![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Department of Neuroscience and Anatomy, G.M. Leader Family Laboratory for Alzheimer’s Disease Research, Penn State University College of Medicine, Hershey, PA 17033, and Nutrition Department, The Pennsylvania State University, University Park, PA 16814 *
2To whom correspondence should be addressed. E-mail: jrc3{at}psu.edu
ABSTRACT
Iron regulatory proteins (IRPs) are proteins involved in the regulation of intracellular iron homeostasis that bind to specific mRNA structures termed iron responsive elements (IREs). Because the target mRNAs for the IRPs are both cytosolic and membrane associated, we hypothesize that movement of IRPs between the cytosolic and the membrane associated subcellular fractions occurs in response to intracellular iron changes. We tested this hypothesis in a cell culture model, using mouse fibroblast cells (NIH 3T3) and macrophage cells (J774), and in a rat model of early iron deficiency and excess. This presented the first opportunity to examine IRP binding activity in rat brain during states of dietary iron deficiency and excess. Binding activity for IRPs was demonstrated in both membrane and cytosolic fractions in the cell lines and the rat brain homogenates. Although IRP binding activity is predominantly located in the cytosol (90%), there was increased IRP/IRE binding activity in both cytosolic and membrane fractions when the cells were treated with deferoxamine, and decreased binding activity after treatment with iron. In the rat study, brain cortex, hippocampus and striatum homogenates had more IRP binding activity in iron-deficient rats and less in iron-supplemented rats in a region- and time-specific manner. The intracellular distribution of IRPs also changed between the cytosolic and membrane fractions of the brain homogenates in conjunction with changes in iron. These in vivo studies are consistent with the cell culture analyses showing intracellular redistribution of IRPs as a function of iron status. The results of these experiments extend our understanding of cytoplasmic mRNA binding protein activity and raise questions regarding the mechanism by which mRNA binding proteins can locate their target mRNAs within cells. The elucidation of this mechanism will have a significant impact on our understanding of eukaryotic gene regulation.
KEY WORDS: • iron regulation • post-transcriptional regulation • rats • iron regulatory proteins • iron deficiency • cell culture
Iron poses a challenge to the cell because of the adverse effects of
both iron deficiency
(ID)3
and excess. Thus, maintenance of intracellular iron homeostasis is
essential for cell survival. Cellular iron homeostasis is achieved
through the controlled synthesis of several proteins involved in the
movement, storage and utilization of iron. The mRNAs of some of these
proteins contain specific sequences termed iron responsive elements
(IREs) that interact with cytoplasmic iron regulatory proteins (IRPs)-1
and -2, which are the key regulators of intracellular iron homeostasis.
The control of IRP/IRE interaction has been the subject of a number of
investigations [see recent reviews by Cairo and Pietrangelo
(1
) and Eisenstein (2
)]. In the cell, IRPs
are thought to exist in three states: 1) bound to an IRE,
2) free and available to bind to an IRE and 3)
free but unavailable to bind IRE (i.e., cytoplasmic aconitase activity
pool). Variations in the size of the intracellular labile iron pool
produce a change in the IRP/IRE binding activity and, as a consequence,
a change in the levels of the proteins involved in iron homeostasis. Of
the target mRNAs for the binding of IRPs, transferrin receptor mRNA is
membrane associated (3
), while ferritin mRNA is found in
both the cytosol and the endoplasmic reticulum (4
, 5
).
Thus, we hypothesize that the IRPs are found in more than one
intracellular compartment and that the post-transcriptional
regulation of IRP/IRE interaction involves intracellular translocation
of IRPs in relation to their target mRNAs between the cytosolic and the
membrane-associated subcellular fractions.
In the brain, the regulation of iron metabolism provides challenges not
matched by any other organ. The brain has an uneven distribution of
iron, with some regions having a high concentration (substantia nigra,
ventral pallidus and globus pallidus) (6
). In the brain,
there is a high requirement for iron during development, as evidenced
in animal studies (7
–9
). If this requirement is not met,
as in early ID, it could have devastating long-lasting effects
(10
–12
). Furthermore, iron excess in the brain has been
associated with neurodegenerative diseases such as Alzheimer’s
(13
–16
) and Parkinson’s (16
–18
).
