Biomedical subjects
Publications and source records attributed to D M Lucas.
We describe recent progress in the development of an ion-trap quantum information processor. We discuss the choice of ion species and describe recent experiments on read-out for a ground-state qubit and photoionization trap loading.
The SH2 domain containing inositol 5'-phosphatase (SHIP) was initially described as a 145-kDa protein phosphorylated on tyrosines upon growth factor and cytokine stimulation. It was shown to be phosphorylated after Fc and B cell receptor activation and plays a role in negative signaling. Different isoforms of the SHIP protein result from alternative mRNA splicing, proteolysis, or a combination of both. The expression of discrete SHIP isoforms changes with the potential developmental-dependent maturation state of myeloid cells, suggesting mechanisms for the regulation of SHIP interactions with other signaling molecules. A p135 (SHIPbeta) spliced isoform is known to be expressed in developing myeloid cells. Now we have identified a new SHIP isoform, SHIPdelta, which is the product of an out-of-frame splice with a deletion of 167 nucleotides in the C-terminal region, resulting in an approximately 110-kDa protein. Biochemically, SHIPdelta differs from SHIPalpha by exhibiting little or no tyrosine phosphorylation or association with the signaling protein Shc after M-CSF activation of FD-Fms cells. In addition, we have characterized the structure of the entire SHIP genomic locus, which provides a basis for understanding the alternative splicing events. SHIP is expressed in hematopoiesis and spermatogenesis, and we also describe the promoter for the SHIP gene, which has potential for explaining the tissue-specific expression pattern.
The inositol 5'-phosphatase, SHIP (also referred to as SHIP-1 or SHIPalpha), is expressed in all cells of the hematopoietic lineage. Depending on the cell type being investigated and the state of differentiation, SHIP isoforms of several different molecular masses (170, 160, 145, 135, 125, and 110 kDa) have been seen in immunoblots. However, the function of the individual isoforms and the effect of expressing multiple isoforms simultaneously are not understood. Some of these SHIP isoforms have recently been characterized at the level of primary sequence. In this report, we investigated the function of the recently characterized 135-kDa SHIP isoform (SHIPbeta), which appears to possess the catalytic domain but lacks some of the protein-protein interaction motifs at the C terminus. By reconstituting SHIP-deficient DT40 B cells with either SHIPbeta or the better-characterized p145 SHIPalpha, we addressed the function of SHIPbeta in the complete absence of SHIPalpha. We observed that SHIPbeta had enzymatic activity comparable with SHIPalpha and that SHIPbeta was able to reconstitute F(c)gammaRIIB1-mediated inhibition of B cell receptor-induced signaling events such as calcium flux and Akt and mitogen-activated protein kinase activation. SHIPbeta was readily phosphorylated in response to B cell receptor cross-linking with the inhibitory receptor F(c)gammaRIIB1 and SHIPbeta also interacted with the adapter protein Shc. During these studies we also observed that the SHIPalpha or SHIPbeta interaction with Grb2 is not required for F(c)gammaRIIB1-mediated inhibition of calcium flux. These data suggest that SHIPbeta, which is normally expressed in B cells along with SHIPalpha, functions comparably with SHIPalpha and that these two isoforms are not likely to be antagonistic in their function in vivo.
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SH2-containing Inositol Phosphatase (SHIP) is a 145 kD protein expressed in hematopoietic cells. SHIP is phosphorylated on tyrosine after receptor binding by several cytokines and has a negative role in hematopoiesis. We cloned a murine complementary DNA (cDNA) sequence for an isoform of SHIP with an internal 183 nucleotide deletion, encoding a protein 61 amino acids shorter than 145 kD SHIP. This deletion eliminates potential SH3-domain binding regions and a potential binding site for the p85 subunit of Phosphatidylinositol 3-Kinase. Using polyclonal anti-SHIP antibodies, we and others have previously observed a 135 kD SHIP isoform that is coexpressed with 145 kD SHIP. Here, we used monoclonal antibodies raised against the region deleted in the spliced form to show that the product of the novel spliced SHIP cDNA is antigenically identical to the 135 kD SHIP isoform. Like 145 kD SHIP, 135 kD SHIP expression was induced on differentiation of bone marrow cells. After macrophage colony-stimulating factor (M-CSF) stimulation of FDC-P1(Fms) myeloid cells, both 145 and 135 kD SHIP forms were tyrosine phosphorylated and could be coimmunoprecipitated with antibodies to Shc and Grb2. However, experiments showed only a weak association of 135 kD SHIP with p85. A potentially analogous 135 kD SHIP species also appears in human differentiated leukocytes.
