of the rat extensor digitorum longus (EDL) muscle with bupivacaine hydrochloride causes muscle degeneration. After injection, rats breathed air with or without periodic HBO [100% O(2) at either 2 or 3 atmospheres absolute (ATA)]. In vitro maximum isometric tetanic force of injured EDL muscles and regenerating myofiber size were unchanged between 2 ATA HBO-treated and untreated rats at 14 days postinjury but were approximately 11 and approximately 19% greater, respectively, in HBO-treated rats at 25 days postinjury. Maximum isometric tetanic force of injured muscles was approximately 27% greater, and regenerating myofibers were approximately 41% larger, in 3 ATA HBO-treated rats compared with untreated rats at 14 days postinjury. These findings demonstrate that periodic HBO inhalation increases maximum force-producing capacity and enhances myofiber growth in regenerating skeletal muscle after myotoxic injury with greater effect at 3 than at 2 ATA.

Animals↗

Evaluation of intestinal permeability and gluten sensitivity in Soft-Coated Wheaten Terriers with familial protein-losing enteropathy, protein-losing nephropathy, or both.

OBJECTIVE: To evaluate intestinal permeability and gluten sensitivity in a family of Soft-Coated Wheaten Terriers (SCWT) affected with protein-losing enteropathy (PLE), protein-losing nephropathy (PLN), or both. ANIMALS: 6 affected adult dogs. PROCEDURE: Intestinal biopsy specimens, urine protein-to-creatinine ratio, serum concentrations of albumin and globulin, and concentration of alpha1-protease inhibitor in feces were evaluated before, during, and 13 weeks after daily administration of 10 g of gluten for 7 weeks. Eosinophils and lymphocytes-plasmacytes were enumerated in intestinal biopsy specimens. Intestinal permeability was evaluated before and during the sixth week of gluten administration via cellobiose-mannitol and chromium-EDTA absorption tests. RESULTS: Serum globulin concentration decreased significantly after prolonged administration of gluten. Although not significant, there was an increase in lymphocytes-plasmacytes and a decrease in eosinophils in intestinal biopsy specimens. Furthermore, these counts were greater than those reported for clinically normal dogs. Gluten administration did not increase intestinal permeability. CONCLUSIONS AND CLINICAL RELEVANCE: Daily administration of gluten was associated with a significant decrease in serum globulin concentration in SCWT affected with PLE or PLN, but other variables remained unchanged. Although enhanced wheat-gluten sensitivity may be one factor involved in the pathogenesis of PLE or PLN in SCWT, this syndrome does not appear to be the result of a specific sensitivity to gluten.

Animals↗

Development and validation of an enzyme-linked immunosorbent assay for feline trypsin-like immunoreactivity.

OBJECTIVE: To develop and validate an ELISA for quantitative analysis of feline trypsin-like immunore-activity (fTLI). SAMPLE POPULATION: Purified feline cationic trypsin (fCT) and rabbit anti-fCT antiserum; blood samples from 63 healthy cats. PROCEDURES: A sandwich capture ELISA was developed, using anti-fCT antiserum purified by affinity chromatography that underwent biotinylation. Purified fCT was used for standards. The assay was validated by determination of sensitivity, working range, linearity, accuracy, precision, and reproducibility. A reference range was established by assaying serum samples from the 63 healthy cats. RESULTS: Sensitivity was 1.23 microg/L; working range was 2 to 567 microg/L. Ratios of observed versus expected results for 4 samples tested at various dilutions ranged from 90.0 to 120.7%. Ratios of observed versus expected results for 5 samples spiked with various concentrations of fCT ranged from 82.0 to 101.8%. Intra- and inter-assay coefficients of variability ranged from 9.9 to 11.1% and from 10.2 to 21.7%, respectively. The reference range for serum fTLI measured with this ELISA was 12 to 82 microg/L. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that an ELISA can be used to measure serum fTLI in cats. The ELISA was sufficiently sensitive, linear, accurate, precise, and reproducible for clinical use.

Animals↗

Serum feline trypsin-like immunoreactivity following administration of ceruletide to healthy cats.