The metabolism of iron in the brain involves the same proteins as
elsewhere in the body but, because of the tightly regulated environment
behind a vascular barrier, it is unclear that the molecular regulation
is similar. IRPs have been demonstrated in human brain
(19
) and the IRP/IRE interaction may be altered in
Alzheimer’s disease (20
). Recently a mouse model in which
IRP2 was rendered null has been reported. These mice may have altered
brain iron status and motor function but this requires more rigorous
evaluation (21
). We previously established a
developmentally sensitive rat model of dietary ID and excess, which
allowed us to report on the changes in brain iron, transferrin,
transferrin receptor and ferritin levels (22
). In this
manuscript, we report the results of the evaluation of the IRP/IRE
binding activities in cortex, hippocampus and striatum homogenates. In
the context of this analysis, an additional question was addressed
regarding the intracellular distribution of the IRPs. This question was
posed because of the compartmentalization of ferritin and transferrin
receptor synthesis.
Because of the constraints of the animal model, namely the developmentally related response to dietary iron changes, and the number of different cells in a given brain region, a cell culture model was developed to more directly examine this intracellular distribution. The main objective of this set of experiments was to characterize the intracellular distribution of IRPs and to correlate this distribution with that of their target mRNAs. We hypothesize that movement of IRPs between the cytosolic and the membrane-associated subcellular fractions occurs in response to intracellular iron changes. We tested this hypothesis in the animal model and the cell culture system.
MATERIALS AND METHODS
Cell culture model.
The following sets of experiments were performed to evaluate the effect of iron levels on the intracellular distribution of IRPs. Mouse fibroblast cells (NIH 3T3) and mouse macrophage cells (J774) were purchased from American Type Culture Collection (Rockville, MD). These cells were chosen to expand the observations from the brain homogenates to non-neural tissues. The cells were grown in a 5% CO2 atmosphere at 37°C in Dulbecco’s modified Eagle’s medium (Gibco, Rockville, MD) supplemented with 10% fetal bovine serum (Biocell, Rancho Dominguez, CA) 4 mmol/L glutamine (Sigma, St. Louis, MO), 1 x 105 U/L penicillin and 1 µg/L streptomycin (Gibco). The cells were plated at 70% confluence, and the experiments were performed 24 h later. To determine the effects of changes in intracellular iron levels, the cells were exposed for 16 h to either ferric ammonium citrate (FAC; 100 mg/L, Sigma) or the iron chelator deferoxamine (DFO; 100 µmol/L, Sigma). The results were consistent for both cell types.
Separation of cytosolic and membrane fraction.
The cells were washed with Hank’s solution (Sigma), trypsinized and collected. To separate membrane bound organelles from cytosol, the cells were washed and homogenized in lysis buffer (10 mm/L HEPES, pH 7.4, 40 mmol/L KCl, 5% glycerol, 3 mmol/L MgCl2, 0.1 mmol/L EDTA, 1 mmol/L dithiothreitol, 1mmol/L phenylmethylsulfonyl fluoride and 0.1 g/L leupeptin, all reagents from Sigma). Homogenization was carried out on ice in a Dounce homogenizer. The homogenates were centrifuged at 4°C and 4,000 x g for 5 min to sediment the nuclei. The resulting supernatant was centrifuged at 4°C and 145,000 x g for 1 h and the cytosolic fraction (supernatant) was collected. The pellet was washed twice in 1 mL lysis buffer, resuspended in 100 µL of lysis buffer and either processed or stored at -70°C. Usually, 50 µL of this solution was treated with 2.5 µL of 10% Triton-X (final concentration 0.5%; Sigma) at 4°C for 1 h and centrifuged at 4°C and 20,000 x g for 5 min and the supernatant collected. The addition of Triton-X was required to obtain optimal binding in the membrane fractions indicating that the IRPs in this fraction are integrally associated with the membrane. This supernatant will be referred to as membrane fraction.
