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D A Williams

Publications and source records attributed to D A Williams.

At least 55 records · Page 3Linked to original sources
PubMed2001

Preliminary studies of a canine 13C-aminopyrine demethylation blood test.

The objectives of this study were to determine whether a 13C-aminopyrine demethylation blood test is technically feasible in clinically healthy dogs, whether oral administration of 13C-aminopyrine causes a detectable increase in percent dose/min (PCD) of 13C administered as 13C-aminopyrine and recovered in gas extracted from blood, and whether gas extraction efficiency has an impact on PCD. A dose of 2 mg/kg body weight of 13C-aminopyrine dissolved in deionized water was administered orally to 6 clinically healthy dogs. Blood samples were taken from each dog 0, 30, 60, and 120 min after administration of the 13C-aminopyrine. Carbon dioxide was extracted from blood samples by addition of acid and analyzed by fractional mass spectrometry. None of the 6 dogs showed any side effects after 13C-aminopyrine administration. All 6 dogs showed a measurable increase of the PCD in gas samples extracted from blood samples at 30 min, 60 min, and 120 min after 13C-aminopyrine administration. Coefficients of variation between the triplicate samples were statistically significantly higher for the %CO2, a measure of extraction efficiency, than for PCD values (P < 0.0001). The 13C-aminopyrine demethylation blood test described here is technically feasible. Oral administration of 13C-aminopyrine did not lead to gross side effects in the 6 dogs. Clinically healthy dogs show a measurable increase of PCD in gas extracted from blood samples after oral administration of 13C-aminopyrine. Efficiency of CO2 extraction from blood samples does not have an impact on PCD determined from these blood samples. This test may prove useful to evaluate hepatic function in dogs.

Aminopyrine↗
PubMed2000

Differential stimulation of c-Kit mutants by membrane-bound and soluble Steel Factor correlates with leukemic potential.

The authors investigated the roles of PI3-kinase and PLC-gamma in stimulation by Steel Factor (SLF) through c-Kit. c-Kit mutants YF719, YF728, and a YF719/YF728 double mutant were expressed in 32D myelomonocytic cells. KitYF719 fails to recruit PI3-kinase after stimulation with SLF, whereas KitYF728 fails to stimulate PLC-gamma phosphorylation or mobilize Ca(++). Both single mutants responded mitogenically to soluble SLF (sSLF) in a manner indistinguishable from wild type (WT), although sSLF failed to stimulate or promote the survival of cells expressing the double mutant. In contrast, although cells expressing WT or YF719 were mitogenically stimulated by membrane-bound SLF (mSLF), stimulation of cells expressing KitYF728 was impaired. Similarly, cells expressing WT or YF719 receptors were stimulated by plate-bound anti-Kit antibodies, whereas cells expressing the YF728 receptor were not stimulated. Neomycin sulfate, a PLC antagonist, inhibited cells expressing YF719 receptors stimulated by sSLF. Neomycin also inhibited cells expressing the WT receptor that were stimulated by mSLF or immobilized anti-Kit antibodies but did not inhibit stimulation of cells expressing WT or YF719 receptors by sSLF. 32D cells expressing KitWT, KitYF719, or KitYF728 were injected into mice and the presence of cells was evaluated by colony assays 6 to 7 weeks later. Although both KitWT and KitYF719 expressing cells could be recovered from the spleen and bone marrow, recovery of KitYF728 cells from these organs was severely reduced. These results indicate that Kit tyrosine 728 is of particular importance for mitogenic stimulation by mSLF or immobilized ligand and is required for full maintenance of cells in vivo, likely through activation of PLC-gamma. (Blood. 2000;96:3734-3742)

Animals↗
PubMed2000

Progress in the use of gene transfer methods to treat genetic blood diseases.

