PubMed Health⌕ Search

Biomedical subjects

H Franklin Bunn

Publications and source records attributed to H Franklin Bunn.

12 recordsLinked to original sources
PubMed2006

New agents that stimulate erythropoiesis.

Recombinant human erythropoietin (rhEpo) has proven to be remarkably safe and effective for treatment of anemias, primarily those secondary to renal disease and malignancy. Despite the worldwide use of rhEpo, concerns about its cost, the need for frequent parenteral administration, and the development of anti-Epo antibodies have prompted development of improved agents to stimulate erythropoiesis. Three strategies appear to be particularly promising. The half-life of Epo in the circulation can be prolonged by the addition of N-linked carbohydrate groups, by formation of adducts with polyethylene glycol, and by preparation of Epo multimers. Second, mimetic peptides can effectively trigger signal transduction at the Epo receptor, thereby boosting red-cell production. Finally, the hypoxia inducible transcription factor (HIF) can be pharmacologically induced by oral agents, resulting in enhanced expression not only of endogenous Epo but also of other genes important in the regulation of erythropoiesis.

Erythropoiesis↗
PubMed2006

Promoter characterization and transcriptional regulation of Ncb5or, a novel reductase necessary for pancreatic beta-cell maintenance.

Ncb5or is a ubiquitously expressed gene required for beta-cell survival in mice. Examination of mouse tissues demonstrated high levels of expression in the pancreas, heart and kidney. A transcription start site was identified 149 bp upstream from the start codon and transient expression analysis in betaTC3 cells indicated the presence of a core promoter located within 348 bp upstream of this site. Deletion of Region C (-216/-157) resulted in a significant decrease in promoter activity and specific nucleotides located in a region designated C2 were demonstrated to be critical for complex binding. Deletion of Region D (-60/-33), which contains multiple consensus Sp1 sites, resulted in an additional loss of promoter activity. The data presented here identify and characterize the previously unknown promoter of Ncb5or, a reductase critical for beta-cell survival.

Animals↗
PubMed2004

Absence of a reductase, NCB5OR, causes insulin-deficient diabetes.

NCB5OR is a highly conserved NAD(P)H reductase that contains a cytochrome b5-like domain at the N terminus and a cytochrome b5 reductase-like domain at the C terminus. The enzyme is located in the endoplasmic reticulum (ER) and is widely expressed in organs and tissues. Targeted inactivation of this gene in mice has no impact on embryonic or fetal viability. At 4 weeks of age, Ncb5or-/- mice have normal blood glucose levels but impaired glucose tolerance. Isolated Ncb5or-/- islets have markedly impaired glucose- or arginine-stimulated insulin secretion. By 7 weeks of age, these mice develop severe hyperglycemia with markedly decreased serum insulin levels and nearly normal insulin tolerance. As the animals age, there is a progressive loss of beta cells in pancreatic islets, but there is no loss of alpha, delta, or PP cells. Electron microscopy reveals degranulation of beta cells and hypertrophic and hyperplastic mitochondria, some of which contain electron dense inclusions. Four-week-old Ncb5or-/- mice have enhanced sensitivity to the diabetogenic agent streptozotocin. NCB5OR appears to play a critical role in protecting pancreatic beta cells against oxidant stress.

Animals↗
PubMed2004

Hypoxia-induced synthesis of hemoglobin in the crustacean Daphnia magna is hypoxia-inducible factor-dependent.

Of the four known globin genes that exist in the fresh-water crustacean Daphnia magna, several are individually induced by hypoxia, lending pale normoxic animals a visible red color when challenged by oxygen deprivation. The promoter regions of the Daphnia globin genes each contain numerous hypoxia response elements (HREs) as potential binding sites for hypoxia-inducible factors (HIFs). Daphnia HIF, bound to human HRE sequences, was detected in extracts from hypoxic (red), but not normoxic (pale), animals. Taking advantage of the phylogenetically conserved HIF/HRE recognition, we employed heterologous transfections of HIF-expressing human and Drosophila cells to model HIF signaling in Daphnia. These experiments revealed that three functional HREs within the promoter of the D. magna globin-2 gene cooperate for maximal hypoxic induction of a downstream luciferase reporter gene. Two of these three cis-elements, at promoter positions -258 and -107, are able to specifically bind human, Drosophila, or Daphnia HIF complexes in vitro. The same two sites are also necessary for maximal induction of reporter transcription under low oxygen tension in the presence of either endogenous human or overexpressed Drosophila HIF proteins. The third motif of the globin-2 gene promoter, a CACGTG palindrome at position -146, functions as a docking site for an unknown constitutive transcription factor. In human cells, this -146 complex interferes with HIF occupancy at the adjacent -107 HRE and thus controls the extent of HIF-mediated hypoxic activation of the downstream target.

