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D K Johnson

Publications and source records attributed to D K Johnson.

At least 37 records · Page 2Linked to original sources
PubMed1998

Unlike other chemicals, etoposide (a topoisomerase-II inhibitor) produces peak mutagenicity in primary spermatocytes of the mouse.

The cancer chemotherapy agent, and topoisomerase-II inhibitor, etoposide (VP-16) produced both recessive mutations at specific loci and dominants at other loci with peak frequencies in primary spermatocytes, a cell type in which the topo-II gene has been shown to be activated. Etoposide thus differs from all other chemicals whose germ-cell-stage specificity has been analyzed. No effects of etoposide exposure of spermatogonial stem cells ( approximately 15, 000 offspring scored) were detectable by either mutagenicity or productivity endpoints. The significant mutagenic response that followed exposure of poststem-cell stages ( approximately 25,000 offspring scored) showed a clear peak, with three of four specific-locus mutants, and three of four dominant mutants conceived during weeks 4 or 5 (days 22-35) post-injection, a period that also encompassed the dominant-lethal peak. For this period, the induced specific-locus rate (with 95% confidence limits) at a weighted-average exposure of 75.1 mg etop/kg was 59.5 (14.6, 170. 9)x10-6/locus. At least 3 of the 4 specific-locus mutations were deletions, paralleling findings with etoposide or analogs in other test systems where a recombinational origin of the deletions has been suggested. Because, unlike other chemicals that induce deletions in male germ cells, etoposide is effective in stages normally associated with recombinational events, it will be of interest to determine whether this chemical can affect meiotic recombination.

Animals↗
PubMed1998

Quantitation of white cell subpopulations by polymerase chain reaction using frozen whole-blood samples. Viral Activation Transfusion Study.

BACKGROUND: Previous methods for processing whole blood (WB) for nucleic acid analyses of white cells (WBCs) required fresh blood samples. A simple protocol that involves the freezing of WB for quantitative polymerase chain reaction (PCR) analyses was evaluated. STUDY DESIGN AND METHODS: Controlled studies were conducted in which paired fresh and frozen WB preparations were analyzed. The integrity of WBCs in the frozen WB samples was first assessed by flow cytometry using CD45 fluorescence, and calibration beads to quantitate recovery of WBC subsets. PCR of an HLA-DQ-A sequence was used to quantitate residual WBCs in a double-filtered red cell (RBC) component spiked with serial dilutions of WBCs, as well as in 51 filtered RBCs and 19 filtered platelet concentrates. Y-chromosome-specific PCR was used to quantitate male WBCs in five female WB samples spiked with serial dilutions of male WBCs and in serially collected frozen WB samples from four females transfused with male blood components. RESULTS: By flow cytometry, all major WBC subpopulations in frozen-thawed WB were quantitatively recovered and immunologically intact, although they were nonviable. HLA-DQ-A PCR quantitation of a dilution series from 8 to 16,700 per mL of WBCs spiked into double-filtered RBCs showed linear correlation of the results with both fresh and frozen preparations of the expected WBC concentrations (r2 = 0.98, p<0.0001 for both), without significant difference between observed and expected values (p>0.05). Y-chromosome-specific PCR results in female WB samples spiked with male WBCs were not significantly different in fresh and frozen preparations over a 3 log10 range of male cells. The results of WBC survival studies on frozen WB samples were consistent with previous observations in fresh blood samples. CONCLUSION: Direct freezing of WB enables subsequent recovery of WBCs for quantitative PCR analyses, with results comparable to those of fresh preparations.This protocol should facilitate wider implementation of nucleic acid-based analyses for quality control of WBC-reduced components, as well as for prospective clinical studies of microchimerism in transfusion and transplant recipients.

Blood Transfusion↗
PubMed1998

Thrombin stimulated reactive oxygen species production in cultured human endothelial cells.

