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D A Bell

Publications and source records attributed to D A Bell.

At least 145 records · Page 8Linked to original sources
PubMed1993

CYP1A1 mRNA levels as a human exposure biomarker: use of quantitative polymerase chain reaction to measure CYP1A1 expression in human peripheral blood lymphocytes.

Accurate human risk assessment requires sensitive methods to evaluate dose-response relationships, especially following low level exposures. We have developed a reverse transcriptase polymerase chain reaction (RT-PCR) method to quantitative cytochrome P450-1A1 (CYP1A1) mRNA levels in human blood lymphocytes. Many polycyclic aromatic hydrocarbons (PAH) such as benzo[a]pyrene, and chlorinated PAH such as polychlorinated dibenzodioxins, dibenzofurans and biphenyls induce CYP1A1 expression through activation of an endogenous protein, the Ah receptor. Using a quantitative competitive RT-PCR method that included a synthetic internal standard we determined copy numbers of CYP1A1 mRNA in resting as well as mitogen-stimulated human blood lymphocytes. In mitogen-stimulated human blood lymphocytes assay variation was approximately 10% for measurement of this low expression gene and mRNA levels correlated well with ethoxyresorufin-O-deethylase (EROD) activity. The expression of mRNA was induced 20-fold upon culturing human lymphocytes with 10 nM TCDD. In nonstimulated, uninduced lymphocytes CYP1A1 levels are extremely low (1000 copies mRNA/10(4) cells) and cannot be measured by EROD activity. Studies of CYP1A1 mRNA expression in chemically-exposed populations are in progress.

Base Sequence↗
PubMed1993

Genotype/phenotype discordance for human arylamine N-acetyltransferase (NAT2) reveals a new slow-acetylator allele common in African-Americans.

Carcinogenic arylamines are acetylated by the hepatic N-acetyltransferase. This enzyme is polymorphic in humans and in some epidemiological studies, the slow-acetylator phenotype has been associated with higher risk of bladder cancer and lower risk of colorectal cancer. The presence of two germline copies of any of several mutant alleles of the NAT2 gene produces a slow-acetylation phenotype. We used a PCR-RFLP technique to identify three known slow-acetylator alleles (M1, M2 and M3). Comparison of results from PCR-RFLP genotyping with caffeine metabolism phenotyping in 42 individuals suggested that an additional slow-acetylator allele was present in our sampled population. We sequenced the NAT2 gene for several discordant slow-acetylator individuals and found a G > A base-change in codon 64 that caused a Arg > Glu amino acid substitution. This sequence change, termed the 'M4' allele, was found in all of the discordant individuals in our population and apparently causes a slow-acetylation phenotype. In addition, we have determined that NAT2 allele frequencies in 372 Caucasian-Americans (WT = 0.25, M1 = 0.45, M2 = 0.28, M3 = 0.02, and M4 = 0.00) and in 128 African-Americans (WT = 0.36, M1 = 0.30, M2 = 0.22, M3 = 0.02 and M4 = 0.09) are significantly different (P < 0.0001). The M4 allele was not found in 372 unrelated Caucasians and appears to be of African origin.

Acetylation↗
PubMed1993

Molecular analysis of mutations induced at the hisD3052 allele of Salmonella by single chemicals and complex mixtures.

More single chemicals and complex environmental mixtures have been evaluated for mutagenicity at the hisD3052 allele of Salmonella, primarily in strain TA98, than in any other mutation assay. The development of colony probe hybridization procedures and the application of the polymerase chain reaction and direct DNA sequencing has permitted rapid molecular access to this allele. We discuss these techniques and the resulting mutation spectra that have been induced by a variety of environmental mutagens and complex mixtures. A common GC or CG deletion within a hot-spot region of the sequence dominates most of the spectra. In addition to this two-base deletion, we have recovered about 200 other types of mutations within the 72-base target for reversion of the hisD3052 allele. These include a variety of deletions (as large as 35 bases), duplications (as large as 46 bases), and complex mutations involving base substitutions. The quasipalindromic nature of the target sequence and its potential to form DNA secondary structures and slippage mismatches appear to be an important basis for the mutability of this allele.

Alleles↗
PubMed1993

Construction of recombinant RNA templates for use as internal standards in quantitative RT-PCR.