BACKGROUND: Antifibrinolytic medications administered before skin incision decrease bleeding after cardiac surgery. Numerous case reports indicate thrombus formation with administration of epsilon-aminocaproic acid (epsilon-ACA). The purpose of this study was to examine the efficacy of epsilon-ACA administered after heparinization but before cardiopulmonary bypass in reducing bleeding and transfusion requirements after primary coronary artery bypass surgery. METHODS: Seventy-four adult patients undergoing primary coronary artery bypass surgery were randomized to receive 125 mg/kg epsilon-ACA followed by an infusion of 12.5 mg x kg(-1) x h(-1) or an equivalent volume of saline. Coagulation studies, thromboelastography, and platelet aggregation tests were performed preoperatively, after bypass, and on the first postoperative day. Mediastinal drainage was recorded during the 24 h after surgery. Homologous blood transfusion triggers were predefined and transfusion amounts were recorded. RESULTS: One patient was excluded for surgical bleeding and five patients were excluded for transfusion against predefined criteria One patient died from a dysrhythmia 2 h postoperatively. Among the remaining 67, the epsilon-ACA group had less mediastinal blood loss during the 24 h after surgery, 529+/-241 ml versus 691+/-286 ml (mean +/- SD), P < 0.05, despite longer cardiopulmonary bypass times and lower platelet counts, P < 0.05. Platelet aggregation was reduced in both groups following cardiopulmonary bypass but did not differ between groups. Homologous blood transfusion was similar between both groups. CONCLUSIONS: Prophylactic administration of epsilon-ACA after heparinization but before cardiopulmonary bypass is of minimal benefit for reducing blood loss postoperatively in patients undergoing primary coronary artery bypass grafting.
We previously demonstrated that the macrophage cell lines RAW 264.7 and WEHI-3 exhibit distinct patterns of gene expression in response to IFN-gamma. This difference is controlled at the transcriptional level and results from a specific inability of the less mature WEHI-3 cells to utilize either the IFN-stimulated response element or the gamma-activated sequence DNA regulatory element in response to stimulation with IFN-gamma, while other aspects of IFN-gamma gene induction remain intact. In the work described here, we examined the components of the IFN-gamma signal transduction pathway in RAW 264.7 and WEHI-3 cells to determine whether differences in pathway components or activity exist in WEHI-3 cells that could give rise to this difference in transcriptional response. Reverse transcriptase-PCR (RT-PCR) and flow cytometric analyses indicated that the levels of IFN-gamma receptor mRNA accumulation and protein expression are comparable for RAW 264.7 and WEHI-3 cells. RT-PCR and immunoblot analyses revealed that the principal components of this signaling pathway, including JAK1, JAK2, and STAT1, are present in both RAW 264.7 and WEHI-3 cells. However, analysis of STAT1 DNA-binding activity by electrophoretic mobility shift assay and of STAT1 phosphorylation by immunoblot revealed that this DNA-binding factor is active in RAW 264.7, but not in WEHI-3, cells after IFN-gamma stimulation. These results demonstrate that the components of the IFN-gamma signal transduction pathway are intact in WEHI-3 cells, but stimulation of these cells by IFN-gamma does not result in STAT1 activation.
BACKGROUND: In the United States, there is a shortage of blood group phenotype-matched red cells (RBCs) for patients with sickle cell disease (SCD). A protocol designed to supply phenotype-matched RBCs for these patients by combining the recruitment of African American blood donors and automated testing of RBCs for these patients for the presumptive Fy(a-b-) phenotype using monoclonal anti-Fy3 was evaluated. STUDY DESIGN AND METHODS: African American donors were recruited, to increase the likelihood of phenotype matches in the donor population. Samples of RBCs were tested for the presumptive Fy(a-b-) phenotype by using monoclonal anti-Fy3 and an automated blood typing analyzer. RBCs confirmed to be Fy(a-b-) were retyped for selected Rh, MNS, Kell, Duffy, and Kidd blood system antigens. The extended phenotypes were matched with those of 41 SCD patients requiring transfusions. RESULTS: Of 8323 blood donations during the study, approximately 40 percent (3329) were made by African Americans. Approximately 22 percent (737) of African Americans were identified as Fy(a-b-) by this protocol and 12 percent (410) were phenotype matches for the 41 SCD patients. CONCLUSION: Combining the recruitment of African American blood donors and automated phenotyping using monoclonal anti-Fy3 offers a practical, relatively low-cost strategy for supplying phenotype-matched RBCs for SCD patients. This protocol increases the options for addressing the shortage of phenotype-matched RBCs for SCD patients.