In order to study the major cellular source of reactive oxygen species (ROS) in perturbed human endothelial cells (EC), the effect of thrombin, a phospholipase A2 activator, on cultured EC ROS generation has been investigated. EC were incubated with 0.1-1 unit/ml thrombin and cellular superoxide anion (O(-)2) release and hydrogen peroxide (H2O2) production measured. Thrombin exposure caused an elevation in EC O(-)2 release and H2O2 production. The effects of protein kinase C, arachidonic acid metabolism, NADPH oxidase, and phospholipase A2 inhibitors on thrombin-induced EC H2O2 production were examined. EC were exposed to 0.5 unit/ml thrombin and cellular H2O2 production measured in the presence and absence of the protein kinase C inhibitor, H-7; arachidonic acid metabolism inhibitors, indomethacin, nordihydroguaiaretic acid, and SKF525A; NADPH oxidase inhibitor, apocynin; and phospholipase A2 inhibitor, 4-bromophenacyl bromide. All inhibitors, with the exception of H-7 and indomethacin, suppressed thrombin-induced EC H2O2 production. The pattern of effects of these metabolic antagonists on thrombin-induced EC ROS production is similar to that previously reported on ROS production in EC exposed to high low-density lipoprotein levels, and in stimulated leukocytes. These findings further implicate NADPH oxidase as a major ROS source in EC.

Cells, Cultured↗
PubMed1997

Lymphocyte subset analysis on frozen whole blood.

An approach to perform lymphocyte subset analysis on frozen-thawed whole blood (F/T WB) is described. WB from 24 human immunodeficiency virus type 1 (HIV-1) seropositive individuals and 21 controls was analyzed fresh and after frozen storage (with or without dimethyl sulfoxide) at -80 degrees C, in liquid nitrogen (LN2), and at -20 degrees C. Analysis of F/T WB utilized 3-color flow cytometry with CD45 and right angle light scatter gating. Absolute cell counts were obtained for 30 samples by using staining tubes containing internal bead standards [TruCount, Becton Dickinson Immunocytometry Systems (BDIS), San Jose, CA]. The mean difference between CD3+4+ percentages for F/T (-80 degrees C storage for up to 1 year) and fresh WB was less than -0.2% (95% limits +/-3%, P = 0.5) with 39 of 45 (87%) results falling within 2% of the fresh values (P = 0.74). Absolute CD3+4+ cell counts for F/T WB were generally lower than corresponding results for fresh aliquots (median difference was 33 cells/microl, P < 0.0001), but the results were highly correlated (r2 = 0.975, P < 0.0001). Results were more variable, although still highly correlated, for CD3+8+ cells, and with other freezing and storage conditions. It is concluded that lymphocyte subset analysis using F/T WB yields comparable results to fresh samples, which should prove useful for a number of practical applications.

Blood Preservation↗
PubMed1997

Mutations in the murine fitness 1 gene result in defective hematopoiesis.

Identification and characterization of mutations that disrupt normal hematopoiesis are essential for understanding the genetic pathways that control the development and regulation of the mammalian hematopoietic system. Previously, the fitness 1 gene was identified by five, independent mutations in N-ethyl-N-nitrosourea (ENU) saturation mutagenesis experiments within the albino (c) region of mouse chromosome 7 (MMU7). We report here that fit1 mutants are anemic, display numerous peripheral blood defects, and are deficient in early hematopoietic progenitor cell populations. The number of both erythroid and myeloid progenitors, as well as B cells, are reduced. These results implicate fit1 involvement in normal hematopoiesis and suggest that further characterization of the fit1 gene, and the five presumed point mutations of the gene, will lead to an improved understanding of normal hematopoiesis in the mouse.

Animals↗
PubMed1997

Mapping individual cosmid DNAs by direct AFM imaging.

Individual cosmid clones have been restriction mapped by directly imaging, with the atomic force microscope (AFM), a mutant EcoRI endonuclease site-specifically bound to DNA. Images and data are presented that locate six restriction sites, predicted from gel electrophoresis, on a 35-kb cosmid isolated from mouse chromosome 7. Measured distances between endonuclease molecules bound to lambda DNA, when compared to known values, demonstrate the accuracy of AFM mapping to better than 1%. These results may be extended to identify other important site-specific protein-DNA interactions, such as transcription factor and mismatch repair enzyme binding, difficult to resolve by current techniques.

