Search PubMed⌕ Search

Biomedical subjects

H F Bunn

Publications and source records attributed to H F Bunn.

At least 37 records · Page 2Linked to original sources
PubMed1995

Use of a marked erythropoietin gene for investigation of its cis-acting elements.

To examine the function of conserved noncoding regions in the erythropoietin (Epo) gene, we have prepared clones and pools of Hep3B cells stably transfected with a marked 4.1-kilobase Epo gene and deletions thereof. The marked transcripts had single base substitutions at three sites in the coding portion of Exon 5, enabling them to be distinguished from endogenous Epo mRNA by ribonuclease protection and competitive polymerase chain reaction. The basal expression and hypoxic induction of the marked Epo gene that had no deletions were indistinguishable from that of the endogenous Epo gene. Likewise, deletion of conserved intervening sequence 1 had minimal effect on hypoxic induction. In contrast, a 3'-deletion that included the conserved 3'-enhancer element resulted in a substantial, but not complete, suppression of hypoxic induction while a 3'-deletion downstream of the enhancer resulted in enhancement. A 188-base pair deletion of a conserved 3'-untranslated region in Exon 5 had minimal effect on hypoxic induction. However, the truncated Epo mRNA had a markedly prolonged half-life (15 h) in comparison to the endogenous Epo mRNA (2.0 h) or the marked full-length Epo mRNA (2.1 h). Further deletions in the 3'-UTR showed that a relatively small region of approximately 50 bases is responsible for the relatively rapid turnover of Epo mRNA. These experiments provide information on cis-acting elements of the Epo gene that cannot be obtained from conventional reporter gene transfection experiments.

Base Sequence↗
PubMed1995

Comments on the physics and chemistry of trehalose as a storage medium for hemoglobin-based blood substitutes: "from Kramers Theory to the Battlefield".

A glass of the naturally-occurring sugar trehalose may be a suitable medium for the storage of hemoglobin-based blood substitutes. Trehalose has many or possibly all of the properties required for this purpose, including solubilization of hemoglobin to a very high concentration, lack of toxicity, slowing of oxidation to the non-oxygen binding methemoglobin, stability at room temperature and above, and ease of transport. It should also be possible to prepare hemoglobin extremely rapidly for injection into the circulation in situations where blood replacement is required immediately, as in a domestic emergency room or on the battlefield. These practical considerations are briefly discussed, as well as the theoretical reasons for slowing of chemical reactions in the glassy state.

Blood Substitutes↗
PubMed1995

The role of hemoglobin based blood substitutes in transfusion medicine.

A cell-free oxygen transporting blood substitute would obviate many of the current concerns about conventional red cell transfusion therapy. Moreover, a stable oxygen-carrying solution could have benefits and applications not possible with red cell transfusions, such as the treatment of acute hypovolemic shock in acute care settings, the treatment of patients such as Jehovah's Witnesses who refuse blood transfusions, the priming of blood oxygenation pumps, ex vivo organ perfusion prior to transplantation, and in vivo perfusion in order to enhance sensitivity to radiation therapy. Among potential blood substitutes that transport oxygen, attention has focused on perfluorocarbons and a variety of hemoglobin preparations, either in free solution or encapsulated into lipid vesicles. In the design and production of hemoglobin solutions the following criteria must be met: low toxicity and antigenicity; efficacy as a plasma expander; prolonged survival in the circulation; adequate oxygen carrying capability and efficient oxygen unloading to tissues; long shelf life. Extensive preclinical testing and recent clinical trials have been performed on human and bovine hemoglobin chemically crosslinked to present rapid leakage of hemoglobin through the kidneys. Bovine hemoglobin has intrinsically low oxygen affinity simulating that of human hemoglobin in red cells. An alternative and attractive strategy is the production of human hemoglobin in E. Coli, thus enabling appropriate genetic mutations to optimize function. These include creation of peptide linkers to enhance plasma survival and amino acid replacements that permit a finely regulated lowering of oxygen affinity.

Animals↗
PubMed1995

Regulation of erythropoietin gene expression.