The PCR has proven to be useful in the analysis of gene expression of specific mRNAs. Although PCR is able to detect rare mRNA transcripts following reverse transcription PCR, determining relative or absolute copy number can be difficult due to sample-to-sample variation. The use of a recombinant mRNA internal standard that contains target mRNA primer sequences greatly improves reproducibility of quantitation. Reverse transcription PCR products generated from the internal standard can be distinguished from the product generated from the target gene mRNA because of their size difference. In this report we present a facile and general PCR-based method for synthesis of internal standards that may be used as competitive or co-amplified templates for quantitative reverse transcription PCR.

Animals↗
PubMed1993

Immunoglobulin V region heavy and light chain gene sequences of the lymphoblastoid cell line GM 4672.

GM 4672 is an IgG2 kappa-producing lymphoblastoid cell line derived from a patient with multiple myeloma. It has been used by many laboratories as a fusion partner for the production of human-human hybridoma monoclonal antibodies. GM 4672 immunoglobulin variable region heavy and light chain family usage was originally assigned to VH1 and VK1, respectively. This assignment was based on the positions of [3H]leucine of the heavy and light chain proteins using the Edman degradation method. Using the polymerase chain reaction and variable region leader primers and constant region primers, we report here the immunoglobulin variable region gene sequence expressed by GM 4672. The VH region belongs to the VH4 family and is most homologous with the V71-2 (87.9%), DK1, and JH4 germline genes. The entire heavy chain V region contained 41 mutations in 36 codons and included 11 N nucleotide additions flanking the D region. GM 4672 VK region contained a VK1 gene rearranged with a JK4 gene. The VK germline gene used by GM 4672 light chain was not identified but showed the most homology with Vb' germline gene (87.7%). When compared to Vb' and JK4 genes, there were 37 mutations in 30 codons with evidence of antigen selection as determined by the replacement to silent mutation ratio in the complementarity-determining regions. The high frequency of mutations in the V region genes of GM 4672 is comparable to the sequences of other myeloma proteins.

Amino Acid Sequence↗
PubMed1993

Intravenous administration of gadolinium in the evaluation of rheumatoid arthritis of the shoulder.

The authors sought to determine the utility of intravenously administered gadolinium chelates in magnetic resonance imaging (MRI) of patients with rheumatoid arthritis of the shoulder. One shoulder was examined for each of 12 patients--6 men and 6 women, ranging in age from 48 to 71 (average 63) years--with well-established disease. The patients had had the disease for 1 to 25 (average 13) years. Static MRI was performed before and after infusion of gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA) (0.1 mmol/kg body weight); T1-weighted or proton-density images (repetition time [TR] 600 to 1000 ms, echo time [TE] 15 to 30 ms) were obtained. Images obtained after enhancement were particularly useful in the differentiation of pannus from fluid and also allowed improved delineation of tears of the supraspinatus tendon. In addition, contrast-enhanced dynamic gradient-echo spoiled GRASS sequences (TR 50 ms, TE 11 ms, flip angle 70 degrees) were obtained for all patients; for these sequences the same slice was imaged repeatedly at 20-second intervals. The rate of increase of signal intensity in abnormal synovium varied from 0.64 to 5.83 (average 2.30, standard deviation [SD] 1.67) units/second; the enhancement factor ranged from 1.55 to 4.64 (average 2.63, SD 0.98). The authors conclude that for imaging the shoulder enhancement with Gd-DTPA allows improved distinction between synovial thickening and joint effusion and may improve assessment of the rotator cuff. The wide range in the rate of signal increase and total enhancement during dynamic imaging probably reflected heterogeneity in the study population.

Acromioclavicular Joint↗
PubMed1992

DNA flow cytometry of epithelioid sarcoma.

DNA ploidy of paraffin-embedded tissue from 11 patients with epithelioid sarcoma and 20 control specimens (granuloma annulare and sarcoid) was studied. DNA aneuploidy was found in 64% of the epithelioid sarcomas and in none of the control samples. Aneuploidy did not correlate with necrosis, vascular invasion, or mitotic rate. There was also no association of aneuploidy with adverse outcome.

Aneuploidy↗
PubMed1992

A human monoclonal autoantibody to platelet glycoprotein IIb derived from normal human lymphocytes.