Animals↗
PubMed1997

Inherited somatic mosaicism caused by an intracisternal A particle insertion in the mouse tyrosinase gene.

A recessive, fully penetrant mutation (c(m1OR)) at the mouse albino locus that results in coat-color mottling has been characterized at the molecular level. Restriction mapping and DNA sequencing analyses provide evidence that mutants carry a 5.4-kb intracisternal A particle (IAP) element insertion upstream of the tyrosinase (Tyr) promoter. Northern blot analysis and reverse transcription-PCR results show that the tyrosinase gene is expressed at much lower levels in mutant than in wild-type mice. The mutant Tyr gene still retains the tissue-specific expression pattern, and the Tyr transcript is not initiated from the IAP long terminal repeat promoter. We propose that the IAP insertion isolates the promoter of the tyrosinase gene from upstream cis-acting regulatory elements, leading to a substantially decreased level of Tyr gene expression in mutants.

Animals↗
PubMed1997

GABAA-receptor alpha-subunit is an essential prerequisite for receptor formation in vivo.

The mechanisms governing the assembly of alpha-, beta- and gamma-subunits to form GABAA-receptors are poorly understood. Here, we report that the alpha-subunit is essential for receptor assembly. In mice homozygous for a deletion on chromosome 7 spanning the alpha 5- and gamma 3-subunit genes, zolpidem-insensitive benzodiazepine binding sites, corresponding to GABAA-receptors containing the alpha 5-subunit, were absent in the hippocampus. This loss of alpha 5-GABAA-receptor binding was also apparent as a 21% decrease in the total number of benzodiazepine binding sites in the hippocampus. In addition, immunoreactivity for the beta 2,3- and gamma 2-subunit was decreased exclusively in neurons which normally express the alpha 5-subunit, such as olfactory bulb granule cells and hippocampal pyramidal cells. In other brain regions of the mutants, the beta 2,3- and gamma 2-subunit staining was unaffected. Controls included two lines of mice homozygous for a shorter chromosomal deletion, that either included or excluded the gamma 3-subunit gene. These two lines were indistinguishable with regard to numbers of benzodiazepine binding sites and levels alpha 5-, beta 2,3- and gamma 2-subunit immunoreactivity, indicating that the lack of gamma 3-subunit gene did not contribute to the observed deficit in receptor formation. These results demonstrate that the absence of the alpha 5-subunit gene prevents the formation of the entire respective receptor complex in adult mouse brain. Thus, the alpha-subunit, unlike the gamma 2-subunit, might play a major role in the assembly or targeting of GABAA-receptor complexes.

Animals↗
PubMed1997

Automatic volumetric capillary cytometry for counting white cells in white cell-reduced plateletpheresis components.

BACKGROUND: As the benefits of white cell (WBC)-reduced blood components become increasingly apparent, the need has arisen for a simple, automated WBC-counting technique that is sensitive to low WBC concentrations. Automated volumetric capillary cytometry was evaluated for its ability to quantify residual WBCs in WBC-reduced plateletpheresis components. STUDY DESIGN AND METHODS: The volumetric capillary cytometry system evaluated uses a laser to excite fluorescent dye-labeled nucleated cells. The number of nucleated cells per microliter is reported. Four studies were performed: linearity, precision of results near the value of 5 x 10(6) WBCs per unit, the limit of detection, and correlation to the Nageotte manual counting method. RESULTS: Assay values correlated to expected values (range, 0-125 WBC/microliter) with an r2 > 0.99. In the range of 5 x 10(5) WBCs per unit the CV was 8.5 percent, and concentration differences of 0.15 log10 were detectable. The limit of detection was 1.0 WBCs per microliter (95% upper confidence limit). The assay correlated to the Nageotte method with an r2 of 0.98, slope of 1.0, and y-intercept of 2.0 WBCs per microliter. Assay results were 10 to 15 percent higher than Nageotte results, in samples with values near 5 x 10(6) WBCs per unit. Technician time per sample was 2 to 3 minutes. CONCLUSION: Volumetric capillary cytometry is precise and sensitive to small differences in WBC concentration in the range of clinical interest. The device provides an efficient new method for quality assurance and control of WBC-reduced plateletpheresis products.