The study of erythropoietin gene expression provides a paradigm for understanding gene regulation in response to hypoxia. The sensor for detecting alterations in oxygen tension appears to be a heme protein. Ongoing transcription and protein synthesis are necessary for hypoxic induction of erythropoietin messenger RNA. In the past few years, considerable progress has been made in the identification and characterization of cis-acting elements and trans-acting factors that contribute to erythropoietin gene expression. The erythropoietin promoter and 3' enhancer function synergistically in response to hypoxia. Whereas hypoxia-inducible factor 1 specifically binds to the 3' enhancer conferring hypoxic induction, hepatic nuclear factor 4 interacts with the promoter as well as the 3' enhancer for stimulus- and tissue-specific induction of the erythropoietin gene. In addition, a segment in the 3' untranslated region contributes to the relatively rapid turnover of erythropoietin messenger RNA.

Animals↗
PubMed1995

The orphan receptor hepatic nuclear factor 4 functions as a transcriptional activator for tissue-specific and hypoxia-specific erythropoietin gene expression and is antagonized by EAR3/COUP-TF1.

The erythropoietin (Epo) gene is regulated by hypoxia-inducible cis-acting elements in the promoter and in a 3' enhancer, both of which contain consensus hexanucleotide hormone receptor response elements which are important for function. A group of 11 orphan nuclear receptors, transcribed and translated in vitro, were screened by the electrophoretic mobility shift assay. Of these, hepatic nuclear factor 4 (HNF-4), TR2-11, ROR alpha 1, and EAR3/COUP-TF1 bound specifically to the response elements in the Epo promoter and enhancer and, except for ROR alpha 1, formed DNA-protein complexes that had mobilities similar to those observed in nuclear extracts of the Epo-producing cell line Hep3B. Moreover, both anti-HNF-4 and anti-COUP antibodies were able to supershift complexes in Hep3B nuclear extracts. Like Epo, HNF-4 is expressed in kidney, liver, and Hep3B cells but not in HeLa cells. Transfection of a plasmid expressing HNF-4 into HeLa cells enabled an eightfold increase in the hypoxic induction of a luciferase reporter construct which contains the minimal Epo enhancer and Epo promoter, provided that the nuclear hormone receptor consensus DNA elements in both the promoter and the enhancer were intact. The augmentation by HNF-4 in HeLa cells could be abrogated by cotransfection with HNF-4 delta C, which retains the DNA binding domain of HNF-4 but lacks the C-terminal activation domain. Moreover, the hypoxia-induced expression of the endogenous Epo gene was significantly inhibited in Hep3B cells stably transfected with HNF-4 delta C. On the other hand, cotransfection of EAR3/COUP-TF1 and the Epo reporter either with HNF-4 into HeLa cells or alone into Hep3B cells suppressed the hypoxia induction of the Epo reporter. These electrophoretic mobility shift assay and functional experiments indicate that HNF-4 plays a critical positive role in the tissue-specific and hypoxia-inducible expression of the Epo gene, whereas the COUP family has a negative modulatory role.

Anaerobiosis↗
PubMed1994

Erythropoietin structure-function relationships. Identification of functionally important domains.

In order to delineate functionally important domains in erythropoietin (Epo), we have prepared and tested a series of amino acid replacements at 51 conserved sites predicted to be on the surface of the molecule. Alanine replacements permitted preservation of alpha-helical structure. Wild type and mutant Epo cDNAs were transiently expressed at high levels in COS1 and COS7 cells. The biological activity of wild type and mutant Epos was assayed in three Epo-responsive cell types: primary murine erythroid spleen cells, the murine HCD57 erythroleukemia cell line, and the human UT7-EPO leukemia cell line. When Arg14 on predicted Helix A was replaced by Ala, biological activity was substantially reduced, whereas replacement with Glu resulted in total loss of specific bioactivity. In a similar manner, the mutein Arg103-->Ala in Helix C was completely lacking in biological activity, whereas both Ser104-->Ala and Leu108-->Ala had decreased bioactivity. In Helix D, the mutein Gly151-->Ala had markedly decreased bioactivity, whereas that of the adjacent Lys152-->Ala mutein was moderately impaired. In contrast, Ala replacements at three nearby sites on Helix D (147, 146, and 143) resulted in muteins with increased bioactivity. In conclusion, our mutagenesis experiments have identified functionally important domains on the surface of the Epo molecule, at sites comparable with those established for other cytokines.

Amino Acid Sequence↗
PubMed1993

Comparison of the human and mouse erythropoietin genes shows extensive homology in the flanking regions.

