Abnormal body iron distribution and erythropoiesis in a novel mouse model with inducible gain of iron regulatory protein (IRP)-1 function.

Disorders of iron metabolism account for some of the most common human diseases. Cellular iron homeostasis is maintained by iron regulatory proteins (IRP)-1 and 2 through their binding to cis-regulatory iron-responsive elements (IREs) in target mRNAs. Mouse models with IRP deficiency have yielded valuable insights into iron biology, but the physiological consequences of gain of IRP function in mammalian organisms have remained unexplored. Here, we report the generation of a mouse line allowing conditional expression of a constitutively active IRP1 mutant (IRP1) using Cre/Lox technology. Systemic activation of the IRP1 transgene from the Rosa26 locus yields viable animals with gain of IRE-binding activity in all the organs analyzed. IRP1 activation alters the expression of IRP target genes and is accompanied by iron loading in the same organs. Furthermore, mice display macrocytic erythropenia with decreased hematocrit and hemoglobin levels as well as impaired erythroid differentiation. Thus, inappropriately high IRP1 activity causes disturbed body iron distribution and erythropoiesis. This new mouse model further highlights the importance of appropriate IRP regulation in central organs of iron metabolism. Moreover, it opens novel avenues to study diseases associated with abnormally high IRP1 activity, such as Parkinson's disease or Friedreich's ataxia.

Self-rated sleep characteristics and bother from nocturia.

PURPOSE: The aim of this study was to evaluate if men with varying degrees of bother from a similar number of nocturia episodes differ with respect to self-rated sleep characteristics and fatigue. MATERIALS AND METHODS: As part of the baseline assessments during a nocturia treatment trial, 55 participants reported frequency and bother of nocturia using the AUA Symptom Inventory and completed 7-day sleep diaries prior to treatment. Participants who reported moderate nocturia (either two or three episodes nightly) were further grouped into categories of LOW (nocturia is no problem or a very small problem) or HIGH bother (nocturia is a big problem). Information from the participant completed sleep diaries was abstracted, including information on daytime napping, total sleep time, mean time needed to return to sleep, nighttime ratings of fatigue, and daytime ratings of fatigue. RESULTS: Of the 55 individuals who completed the pilot study, 24 study participants reported two or three episodes of nocturia and had either HIGH (n = 11) or LOW (n = 13) bother. Participants categorised with HIGH bother were significantly more likely than those with LOW bother to report difficulty initiating sleep (47.7 ± 34.4 vs. 23.5 ± 13.6 min, p = 0.05), difficulty returning to sleep after an awakening (28.9 ± 16.1 vs. 15.4 ± 9.6 min, p = 0.03) and greater morning fatigue (3.3 ± 0.7 vs. 2.5 ± 1.0, p = 0.04 on a 7-point scale). CONCLUSIONS: Since bother related to nocturia is linked to sleep quality, interventions targeting fatigue and sleep maintenance may provide useful targets in the management of nocturia in men.

Direct measurement of the 7Be solar neutrino flux with 192 days of borexino data.

We report the direct measurement of the 7Be solar neutrino signal rate performed with the Borexino detector at the Laboratori Nazionali del Gran Sasso. The interaction rate of the 0.862 MeV 7Be neutrinos is 49+/-3stat+/-4syst counts/(day.100 ton). The hypothesis of no oscillation for 7Be solar neutrinos is inconsistent with our measurement at the 4sigma C.L. Our result is the first direct measurement of the survival probability for solar nu(e) in the transition region between matter-enhanced and vacuum-driven oscillations. The measurement improves the experimental determination of the flux of 7Be, pp, and CNO solar nu(e), and the limit on the effective neutrino magnetic moment using solar neutrinos.

Effects of iron supplementation on binding activity of iron regulatory proteins and the subsequent effect on growth performance and indices of hematological and mineral status of young pigs.