Blood Component Removal↗
PubMed1997

Environmental endotoxin measurement: interference and sources of variation in the Limulus assay of house dust.

Studies suggest that endotoxin levels in offices and homes may be associated with sick building syndrome or increased asthma severity. However, reported endotoxin levels in these studies were highly inconsistent, suggesting incompatible measurements from various laboratories. Therefore, an investigation of Limulus assay for endotoxin in house dust was undertaken. Interference with the assay was common and could produce endotoxin estimates varying by a factor of > 100, depending on the dilution used. Analysis of dose-response curves allowed detection of two types of interference: dilution-dependent and dilution-independent. Dilution-dependent interference persisted when samples were reassayed, but valid estimates could be obtained by appropriate dilution and data analysis. Valid estimates could not be obtained from assay showing dilution-independent interference. However, dilution-independent interference was frequently overcome by repeating the assay. Estimates based on a single sample dilution produced reasonable results on average when a dilution factor of 900 was used. However, the results were less precise from a single dilution (median coefficient of variation [CV] 40%) than from the complete dose-response curve (median CV 23%) and ranged from 11 to 240% of the estimates based on complete dose-response data. A comparison of extraction methods found that sonication in a phosphate-triethylamine buffer produced greater endotoxin activity than extraction in buffer with addition of saponin or sodium dodecyl sulfate. Endotoxin activity was stable for 8 to 10 weeks in dust samples stored at 4 and -20 degrees C but was not stable in extracts. The lot of Limulus amebocyte lysate reagent and method of detecting a response in the kinetic Limulus assay had significant effects on endotoxin estimates.

Analysis of Variance↗
PubMed1997

Murine albino-deletion complex: high-resolution microsatellite map and genetically anchored YAC framework map.

The murine albino-deletion complex developed as part of the Oak Ridge specific-locus test covers 6-11 cM of chromosome 7. This complex has proven to be a valuable resource for localizing traits to a small target region for positional cloning. In this study, we mapped the endpoints of deletions in this complex using all of the available Mit simple-sequence length polymorphism (SSLP) markers. Concurrently, this mapping has determined the map order of nearly all of the SSLP markers, most of which were previously unresolved. The SSLP-based deletion map was confirmed and genetic distances were determined using the European Collaborative Interspecific Backcross panel of nearly a thousand mice. The average SSLP marker resolution is 0.3-0.4 cM, comparable to the cloning capacity of yeast artificial chromosomes (YACs). The SSLP markers were then used to construct a genetically anchored YAC framework map that further confirms the deletion map. We find that the largest deleted region distal to Tyr is about two to three times larger than the largest proximal deletion region, and the original C3H/101 regions flanking the deletions (moved to an St2A cch/cch background) are smaller than anticipated, which we suggest may result from increased recombination rates immediately flanking the deleted regions.

Animals↗
PubMed1997

Low-density lipoprotein stimulated peroxide production and endocytosis in cultured human endothelial cells: mechanisms of action.

The effects of arachidonic acid metabolism and NADPH oxidase inhibitor on the hydrogen peroxide (H2O2) generation and endocytotic activity of cultured human endothelial cells (EC) exposed to atherogenic low-density lipoprotein (LDL) levels have been investigated. EC were incubated with 240 mg/dl LDL cholesterol and cellular H2O2 production and endocytotic activity measured in the presence and absence of the arachidonic acid metabolism inhibitors, indomethacin, nordihydroguaiaretic acid, and SKF525A, and NADPH oxidase inhibitor, apocynin. All inhibitors, with the exception of indomethacin, markedly reduced high LDL-induced increases in EC H2O2 generation and endocytotic activity. EC exposed to exogenously applied arachidonic acid had cellular functional changes similar to those induced by high LDL concentrations. EC incubated with 1-25 uM arachidonic acid had increased H2O2 production and heightened endocytotic activity. Likewise, EC pre-loaded with [3H]arachidonic acid when exposed to increasing LDL levels (90-330 mg/dl cholesterol) had a dose-dependent rise in cytosolic [3H]arachidonic acid. The phospholipase A2 inhibitors, 4-bromophenacyl bromide and 7,7-dimethyleicosadienoic acid, markedly inhibited H2O2 production in EC exposed to 240 mg/dl LDL cholesterol. These findings suggest that arachidonic acid contributes mechanistically to high LDL-perturbed EC H2O2 generation and heightened endocytosis. Such cellular functional changes add to our understanding of endothelial perturbation, which has been hypothesized to be a major contributing factor in the pathogenesis of atherosclerosis.