RNA band shift assay.
A synthetic RNA transcript containing the IRE was generated from the
oligonucleotide template
5'-ttatgctgagtgatatccctctcctaggacgaagttgtcacgaacctgcctagg-3' with T7
polymerase in the presence of 32P-CTP (NEN Life Sciences,
Boston, MA). Binding reactions were carried out as described previously
(19
). Briefly, equal amounts of protein for the
cytoplasmic and membrane fractions were incubated with the synthetic
radiolabeled IRE probe. To measure total IRP binding activity, a subset
of samples were treated with 2% ß-mercaptoethanol (BME, Sigma) for 5
min before the addition of the probe. The RNA-protein complexes
were separated on a 6% native polyacrylamide gel and visualized by
autoradiography. The autoradiograms were scanned and the bands were
subjected to densitometric analysis using the Collage Image Analysis
System (Fotodyne, Hartland, WI).
Analysis of polysomal distributions.
To investigate the relationship between IRPs and ferritin mRNA in the ribosomal and nonribosomal fractions, analysis of ribosomal distribution was performed in both cell types. After treatment, the cells were lysed by the addition of a solution containing 10 mmol/L Tris-HCl, pH 7.4, 10 mmol/L NaCl and 1.5 mmol/L MgCl2 and then homogenized in a Dounce homogenizer. The lysates were centrifuged at 10,000 x g and the resulting postmitochondrial supernatants were collected. An aliquot of these supernatants (500 µL containing 1 mg total protein) was loaded on top of 11 mL of a 0.22- to 0.66-mol/L linear sucrose gradient and centrifuged at 145,000 x g for 2 h. Twenty fractions were collected with an ISCO fractionator (Lincoln, NE), which resolves the gradient from the top, i.e., the first fraction represents the top of the gradient and the last fraction represents the bottom of the gradient. Ribosomal distribution was monitored at 254 nm during collection. The relative amount of ribosomal mRNA was determined in each fraction using 32P-labeled cDNA to the 28S ribosomal mRNA. Free and total IRP binding activities were measured in the same fractions by RNA band shift assay as described above.
Northern blot analysis of the distribution of L-ferritin mRNA in the sucrose gradient fractions.
Total RNA was isolated in each of the fractions by the method described
by Chomczynski and Sacchi (23
). Equal amounts of total RNA
were separated on a 1% agarose gel and transferred overnight to a
nylon hybridization membrane (Amersham, Piscataway, NJ) by capillary
elution. After RNA was immobilized by UV crosslinking, the membrane was
placed between two 586 filter papers (Schleicher & Schuell, Keene, NH)
saturated in hybridization solution containing 5x saline sodium
citrate buffer, 11 mol/L formamide, 17 mmol/L SDS and 2x Denhardt’s
solution (all reagents from Sigma). For RNA measurement, an aliquot of
the fraction was treated with 17 mmol/L SDS and the RNA was extracted
with phenol and precipitated with ethanol. Ferritin probes were derived
from human ferritin cDNA clones (provided by J. Drysdale, Tufts
University, Boston, MA), labeled with 32P-CTP, added to the
hybridization buffer and incubated overnight at 42°C. After
sequential washing, the membranes were exposed to X-ray film at
-70°C.
Animal model.
The animals, dietary treatment and specimen collection are the same as
in Piñero et al. (22
) and the details for each are
presented in that manuscript. In brief, the experimental design allowed
us to obtain rats that had early ID/excess, between postnatal day (PND)
10 and 21, late ID/excess, between PND 21 and 35, or long-term
ID/excess, between PND 10 and 35. At PND 21 or PND 35 the rats were
killed by exsanguination while being perfused, and the brains removed
and dissected into several regions. These regions were suspended in
0.32 mol/L sucrose solution, homogenized and saved at -70°C for
further analyses. The intracellular distribution of IRP1 was evaluated
in three different brain regions (cortex, hippocampus and striatum) of
early ID/excess and long-term ID/excess rats. These regions and
times were chosen because they showed the most dramatic responses in
iron, transferrin, transferrin receptor (TfR) and ferritin
concentrations to changes in dietary iron status (22
).