Two experiments were conducted to determine whether administering a dopamine antagonist to steers fed endophyte-infected (E+) tall fescue would increase serum prolactin (PRL) and reduce rectal temperature. Steers in both experiments were housed in environmentally controlled chambers (32 degrees C; 50% relative humidity). In Exp. 1, 10 steers were allotted randomly to receive s.c. injections of either 0, .006, .03, or .06 mg of Ro 24-0409 (dopamine antagonist)/kg BW. The experiment was designed in four phases: endophyte-free seed (E-) without antagonist (d -11 to 0); E- with antagonist (d 0 to 7); E+ with antagonist (d 7 to 28); E+ without antagonist (d 28 to 38). In Exp. 2, 22 Holstein steers were allotted randomly to the same treatments and design, except three steers were maintained on E- without antagonist. Steers were fed individually with intakes measured daily. In Exp. 1, feed intake and rectal temperature were not improved (P > .05) by antagonist injection. In Exp. 2, antagonist injections increased (P < .05) PRL. Ingestion of E+ decreased (P < .05) feed intake and serum PRL. Antagonist injection decreased (P < .05) rectal temperature and increased (P < .05) serum PRL.
We have characterized the transcriptional response to IFN-gamma in two maturationally distinct macrophage populations: the mature RAW 264.7 cell line, phenotypically identical to thioglycollate-elicited peritoneal macrophages, and the less mature WEHI-3 cell line. We first investigated the use of two IFN-gamma-responsive regulatory elements, the interferon-stimulated response element (ISRE) and the gamma-activated sequence (GAS), in these cells. Transient transfection assays revealed that synthetic promoter constructs containing either the ISRE or GAS regulatory motif fused to a luciferase reporter gene were transcriptionally inactive in the WEHI-3 cell line. We then analyzed the expression in the two cell lines of a panel of known IFN-gamma-responsive genes that are transcriptionally controlled by different regulatory elements. RT-PCR analysis revealed that both cell lines responded to IFN-gamma treatment by up-regulating genes that are transcriptionally controlled by kappa B or W box DNA binding motifs. However, genes regulated by ISRE or GAS elements were induced by IFN-gamma only in the RAW 264.7 cell line. Kinetic analysis of the transcriptional activity of synthetic promoter constructs in the RAW 264.7 cell line showed rapid IFN-gamma induction through both the ISRE and GAS motifs, indicating that both elements are utilized early after IFN-gamma stimulation in mature macrophages. These results suggest that cis-acting DNA response element utilization, and the subsequent profiles of IFN-gamma-induced gene expression, differ in macrophages at different stages of maturation.
A comprehensive epidemiological study of the involvement of cannabis and ethanol in motor vehicle fatalities in the Province of Ontario, Canada, is described. The study is based on toxicological analyses of blood and, when available, urine specimens. Ethanol was determined by headspace gas chromatography (GC). For cannabis, the methods employed were radioimmunoassays (RIAs) for screening and gas chromatography/mass spectrometry (GC/MS) for the determination of delta-9-tetrahydrocannabinol (THC) in blood. The study sample consisted of 1169 drivers and 225 pedestrians. THC was detected in the blood of 127 driver victims (10.9%) in concentrations ranging from 0.2 to 37 ng/mL, with a mean of 3.1 +/- 5.0 ng/mL. Ethanol was found in 667 driver victims (57.1%), in concentrations ranging from 9 to 441 mg/100 mL, with a mean of 165.8 +/- 79.5 mg/100 mL. For pedestrians, the incidence of THC and ethanol in the blood was 7.6 and 53.3%, respectively. The incidence of THC in the driver victims in this study constitutes an approximately threefold increase over the results of an Ontario study completed in 1979. At least a part of the increase may be attributed to interstudy differences in analytical methodology for cannabinoids.