OBJECTIVE: To determine changes in serum feline trypsin-like immunoreactivity (fTLI) in response to administration of ceruletide to healthy cats. ANIMALS: 11 healthy cats. PROCEDURES: Serum fTLI was determined, using a radioimmunoassay, before and 10, 20, 30, 40, and 50 minutes after IM administration of ceruletide (0.3 mg/kg [0.14 mg/lb]). RESULTS: Mean +/- SD baseline serum fTLI was 23.1 +/- 4.1 mg/L. There was a statistically significant, but clinically unimportant, increase in serum fTLI 10 and 30 minutes after ceruletide administration. CONCLUSIONS AND CLINICAL RELEVANCE: In healthy cats, administration of ceruletide induced a statistically significant, but clinically unimportant, increase in serum fTLI. Whether responses in cats with exocrine pancreatic disorders would be different is unknown, but results suggest that a ceruletide stimulation test would likely not be useful for differentiating between healthy cats and cats with subclinical chronic exocrine pancreatic disorders.

Animals↗

Deleterious effects of chronic clenbuterol treatment on endurance and sprint exercise performance in rats.

The beta(2)-adrenergic agonist, clenbuterol, has powerful muscle anabolic and lipolytic effects and is used by athletes to improve exercise performance; however, its use in conjunction with different forms of exercise training has received limited attention. Since previous studies have reported that chronic use of other beta(2)-adrenergic agonists has deleterious effects on cardiac muscle structure and function, the aim of the present study was to determine whether chronic clenbuterol administration would reduce the exercise capabilities of rats subjected to long-term treadmill sprint running, endurance swimming or voluntary wheel running training. The effect of clenbuterol treatment on exercise performance in rats was evaluated in three separate studies. Different groups of male rats were assigned to an endurance swimming (2 h/day, 5/7 days, 18 weeks) group, a treadmill sprint running (8x1 min bouts, 1.05 m/s, 20 weeks) group, or a voluntary wheel running (16 weeks) group. In each study, rats were allocated into either a treated group that received clenbuterol (2 mg.kg(-1).day(-1)) in their drinking water or an untreated control group. In each of the three studies, treated rats exhibited a reduction in exercise performance compared with untreated rats. Treated rats ran approximately 57% less total distance than untreated rats in the voluntary running programme and were unable to complete the swimming and sprinting protocols performed by the untreated rats. In each of the studies, the treated rats exhibited cardiac hypertrophy, with absolute heart mass increased by approximately 19% and heart mass relative to body mass increased by approximately 20%. The hearts of sedentary rats treated with clenbuterol exhibited extensive collagen infiltration surrounding blood vessels and in the wall of the left ventricle. The results indicate strongly that chronic clenbuterol administration deleteriously affects exercise performance in rats, potentially due to alterations in cardiac muscle structure and function.

Adrenergic beta-Agonists↗

Development and validation of a method for simultaneous separation and quantification of 5 different sugars in canine urine.

The objective of this project was to develop and validate a method for concurrent separation and quantification of methylglucose, rhamnose, xylose, sucrose, and lactulose in canine urine by using high pressure anion exchange liquid chromatography and pulsed amperometric detection. The method was validated by evaluating dilutional parallelism, spiking recovery, intra-assay variability, and inter-assay variability. Observed to expected ratios for 3 urine samples, and all sugars, ranged from 77.6% to 106.9% for a 1:2 dilution, 85.2% to 121.4% for a 1:4 dilution, and 91.6% to 163.7% for a 1:8 dilution. Observed to expected ratios for spiking recovery of 3 urine samples, all sugars, and 5 different spiking solutions, ranged from 85.5% to 116.7 % (mean +/- SD, 100.5 +/- 6.0%). The intra-assay coefficients of variation were 1.6%, 3.4%, and 4.7% for methylglucose; 1.6%, 2.0%, and 3.6% for rhamnose; 2.7%, 1.4%, and 1.1% for xylose; 9.8%, 3.4%, and 4.0% for sucrose; and 3.2%, 3.3%, and 3.3% for lactulose. Inter-assay coefficients of variation were 3.2%, 5.7%, and 4.2% for methylglucose; 4.3%, 5.4%, and 6.4% for rhamnose; 3.3%, 5.0%, and 4.2% for xylose; 9.4%, 9.9%, and 9.4% for sucrose; and 6.1%, 4.9%, and 2.7% for lactulose. In conclusion, a method for simultaneous separation and quantification of 5 sugars in canine urine was established and found to be linear, accurate, precise, and reproducible. This method may prove useful in the simultaneous evaluation of gastric permeability, small intestinal permeability, and small intestinal mucosal function in dogs with gastrointestinal disorders.