Animals↗
PubMed2004

Regulation of Drosophila hypoxia-inducible factor (HIF) activity in SL2 cells: identification of a hypoxia-induced variant isoform of the HIFalpha homolog gene similar.

Although hypoxia-inducible factor-alpha (HIFalpha) subunit-specific hydroxylation and proteolytic breakdown explain the binary switch between the presence (hypoxia) and absence (normoxia) of HIFs, little is known of the mechanisms that fine-tune HIF activity under constant, rather than changing, oxygen tensions. Here, we report that the Drosophila HIFalpha homolog, the basic helix-loop-helix/PAS protein Sima (Similar), in hypoxic cultures of SL2 cells is expressed in full-length (fl) and splice variant (sv) isoforms. The following evidence supports the role of flSima as functional HIFalpha and the role of SL2 HIF as a transcriptional activator or suppressor. The pO(2) dependence of Sima abundance matched that of HIF activity. HIF-dependent changes in candidate target gene expression were detected through variously effective stimuli: hypoxia (strong) > iron chelation, e.g. desferrioxamine (moderate) >> transition metals, e.g. cobalt approximately normoxia (ineffective). Sima overexpression augmented hypoxic induction or suppression of different targets. In addition to the full-length exon 1-12 transcript yielding the 1510-amino acid HIFalpha homolog, the sima gene also expressed, specifically under hypoxia, an exon 1-7/12 splice variant, which translated into a 426-amino acid Sima truncation termed svSima. svSima contains basic helix-loop-helix and PAS sequences identical to those of flSima, but, because of deletion of exons 8-11, lacks the oxygen-dependent degradation domain and nuclear localization signals. Overexpressed svSima failed to transactivate reporter genes. However, it attenuated HIF (Sima.Tango)-stimulated reporter expression in a dose-dependent manner. Thus, svSima has the potential to regulate Drosophila HIF function under steady and hypoxic pO(2) by creating a cytosolic sink for the Sima partner protein Tango.

Amino Acid Sequence↗
PubMed2004

NCB5OR is a novel soluble NAD(P)H reductase localized in the endoplasmic reticulum.

The NAD(P)H cytochrome b5 oxidoreductase, Ncb5or (previously named b5+b5R), is widely expressed in human tissues and broadly distributed among the animal kingdom. NCB5OR is the first example of an animal flavohemoprotein containing cytochrome b5 and chrome b5 reductase cytodomains. We initially reported human NCB5OR to be a 487-residue soluble protein that reduces cytochrome c, methemoglobin, ferricyanide, and molecular oxygen in vitro. Bioinformatic analysis of genomic sequences suggested the presence of an upstream start codon. We confirm that endogenous NCB5OR indeed has additional NH2-terminal residues. By performing fractionation of subcellular organelles and confocal microscopy, we show that NCB5OR colocalizes with calreticulin, a marker for endoplasmic reticulum. Recombinant NCB5OR is soluble and has stoichiometric amounts of heme and flavin adenine dinucleotide. Resonance Raman spectroscopy of NCB5OR presents typical signatures of a six-coordinate low-spin heme similar to those found in other cytochrome b5 proteins. Kinetic measurements showed that full-length and truncated NCB5OR reduce cytochrome c actively in vitro. However, both full-length and truncated NCB5OR produce superoxide from oxygen with slow turnover rates: kcat = approximately 0.05 and approximately 1 s(-1), respectively. The redox potential at the heme center of NCB5OR is -108 mV, as determined by potentiometric titrations. Taken together, these data suggest that endogenous NCB5OR is a soluble NAD(P)H reductase preferentially reducing substrate(s) rather than transferring electrons to molecular oxygen and therefore not an NAD(P)H oxidase for superoxide production. The subcellular localization and redox properties of NCB5OR provide important insights into the biology of NCB5OR and the phenotype of the Ncb5or-null mouse.

Animals↗
PubMed2004

Variation in NCB5OR: studies of relationships to type 2 diabetes, maturity-onset diabetes of the young, and gestational diabetes mellitus.