Homogenates were kept at -70°C until thawed for IRP binding analyses
in the present study. The homogenates were separated into cytosolic and
membrane fractions using the method described above. IRP binding
activity was determined using autoradiography. Each sample used to
separate the cytosolic and membrane fractions was the result of pooling
samples from three different animals from each group. The
autoradiograms were scanned and the bands subjected to densitometric
analysis with the Collage Image Analysis System.
The results of the IRP binding activity for each brain region and
subcellular fraction are presented as percentage of the corresponding
control group. The data were analyzed using the SAS System for Windows,
version 6.12. One-way ANOVA with one between-group factor
followed by Dunnett’s post hoc test was used to evaluate if the
experimental treatments were different from their adequate controls.
The 
level for the analyses was set at P < 0.05.
RESULTS
Effect of iron on IRP binding activity.
The results of the gel shift assay (Fig. 1
) showed that IRP binding activity was found in both the cytosolic and
membrane fractions of the cells. Exposing the cells to FAC decreased
the IRP binding activity in each fraction, while DFO treatment
increased the binding in both fractions. There was an increase in
binding activity of both IRP1 and IRP2 in the membrane fraction after
DFO treatment compared with the untreated membrane fraction. The total
IRP binding activity measured after treatment with BME remained
unchanged after treatment in the cytosolic fraction. In the membrane
fraction, by contrast, total IRP binding activity was decreased after
iron treatment and increased after DFO treatment with respect to the
untreated cells. IRP2 was not detected in the membrane fraction of the
untreated control and DFO-treated samples, except after BME
treatment. The uneven distribution of total binding activity in the
membrane fraction between the three conditions suggests movement of the
IRPs between membrane and cytosolic fractions in response to treatment.
|
A sucrose gradient was obtained to demonstrate the distribution of IRP
(measured by binding activity) and a target mRNA
(L-ferritin) as a function of iron status. The
postmitochondrial fraction of the cytosol was separated on a sucrose
gradient and the non–ribosome- and ribosome-containing fractions
were identified using 28S ribosomal RNA as a probe. Neither the
percentage of total protein in each fraction nor the ribosomal
distribution were altered by treatment (data not shown). The IRP
binding activity in the different fractions was measured by gel
shift assay, and the results are shown in Figure 2
. Panels A and B are the same samples but the
films were exposed for 12 and 48 h, respectively. The exposure
times were modified in an attempt to detect IRP2 and also to detect the
presence of even minimal amounts of IRP in the various fractions. IRP
binding activity (for both IRP1 and IRP2) was most prominent in the
ribosome free fractions in the untreated samples. Deferoxamine
treatment was associated with more IRP-binding activity in both the
non–ribosome- and ribosome-containing fractions than in the
control samples. FAC-treated samples had less IRP binding activity
than control or DFO-treated groups in all of the fractions. Only in
the DFO-treated group was IRP binding activity observed in the
ribosome-containing fractions. These data indicate a shift in the
IRPs in the cytosolic fractions as a function of iron availability.
Total IRP binding activity was dramatically increased in the
nonribosomal fraction in both the iron-treated and DFO-treated
samples (Fig. 2
C).
|
A and B). After iron treatment, 60% of the
L-ferritin mRNA was found within the
ribosomal-containing fraction, while iron chelation with DFO
resulted in the majority (70%) of the L-ferritin
mRNA appearing in the non–ribosome-containing fractions (Fig. 3
C). The intracellular redistribution of
L-ferritin mRNA demonstrates that our treatment
induced changes in a manner consistent with synthesis of ferritin in
response to iron status.