Animals↗

Stevens-Johnson syndrome after erythromycin therapy while deployed at sea.

Stevens-Johnson syndrome is a cutaneous reaction pattern that represents the progression of symptoms of erythema multiforme. These reactions can range from mild (EM minor) to severe (EM major) and even life-threatening (Stevens-Johnson syndrome or toxic epidermal necrolysis). The difference between Stevens-Johnson syndrome and toxic epidermal necrolysis is the percentage of body surface area involved; toxic epidermal necrolysis involves widespread skin necrosis and bullous formation with epidermal detachment resembling scalded skin. The three most common triggers for Stevens-Johnson syndrome are herpes simplex infection, Mycoplasma infection, and drug reactions. This is a case of Stevens-Johnson syndrome occurring after erythromycin treatment aboard an aircraft carrier while deployed at sea in the Persian Gulf.

Adult↗

The presence of novel amino acids in the cytoplasmic domain of stem cell factor results in hematopoietic defects in Steel(17H) mice.

Stem cell factor (SCF) is expressed as an integral membrane growth factor that may be differentially processed to produce predominantly soluble (S) (SCF(248)) or membrane-associated (MA) (SCF(220)) protein. A critical role for membrane presentation of SCF in the hematopoietic microenvironment (HM) has been suggested from the phenotype of the Steel-dickie (Sl(d)) mice, which lack MA SCF, and by studies performed in our laboratory (and by others) using long-term bone marrow cultures and transgenic mice expressing different SCF isoforms. Steel(17H) (Sl(17H)) is an SCF mutant that demonstrates melanocyte defects and sterility in males but not in females. The Sl(17H) allele contains a intronic mutation resulting in the substitution of 36 amino acids (aa's) in the SCF cytoplasmic domain with 28 novel aa's. This mutation, which affects virtually the entire cytoplasmic domain of SCF, could be expected to alter membrane SCF presentation. To investigate this possibility, we examined the biochemical and biologic properties of the Sl(17H)-encoded protein and its impact in vivo and in vitro on hematopoiesis and on c-Kit signaling. We demonstrate that compound heterozygous Sl/Sl(17H) mice manifest multiple hematopoietic abnormalities in vivo, including red blood cell deficiency, bone marrow hypoplasia, and defective thymopoiesis. In vitro, both S and MA Sl(17H) isoforms of SCF exhibit reduced cell surface expression on stromal cells and diminished biological activity in comparison to wild-type (wt) SCF isoforms. These alterations in presentation and biological activity are associated with a significant reduction in the proliferation of an SCF-responsive erythroid progenitor cell line and in the activation of phosphatidylinositol 3-Kinase/Akt and mitogen-activated protein-Kinase signaling pathways. In vivo, transgene expression of the membrane-restricted (MR) (SCF(X9/D3)) SCF in Sl/Sl(17H) mutants results in a significant improvement in peripheral red blood cell counts in comparison to Sl/Sl(17H) mice.

Amino Acid Sequence↗

Costimulation of transduced T lymphocytes via T cell receptor-CD3 complex and CD28 leads to increased transcription of integrated retrovirus.