A report by French physician-scientists suggests a successful application of gene transfer methods in the treatment of two children with severe combined immunodeficiency (SCID) due to defective interleukin 2 receptor common gamma chain. The protocol used in this clinical trial was derived from a number of preclinical and basic studies leading to improved transduction of hematopoietic stem and primitive progenitor cells using retrovirus vectors. These improvements have also been shown to impact transduction of a long-lived progenitor cell in a chemotherapy protocol in cancer patients. The improved results of these human trials come during a period of increased scrutiny and criticism of human gene therapy trials, due, in part, to significant toxicities in some trials using adenovirus-based vectors. The potential efficacy versus toxicity of phase I trials of human gene therapy is also under question. After many years of research, however, there appears to be real evidence that genetic diseases may be successfully treated by gene transfer techniques. Future clinical studies should be based on continued progress in the understanding of the toxicology of gene delivery systems, vector technology, and target cell manipulation.

Adenoviridae↗
PubMed2000

Direct reversal of DNA damage by mutant methyltransferase protein protects mice against dose-intensified chemotherapy and leads to in vivo selection of hematopoietic stem cells.

Direct reversal of O6 adducts caused by chemotherapy agents is accomplished in mammalian cells by the protein O6-methylguanine DNA methyltransferase (MGMT). Some tumors overexpress MGMT and are resistant to alkylator therapy. One future approach to treatment of these tumors may rely on concurrent pharmacological depletion of tumor MGMT with O6-benzylguanine (6-BG) and protection of sensitive tissues, such as hematopoietic stem and progenitor cells, using genetic modification with 6-BG-resistant MGMT mutants. We have used retroviral-mediated gene transfer to transduce murine hematopoietic bone marrow cells with MGMT point mutants showing resistance to 6-BG depletion in vitro. These mutants include proline to alanine and proline to lysine substitutions at the 140 position (P140A and P140K, respectively), which show 40- and 1000-fold resistance to 6-BG compared with wild-type (WT) MGMT. Lethally irradiated mice were reconstituted with murine stem cells transduced with murine stem cell virus retrovirus expressing each mutant, WT MGMT, or mock-infected cells and then treated with a combination of 30 mg/kg 6-BG and 10 mg/kg 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) or with 40 mg/kg BCNU alone. Compared with mice treated with BCNU alone, significant myeloid toxicity and death occurred in mice reconstituted with mock-infected or WT MGMT (<0.1 probability of survival) or the P140A mutant (0.13 probability of survival) MGMT cDNAs. In contrast, after an initial period of mild cytopenia, mice reconstituted with the P140K mutant (0.83 probability of survival) recovered nearly normal blood counts, even during continued treatment. Comparison of peripheral blood neutrophils after completion of 5 weekly treatments in these animals showed a direct correlation between the treatment and in vivo selection for progeny of transduced cells (pretreatment, approximately 8-12% transduced cells; no treatment, approximately 6% transduced cells; BCNU only, 51% transduced cells; 6-BG/BCNU, 93% transduced cells). To determine whether this selection occurred at the stem cell level, bone marrow from each treatment group was infused into secondary recipients. Whereas animals that received bone marrow from untreated animals reconstituted with 2% transduced cells, animals receiving marrow from 6-BG/BCNU-treated animals reconstituted with 94% transduced cells, demonstrating nearly complete selection for stem cells in the primary animals. Mice reconstituted with marrow from animals treated with BCNU only demonstrated 23% transduced cells, consistent with partial selection of stem cells in the primary mice. The levels of transduced cells also correlated with survival during a second round of intensive combination chemotherapy (probability of survival: 6-BG/BCNU, 1.0; BCNU alone, >0.70; no treatment, <0.1). These data demonstrate that mutant MGMT expressed in the bone marrow can protect mice from time- and dose-intensive chemotherapy and that the combination of 6-BG and BCNU leads to uniform selection of transduced stem cells in vivo in mice.

3T3 Cells↗
PubMed2000

Dominant negative mutation of the hematopoietic-specific Rho GTPase, Rac2, is associated with a human phagocyte immunodeficiency.