Recent data show that homozygous Ncb5or(-/-) knock-out mice present with an early-onset nonautoimmune diabetes phenotype. Furthermore, genome-wide scans have reported linkage to the chromosome 6q14.2 region close to the human NCB5OR. We therefore considered NCB5OR to be a biological and positional candidate gene and examined the coding region of NCB5OR in 120 type 2 diabetic patients and 63 patients with maturity-onset diabetes of the young using denaturing high-performance liquid chromatography. We identified a total of 22 novel nucleotide variants. Three variants [IVS5+7del(CT), Gln187Arg, and His223Arg] were genotyped in a case-control design comprising 1,246 subjects (717 type 2 diabetic patients and 529 subjects with normal glucose tolerance). In addition, four rare variants were investigated for cosegregation with diabetes in multiplex type 2 diabetic families. The IVS5+7del(CT) variant was associated with common late-onset type 2 diabetes; however, we failed to relate this variant to any diabetes-related quantitative traits among the 529 control subjects. Thus, variation in the coding region of NCB5OR is not a major contributor in the pathogenesis of nonautoimmune diabetes.

Animals↗
PubMed2002

Detecting and responding to hypoxia.

Adaptation to hypoxia is a topic of considerable clinical relevance, as it influences the pathophysiology of anaemia, polycythaemia, tissue ischaemia and cancer. A growing number of physiologically relevant genes are regulated in response to changes in intracellular oxygen tension. These include genes encoding erythropoietin, vascular endothelial growth factor and tyrosine hydroxylase. Studies on the regulation of the erythropoietin gene have provided insights into the common mechanism of oxygen sensing and signal transduction, leading to activation of the hypoxia-inducible transcription factor 1 (HIF-1). Activation of HIF-1 by hypoxia depends on rescue of its alpha-subunit from oxygen-dependent degradation in the proteasome, allowing it to form a heterodimer with HIF-1 beta. This then translocates to the nucleus. There, HIF-1 assembles with a highly conserved orphan nuclear receptor, HNF-4, and a critical transcriptional adaptor, p300. This complex binds to a 3' enhancer on the erythropoietin gene, enabling transcription of erythropoietin. HIF-1 also activates other genes, the cis-acting elements of which contain cognate hypoxia response elements. There is growing evidence that the oxygen sensor is a flavohaem protein and that the signal transduction pathway involves changes in the level of intracellular reactive oxygen intermediates. We have recently cloned a novel fusion protein called cytochrome b5/b5 reductase, which is a cyanide-insensitive NADPH oxidase and, therefore, a candidate to be the oxygen sensor. This flavohaem protein is widely expressed in cell lines and tissues, with localization in the perinuclear space. In the presence of oxygen and iron, it may induce oxidative modifications that target HIF-1 alpha for ubiquitination and degradation.

Adaptation, Physiological↗
PubMed2000

Erythropoietin induction in Hep3B cells is not affected by inhibition of heme biosynthesis.

Erythropoietin (Epo) is one of the physiologically important genes whose transcription is up-regulated by hypoxia. Our laboratory previously proposed that the sensor of this event is a heme protein which turns over rapidly. We have investigated the effects of four inhibitors of heme synthesis (4,6-dioxoheptanoic acid (DHA), isoniazid (INH), N-methyl protoporphyrin IX (MPP), and deferoxamine mesylate (DSF)) on hypoxia-, cobalt-, and DSF-induced erythropoietin (Epo) mRNA expression, heme biosynthesis, and cell viability in Hep3B cells. DHA (0.1-1.0 mM) inhibited heme biosynthesis more than 85%, but did not suppress Epo mRNA expression. Epo mRNA expression was inhibited only at higher concentrations of DHA (2, 4 mM) which also inhibited cell viability. No suppression of Epo mRNA expression by INH was observed at doses known to inhibit heme biosynthesis. MPP did not suppress Epo mRNA expression although it showed an inhibitory effect on heme biosynthesis without any decreased cell viability. 130 microM DSF, a dose which inhibited heme biosynthesis without cell toxicity, suppressed hypoxia-induced Epo mRNA expression, but enhanced cobalt-induced Epo mRNA expression. These results show that although the oxygen sensor is probably a heme protein it does not turn over rapidly. Therefore, cobalt is unlikely to act by substituting for heme iron.

Cell Survival↗