Two experiments were conducted to evaluate the effects of supplemental Fe on the binding activity of iron regulatory proteins (IRP) and the subsequent effect on growth performance and indices of hematological and mineral status of young pigs. In Exp. 1, male pigs (n = 10; 1.8 kg; age = 14 +/- 1 h) were allotted by BW to two treatments (five pigs per treatment). Treatments administered by i.m. injection were as follows: 1) 1 mL of sterile saline solution (Sal); and 2) 1 mL of 200 mg Fe as Fe-dextran (Fe). Pigs were bled (d 0 and 13) to determine hemoglobin (Hb), hematocrit (Hct), transferrin (Tf), and plasma Fe (PFe), and then killed (d 13) to determine spontaneous and 2-mercaptoethanol (2-ME)-inducible IRP RNA binding activity in liver and liver and whole-body mineral concentrations. Contemporary pigs (n = 5; 2.2 kg; age = 14 +/- 2 h) were killed at d 0 to establish baseline (BL1) measurements. In Exp. 2, pigs (six pigs per treatment; 6.5 kg; age = 19 +/- 3 d) were fed a basal diet (Phase 1 = d 0 to 7; Phase 2 = d 7 to 21; Phase 3 = d 21 to 35) supplemented with 0 or 150 mg/kg of Fe as ferrous sulfate and killed at d 35 (18.3 kg; age = 54 +/- 3 d). In addition, pigs (n = 5; 5.9 kg; age = 19 +/- 3 d) were killed at the start of Exp. 2 to establish baseline (BL2) measurements, and liver samples were collected and analyzed for IRP RNA binding activity. In Exp. 1, no difference (P = 0.482) was observed in ADG. On d 13, Fe-treated pigs had greater (P = 0.001) Hb, Hct, and PFe and less (P = 0.002) Tf than Sal-treated pigs. Whole-body Fe concentration was greater (P = 0.002) in Fe- vs. Sal-treated pigs. Treated pigs (Fe or Sal) had greater (P = 0.006) whole-body Cu and less (P = 0.002) whole-body Ca, Mg, Mn, P, and Zn concentrations than BL1. Liver Fe concentration was greater (P = 0.001) in Fe- vs. Sal-treated pigs, but liver Fe concentration of Sal-treated pigs was less (P = 0.001) than that of BL1 pigs. Sal-treated pigs had greater (P = 0.004) spontaneous IRP binding activity than Fe-treated pigs. In Exp. 2, spontaneous and 2-ME inducible IRP binding activities were greater (P = 0.013 and 0.005, respectively) in pigs fed diets containing 0 vs. 150 mg of added Fe/kg of diet. Moreover, pigs fed either treatment for 35 d had greater (P = 0.001) 2-ME inducible IRP binding activity than BL2 pigs. Results indicate that IRP binding activity is influenced by Fe supplementation. Subsequently, other indicators of Fe status are affected via the role of IRP in posttranscriptional expression of Fe storage and transport proteins.

Iron regulatory proteins and the molecular control of mammalian iron metabolism.

Mammalian iron homeostasis is maintained through the concerted action of sensory and regulatory networks that modulate the expression of proteins of iron metabolism at the transcriptional and/or post-transcriptional levels. Regulation of gene transcription provides critical developmental, cell cycle, and cell-type-specific controls on iron metabolism. Post-transcriptional control through the action of iron regulatory protein 1 (IRP1) and IRP2 coordinate the use of messenger RNA-encoding proteins that are involved in the uptake, storage, and use of iron in all cells of the body. IRPs may also provide a link between iron availability and cellular citrate use. Multiple factors, including iron, nitric oxide, oxidative stress, phosphorylation, and hypoxia/reoxygenation, influence IRP function. Recent evidence indicates that there is diversity in the function of the IRP system with respect to the response of specific IRPs to the same effector, as well as the selectivity with which IRPs modulate the use of specific messenger RNA.

Angiogenesis in the latissimus dorsi muscle using different regimens of electrical stimulation and pharmaceutical support.