|
There was an increase in the IRP binding activity in brain regions of
iron-deficient rats compared with controls in both the cytosolic
and membrane fractions in rats of all ages and brain regions examined
except for the cytosolic fraction of striatum at PND35 (Fig. 4
). The only change produced by dietary iron supplementation was a
decrease in the cytosolic fraction of the cortex at PND 35 and in the
membrane fraction of the hippocampus at PND 21.
|
DISCUSSION
The main objective of this set of experiments was to characterize the intracellular distribution of IRPs as a function of iron status and compare this distribution with that of a target mRNA. We used both an in vivo and a cell culture approach, and the results show that IRP binding activity is found in two subcellular locations, a cytosolic fraction and a membrane-associated fraction. Furthermore, our results demonstrate that the amount of binding activity in each subcellular location can be altered by cellular iron status. An additional question raised in this study is the relationship between the IRP and their target IREs. We showed that distribution of L-ferritin mRNA is altered between the ribosomal and nonribosomal fractions as a function of iron status, so that when iron is high, more L-ferritin mRNA is found in the ribosomal fraction, and when iron is low, ferritin mRNA is more abundant in the nonribosomal fraction. In the same samples, IRP binding activity was predominantly in the nonribosomal fractions in the untreated group and was only seen in the ribosome-containing fractions after iron chelation. The IRP binding activity shifted to the non–ribosome-containing fraction after exposure to iron, which is consistent with the shift of ferritin mRNA into the translatable pool. The redistribution to the ribosome-containing fraction after iron chelation is consistent with the concept that IRP binds to IREs on the 3' UTR of transferrin receptor mRNA that is being translated in the rough endoplasmic reticulum as a result of cellular ID. These data support our hypothesis that the subcellular distribution of IRPs is influenced by iron availability, and that the distribution is consistent with that thought to occur for the target mRNAs.
In the rat studies, we have showed that IRP binding activity is present
in the rat brain and is altered in brain subsequent to dietary ID and
excess. These data agree with the reports by Chen et al.
(24
, 25
) that the activity of liver IRP responds to changes
in dietary iron levels in rats. However, in the brain, the change in
binding activity is region specific and developmentally sensitive.
Also, in our study, excess dietary iron had a limited effect on IRP
binding activity in the brain, but IRP binding activity was
dramatically decreased in liver after excess dietary iron in the
studies of Chen et al. (24
, 25
). The changes in IRP binding
activity in the brain regions analyzed are consistent with the changes
in TfR, ferritin, transferrin and iron status found in
the same brain regions (22
). For example, during ID, there
is an increase in TfR associated with the increased IRP binding
activity for each of the different brain regions. The lack of an effect
of dietary iron on brain IRP binding activity probably reflects the
action of the blood-brain barrier to limit iron acquisition by the
brain. The changes in IRP binding activity in this study indicate that
regional and age-related changes in iron metabolism found in the
previous report are regulated at the molecular level. The data further
suggest that the molecular regulatory mechanism is age and brain region
sensitive.
Because of the number of different cell types in the brain and the
heterogeneous distribution of iron in the brain, the layers of
complexity in this organ are too many to understand the mechanism
underlying redistribution. Since we began this study, the cellular and
regional distribution of IRPs in the mouse brain have been reported and
confirm our notion that the IRP expression in brain is heterogenous
(26
). Thus, the cell culture models not only complement
the brain analyses but are essential to identifying the intracellular
relationship between IRPs and their target mRNAs.
In summary, we have shown that the IRPs exist in different subcellular locations and the location is associated with cellular iron status. These results suggest that intracellular movement of IRPs may be critical to post-transcriptional regulation of ferritin and transferrin receptor mRNAs. We also demonstrate that IRPs exist in rat brain and are responsive to dietary iron changes in a manner that is consistent with changes in TfR and ferritin. Thus, it appears that IRPs are involved in regulation of brain iron status, but the region and age sensitivity suggest that the IRPs may themselves be subjected to currently unidentified regulatory mechanisms. Furthermore, because IRPs regulate iron acquisition in the brain, elucidation of the regulation of these proteins may provide insight into the underlying defects in regulation that permits excess iron to accumulate in Alzheimer’s and Parkinson’s diseases.