Considerable insights into important cis regulatory elements in a gene can be gleaned from the identification of sequence homologies among different species. To extend and optimize the sequence comparison between human and mouse erythropoietin (Epo) genes, we have obtained new human sequence from 5,547 to 385 bp upstream of the cap site and extended the 3' flank by 489 bp. In addition, we have obtained new sequence information on the mouse Epo gene extending from within the 3' untranslated region (UTR) to 1,001 bp downstream of the polyadenylation site. Analysis of these additional sequences shows considerable homology between human and mouse Epo genes as far as 4 kb (human) or 3 kb (mouse) upstream of the cap sites, as well as far more homology at the 3' end than was previously realized. In addition, both species were found to have a high frequency of short interspersed (SINE) repetitive sequences that interrupt homologies in both the 5' flank and within the transcription unit.

Amino Acid Sequence↗
PubMed1993

Erythropoietin structure-function relationships: high degree of sequence homology among mammals.

To investigate structure-function relationships of erythropoietin (Epo), we have obtained cDNA sequences that encode the mature Epo protein of a variety of mammals. A first set of primers, corresponding to conserved nucleotide sequences between mouse and human DNAs, allowed us to amplify by polymerase chain reaction (PCR) intron 1/exon 2 fragments from genomic DNA of the hamster, cat, lion, dog, horse, sheep, dolphin, and pig. Sequencing of these fragments permitted the design of a second generation of species-specific primers. RNA was prepared from anemic kidneys and reverse-transcribed. Using our battery of species-specific 5' primers, we were able to successfully PCR-amplify Epo cDNA from Rhesus monkey, rat, sheep, dog, cat, and pig. Deduced amino acid sequences of mature Epo proteins from these animals, in combination with known sequences for human, Cynomolgus monkey, and mouse, showed a high degree of homology, which explains the biologic and immunological cross-reactivity that has been observed in a number of species. Human Epo is 91% identical to monkey Epo, 85% to cat and dog Epo, and 80% to 82% to pig, sheep, mouse, and rat Epos. There was full conservation of (1) the disulfide bridge linking the NH2 and COOH termini; (2) N-glycosylation sites; and (3) predicted amphipathic alpha-helices. In contrast, the short disulfide bridge (C29/C33 in humans) is not invariant. Cys33 was replaced by a Pro in rodents. Most of the amino acid replacements were conservative. The C-terminal part of the loop between the C and D helices showed the most variation, with several amino acid substitutions, deletions, and/or insertions. Calculations of maximum parsimony for intron 1/exon 2 sequences as well as coding sequences enabled the construction of cladograms that are in good agreement with known phylogenetic relationships.

Animals↗
PubMed1993

Erythropoietin structure-function relationships. Mutant proteins that test a model of tertiary structure.

On the basis of its primary sequence and the location of its disulfide bonds, we propose a structural model of the erythropoietic hormone erythropoietin (Epo) which predicts a four alpha-helical bundle motif, in common with other cytokines. In order to test this model, site-directed mutants were prepared by high level transient expression in Cos7 cells and analyzed by a radioimmuno assay and by bioassays utilizing mouse and human Epo-dependent cell lines. Deletions of 5 to 8 residues within predicted alpha-helices resulted in the failure of export of the mutant protein from the cell. In contrast, deletions at the NH2 terminus (delta 2-5), the COOH terminus (delta 163-166), or in predicted interhelical loops (AB: delta 32-36, delta 53-57; BC: delta 78-82; CD: delta 111-119) resulted in the export of immunologically detectable Epo muteins that were biologically active. The mutein delta 48-52 could be readily detected by radioimmunoassay but had markedly decreased biological activity. However, replacement of each of these deleted residues by serine resulted in Epo muteins with full biological activity. Replacement of Cys29 and Cys33 by tyrosine residues also resulted in the export of fully active Epo. Therefore, this small disulfide loop is not critical to Epo's stability or function. The properties of the muteins that we tested are consistent with our proposed model of tertiary structure.

Amino Acid Sequence↗
PubMed1993

In vivo and in vitro regulation of erythropoietin mRNA: measurement by competitive polymerase chain reaction.

