Biomedical subjects
R B Wilson
Publications and source records attributed to R B Wilson.
Normal serum iron and ferritin concentrations in patients with Friedreich's ataxia.
Friedreich's ataxia (FRDA) is caused by point mutations or trinucleotide repeat expansions in both alleles of the gene encoding frataxin. Studies of frataxin homologues in lower eukaryotes suggest that mitochondrial iron accumulation may underlie the pathophysiology of FRDA. To evaluate the possible role of iron-chelation therapy for FRDA, we measured serum iron and ferritin concentrations in 10 FRDA patients. The measurements were within normal limits, suggesting that iron-chelation therapy for FRDA may be problematic.
Novel bcl-2 breakpoints in patients with follicular lymphoma.
Using genomic DNA from patients with follicular lymphoma, we performed polymerase chain reaction (PCR) amplifications to detect t(14;18) translocations. Unexpectedly large products of approximately 1 kilobase (kb) were detected by gel electrophoresis in 2 of 50 positive cases. In these 2 cases, sequence analyses showed novel breakpoints in the 3' untranslated region of bcl-2, approximately 800 bp downstream of the major breakpoint region (mbr). The breakpoints in IgH occurred in JH4 in one patient and JH5 in the other. Sequences just upstream of the new bcl-2 breakpoints suggest a mechanism of translocation that may include minisatellite core-mediated recombination. In one of our two patients with novel bcl-2 breakpoints, the approximately 1 kb product obtained using conventional mbr primers was detectable only when a nested PCR was performed. These findings have important implications for diagnosis and minimal residual disease detection in t(14;18)-positive lymphomas.
Ethylene glycol poisoning: toxicokinetic and analytical factors affecting laboratory diagnosis.
Ethylene glycol poisoning is an important toxicological problem in medical practice because early diagnosis and treatment can prevent considerable morbidity and mortality. When ingested in the form of antifreeze or other automotive products, ethylene glycol results in central nervous system depression, cardiopulmonary compromise, and renal insufficiency. Metabolism of ethylene glycol to organic acids is required for metabolic derangement and organ damage. Laboratory features of ethylene glycol poisoning include increased anion gap metabolic acidosis, increased osmolal gap, calcium oxalate crystalluria, and detectable ethylene glycol in serum. This Case Conference integrates discussion of the toxicokinetic and analytical variables that affect the laboratory diagnosis of ethylene glycol intoxication.
An evaluation of the Factor V Leiden mutation in a cohort of African-American pregnant women.
The objective of this work was to study the prevalence of the Factor V Leiden mutation in an obstetrical clinic largely comprised of African-American women. A cross-sectional study was performed evaluating a total of 231 consecutive women of African-American origin. Of these patients, 21 were considered at high risk for thrombosis, but none was found to carry the mutation. One patient (0.4 per cent) of the total was heterozygous for the Factor V Leiden mutation. African-American women do not appear to be at an increased risk of being heterozygous or homozygous for the Leiden mutation. This low prevalence may be confounded by ascertainment bias in a population of pregnant women.
Interaction of erythropoietin RNA binding protein with erythropoietin RNA requires an association with heat shock protein 70.
Synthesis of erythropoietin (Epo), the glycoprotein hormone that regulates red blood cell formation, is induced in response to low oxygen stress (hypoxia), and is regulated at both transcriptional and post-transcriptional levels. We have previously described an Epo RNA binding protein (ERBP) which specifically binds to the 3'-untranslated region of Epo mRNA and is likely involved in the regulation of Epo mRNA stability. Since heat shock proteins (hsps) are induced in response to a variety of stresses, including hypoxia, we tested the possibility that hsps are involved in ERBP-Epo RNA complex formation. When human anti-hsp70 antibody was added to ERBP-containing human hepatoma cell (Hep3B) lysates, the ERBP-Epo RNA complex was inhibited in an electrophoretic mobility band shift assay. In addition, the anti-hsp70 antibody-inhibited complex could be rescued if lysates were pretreated with purified inducible hsp70, but not with bovine serum albumin (BSA). In vivo studies using quercetin to inhibit hsp70 induction support the notion that hsp70 is involved in ERBP-Epo RNA complex formation. Taken together, these findings suggest involvement of hsp70 in ERBP-Epo mRNA complex formation, and our model suggests a novel role for hsps in the regulation of EPO mRNA stability.
Respiratory deficiency due to loss of mitochondrial DNA in yeast lacking the frataxin homologue.
Friedreich's ataxia (FRDA) is an autosomal recessive degenerative disorder that primarily affects the nervous system and heart. Patients with FRDA have point mutations or trinucleotide repeat expansions in both alleles of FRDA, which encodes a protein termed frataxin. We show that the yeast frataxin homologue, which we have named YFH1, localizes to mitochondria and is required to maintain mitochondrial DNA. The YFH1-homologous domain of frataxin functions in yeast and a disease-associated missense mutation of this domain, or the corresponding domain in YFH1, reduces function. Our data suggest that mitochondrial dysfunction contributes to FRDA pathophysiology.