Tonsillar lymphocytes from an otherwise healthy nonthrombocytopenic male child were fused with the lymphoblastoid cell line GM 4672. Twenty of 472 (4%) hybridomas had antiplatelet reactivity detected using intact platelets in an enzyme-linked immunosorbent assay. One hybridoma (STO 171) reacted to platelet glycoprotein IIb (integrin alpha IIb) as determined by radioimmunoprecipitation and immunoblotting. Antibody specificity was confirmed using immunodepletion experiments with isotypic antibodies derived from a mutlitransfused Glanzmann's thrombasthenic patient. The antibody reactivity was restricted to platelets and did not react with other integrin alpha-chain proteins expressed on granulocytes or cultured human brain-derived microvascular endothelial cells. These studies indicate that lymphocytes of normal, nonthrombocytopenic individuals have the genetic potential to produce antiplatelet autoantibodies.

Antibodies, Monoclonal↗
PubMed1992

DNA content in juvenile granulosa cell tumors of the ovary: a study of early- and advanced-stage disease.

Paraffin-embedded tissue from 38 patients with early- and advanced-stage juvenile granulosa cell tumor (JGCT) of the ovary was analyzed by flow cytometry to investigate whether the DNA content is related to the histologic features, stage, or clinical outcome. Thirty-three cases were suitable for analysis: twenty-seven Stage Ia, two Stage Ic, and four Stage III. Eighteen (55%) tumors were DNA diploid and fifteen (45%) tumors were DNA aneuploid with a mean S-phase fraction (SPF) of 13.6% and a range of DNA indices from 1.0 to 2.2. Neither the DNA ploidy nor the SPF was associated with the stage of the disease. An analysis of the relation between DNA content and histopathologic features revealed that aneuploidy was associated with high mitotic rates and to a lesser extent with high-grade nuclear atypia. DNA aneuploidy was not associated with aggressive behavior of Stage Ia JGCTs. However, among the four patients with Stage III tumors, the two with diploid, low-SPF tumors were alive and well, whereas the two with aneuploid, high-SPF tumors developed recurrences or died. These data suggest that further studies on the prognostic significance of flow cytometric analysis of DNA content in advanced-stage JGCTs are warranted.

Adolescent↗
PubMed1992

Preoperative serum CA-125 levels in borderline tumors of the ovary.

Preoperative serum CA-125 levels were evaluated in 38 patients who underwent primary surgery for epithelial ovarian tumors of borderline malignancy at the Brigham and Women's Hospital and Massachusetts General Hospital between 1981 and 1990. Surgical staging was Stage I in 25 (66%) patients, Stage II in 2 (5%) patients, Stage III in 10 (26%) patients, and Stage IV in 1 (3%) patient. The mean sizes of mucinous and serous ovarian tumors were 21.9 and 10.3 cm, respectively (P = 0.0002). All 13 patients (100%) with mucinous tumors had Stage I disease, while 12 (50%) of 24 patients with serous tumors were Stage I. Combining all cell types, 10 (40%) of 25 patients with Stage I disease had an elevated preoperative CA-125 level, while 2 (100%) of 2, 9 (90%) of 10, and 1 (100%) of 1 patient with Stage II, III, and IV disease, respectively, had increased preoperative levels. Among patients with serous tumors, 3 (25%) of 12 Stage I patients had an elevated preoperative CA-125 level, while 11 (92%) of 12 Stage II-IV tumors had elevated levels (P less than 0.001). These data suggest that preoperative CA-125 level correlates with stage of disease in patients with serous borderline ovarian tumors.

Adult↗
PubMed1992

Production and characterization of human hybridoma monoclonal anti-Sm and anti-U1RNP antibodies spontaneously produced from normal human lymphocytes.

A panel of human:human hybridomas secreting monoclonal anti-Sm/RNP antibodies was established by the fusion of normal human tonsillar lymphocytes to the lymphoblastoid B cell line GM4672. The specificity of these antibodies was studied by direct binding and competitive inhibition in enzyme linked immunosorbent assays (ELISAs), and by immunoblotting. The stable subclones of these hybridoma monoclonal anti-Sm/RNP antibodies could be classified into four groups according to ELISA. Group I bound Sm/RNP only, group II bound Sm-RNP, Ro/SS-A and La/SS-B, group III bound Sm/RNP and ssDNA, and group IV bound Sm/RNP, Ro/SS-A, La/SS-B and ssDNA. When antibodies from each of the groups were tested by immunoblotting, the following pattern of reactivity emerged. Group II and IV antibodies reacted with U1RNP-A, Sm-B'/B, Sm-D and Sm-E proteins, as well as the Ro/SS-A and La/SS-B proteins. In contrast, group I and III antibodies did not bind to any individual protein components of Sm/RNP,Ro/SS-A or La/SS-B antigens, but recognized their conformational epitopes. These results, therefore, directly demonstrate for the first time that normal-derived B cells have the genetic potential, revealed here by somatic cell hybridization, to produce anti-Sm/RNP antibody responses which are ordinarily only associated with systemic lupus erythematosus (SLE) and related connective tissue diseases.