It is our contention that the prevention of ischemia-reperfusion injuries immediately after latissimus dorsi muscle (LDM) mobilization and enhancement of angiogenesis will be effective in improving cardiomyoplasty results. The investigations were performed on adult sheep. Three hours after LDM mobilization, various stages of leukocyte-endothelium interaction were revealed: leukocytes binding to the endothelium, leukocyte destruction of endothelium, and leukocytes leaving capillaries through gaps in the endothelium. Fifty-six days after mobilization various stages of necrosis were discernible. The area occupied by capillaries was 3.45 +/- 0.26% vs. 3.99 +/- 0.24% in control muscle; most of the endothelial cells exhibited morphologic degeneration. Electrical stimulation with 60 CPM actually decreased the capillary density to 2.15 +/- 0.7%, and most of the endothelial cells were damaged, with disrupted plasma membranes. Muscle subjected to 15 CPM increased the percent of capillaries to 5.01 +/- 0.56%, and endothelial cells appeared normal in ultrastructure. Pharmaceutical support prevented muscle damage and accelerated revascularization. After 56 days of autologous biological glue (ABG) application, the area occupied by capillaries was 5.57 +/- 0.24%. This increased to 8.47 +/- 0.72% when aprotinin (proteinase inhibitor) was added to ABG, and to 9.40 +/- 1.24% with pyrrolostatin (free radical scavenger). Both ABG application with aprotinin and electrical stimulation at 15 CPM prevent the LDM from postmobilization damage, and increase angiogenic potential.

Discovery of the ceruloplasmin homologue hephaestin: new insight into the copper/iron connection.

Approximately 75 years ago Hart and colleagues discovered that copper deficiency impaired mammalian iron metabolism. Discovery of hephaestin identifies a critical new component of the copper and iron connection in mammals. Hephaestin appears to be a multicopper oxidase required for efficient export of iron from the intestine.

Novel role of phosphorylation in Fe-S cluster stability revealed by phosphomimetic mutations at Ser-138 of iron regulatory protein 1.

Animals regulate iron metabolism largely through the action of the iron regulatory proteins (IRPs). IRPs modulate mRNA utilization by binding to iron-responsive elements (IRE) in the 5' or 3' untranslated region of mRNAs encoding proteins involved in iron homeostasis or energy production. IRP1 is also the cytosolic isoform of aconitase. The activities of IRP1 are mutually exclusive and are modulated through the assembly/disassembly of its [4Fe-4S] cluster, reversibly converting it between an IRE-binding protein and cytosolic aconitase. IRP1 is also phosphoregulated by protein kinase C, but the mechanism by which phosphorylation posttranslationally increases IRE binding activity has not been fully defined. To investigate this, Ser-138 (S138), a PKC phosphorylation site, was mutated to phosphomimetic glutamate (S138E), aspartate (S138D), or nonphosphorylatable alanine (S138A). The S138E IRP1 mutant and, to a lesser extent, the S138D IRP1 mutant were impaired in aconitase function in yeast when grown aerobically but not when grown anaerobically. Purified wild-type and mutant IRP1s could be reconstituted to active aconitases anaerobically. However, when exposed to oxygen, the [4Fe-4S] cluster of the S138D and S138E mutants decayed 5-fold and 20-fold faster, respectively, than was observed for wild-type IRP1. Our findings suggest that stability of the Fe-S cluster of IRP1 can be regulated by phosphorylation and reveal a mechanism whereby the balance between the IRE binding and [4Fe-4S] forms of IRP1 can be modulated independently of cellular iron status. Furthermore, our results show that IRP1 can function as an oxygen-modulated posttranscriptional regulator of gene expression.

Iron regulatory proteins, iron responsive elements and iron homeostasis.

The discovery of iron regulatory proteins (IRPs) has provided a molecular framework from which to more fully understand the coordinate regulation of vertebrate iron metabolism. IRPs bind to iron responsive elements (IREs) in specific mRNAs and regulate their utilization. The targets of IRP action now appear to extend beyond proteins that function in the storage (ferritin) or cellular uptake (transferrin receptor) of iron to include those involved in other aspects of iron metabolism as well as in the tricarboxylic acid cycle. To date, it appears that IRPs modulate the utilization of six mammalian mRNAs. Current studies are aimed at defining the mechanisms responsible for the hierarchical regulation of these mRNAs by IRPs. In addition, much interest continues to focus on the signaling pathways through which IRP function is regulated. Multiple factors modulate the RNA binding activity of IRP1 and/or IRP2 including iron, nitric oxide, phosphorylation by protein kinase C, oxidative stress and hypoxia/reoxygenation. Because IRPs are key modulators of the uptake and metabolic fate of iron in cells, they are focal points for the modulation of cellular iron homeostasis in response to a variety of agents and circumstances.