Localization of BRCA1 and a splice variant identifies the nuclear localization signal.
Inherited mutations in BRCA1 confer susceptibility to breast and ovarian neoplasms. However, the function of BRCA1 and the role of BRCA1 in noninherited cancer remain unknown. Characterization of alternately spliced forms of BRCA1 may identify functional regions; thus, we constructed expression vectors of BRCA1 and a splice variant lacking exon 11, designated BRCA1 delta 672-4095. Immunofluorescence studies indicate nuclear localization of BRCA1 but cytoplasmic localization of BRCA1 delta 672-4095. Two putative nuclear localization signals (designated NLS1 and NLS2) were identified in exon 11; immunofluorescence studies indicate that only NLS1 is required for nuclear localization. RNA analysis indicates the expression of multiple, tissue-specific forms of BRCA1 RNAs; protein analysis with multiple antibodies suggests that at least three BRCA1 isoforms are expressed, including those lacking exon 11. The results suggest that BRCA1 is a nuclear protein and raise the possibility that splicing is one form of regulation of BRCA1 function by alteration of the subcellular localization of expressed proteins.
Protein kinase C beta from Friend erythroleukemia cells is associated with chromatin and DNA.
Certain protein kinase C (PKC) isotypes are localized to the nucleus during cellular proliferation in murine erythroid cells, as well as in human promyelocytic leukemia and erythroleukemia cells. Because the structure of these PKC isotypes contains a conserved cysteine-rich region that contains the zinc finger DNA binding motif, we tested the hypothesis that selected PKC isotypes found in Friend erythroleukemia cells can bind to DNA. Cell lysates from murine Friend erythroleukemia cells, which express alpha, beta I, and beta II PKC, expressed greater amounts of the beta isoforms than the alpha isoform of PKC in their nuclei, and PKC beta I was found in the chromatin of these cells. Lysates of these cells were tested for their ability to bind to a DNA-cellulose column. Bound proteins were eluted with a step gradient of increasing KCl concentrations, and eluant fractions were then subjected to immunoblot analysis using isotype-specific antibodies to the alpha and beta I isotypes of PKC. DNA binding was detected for the PKC beta I isotype, which is present in the nucleus, but not for the more abundant PKC alpha isotype, which resides primarily in the cytoplasm. These results demonstrate that PKC can associate with DNA, and that this association is isotype specific in Friend erythroleukemia cells.
Inhibitory effects of zidovudine in erythroid progenitor cells. Reversal with a combination of erythropoietin and interleukin-3.
To investigate the mechanisms that may be involved in zidovudine (AZT)-induced hematopoietic toxicity, spleen cells isolated from phenylhydrazine-treated anemic mice or murine bone marrow erythroid progenitor cells were treated with AZT (1-10 microM) for 24 hr. A concentration-dependent inhibition of the binding of 125I-labeled erythropoietin (Epo) was observed, suggesting down-regulation of Epo receptors. To determine if this effect is due to modulation of the levels of Epo receptor mRNA and to assess the effect of AZT on the expression of protooncogenes, mRNA levels were monitored by the slot blot hybridization technique. AZT caused a concentration-dependent inhibition in the levels of the mRNA of Epo receptors and c-fos, whereas the level of c-myc mRNA was unaffected. AZT also inhibited protein kinase C (PKC) activity in a concentration- and time-dependent manner, causing 50% inhibition at 10 microM within 3 hr. Simultaneous addition of Epo or interleukin-3 (IL-3) partially reversed the inhibitory effects of AZT on the levels of the mRNAs and on PKC activity; however, a combination of Epo and IL-3 was significantly more effective. These studies demonstrate that (i) AZT-induced down-regulation of Epo receptors and c-fos expression coupled with inhibition of Epo receptor-mediated signal transduction through PKC are significant contributory factors to AZT-induced erythroid toxicity, and (ii) these inhibitory effects can be overcome by treatment with a combination of Epo and IL-3.
Wild type PAX3 protein and the PAX3-FKHR fusion protein of alveolar rhabdomyosarcoma contain potent, structurally distinct transcriptional activation domains.
Alveolar rhabdomyosarcoma (ARMS) is characterized cytogenetically by a t(2;13)(q35;q14) chromosomal translocation involving two transcription factor genes: PAX3 and FKHR. ARMS cells express a PAX3-FKHR fusion protein containing the complete N-terminal, DNA-binding domain of PAX3 and the C-terminus of FKHR. Recently we demonstrated that PAX3-FKHR is a more potent transcriptional activator than PAX3 despite impaired binding to canonical PAX3 binding sites. Therefore, we propose that the gene fusion results in switching of PAX3 and FKHR transactivation domains with distinct structure, potency or function. To compare the PAX3 and putative PAX3-FKHR transactivation domains, we fused C-terminal test fragments to the heterologous GAL4 DNA-binding domain and tested activation of a reporter gene co-transfected into four cell types. GAL4-PAX3 and GAL4-PAX3-FKHR were found to be potent activators exhibiting different concentration-dependent transactivation profiles and distinct structural motifs. Deletion mapping demonstrated essential acidic and/or serine/threonine-rich domains in the extreme 3' ends of their respective coding regions and positive modifying elements in adjacent 5' sequences. These data demonstrate that PAX3 and PAX3-FKHR contain structurally distinct transcriptional activation domains and suggest that a consequent difference in function is important for oncogenesis.