Rho GTPases control a variety of cellular processes, including actin polymerization, integrin complex formation, cell adhesion, gene transcription, cell cycle progression, and cell proliferation. A patient is described who has recurrent infections and defective neutrophil cellular functions similar to those found in Rac2-deficient mice. Molecular methods were used to clone the expressed Rac2 cDNA from this patient, and a single base pair change (G-->A at nucleotide 169) in the coding sequence was identified. This results in an asparagine for aspartic acid mutation at amino acid 57 (D57N), a residue that is involved in nucleotide binding and is conserved in all mammalian Rho GTPases. The cloned cDNA was then introduced into normal bone marrow cells through retrovirus vectors, and neutrophils expressing this mutant exhibited decreased cell movement and production of superoxide in response to fMLP. The expressed recombinant protein was also analyzed biochemically and exhibited defective binding to GTP. Functional studies demonstrated that the D57N mutant behaves in a dominant-negative fashion at the cellular level. The syndrome of Rac2 dysfunction represents a human condition associated with mutation of a Rho GTPase and is another example of human disease associated with abnormalities of small G protein signaling pathways. (Blood. 2000;96:1646-1654)

3T3 Cells↗
PubMed2000

Role of the guanosine triphosphatase Rac2 in T helper 1 cell differentiation.

T helper 1 (TH1) cells mediate cellular immunity, whereas TH2 cells potentiate antiparasite and humoral immunity. We used a complementary DNA subtraction method, representational display analysis, to show that the small guanosine triphosphatase Rac2 is expressed selectively in murine TH1 cells. Rac induces the interferon-gamma (IFN-gamma) promoter through cooperative activation of the nuclear factor kappa B and p38 mitogen-activated protein kinase pathways. Tetracycline-regulated transgenic mice expressing constitutively active Rac2 in T cells exhibited enhanced IFN-gamma production. Dominant-negative Rac inhibited IFN-gamma production in murine T cells. Moreover, T cells from Rac2-/- mice showed decreased IFN-gamma production under TH1 conditions in vitro. Thus, Rac2 activates TH1-specific signaling and IFN-gamma gene expression.

Animals↗
PubMed2000

Galileo at Io: results from high-resolution imaging.

During late 1999/early 2000, the solid state imaging experiment on the Galileo spacecraft returned more than 100 high-resolution (5 to 500 meters per pixel) images of volcanically active Io. We observed an active lava lake, an active curtain of lava, active lava flows, calderas, mountains, plateaus, and plains. Several of the sulfur dioxide-rich plumes are erupting from distal flows, rather than from the source of silicate lava (caldera or fissure, often with red pyroclastic deposits). Most of the active flows in equatorial regions are being emplaced slowly beneath insulated crust, but rapidly emplaced channelized flows are also found at all latitudes. There is no evidence for high-viscosity lava, but some bright flows may consist of sulfur rather than mafic silicates. The mountains, plateaus, and calderas are strongly influenced by tectonics and gravitational collapse. Sapping channels and scarps suggest that many portions of the upper approximately 1 kilometer are rich in volatiles.

Extraterrestrial Environment↗
PubMed2000

HIV-1 gp120 and chemokines activate ion channels in primary macrophages through CCR5 and CXCR4 stimulation.

HIV type 1 (HIV-1) uses the chemokine receptors CCR5 and CXCR4 as coreceptors for entry into target cells. Here we show that the HIV-1 envelope gp120 (Env) activates multiple ionic signaling responses in primary human macrophages, which are important targets for HIV-1 in vivo. Env from both CCR5-dependent JRFL (R5) and CXCR4-dependent IIIB (X4) HIV-1 opened calcium-activated potassium (K(Ca)), chloride, and calcium-permeant nonselective cation channels in macrophages. These signals were mediated by CCR5 and CXCR4 because macrophages lacking CCR5 failed to respond to JRFL and an inhibitor of CXCR4 blocked ion current activation by IIIB. MIP-1beta and SDF-1alpha, chemokine ligands for CCR5 and CXCR4, respectively, also activated K(Ca) and Cl(-) currents in macrophages, but nonselective cation channel activation was unique to gp120. Intracellular Ca(2+) levels were also elevated by gp120. The patterns of activation mediated by CCR5 and CXCR4 were qualitatively similar but quantitatively distinct, as R5 Env activated the K(Ca) current more frequently, elicited Cl(-) currents that were approximately 2-fold greater in amplitude, and elevated intracellular Ca(+2) to higher peak and steady-state levels. Env from R5 and X4 primary isolates evoked similar current responses as the corresponding prototype strains. Thus, the interaction of HIV-1 gp120 with CCR5 or CXCR4 evokes complex and distinct signaling responses in primary macrophages, and gp120-evoked signals differ from those activated by the coreceptors' chemokine ligands. Intracellular signaling responses of macrophages to HIV-1 may modulate postentry steps of infection and cell functions apart from infection.