The regulation of erythropoietin (Epo) production was investigated by competitive polymerase chain reaction, a highly sensitive and accurate means of measuring Epo mRNA levels. Co-amplification of the test sample with added mutant Epo cDNA template corrects for variability in the efficiency of amplification. Epo mRNA levels were determined in tissues of normal rats and in animals with varying degrees of anemia. Reduction of the hematocrit level from 0.40 to 0.15-0.20 resulted in a 300-fold increase in kidney Epo mRNA, which comprised 80% of the total Epo mRNA versus 20% from the liver. In contrast, very low levels detected in lung and spleen were not significantly increased by anemia. The human hepatoma cell line, Hep3B, secretes high levels of Epo in response to hypoxia. This regulation is, to a large extent, transcriptional. When Hep3B cells were incubated in the presence of decreasing O2 tension from 160 to 7 mm Hg, there was a monotonic increase in Epo mRNA to 50 to 100 times the normoxic level. Hyperoxia did not suppress basal expression. When cells were incubated at a PO2 of 7 mm Hg, induction of Epo mRNA was first noted at 30 minutes and was maximal at 5 to 6 hours. After Epo mRNA was boosted by a 4-hour hypoxic incubation, cells were then exposed to normoxia, which shut off further transcription of the Epo gene. The decay of Epo mRNA levels closely followed first order kinetics with a half-life of 2 hours, an effective measurement of message stability.

Animals↗
PubMed1993

Reversing ontogeny.

Explore the source record for details and available documents.

Anemia, Sickle Cell↗
PubMed1993

Regulation of the erythropoietin gene.

Erythropoietin (Epo), the hormone that stimulates red blood cell production, is induced by hypoxia. We have utilized the human hepatoma cell line, Hep3B, to investigate the regulation of the Epo gene. We present evidence that the oxygen sensor in Hep3B cells is a heme protein. Hypoxic and cobalt induction of Epo protein is paralleled by a 50- to 100-fold increase in Epo mRNA which we have accurately quantified by means of an assay based on competitive polymerase chain reaction. This increase in Epo mRNA is due primarily to increased transcription. Transfection experiments utilizing the sensitive luciferase reporter gene show that the minimal portions of the Epo gene required for hypoxic induction include a 53 bp promoter element and a 43 bp enhancer located downstream from the polyadenylation site. Gel shift experiments show that these two regions cross-compete for specific DNA binding proteins. The enhancer contains a hexanucleotide direct repeat with a two bp insert which footprints with nuclear extracts from Hep3B cells and, when mutated, results in loss of hypoxic induction. This sequence is likely to bind to a member of the steroid/thyroid hormone receptor family of DNA binding proteins. These enhancer and promoter elements appear to cooperate in enabling the Epo gene to respond to hypoxia in a physiologically appropriate manner.

Erythropoietin↗
PubMed1992

Clonality in myeloproliferative disorders.

The myeloproliferative disorders are a group of hematologic diseases that are believed to arise from somatic mutations in an early hematopoietic stem cell. This statement is based on the demonstration of monoclonal involvement of terminally differentiated myeloid and lymphoid elements. The techniques for establishing clonal derivation of cells are discussed and the application of these techniques to myeloproliferative diseases is reviewed. The evidence for limited myeloid involvement, lineage heterogeneity, in some patients with myeloproliferative disorders is summarized.

Blood Cell Count↗
PubMed1992

Hypoxic induction of the human erythropoietin gene: cooperation between the promoter and enhancer, each of which contains steroid receptor response elements.

Transcription of the human erythropoietin (Epo) gene is stimulated by exposure to hypoxia and/or cobalt in whole animals and in Hep3B cells. We have systematically investigated the promoter and 3' enhancer elements necessary for this induction by transient transfection of Hep3B cells. We define a promoter region of 53 bp and an enhancer region of 43 bp that confer hypoxia and cobalt inducibility. Each element gives rise to a 6- to 10-fold induction alone. In combination they produce a 50-fold induction after stimulation, similar to the 50- to 100-fold induction of the endogenous Epo gene. Two areas of DNA sequence homology are present in these regions. We demonstrate specific DNA-protein interactions in the enhancer and the ability of the promoter element to compete with these interactions in electrophoretic mobility shift assays. DNase I footprinting and methylation interference data further refine the cis-acting element in the 43-bp enhancer to a short region containing a direct repeat of a steroid/thyroid hormone receptor response element half-site separated by a 2-bp gap. Two half-site consensus sequences are also present in the 53-bp promoter. Site-specific mutation of the half-site sequences in the enhancer destroys the functional activity of the enhancer.

Base Sequence↗