Dietary iron intake rapidly influences iron regulatory proteins, ferritin subunits and mitochondrial aconitase in rat liver.

Iron regulatory protein 1 (IRP1) and IRP2 are cytoplasmic RNA binding proteins that are central regulators of mammalian iron homeostasis. We investigated the time-dependent effect of dietary iron deficiency on liver IRP activity in relation to the abundance of ferritin and the iron-sulfur protein mitochondrial aconitase (m-acon), which are targets of IRP action. Rats were fed a diet containing 2 or 34 mg iron/kg diet for 1-28 d. Liver IRP activity increased rapidly in rats fed the iron-deficient diet with IRP1 stimulated by d 1 and IRP2 by d 2. The maximal activation of IRP2 was five-fold (d 7) and three-fold (d 4) for IRP1. By d 4, liver ferritin subunits were undetectable and m-acon abundance eventually fell by 50% (P < 0.05) in iron-deficient rats. m-Acon abundance declined most rapidly from d 1 to 11 and in a manner that was suggestive of a cause and effect type of relationship between IRP activity and m-acon abundance. In liver, iron deficiency did not decrease the activity of cytosolic aconitase, catalase or complex I of the electron transport chain nor was there an effect on the maximal rate of mitochondrial oxygen consumption with the use of malate and pyruvate as substrates. Thus, the decline in m-acon abundance in iron deficiency is not reflective of a global decrease in liver iron-sulfur proteins nor does it appear to limit ATP production. Our results suggest a novel role for m-acon in cellular iron metabolism. We conclude that, in liver, iron deficiency preferentially affects the activities of IRPs and the targets of IRP action.

Iron differentially stimulates translation of mitochondrial aconitase and ferritin mRNAs in mammalian cells. Implications for iron regulatory proteins as regulators of mitochondrial citrate utilization.

Utilization of mRNAs containing iron-responsive elements (IREs) is modulated by iron-regulated RNA-binding proteins (iron regulatory proteins). We examine herein whether iron differentially affects translation of ferritin and mitochondrial aconitase (m-Acon) mRNAs because they contain a similar but not identical IRE in their 5'-untranslated regions. First, we demonstrate that m-Acon synthesis is iron-regulated in mammalian cells. In HL-60 cells, hemin (an iron source) stimulated m-Acon synthesis 3-fold after 4 h compared with cells treated with an iron chelator (Desferal). Furthermore, hemin stimulated m-Acon synthesis 2-4-fold in several cell lines. Second, we show that iron modulates the polysomal association of m-Acon mRNA. We observed m-Acon mRNA in both ribonucleoprotein and polyribosomal fractions of HL-60 cells. Hemin significantly increased the polyribosomal association and decreased the ribonucleoprotein abundance of m-Acon mRNA in HL-60 cells. Third, our results indicate that iron differentially regulates translation of m-Acon and ferritin mRNAs. A dose response to hemin in HL-60 cells elicited a 2-2.4-fold increase in m-Acon synthesis within 5 h compared with untreated cells, whereas ferritin synthesis was stimulated 20-100-fold. We conclude that iron modulates m-Acon synthesis at the translational level and that iron regulatory proteins appear to differentially affect translation of IRE-containing mRNAs.

Interaction of the hemochromatosis gene product HFE with transferrin receptor modulates cellular iron metabolism.

Mutations of a novel MHC class I-like protein, termed HFE, have been found in the vast majority of patients with the iron overload disease heredity hemochromatosis. Identification of HFE is likely to shed light on one of the major enigmas of mammalian iron homeostasis: How is intestinal iron absorption regulated?

Iron regulatory protein 1 is not required for the modulation of ferritin and transferrin receptor expression by iron in a murine pro-B lymphocyte cell line.