Calcium↗
PubMed2000

The effect of ex vivo refrigerated storage and cell preservation solution (Cyto-Chex II) on CD11b expression and oxidative burst activity of dog neutrophils.

The expression of CD11b and oxidative burst activity of dog neutrophils undergoing ex vivo refrigerated storage was studied using flow-cytometry . Additionally, the effect of a proprietary cell stabilization reagent (Cyto-Chex) on the expression of CD11b and oxidative burst activity was studied. Expression of CD11b was very high (>90% positive) on dog neutr ophils isolated from peripheral blood. Dog neutrophils showed a rapid and sustained increase in CD11b antigen density (P<0.01) during refrigerated storage, this increase was prevented by treatment with Cyto-Chex but was not completely blocked on the first day. There were no significant differences in mean antigen density between any days in the non-preserved group or between Days 1 to 4 in the Cyto-Chex treated group. The non-treated group showed significantly greater mean antigen density at all time points when compared to the preservative treated group (P<0.0001). Treatment with Cyto-Chex did not interfere with measurement of oxidative burst function on the first 2 days. Alterations of both resting oxidative activity and stimulated response were observed over time in both treated and untreated blood samples. Cyto-Chex treated samples showed a dramatic, significant decline in stimulated response after the third day of storage (P<0.001), while non-treated cells showed steadily increasing, but non-significant differences in stimulated response. Cyto-Chex was demonstrated to be a useful reagent for stabilization of dog neutrophil membrane antigens during storage, however this reagent is not recommended for preservation of cells for functional assays.

Animals↗
PubMed2000

Genetic and biochemical evidence that haploinsufficiency of the Nf1 tumor suppressor gene modulates melanocyte and mast cell fates in vivo.

Neurofibromatosis type 1 (NF1) is a common autosomal-dominant disorder characterized by cutaneous neurofibromas infiltrated with large numbers of mast cells, melanocyte hyperplasia, and a predisposition to develop malignant neoplasms. NF1 encodes a GTPase activating protein (GAP) for Ras. Consistent with Knudson's "two hit" model of tumor suppressor genes, leukemias and malignant solid tumors in NF1 patients frequently demonstrate somatic loss of the normal NF1 allele. However, the phenotypic and biochemical consequences of heterozygous inactivation of Nf1 are largely unknown. Recently neurofibromin, the protein encoded by NF1, was shown to negatively regulate Ras activity in Nf1-/- murine myeloid hematopoietic cells in vitro through the c-kit receptor tyrosine kinase (dominant white spotting, W). Since the W and Nf1 locus appear to function along a common developmental pathway, we generated mice with mutations at both loci to examine potential interactions in vivo. Here, we show that haploinsufficiency at Nf1 perturbs cell fates in mast cells in vivo, and partially rescues coat color and mast cell defects in W(41) mice. Haploinsufficiency at Nf1 also increased mast cell proliferation, survival, and colony formation in response to Steel factor, the ligand for c-kit. Furthermore, haploinsufficiency was associated with enhanced Ras-mitogen-activated protein kinase activity, a major downstream effector of Ras, via wild-type and mutant (W(41)) c-kit receptors. These observations identify a novel interaction between c-kit and neurofibromin in vivo, and offer experimental evidence that haploinsufficiency of Nf1 alters both cellular and biochemical phenotypes in two cell lineages that are affected in individuals with NF1. Collectively, these data support the emerging concept that heterozygous inactivation of tumor suppressor genes may have profound biological effects in multiple cell types.