Primary human T lymphocytes were transduced at high efficiency with the Moloney murine leukemia virus (Mo-MuLV) vector, LNC-mB7-1, in which an internal cytomegalovirus (CMV) promoter drives expression of the murine B7-1 cDNA. Compared with transduced T cells expanded in IL-2 or reactivated with soluble antibodies to CD3 or CD28, transgene expression was significantly increased after activation on immobilized anti-CD3 antibodies (CD3i) or by simultaneous activation on immobilized anti-CD3 and anti-CD28 antibodies (CD3i/CD28i). A similar pattern of transgene expression was observed in T cells transduced with Mo-MuLV LNC-EGFP. Proviral copy number was maintained in LNC-mB7-1-transduced T cells expanded in IL-2 or reactivated on CD3i/CD28i. Substantial increases in LNC-mB7-1 steady state mRNA in reactivated T lymphocytes, compared with those maintained in IL-2, correlated with increased transcription of the LNC-mB7-1 proviral DNA. Furthermore, T cells transduced with the Mo-MuLV ZIPPGK-mADA, in which the mADA cDNA is driven by an internal human phosphoglycerate kinase (PGK) promoter, showed increases in steady state ZIPPGK-mADA RNA on reactivation. High levels of transgene expression were evident irrespective of cell cycle position in both CD4+ and CD8+ lymphocytes. After reactivation, increases in LNC-mB7-1 mRNA were observed in the presence of the protein synthesis inhibitor cycloheximide, indicating that proteins involved in upregulating transgene expression preexisted in transduced lymphocytes. Induction of transgene expression on CD3i/CD28i showed a dose-dependent decrease in transgene expression when incubated with selective protein kinase inhibitors. These data provide new insights into the mechanisms governing transgene expression driven by Mo-MuLV constructs containing internal promoters in transduced primary T lymphocytes.

Antibodies↗

Contractile activation characteristics of single permeabilized fibres from levator palpebrae superioris, orbicularis oculi and vastus lateralis muscles from humans.

1. We investigated the contractile activation characteristics of single membrane-permeabilized fibres from the following muscles from humans: the levator palpebrae superioris (LPS), an extraocular muscle; the orbicularis oculi (OO), a facial muscle; and the vastus lateralis (VL), a major muscle of the thigh. 2. Single permeabilized muscle fibres were isolated from each of the different muscles, attached to a sensitive force transducer and activated by rapid immersion in buffered solutions of varying [Ca2+] and [Sr2+]. Fibres were allocated into discrete populations based on their contractile characteristics, including their differential force responses during Ca2+ and Sr2+ activation. 3. With the exception of one fibre from the LPS, all 152 fibres sampled from the three different human muscles could be classified into either population I (slow, type I) or population II (fast, type II) based on their force-pCa(pSr) relations. The LPS muscle fibre which was unable to be classified into the two major fibre populations displayed a combination of the typical force-pCa(pSr) relations for mammalian fast and slow muscle fibres. 4. Although fibres from the LPS, OO and VL muscles had similar differential sensitivities to Ca2+and Sr2+, the steepness of the force-pCa(pSr) curves for fibres from the LPS and OO muscles were highly variable compared with those for fibres from the VL muscle. Specific forces (N cm-2) of the smaller diameter fibres from the LPS and OO muscles were significantly lower than those of fibres from the VL muscle. 5. The differences in the contractile activation characteristics between fibres from the VL muscle and those of fibres from facial (OO) muscles and extraocular (LPS) muscles, reflect the differences in their fibre composition that are responsible for their functional specificity.

Algorithms↗

Retroviral-fibronectin interactions in transduction of mammalian cells.

Hematopoiesis occurs in a complex environment in the medullary cavity in close proximity to stromal cells, fibroblasts, endothelial cells and matrix molecules. Hematopoietic cell interactions in this environment appear to involve both integrin and proteoglycan-mediated cell-cell and cell-matrix interactions. Genetic transduction of hematopoietic stem cells via retroviral vectors has been hampered by low efficiency of gene transfer. Recently, hematopoietic stem cell adhesion to the extracellular matrix molecule fibronectin has been shown to increase transduction of these target cells using retrovirus vectors. The mechanism of increased transduction appears to involve colocalization of virus particles and target cells. These data are reviewed in this paper.

Animals↗