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H F Bunn

Bio: H F Bunn is an academic researcher from Brigham and Women's Hospital. The author has contributed to research in topics: Hemoglobin & Erythropoietin. The author has an hindex of 59, co-authored 118 publications receiving 19438 citations. Previous affiliations of H F Bunn include Boston University & United States Department of the Army.


Papers
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Journal ArticleDOI
TL;DR: The identification of an oxygen-dependent degradation (ODD) domain within HIF-1alpha that controls its degradation by the ubiquitin-proteasome pathway is reported and may provide a means of controlling gene expression by changes in oxygen tension.
Abstract: Hypoxia induces a group of physiologically important genes such as erythropoietin and vascular endothelial growth factor. These genes are transcriptionally up-regulated by hypoxia-inducible factor 1 (HIF-1), a global regulator that belongs to the basic helix-loop-helix PAS family. Although HIF-1 is a heterodimer composed of α and β subunits, its activity is primarily determined by hypoxia-induced stabilization of HIF-1α, which is otherwise rapidly degraded in oxygenated cells. We report the identification of an oxygen-dependent degradation (ODD) domain within HIF-1α that controls its degradation by the ubiquitin-proteasome pathway. The ODD domain consists of ≈200 amino acid residues, located in the central region of HIF-1α. Because portions of the domain independently confer degradation of HIF-1α, deletion of this entire region is required to give rise to a stable HIF-1α, capable of heterodimerization, DNA-binding, and transactivation in the absence of hypoxic signaling. Conversely, the ODD domain alone confers oxygen-dependent instability when fused to a stable protein, Gal4. Hence, the ODD domain plays a pivotal role for regulating HIF-1 activity and thereby may provide a means of controlling gene expression by changes in oxygen tension.

2,113 citations

Journal ArticleDOI
TL;DR: An adaptation of the polymerase chain reaction (PCR) for highly accurate quantitation of mRNA or DNA from a small number of cells for expression of two cytokines and the copy number of the human GM-CSF gene in normal human cells is described.
Abstract: The expression of two cytokines, granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 3 (IL-3), has been investigated in MLA-144 cells before and after induction with phorbol 12-myristate 13-acetate. We describe an adaptation of the polymerase chain reaction (PCR) for highly accurate quantitation of mRNA or DNA from a small number of cells. Aliquots of the PCR mixture containing cDNA copies of the RNA to be assayed were added to serial dilutions of a competitor DNA fragment that differed from the cDNA of interest by having either a small intron or a mutated internal restriction enzyme site. Therefore, the same primers were used to coamplify the unknown and the competitor. The ratio of products remains constant through the amplification and can be readily quantitated. In unstimulated cells, no GM-CSF or IL-3 mRNA could be detected. However, with appropriate induction, mRNA for both cytokines was detected and quantitated in as few as 200 cells. Competitive PCR was also used to accurately quantitate the copy number of the human GM-CSF gene in normal human cells, in a clonal population of cells from a patient with 5q- syndrome, and in a human-hamster cell line known to have only one copy of the human GM-CSF gene.

1,564 citations

Journal ArticleDOI
TL;DR: Redox chemistry appears to play a critical role both in the trans-activation of oxygen-responsive genes in unicellular organisms as well as in the activation of HIF-1, which is required for the hypoxic induction of physiologically important genes.
Abstract: This review focuses on the molecular stratagems utilized by bacteria, yeast, and mammals in their adaptation to hypoxia. Among this broad range of organisms, changes in oxygen tension appear to be sensed by heme proteins, with subsequent transfer of electrons along a signal transduction pathway which may depend on reactive oxygen species. These heme-based sensors are generally two-domain proteins. Some are hemokinases, while others are flavohemoproteins [flavohemoglobins and NAD(P)H oxidases]. Hypoxia-dependent kinase activation of transcription factors in nitrogen-fixing bacteria bears a striking analogy to the phosphorylation of hypoxia inducible factor-1 (HIF-1) in mammalian cells. Moreover, redox chemistry appears to play a critical role both in the trans-activation of oxygen-responsive genes in unicellular organisms as well as in the activation of HIF-1. In yeast and bacteria, regulatory operons coordinate expression of genes responsible for adaptive responses to hypoxia and hyperoxia. Similarly, in mammals, combinatorial interactions of HIF-1 with other identified transcription factors are required for the hypoxic induction of physiologically important genes.

1,167 citations

Journal ArticleDOI
TL;DR: A wealth of information is produced on the mechanisms by which a single base substitution in the gene encoding the human β-globin subunit, with the resulting replacement of β6 glutamic acid by valine, leads to the protean and devastating clinical manifestations of sickle cell disease.
Abstract: In 1949, the discovery that sickle hemoglobin (α2 βS2) has an abnormal electrophoretic mobility prompted Linus Pauling and his colleagues to christen sickle cell anemia “a molecular disease.”1 The ensuing five decades have produced a wealth of information on the mechanisms by which a single base substitution in the gene encoding the human β-globin subunit, with the resulting replacement of β6 glutamic acid by valine, leads to the protean and devastating clinical manifestations of sickle cell disease. Until recently there was a disappointing lag in the application of this knowledge to the design of safe and effective . . .