Animals↗
PubMed2000

Functional properties of regenerating skeletal muscle following LIF administration.

We tested the hypothesis that periodic systemic administration of the myogenic cytokine leukemia inhibitory factor (LIF) enhances the functional recovery of regenerating skeletal muscle following bupivacaine-induced degeneration. LIF had no effect on functional capacity or regenerating myofiber size in rat muscles at 7, 14, or 21 days post-injury. The results do not support exogenous administration of LIF as a treatment for acute muscle injury, but a more frequent dosing regimen should be tested.

Animals↗
PubMed2000

Determination of the kinetics of rat UDP-glucuronosyltransferases (UGTs) in liver and intestine using HPLC.

Uridinediphosphoglucuronosyl transferases (UGTs) are a group of membrane bound proteins which catalyze the transfer of glucuronic acid from UDP-glucuronic acid to a wide variety of xenobiotics and drug molecules enabling them to be eliminated. The major UGT isoforms found in the rat are 1A1, 1A6, 2B1 and 2B12. Conventional methods for the assay of glucuronides (GLs) include TLC, extraction and colorimetry or quantification of the aglycone, liberated after hydrolyzing the GL with beta-glucuronidase. However these techniques cannot distinguish between isomeric GLs or GLs of multiple acceptor site substrates. Therefore the purpose of this study was to develop simple and sensitive HPLC methods for the direct and simultaneous analysis of the GL(s) and their aglycones without the drawbacks of the conventional methods. The three classical substrates we chose were 4-methylumbelliferone (4MU), testosterone (TES) and 8-hydroxyquinoline (8HOQ) representing UGT isoforms 1A6, 2B1 and 2B12 of the rat family, respectively. Here we report the validated HPLC conditions, for the detection and separation of 4-methylumbelliferone glucuronide (4MUG), testosterone glucuronide (TESG) and 8-hydroxyquinoline glucuronide (8HOQG) and their aglycones in incubation media containing male Sprague-Dawley rat liver and intestinal microsomal preparations. The separations were achieved on a Zorbax SB-CN column (150 x 4.6 mm, 5 micron). The analysis time for the separation of TES, 8HOQ and 4MU and their glucuronides were 17, 12 and 30 min, respectively. The methods showed excellent linearity (r2 > 0.99) over the concentration ranges tested (0.25-5.0 nmoles of TESG; 0.125-18.75 nmoles of 8HOQG and 0.125-12.5 nmoles of 4MUG), good precision and accuracy (RSD<2.5%). Inter-day variability studies (n = 3) showed no significant difference between the regression lines obtained on the three days. Recoveries were good ( > 90%) at all three points (low, mid-point, high) of the standard curve. The limits of detection were 0.125, 0.1 and 0.1 nmole for TESG, 8HOQG and 4MUG. respectively. The above methods were used to estimate kinetic parameters such as Vmax and Km for the GLs of the three substrates in both liver and intestinal tissue preparations and the values were comparable with previously reported results. UGT2B1 was found primarily in the liver while UGTs 1A6 and 2B12 were present in comparable amounts in both tissues.

Animals↗
PubMed2000

Rac2 stimulates Akt activation affecting BAD/Bcl-XL expression while mediating survival and actin function in primary mast cells.

Mast cells generated from Rac2-deficient (-/-) mice demonstrated defective actin-based functions, including adhesion, migration, and degranulation. Rac2(-/-) mast cells generated lower numbers and less mast cell colonies in response to growth factors and were deficient in vivo. Rac2(-/-) mast cells demonstrated a significant reduction in growth factor-induced survival, which correlated with the lack of activation of Akt and significant changes in the expression of the Bcl-2 family members BAD and Bcl-XL, in spite of a 3-fold induction of Rac1 protein. These results suggest that Rac2 plays a unique role in multiple cellular functions and describe an essential role for Rac2 in growth factor-dependent survival and expression of BAD/Bcl-XL.