Two experiments were conducted to determine the effects of salinomycin and lasalocid on metabolism and growth of growing steers. In Exp. 1, 80 Angus steers (228 kg) were assigned to the following treatments: 1) control, 2) 50 mg salinomycin.hd-1.d-1, 3) 100 mg salinomycin.hd-1.d-1 and 4) 250 mg lasalocid.hd-1.d-1. Steers were fed corn silage once daily with allotments based on the amount of silage that each pen of five steers would consume in a 24-h period. In addition, .81 kg/hd of a corn-soybean meal supplement was fed daily during the 112-d study. Daily gains were similar across treatments, but feed intake was lower (P less than .05) for steers fed ionophores. Molar proportions of ruminal acetate were lower (P less than .05) in steers fed ionophores at 28 and 90 d. Ruminal propionate was lower (P less than .05) in control steers at 28 d, but values were similar across treatments on d 90. Plasma copper (Cu) was lower (P less than .05) in control steers on both sampling days. In Exp. 2, 16 Hereford steers were allotted to two blocks of eight animals each and assigned to one of three treatments: 1) control (n = 6), 2) 11 mg salinomycin/kg diet (n = 6) and 3) 33 mg lasalocid/kg diet (n = 4). Following a 28-d adjustment period, apparent absorption and retention of macrominerals and nitrogen (N) were determined during a 5-d collection period. Apparent absorption and retention of N did not differ among treatments when data were analyzed using N intake as a covariate.(ABSTRACT TRUNCATED AT 250 WORDS)
Grazing trials were conducted for 2 yr using weanling Brahman crossbred beef steers to evaluate graded levels of salinomycin (0, 50, 100 or 150 mg. head-1.d-1) for 161 d and to evaluate salinomycin in a free-choice mineral supplement (99 d). The 40 and 48 steers in trials 1 and 2 had average initial weights of 198 and 285 kg, respectively. In trial 1, steers were group-fed to consume either 0, 50, 100 or 150 mg of salinomycin.head-1.d-1 in .9 kg ground corn while grazing bermudagrass pastures. Both linear (P less than .01) and quadratic (P less than .05) effects were observed for steer performance as salinomycin level increased from 0 to 150 mg.head-1.d-1. Linear increases (P less than .01) in ruminal NH3-N (mg/100 ml) and in the molar proportion of propionate and decreases (P less than .01) in butyrate and acetate/propionate were detected. In trial 2, mineral supplements with and without salinomycin were fed free-choice to steers on bermudagrass pasture. The mean salinomycin intake of 38 mg.head-1.d-1 was lower than anticipated as a result of the instability of salinomycin in the mineral supplement and the slightly lower intake (65 g/d) than anticipated (75 g/d). Performance of steers was not influenced by salinomycin supplementation in trial 2. The ionophore salinomycin at intakes over 50 mg.head-1.d-1 appears to increase the performance of steers grazing bermudagrass pasture.
Six trials involving 586 pigs initially averaging 9 kg were conducted at three locations (two trials/location) to evaluate the effects of the dietary addition of 0, 27.5, 55, 82.5 and 110 ppm of a monocarboxylic polyether antibiotic, salinomycin, on feed intake, growth rate and feed efficiency of swine fed corn-soybean meal-based diets. Over the total trial (9 to 97 kg), quadratic improvements in gain (P less than .01) and feed:gain (P less than .04) occurred with increasing level of salinomycin, but feed intake was not affected (P greater than .25). Mean improvements for all drug levels over control values for the total trial were 4.3% for daily gain (P less than .01) and 3.4% for feed:gain (P less than .01). For the total trial, rate and efficiency of gain were optimized in pigs fed the 82.5-ppm level of salinomycin with an improvement of 5.2% in daily gain and 4.8% in feed:gain over pigs fed the control diet. The absence of any significant trial X treatment interaction suggests that the nature of the response was not dependent on differences in environment, management or other factors that existed among trial locations.
Results are presented of a comprehensive drug study carried out on specimens from drivers and pedestrians fatally injured in Ontario. Toxicological analyses were regularly performed on blood and urine and occasionally on vitreous humor, stomach contents, and liver. The analytical procedures could detect and quantitate a wide variety of drugs including such illicit drugs as Cannabis. With respect to drivers, alcohol was found in 57% of the study sample and drugs other than alcohol, in 26%. However, in only 9.5% of the drivers were psychoactive drugs (other than alcohol) detected in the blood in concentrations that may adversely affect driving skills. Delta-9-Tetrahydrocannabinol and diazepam accounted for a majority of the findings in this category.
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