1,008 citations

Journal ArticleDOI
09 Dec 1988-Science
TL;DR: A model is proposed in which a ligand-dependent conformational change in a heme protein accounts for the mechanism by which hypoxia as well as cobalt and nickel stimulate the production of Epo.
Abstract: Erythropoietin (Epo), the hormone that stimulates red blood cell production, is synthesized in the kidney and liver in response to hypoxia. The human hepatoma cell line Hep3B regulates its production of Epo in a physiologic manner. Either hypoxia or cobalt chloride markedly increases expression of Epo mRNA as well as production of biologically active and immunologically distinct Epo protein. New protein synthesis is required before the induction of increased levels of hypoxia- or cobalt-induced Epo mRNA. Hypoxia, cobalt chloride, and nickel chloride appear to stimulate Epo production through a common pathway. The inhibition of Epo production at low partial pressures of oxygen by carbon monoxide provides evidence that a heme protein is integrally involved in the oxygen-sensing mechanism. This hypothesis is further supported by the finding that when heme synthesis is blocked, hypoxia-, cobalt-, and nickel-induced Epo production are all markedly inhibited. A model is proposed in which a ligand-dependent conformational change in a heme protein accounts for the mechanism by which hypoxia as well as cobalt and nickel stimulate the production of Epo.

1,003 citations


Cited by
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Journal ArticleDOI
TL;DR: There is growing evidence that aging involves, in addition, progressive changes in free radical-mediated regulatory processes that result in altered gene expression.
Abstract: At high concentrations, free radicals and radical-derived, nonradical reactive species are hazardous for living organisms and damage all major cellular constituents. At moderate concentrations, how...

9,131 citations

Journal ArticleDOI
TL;DR: This review summarizes the current state of knowledge of the functions of NOX enzymes in physiology and pathology.
Abstract: For a long time, superoxide generation by an NADPH oxidase was considered as an oddity only found in professional phagocytes. Over the last years, six homologs of the cytochrome subunit of the phag...

5,873 citations

Journal ArticleDOI
20 Apr 2001-Science
TL;DR: It is shown that the interaction between human pVHL and a specific domain of the HIF-1α subunit is regulated through hydroxylation of a proline residue by an enzyme the authors have termed Hif-α prolyl-hydroxylase (HIF-PH).
Abstract: Hypoxia-inducible factor (HIF) is a transcriptional complex that plays a central role in the regulation of gene expression by oxygen. In oxygenated and iron replete cells, HIF-alpha subunits are rapidly destroyed by a mechanism that involves ubiquitylation by the von Hippel-Lindau tumor suppressor (pVHL) E3 ligase complex. This process is suppressed by hypoxia and iron chelation, allowing transcriptional activation. Here we show that the interaction between human pVHL and a specific domain of the HIF-1alpha subunit is regulated through hydroxylation of a proline residue (HIF-1alpha P564) by an enzyme we have termed HIF-alpha prolyl-hydroxylase (HIF-PH). An absolute requirement for dioxygen as a cosubstrate and iron as cofactor suggests that HIF-PH functions directly as a cellular oxygen sensor.

5,186 citations

Journal ArticleDOI
20 May 1999-Nature
TL;DR: It is indicated that the interaction between HIF-1 and pVHL is iron dependent, and that it is necessary for the oxygen-dependent degradation of HIF α-subunits, which may underlie the angiogenic phenotype of VHL-associated tumours.
Abstract: Hypoxia-inducible factor-1 (HIF-1) has a key role in cellular responses to hypoxia, including the regulation of genes involved in energy metabolism, angiogenesis and apoptosis. The alpha subunits of HIF are rapidly degraded by the proteasome under normal conditions, but are stabilized by hypoxia. Cobaltous ions or iron chelators mimic hypoxia, indicating that the stimuli may interact through effects on a ferroprotein oxygen sensor. Here we demonstrate a critical role for the von Hippel-Lindau (VHL) tumour suppressor gene product pVHL in HIF-1 regulation. In VHL-defective cells, HIF alpha-subunits are constitutively stabilized and HIF-1 is activated. Re-expression of pVHL restored oxygen-dependent instability. pVHL and HIF alpha-subunits co-immunoprecipitate, and pVHL is present in the hypoxic HIF-1 DNA-binding complex. In cells exposed to iron chelation or cobaltous ions, HIF-1 is dissociated from pVHL. These findings indicate that the interaction between HIF-1 and pVHL is iron dependent, and that it is necessary for the oxygen-dependent degradation of HIF alpha-subunits. Thus, constitutive HIF-1 activation may underlie the angiogenic phenotype of VHL-associated tumours. The pVHL/HIF-1 interaction provides a new focus for understanding cellular oxygen sensing.

4,845 citations

Journal ArticleDOI
29 Oct 1992-Nature
TL;DR: It is shown that vascular endothelial growth factor (VEGF) probably functions as a hypoxia-inducible angiogenic factor and is specifically induced in a subset of glioblastoma cells distinguished by their immediate proximity to necrotic foci and the clustering of capillaries alongside VEGF-producing cells.
Abstract: Inefficient vascular supply and the resultant reduction in tissue oxygen tension often lead to neovascularization in order to satisfy the needs of the tissue. Examples include the compensatory development of collateral blood vessels in ischaemic tissues that are otherwise quiescent for angiogenesis and angiogenesis associated with the healing of hypoxic wounds. But the presumptive hypoxia-induced angiogenic factors that mediate this feedback response have not been identified. Here we show that vascular endothelial growth factor (VEGF; also known as vascular permeability factor) probably functions as a hypoxia-inducible angiogenic factor. VEGF messenger RNA levels are dramatically increased within a few hours of exposing different cell cultures to hypoxia and return to background when normal oxygen supply is resumed. In situ analysis of tumour specimens undergoing neovascularization show that the production of VEGF is specifically induced in a subset of glioblastoma cells distinguished by their immediate proximity to necrotic foci (presumably hypoxic regions) and the clustering of capillaries alongside VEGF-producing cells.

4,627 citations