FOOTNOTES
1 Supported by the grants NS34280 and DK53430 from
the National Institutes of Health. 
3 Abbreviations used: BME, ß-mercaptoethanol;
DFO, deferoxamine; FAC, ferric ammonium citrate; ID, iron deficiency;
IRE, iron responsive element; IRP, iron regulatory protein; PND,
postnatal day; TfR, transferrin receptor.
Manuscript received 1 April 2001. Initial review completed 22 May 2001. Revision accepted 14 August 2001.
LITERATURE CITED
1. Cairo, G. & Pietrangelo, A. (2000) Iron regulatory proteins in pathology. Biochem. J. 352:241-250.
2. Eisenstein, R. S. (2000) Iron regulatory proteins and the molecular control of mammalian iron metabolism. Annu. Rev. Nutr. 20:627-662.[Medline]
3. Seiser, C., Posch, M., Thompson, N. & Kuhn, L. C. (1995) Effect of transcription inhibitors on the iron-dependent degradation of transferrin receptor mRNA. J. Biol. Chem. 270:29400-29406.
4. Linder, M. C., Madani, N., Middleton, R., Miremadi, A., Cairo, G., Tacchini, L., Schiaffonati, L. & Rappocciolo, E. (1992) Ferritin synthesis on polyribosomes attached to the endoplasmic reticulum. J. Inorg. Biochem. 47:229-240.[Medline]
5. Madani, N. & Linder, M. C. (1992) Differential effects of iron and inflammation on ferritin synthesis on free and membrane-bound polyribosomes of rat liver. Arch. Biochem. Biophys. 299:206-213.[Medline]
6. Beard, J. L., Connor, J. R. & Jones, B. C. (1993) Brain iron: location and function. Prog. Food Nutr. Sci. 17:183-221.[Medline]
7. Connor, J. R., Pavlick, G., Karli, D., Menzies, S. L. & Palmer, C. (1995) A histochemical study of iron-positive cells in the developing rat brain. J. Comp. Neurol. 355:111-123.[Medline]
8. Roskams, A. J. & Connor, J. R. (1994) Iron, transferrin, and ferritin in the rat brain during development and aging. J. Neurochem. 63:709-716.[Medline]
9. Taylor, E. M. & Morgan, E H. (1990) Developmental changes in transferrin and iron uptake by the brain in the rat. Dev. Brain Res. 55:35-42.[Medline]
10. Felt, B. T. & Lozoff, B. (1996) Brain iron and behavior of rats are not normalized by treatment of iron deficiency anemia during early development. J. Nutr. 126:693-701.
11. Lozoff, B., Jimenez, E., Hagen, J., Mollen, E. & Wolf, A. W. (2000) Poorer behavioral and developmental outcome more than 10 years after treatment for iron deficiency in infancy. Pediatrics 105:E51.
12. Piñero, D. J., Jones, B. C. & Beard, J. L. (2001) Variations in Dietary Iron Alter Behavior in Developing Rats. J Nutr 131:311-318.
13. Bartzokis, G., Sultzer, D., Cummings, J., Holt, L. E., Hance, D. B., Henderson, V. W. & Mintz, J. (2000) In vivo evaluation of brain iron in Alzheimer disease using magnetic resonance imaging. Arch. Gen. Psychiatry. 57:47-53.