Actins↗
PubMed2000

Serum unconjugated bile acids as a test for intestinal bacterial overgrowth in dogs.

Small intestinal bacterial overgrowth (SIBO) has a high incidence in dogs and, as in humans, is difficult to diagnose. The aim of this study was to determine the diagnostic significance of serum unconjugated bile acid concentrations in dogs with bacterial overgrowth. Fasting sera were obtained from 23 dogs: 10 with culture-proven SIBO, 8 with indirectly diagnosed SIBO (normal pancreatic function but small intestinal disease associated with subnormal serum cobalamin and supranormal folate concentrations), and 5 healthy controls. Unconjugated bile acids were determined using gas chromatography-mass spectrometry after isolation by liquid-solid extraction and anion-exchange chromatography. Mean serum unconjugated bile acid concentrations were significantly elevated in dogs with SIBO (mean +/- SD: 0.91 +/- 1.03 micromol/liter), and in dogs with indirectly diagnosed SIBO (2.11 +/- 2.20 micromol/liter) compared to clinically healthy dogs (0.015 +/- 0.015 micromol/liter, P < 0.005). Cholic acid was the predominant unconjugated bile acid in the serum of dogs with SIBO. In conclusion serum unconjugated bile acid concentrations of healthy dogs are significantly lower than reported values for humans, and this fraction represents a relatively small proportion (0-2.3%; mean 0.8%) of the total bile acids in dogs. Unconjugated bile acids increased 10- to 20-fold in dogs with SIBO indicating the clinical utility of serum unconjugated bile acids for diagnosis of intestinal bacterial overgrowth in dogs.

Animals↗
PubMed2000

Hydrogen peroxide modulates Ca2+-activation of single permeabilized fibres from fast- and slow-twitch skeletal muscles of rats.

We examined the effects of redox modulation on single membrane-permeabilized fibre segments from the fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus muscles of adult rats to determine whether the contractile apparatus was the redox target responsible for the increased contractility of muscles exposed to low concentrations of H2O2. The effects of H2O2 on maximum Ca2+-activated force were dose-dependent with 30 min exposure to 5 mM H2O2 causing a progressive decrease by 22+/-3 and 13+/-2% in soleus and EDL permeabilized muscle fibres, respectively. Lower concentrations of exogenous H2O2 (100 microM and 1 mM) had no effect on maximum Ca2+-activated force. Subsequent exposure to the reductant dithiothreitol (DTT, 10 mM, 10 min) fully reversed the H2O2-induced depression of force in EDL, but not in soleus muscle fibres. Incubation with DTT alone for 10 min did not alter Ca2+-activated force in either soleus or EDL muscle fibres. The sensitivity of the contractile filaments to Ca2+ (pCa50) was not altered by exposure to any concentration of exogenous H2O2. However, all concentrations of H2O2 diminished the Hill coefficient in permeabilized fibres from the EDL muscle, indicating that the cooperativity of Ca2+ binding to troponin is altered. H2O2 (5 mM) did not affect rigor force, which indicates that the number of crossbridges participating in contraction was not reduced. In conclusion, H2O2 may reduce the maximum Ca2+ activated force production in skinned muscle fibres by decreasing the force per crossbridge.

Animals↗
PubMed2000

Mechanisms of second-order conditioning with a backward conditioned stimulus.

Five conditioned suppression experiments with rats examined the conditions under which backward pairings endow a first-order conditioned stimulus (CS1) with the ability to serve as a secondary reinforcer. Experiments 2-5B found evidence for excitatory second-order conditioning (SOC) if, during first-order pairings, the US-CS1 interval was 0 s rather than 3 s. Levels of SOC were comparable after forward and backward pairings (Experiments 1-3), and were unaffected by extinction of CS1 after SOC (Experiment 3). These results suggest that forward and backward CS1s support SOC for the same reason, and they call into question the need to invoke any special mechanism such as memory integration.

Analysis of Variance↗