Iron regulatory proteins (IRPs) are cytoplasmic RNA binding proteins that are central components of a sensory and regulatory network that modulates vertebrate iron homeostasis. IRPs regulate iron metabolism by binding to iron responsive element(s) (IREs) in the 5' or 3' untranslated region of ferritin or transferrin receptor (TfR) mRNAs. Two IRPs, IRP1 and IRP2, have been identified previously. IRP1 exhibits two mutually exclusive functions as an RNA binding protein or as the cytosolic isoform of aconitase. We demonstrate that the Ba/F3 family of murine pro-B lymphocytes represents the first example of a mammalian cell line that fails to express IRP1 protein or mRNA. First, all of the IRE binding activity in Ba/F3-gp55 cells is attributable to IRP2. Second, synthesis of IRP2, but not of IRP1, is detectable in Ba/F3-gp55 cells. Third, the Ba/F3 family of cells express IRP2 mRNA at a level similar to other murine cell lines, but IRP1 mRNA is not detectable. In the Ba/F3 family of cells, alterations in iron status modulated ferritin biosynthesis and TfR mRNA level over as much as a 20- and 14-fold range, respectively. We conclude that IRP1 is not essential for regulation of ferritin or TfR expression by iron and that IRP2 can act as the sole IRE-dependent mediator of cellular iron homeostasis.

Isolation, characterization, and functional studies of rat liver iron regulatory protein 1.

Ferritin mRNAs are translationally regulated by the binding of either of two cytosolic proteins, iron regulatory protein 1 (IRP1) or IRP2, to the iron responsive element (IRE) located in their 5' untranslated region (UTR). Rat liver IRP1 was purified by anion exchange, gel filtration, and affinity chromatography using a concatemerized version of the IRE. Two bands with M(r) of 95,000 and 100,000 were observed by reducing SDS-PAGE. A single protein was responsible for both bands since: (1) [32P]IRE RNA specifically cross-linked to both components; (2) alkylation with iodoacetamide resulted in formation of a single species with M(r) of 95,000; and (3) they possessed identical peptide patterns after digestion with cyanogen bromide. The N-terminal sequence of rat liver IRP1 was MKNPFAHLAEPLDPAQPGKKFNLNKLEDSRYGRLPFXIRVLLEAAV which is identical to the sequence deduced from the cDNA. Rat liver IRP1 has an amino acid composition similar to that of bovine liver caconitase. Several species of IRP1 were observed by two-dimensional gel electrophoresis with pIs ranging from 7.5 to 8.0. Rat liver IRP1 bound the IRE with high affinity (K(D) = 0.04 nM) and repressed translation of ferritin mRNA in vitro. IRP1 bound 100-fold less well to an IRE variant and failed to significantly repress translation of a ferritin mRNA containing the mutated IRE. We conclude that decreases in the affinity of interaction between IRP1 and the IRE, of a magnitude similar to that observed when the binding protein in converted to c-aconitase, are sufficient to significantly enhance translation of ferritin mRNA in vitro.

The iron-sulfur cluster of iron regulatory protein 1 modulates the accessibility of RNA binding and phosphorylation sites.

Iron regulatory protein 1 (IRP1) modulates iron metabolism by binding to mRNAs encoding proteins involved in the uptake, storage, and metabolic utilization of iron. Iron regulates IRP1 function by promoting assembly of an iron-sulfur cluster in the apo or RNA binding form, thereby converting it to the active holo or cytoplasmic aconitase form. In continuing our studies on phosphoregulation of IRP1 by protein kinase C (PKC), we noted that the purified apoprotein was more efficiently phosphorylated than was the form partially purified from liver cytosol by chromatography on DEAE-Sepharose which had characteristics of the [3Fe-4S] form of the protein. RNA binding measurements revealed a 20-fold increase in RNA binding affinity and a 4-5-fold higher rate of phosphorylation after removal of the Fe-S cluster from the highly purified [4Fe-4S] form. Phosphorylation of apo-IRP1 by PKC was specifically inhibited by IRE-containing RNA. The RNA binding form had a more open structure as judged by its much greater sensitivity to limited cleavage by a number of proteases. N-Terminal sequencing of chymotryptic peptides of apo-IRP1 demonstrated an increased accessibility to proteolysis of sites (residues 132 and 504) near or within the putative cleft of the protein, including regions that are thought to be involved in RNA binding (residues 116-151) and phosphoregulation (Ser 138). Enhanced cleavage was also observed in the proposed hinge linker region (residue 623) on the surface of the protein opposite from the cleft. Taken together, our results indicate that significant structural changes occur in IRP1 during cluster insertion or removal that affect the accessibility to RNA binding and phosphorylation sites.