Antibodies, Antinuclear↗
PubMed1992

Characterization of human-human hybridoma monoclonal anti-Ro(SS-A) autoantibodies derived from normal tonsil lymphoid cells.

Human-human hybridomas obtained from the separate fusion of tonsillar lymphoid cells from three different normal individuals to the lymphoblastoid cell line GM 4672 were screened by ELISA for the presence of autoantibody to Ro(SS-A). Those anti-Ro(SS-A) reactive hybridomas were then cloned by limiting dilution. Nineteen monoclonal IgM anti-Ro(SS-A) antibodies were obtained, which showed specificity to Ro(SS-A) by ELISA and Western blotting (60 kDa). Some of these monoclonal anti-Ro(SS-A) antibodies showed reactivity to DNA (2/19), cardiolipin (9/19), Sm/RNP (15/19) by ELISA, and to IgG (12/19) and La(SS-B) (19/19) by ELISA and Western blotting. None showed reactivity to the unrelated proteins casein and BSA, nor to RNA. Inhibition studies revealed that the binding to Ro(SS-A) of both IgM hybridoma monoclonal and SLE serum polyclonal IgM anti-Ro(SS-A) antibodies was inhibited with Ro(SS-A), La(SS-B) and Sm/RNP but not with IgG, DNA, RNA and BSA. These data indicate that (1) normal humans have the genetic potential to express antibodies to Ro(SS-A) and (2) the normally derived monoclonal and SLE serum IgM anti-Ro(SS-A) antibodies share similar antigen binding properties and therefore may possibly originate from a common pool of precursor B cells.

Antibodies, Antinuclear↗
PubMed1992

DNA content and proliferative activity in ovarian small cell carcinomas of the hypercalcemic type. Implications for diagnosis, prognosis, and histogenesis.

Ovarian small cell carcinoma of the hypercalcemic type is a rare cancer of young women of nuclear histogenesis. Although usually lethal, a subset of patients with stage I tumors have survived. Twenty-five cases of small cell carcinoma (17 stage I, 1 stage II, and 7 stage III) were evaluated by flow cytometric analysis performed on paraffin-embedded tissue. Forty classifiable histograms from 23 cases were DNA diploid; histograms from two cases could not be interpreted. The mean S-phase fraction was 10.5% (4.7% to 18.4%), and the mean G2/M fraction was 5% (1.5% to 19.5%) in 22 cases. Mean values of mitotic rate, S-phase fraction, G2/M fraction, and proliferation index (%S + %G2/M) were not associated with stage or outcome, nor did any proliferation variable correlate with interval to death. A comparative review of flow cytometric findings in other types of ovarian cancer that may be confused with small cell carcinoma indicates that flow cytometry is an objective diagnostic aid to distinguish small cell carcinoma of the hypercalcemic type from other forms of small cell carcinoma, malignant germ cell tumors, and sex-cord tumors, and that small cell carcinoma probably does not belong in either the surface epithelial or germ cell categories of ovarian cancer. Finally, the flow cytometric findings in this report exemplify the rare phenomenon of a diploid DNA content in a very lethal tumor and indicate that an accurate diagnosis of tumor type is essential before a prognosis on the basis of flow cytometric data can be made.

Adolescent↗
PubMed1992

DNA flow cytometry of primary tumors and their skin metastases. Correlation of DNA histograms.