Acetophenones↗
PubMed1996

Mapping of the ARIX homeodomain gene to mouse chromosome 7 and human chromosome 11q13.

The recently described homeodomain protein ARIX is expressed specifically in noradrenergic cell types of the sympathetic nervous system, brain, and adrenal medulla. ARIX interacts with regulatory elements of the genes encoding the noradrenergic biosynthetic enzymes tyrosine hydroxylase and dopamine beta-hydroxylase, suggesting a role for ARIX in expression of the noradrenergic phenotype. In the study described here, the mouse and human ARIX genes are mapped. Using segregation analysis of two panels of mouse backcross DNA, mouse Arix was positioned approximately 50 cM distal to the centromere of chromosome 7, near Hbb. Human ARIX was positioned through analysis of somatic cell hybrids and fluorescence in situ hybridization of human metaphase chromosomes to chromosome 11q13.3-q13.4. These map locations extend and further define regions of conserved synteny between mouse and human genomes and identify a new candidate gene for inherited developmental disorders linked to human 11q13.

Animals↗
PubMed1995

Initial experience evaluating 90yttrium-radiolabeled anti-carcinoembryonic antigen chimeric T84.66 in a phase I radioimmunotherapy trial.

Chimeric T84.66 (cT84.66) is a high-affinity (5 x 10(10) M-1) anti-carcino-embryonic antigen (CEA) IgG1. In a recently completed pretherapy imaging trial, 111In-labeled cT84.66 demonstrated targeting of CEA-producing metastatic sites and low immunogenicity, with human antichimeric antibody (HACA) response in only 1 of 15 patients after a single administration. The purpose of the present study was to evaluate cT84.66-diethylenetriaminepentaacetic acid labeled with 90Y in a dose-escalation Phase I trial. Patients with metastatic CEA-producing malignancies received imaging doses of 5 mCi 111In-labeled cT84.66 first, followed 1-2 weeks later by 5 mg cT84.66 labeled with the therapeutic dose of 90Y. Immediately following the therapeutic infusion, diethylenetriaminepentaacetic acid was administered by continuous i.v. infusion over 3 days at 250 mg/m2 body surface area/24 h. Biodistribution, tumor targeting, absorbed radiation dose estimates, antibody clearance, and HACA response were evaluated through blood samples, 24-h urine collections, and nuclear images performed at serial time points after infusion. To date, three patients with metastatic colorectal cancer have been evaluated at the first dose level of 5 mCi/m2. No side effects were associated with antibody administration. Localization of the antibody to nonhepatic metastatic sites was observed. Size-exclusion high-performance liquid chromatography demonstrated the formation of CEA:antibody complexes in serum in all three patients. A significant variation among patients in the clearance rate of the antibody and complexes from blood to liver was seen, which resulted in a reciprocal relationship between estimated liver dose and red marrow dose. Patients who demonstrated faster clearance to liver demonstrated greater excretion of a low-molecular-weight metabolite through the urine. Two patients developed HACA response, which persisted at 4 months after therapy. At this first dose level, no tumor responses were seen and reversible grade 1 thrombocytopenia was observed in 2 patients. cT84.66 demonstrated effective localization in CEA-producing tumors. Its low immunogenicity after a single administration makes it attractive for further evaluation as a radioimmunotherapeutic agent. However, further evaluation is needed to determine whether its immunogenicity will remain low after multiple administrations. Additionally, in two of the three patients, we identified rapid clearance of the antibody to the liver. This underscores the need to identify, characterize, and understand further those factors that influence the biodistribution and clearance of anti-CEA antibodies to allow for better selection of patients for therapy and rational planning of radioimmunotherapy.

Carcinoembryonic Antigen↗