Dietary iron intake modulates the activity of iron regulatory proteins and the abundance of ferritin and mitochondrial aconitase in rat liver.

Iron regulatory protein 1 (IRP1) and IRP2 are cytoplasmic RNA binding proteins that coordinate cellular iron homeostasis in mammals. We investigated the effect of dietary iron intake on rat liver IRP activity in relation to the abundance of two targets of IRP action, ferritin and mitochondrial aconitase (m-aconitase). Rats were fed diets containing 2, 11, 20, 37 (control), 72 or 107 mg iron/kg diet for 3 wk. RNA binding activity of IRP1 and IRP2 was enhanced one- to twofold in rats fed 11 or 2 mg iron/kg diet compared with control rats. IRP RNA binding activity was inversely correlated to blood hemoglobin levels (r = -0.787; P < 0.0001). Compared with control rats, liver ferritin levels were depressed in rats fed 20 mg iron/kg diet and were undetectable in rats ingesting diets with 11 or 2 mg iron/kg diet. Ferritin concentrations were biphasically related to IRP RNA binding activity with the regulation of IRP occurring before the onset of ferritin accumulation. Iron deficiency caused up to a 50% decline in m-aconitase abundance. IRP RNA binding activity and m-aconitase abundance were inversely correlated (r = -0.751; P < 0.0001). Our results indicate that (1) liver IRP activity is responsive to a range of dietary iron levels, (2) there appears to be a differential effect of IRPs on ferritin and m-aconitase abundance, and (3) activation of IRPs may contribute to the alterations in energy metabolism in iron deficiency through an impairment of m-aconitase synthesis.

Comparison of different regimens of electrical stimulation applied to nonmobilized and newly mobilized latissimus dorsi muscle.

We investigated the possibility of preventing further aggravation of muscle ischemia and necrosis in newly mobilized, unconditioned latissimus dorsi muscle (LDM) by utilizing short increments of stimulation with intervening rest periods. Adult St. Croix sheep (N = 12) weighing 30 +/- 8 kg were used in this study. Fatigue tests (30 min) using different stimulation regimens before and after LDM mobilization were performed on all animals; the length of time to return to baseline levels was also measured. Our investigation yielded results that contradict the conventional wisdom that any electrical stimulation damages newly mobilized LDM and will cause a considerable decrease in contractile force (CF). Stimulation regimens using continuous contractions at 30 and 60 contractions per minute (CPM) for 30 minutes were damaging to the LDM. CF also dropped significantly and returned slowly to baseline values: at 60 CPM, CF dropped to 50 +/- 4% and did not return to baseline even after 90 minutes of rest; at 30 CPM, CF dropped to 61 +/- 4% and baseline was restored after 80 minutes of rest. Electrical stimulation using continuous contractions at a slower rate (15 CPM) was tolerable, although a 23% decrease in CF was noted (p < 0.05 when compared to 60 CPM). These results did not satisfy us that such a regimen would be useful for cardiac assistance immediately after cardiomyoplasty. The work-rest regimen at 30 CPM also gave poor results: CF decreased to 75 +/- 2% and baseline was restored after 80 minutes of rest. Promising results were seen when utilizing a work-rest regimen at 15 CPM. The newly mobilized LDM showed no visible signs of fatigue: CF decreased minimally to 92 +/- 3% (p < 0.05 when compared to 30 CPM), and light microscopic analysis of biopsies revealed no morphological damage exceeding that typically seen after subtotal mobilization. Such results open avenues for future investigations: beginning electrical stimulation immediately after cardiomyoplasty (using a single impulse and a slow rate of contraction); decreasing the length of time necessary to obtain full cardiac assistance; and beginning partial cardiac assistance immediately after cardiomyoplasty (if needed) for approximately 30 minutes several times a day.