The purpose of this study was to determine whether quantification of DNA content would demonstrate conserved patterns in primary tumors and their metastases and to learn whether this technique would be useful in determining if a tumor was metastatic from a previous primary tumor or represented a new neoplasm. The study comprised of all cases nonmelanoma metastatic skin lesions in which the primary tumor was also resected at this hospital between the years 1984 and 1990 (22 cases). Both the primary and metastatic lesions were examined by the deparaffinized flow cytometric technique for DNA content. DNA-aneuploid tumors were considered to correlate if the DNA indices (ratio of aneuploid to diploid peak channels) were within 10%. Thirty-four tumors were DNA-aneuploid; eight tumors were DNA-diploid. We found agreement in 20 of 22 cases (agree--aneuploid: 16 cases; agree--diploid: four cases). In six of our cases the histograms of the primary and the metastatic lesions did not correlate when only one block was examined; however, when three blocks from each site were studied, we found concordance of DNA indices in four cases. There was disagreement in two of 22 cases (disagree--different DNA indices: two cases). In both of these cases, only one block of the primary tumor was available for examination, and the histograms were of marginal quality. This study demonstrates that the histogram pattern (aneuploid versus diploid) of DNA content is highly conserved between primary and metastatic tumors. There was 91% agreement between DNA indices of primary tumors and their metastases (20 of 22 cases) using a 10% correlation criterion.(ABSTRACT TRUNCATED AT 250 WORDS)

Aneuploidy↗
PubMed1992

The production of human monoclonal antiplatelet auto-antibodies derived from human lymphocytes of normal origin: reactivity to DNA, anionic phospholipids and platelet proteins.

Human hybridoma monoclonal antiplatelet antibodies were produced using tonsillar lymphocytes from a nonthrombocytopenic male fused to the lymphoblastoid cell line GM 4672. Twenty of 472 (4%) IgM producing hybridomas had antiplatelet reactivity as detected by ELISA. Thirteen of these antiplatelet antibody producing hybridomas with clonality ensured by limiting dilution were tested for antigenic specificity. Two different and mutually exclusive groups of antiplatelet antibodies were identified. The first group of antiplatelet antibodies (four clones) showed reactivity that was limited to DNA and anionic phospholipids. Antibodies from the second group (seven clones) showed reactivity by immunoblotting to a variety of platelet proteins including platelet glycoprotein IIb. These antibodies did not bind DNA nor anionic phospholipids. These studies indicate that lymphocytes of normal human origin have the genetic potential to produce antiplatelet autoantibodies. These antiplatelet antibodies segregate on the basis of their target antigens into two major groups, which mimic the target antigens held responsible for antiplatelet autoantibodies in disease. These include glycoproteins (typical of chronic idiopathic thrombocytopenic purpura) and DNA and/or anionic phospholipids (typical of the lupus anticoagulant syndrome).

Antibodies, Monoclonal↗
PubMed1992

Genetic monitoring of human polymorphic cancer susceptibility genes by polymerase chain reaction: application to glutathione transferase mu.

Several genes involved in the metabolism of carcinogens have been found to be polymorphic in human populations and are associated with increased risk of cancer at some sites. This study focuses on the polymorphic enzyme glutathione transferase mu (GT mu). Smokers with low lymphocyte GT mu activity are at an approximately 2-fold higher risk for lung cancer and an approximately 3-fold higher risk for stomach and colon adenocarcinomas. Recent cloning and sequencing of the GST1 gene has allowed the development of convenient genotyping methods based on restriction fragment length polymorphisms (RFLP) or the polymerase chain reaction (PCR). The GST1 polymorphism has been shown to be a deletion of the gene locus. To detect the presence or absence of the gene we amplified exons 4-5 and/or exons 6-7 of the GST1 gene by PCR. PCR amplification produced bands of 215-bp or 273-bp from individuals with one or two copies of the GST1 allele and no band if the individual was homozygously deleted (0/0). In the exon 6-7 PCR, we co-amplified a 268-bp portion of the beta-globin gene as an internal reference standard for quantitative analysis of product yield. This allowed homozygote individuals (+/+) to be distinguished from heterozygotes (+/0). We have compared the GST1 genotype to lymphocyte GT mu activity measured on trans-stilbene oxide (TSO) in the lymphocytes of 45 individuals. Low GT mu activity (< 67 pmole/min/10(7) cells) was strongly associated (24/24) with the GST1 0/0 genotype. With the exception of one individual, activities greater than 67 pmole/min/10(7) were associated with the presence of the GST1 allele (20/21). Individuals with the highest GT-TSO activity were found to be homozygous for GST1. (+/+), while heterozygotes (+/0) generally had lower activity, suggesting a gene dosage effect in lymphocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Gene Deletion↗