14. Connor, J. R., Menzies, S. L., St. Martin, S. M. & Mufson, E. J. (1992) A histochemical study of iron, transferrin and ferritin in Alzheimer’s diseased brains. J. Neurosci. Res. 31:75-83.[Medline]
15. Connor, J. R., Snyder, B. S., Beard, J. L., Fine, R. E. & Mufson, E. J. (1992) Regional distribution of iron and iron-regulatory proteins in the brain in aging and Alzheimer’s disease. J. Neurosci. Res. 31:327-335.[Medline]
16. Connor, J. R., Snyder, B. S., Arosio, P., Loeffler, D. A. & LeWitt, P. (1995) A quantitative analysis of isoferritins in select regions of aged, Parkinsonian, and Alzeimer’s diseased brains. J. Neurochem. 65:717-724.[Medline]
17. Dexter, D. T, Carayon, A., Javoy-Agid, F., Agid, Y., Wells, F. R., Daniel, S. E., Lees, A. J., Jenner, P. & Marsden, C. D. (1991) Alterations in the levels of iron, ferritin and other trace metals in Parkinson’s disease and other neurodegenerative diseases affecting the basal ganglia. Brain 114:1953-1975.
18. Sofic, E., Paulus, W., Jellinger, K., Riederer, P. & Youdim, M.B.H. (1991) Selective increase of iron in substantia nigra zona compacta of Parkinsonian brains. J. Neurochem. 56:978-982.[Medline]
19. Hu, J. & Connor, J. R. (1996) Demonstration and characterization of the iron regulatory protein in human brain. J. Neurochem. 67:838-844.[Medline]
20. Piñero, D. J., Hu, J. & Connor, J. R. (2000) Alterations in the interaction between iron regulatory proteins and their iron responsive element in normal and Alzheimer’s diseased brains. Cell. Mol. Biol. (Noisy-le-grand) 46:761-776.
21. LaVaute, T., Smith, S., Cooperman, S., Iwai, K., Land, W., Meyron-Holtz, E., Drake, S. K., Miller, G., Abu-Asab, M., Tsokos, M., Switzer, R. 3rd, Grinberg, A., Love, P., Tresser, N. & Rouault, T. A. (2001) Targeted deletion of the gene encoding iron regulatory protein-2 causes misregulation of iron metabolism and neurodegenerative disease in mice. Nat. Genet. 27:209-214.[Medline]
22. Piñero, D. J., Li, N.-Q., Connor, J. R. & Beard, J. L. (2000) Variations in dietary iron alter brain iron metabolism in developing rats. J. Nutr. 130:254-263.
23. Chomczynski, P. & Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162:156-159.[Medline]
24. Chen, O. S., Schalinske, K. L. & Eisenstein, R. S. (1997) Dietary iron intake modulates the activity of iron regulatory proteins and the abundance of ferritin and mitochondrial aconitase in rat liver. J. Nutr. 127:238-248.
25. Chen, O. S., Blemings, K. P., Schalinske, K. L. & Eisenstein, R. S. (1998) Dietary iron intake rapidly influences iron regulatory proteins, ferritin subunits and mitochondrial aconitase in rat liver. J. Nutr. 128:525-535.
26. Leibold, E. A., Gahring, L. C. & Rogers, S. W. (2001) Immunolocalization of iron regulatory protein expression in the murine central nervous system. Histochem. Cell Biol. 115:195-203.[Medline]
This article has been cited by other articles:
|
|
 |
|
  M. C. Sammarco, S. Ditch, A. Banerjee, and E. Grabczyk Ferritin L and H Subunits Are Differentially Regulated on a Post-transcriptional Level J. Biol. Chem., February 22, 2008; 283(8): 4578 - 4587. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. S. Golub, C. E. Hogrefe, and S. L. Germann Iron Deprivation during Fetal Development Changes the Behavior of Juvenile Rhesus Monkeys J. Nutr., April 1, 2007; 137(4): 979 - 984. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. M. Patton, D. J. Pinero, N. Surguladze, J. Beard, and J. R. Connor Subcellular localization of iron regulatory proteins to Golgi and ER membranes J. Cell Sci., October 1, 2005; 118(19): 4365 - 4373. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. D. Knutson, M. R. Vafa, D. J. Haile, and M. Wessling-Resnick Iron loading and erythrophagocytosis increase ferroportin 1 (FPN1) expression in J774 macrophages Blood, December 1, 2003; 102(12): 4191 - 4197. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