Changes in redox affect the activity of erythropoietin RNA binding protein.
We have previously identified a cytosolic protein, erythropoietin RNA binding protein (ERBP), which is up-regulated in certain tissues in response to hypoxia. To further characterize the interaction of ERBP and erythropoietin (EPO) mRNA, we have examined the role of reduction-oxidation in the EPO mRNA binding mechanism of ERBP isolated from human hepatoma cells (Hep3B). Reducing agents dithiothreitol (DTT) and 2-mercaptoethanol (2-ME) increased ERBP binding activity in a concentration-dependent manner, whereas the oxidizing agent, diamide, abolished ERBP binding activity. In addition, treatment of Hep3B cell lysates with the irreversible sulfhydryl alkylating agent N-ethylmaleimide resulted in inhibition of the EPO mRNA-ERBP complex. Taken together, these findings suggest that sulfhydryl groups may play a role in vivo in the regulation of EPO production through the modulation of ERBP binding activity.
Presumptive botulism in cattle associated with plastic-packaged hay.
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Guidelines for internal validation of the HLA-DQ alpha DNA typing system.
Validation experiments were performed to evaluate the HLA-DQ alpha DNA typing system for forensic casework. Temperature profiles for two Perkin Elmer TC-1 Thermal Cyclers were measured, and the efficiency of the instruments was tested by amplifying a control sample (DQ alpha 1.2,4) susceptible to allelic drop-out. DNA extraction using chelex was compared to non-organic extraction, and the amplification and hybridization procedures were evaluated at extremes of time and temperature. With the protocol in place, samples exposed to stresses commonly encountered in forensic casework were typed to determine the flexibility of the system. Mixed samples of two different bloods and blood mixed with saliva were typed to determine the threshold at which mixtures could be resolved using the reverse dot blot method. Different storage conditions were evaluated using a set of control bloodstrains, and a set of 12 postmortem blood samples was typed repeatedly over the course of 5 months to determine the effects of natural degradation on the DQ alpha results. Finally, casework stains on a variety of substrates were typed. These experiments demonstrate the flexibility of the HLA-DQ alpha system. Based upon these results, a comprehensive quality assurance program was developed to ensure the integrity of typing results for casework.
The receptor-recycling and lysosome biogenesis mutant TfT1.11 belongs to a new complementation group, End6.
We have previously isolated a Chinese hamster ovary (CHO) mutant with a temperature-dependent pleiotropic defect in receptor recycling. This mutant, TfT1.11, has also been shown to have defects in fluid-phase endocytosis and lysosome biogenesis. Previously isolated CHO cell mutants with defects in endocytosis have been assigned to five recessive complementation groups (End1-End5). We have performed complementation analysis using polyethylene glycol-induced fusion of genetically marked sublines of TfT1.11 with representative mutants from each of the End groups. Complementation of the receptor trapping and lysosome biogenesis defects as well as temperature lethality was observed with all groups, demonstrating that TfT1.11 defines a new complementation group, End6.
Designer red cells.
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The IgE and IgG antibody responses to aerosols of Nephrops norvegicus (prawn) antigens: the association with clinical hypersensitivity and with cigarette smoking.
Raised levels of serum IgE antibodies to prawn antigens were found in 15 of 26 seafood factory process workers with respiratory symptoms and in one of 26 case-matched asymptomatic controls (P < 0.001). Raised IgG antibody titres against the same antigens were found in 18 subjects in each symptom grouping, and the median titres of this antibody did not differ significantly between the groups. The prawn-specific IgE antibody response was significantly associated with atopy (IgE antibody response to common allergens) and with a history of cigarette smoking, confirmed by level of serum cotinine, a major nicotine metabolite. Non-atopic non-smokers were unlikely to become sensitized. The titre of the prawn-specific IgE antibody correlated with the duration of exposure and with the duration of symptoms. Discriminant analysis of the serological profile (anti-prawn IgE, total IgE and cotinine) was sufficient to assign individuals correctly into symptomatic or asymptomatic categories in 77% of subjects. The titres of the IgE and IgG antibody responses to prawn antigens did not correlate, and the main factor which seemed to determine the antibody isotype response to these inhaled antigens was cigarette smoking. IgE antibody was produced mainly by smokers, whereas IgG antibody was the predominant isotype produced by non-smokers.
Laparoscopic cholecystectomy in pregnancy: two case reports.
Laparoscopic cholecystectomy (LC) was previously avoided in pregnancy. Two cases of LC performed in gravid women with recurrent symptoms of biliary disease are reported.