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James R Connor

Publications and source records attributed to James R Connor.

At least 37 records · Page 2Linked to original sources
PubMed2005

Ferritin levels in the cerebrospinal fluid and restless legs syndrome: effects of different clinical phenotypes.

STUDY OBJECTIVE: To determine whether patients with restless legs syndrome (RLS) and controls differ in regard to levels of ferritin and transferrin in the cerebrospinal fluid (CSF) when samples are collected at night, to determine whether patients with early-onset and late-onset RLS show a different outcome for CSF values, and to determine whether the CSF ferritin level correlates with disease severity. DESIGN: Collection of CSF and plasma; assessment of disease severity using objective (periodic limb movements) and subjective (Johns Hopkins Restless Legs Severity Scale) measures of severity. SETTING: General Clinical Research Center. PARTICIPANTS: Thirty subjects with idiopathic RLS (15 early- and 15 late-onset RLS) and 22 age- and sex-matched controls. INTERVENTION: N/A. RESULTS: Nighttime CSF ferritin levels were lower in the total RLS group compared with controls. Further assessment found that the early-onset (less than 45 years of age) but not the late-onset (greater than or equal to 45 years of age) RLS group had significantly lower CSF ferritin levels compared with controls. There was a strong correlation between the age of symptom onset and CSF ferritin values (r = 0.64): the earlier the age, the lower the ferritin level. A regression analysis showed that both sex and RLS subtype had significant effects on the CSF ferritin level, with women with early-onset RLS having substantial lower values than men with late-onset RLS. A comparison between these nighttime CSF values and previously published daytime samples suggests that diurnal changes may have effects on the findings. CONCLUSIONS: This study is distinct in showing that the degree of the CSF-ferritin effect is best defined by the clinical phenotypes of sex and age of symptom onset and by the time of day that samples are collected.

Adult↗
PubMed2004

Development of a fluorescent reporter to assess iron regulatory protein activity in living cells.

Through the insertion of an iron responsive element (IRE) into a pd2ECFP vector, we demonstrate a noninvasive method for determining alterations in iron regulatory protein (IRP) activity that results in changes in protein translation in living cells. This construct takes advantage of the specifically iron-dependent interaction between IRPs that bind IREs on mRNAs to posttranscriptionally regulate protein expression in a manner similar to ferritin production. In this report, we demonstrate, using HEK-293 cells, that an IRE-driven fluorescent reporter can be used to observe changes in cellular iron status that are sufficient to alter protein synthesis. When iron availability was decreased, there was less cyan fluorescent protein (CFP) expression, suggesting that IRPs bind to the IRE and block protein translation. Conversely, exposing the cells to iron increased CFP fluorescence. This construct has advantages over traditionally used dyes and existing IRE driven constructs because it can be used to repeatedly study iron-influenced protein production over extended periods of time. The future applications of this construct include investigation of how mutations in cells may impact cellular iron metabolism and how various types of exogenously applied trophic, stress, and therapeutic agents may impact cellular iron metabolism.

Amino Acid Motifs↗
PubMed2004

Interactions and reactions of ferritin with DNA.

Ferritin, normally considered a cytoplasmic iron-storage protein, is also found in the nuclei of some cells. There is no current agreement about its function(s) in this environment. Proposals include DNA protection, provision of iron to nuclear enzymes, and regulation of transcription initiation, but evidence for these functions is scanty. We have shown previously that H-ferritin subunits can be cross-linked to chromosomal DNA in vivo (Thompson, K. J., Fried, M. G., Ye, Z., Boyer, P., and Connor, J. R. (2002) J. Cell Sci. 115, 2165-2177). Here we describe systematic analyses of DNA binding and the covalent stability of DNA in the presence of ferritins from several different sources. Our data show that the H-subunit of human ferritin binds DNA, whereas neither the L-subunit nor the ferroxidase-deficient 222-mutant of the H-subunit has detectable binding activity. DNA binding is without significant preference for base composition, sequence, or the nature of DNA ends. H- and L-ferritins and ferritins of mixed subunit composition stimulate the conversion of superhelical plasmid DNA to the relaxed form. The sensitivity of this conversion to glycerol suggests that DNA is nicked by a free radical mechanism. The rate of nicking correlates with the iron content of the ferritin and is strongly inhibited by chelators. Ferritin-dependent nicking is characterized by a kinetic lag that is not seen in control reactions containing free iron species. These results suggest that the release of iron from ferritin is an important part of the nicking mechanism. The potential role of ferritin as a protector of the genome is discussed in the context of these results.

Buffers↗
PubMed2004

Iron, brain ageing and neurodegenerative disorders.

There is increasing evidence that iron is involved in the mechanisms that underlie many neurodegenerative diseases. Conditions such as neuroferritinopathy and Friedreich ataxia are associated with mutations in genes that encode proteins that are involved in iron metabolism, and as the brain ages, iron accumulates in regions that are affected by Alzheimer's disease and Parkinson's disease. High concentrations of reactive iron can increase oxidative-stress induced neuronal vulnerability, and iron accumulation might increase the toxicity of environmental or endogenous toxins. By studying the accumulation and cellular distribution of iron during ageing, we should be able to increase our understanding of these neurodegenerative disorders and develop new therapeutic strategies.

Aging↗
PubMed2004

Regulation of the profile of iron-management proteins in brain microvasculature.

The distribution of brain iron is heterogeneous, but the mechanism by which these regional differences are achieved and maintained is unknown. In this study, the authors test two hypotheses related to brain iron transport. The first is that there is regional variability in the profile of proteins associated with iron transport and storage in the brain microvasculature. The second hypothesis is that the iron status of the brain will dictate the response of the protein profile in the microvasculature to changes in systemic iron status. The profile analysis consists of transferrin (iron transport), ferritin (iron storage), transferrin receptor (iron uptake), and divalent metal transporter 1 (release of iron from endosomes). An additional protein involved in cellular iron efflux, ferroportin, was not detected in brain microvasculature. The results show that there are significantly higher levels of these proteins in the microvasculature from each area of the brain compared to a whole brain homogenate, but no regional differences within the microvasculature. The levels of ferritin observed in the microvasculature indicate that the microvascular endothelial cells have significant iron storage capacity. There are no significant changes in the regional protein profiles in response to systemic iron manipulation when brain iron status was normal. In contrast, in Belgrade rats, whose brain is iron deficient, the expression of both divalent metal transporter 1 and transferrin receptor was increased compared with control in almost all brain regions examined, but not transferrin or ferritin. These findings indicate that regional brain iron heterogeneity is not maintained by differences in microvascular iron-management protein levels. The results also indicate that brain iron status dictates the response of the microvascular protein profile to systemic iron manipulation.

Animals↗
PubMed2004

Redox metals in Alzheimer's disease.

Redox metals in the brain play many important roles in maintenance of cellular function. The maintenance of their homeostasis is of paramount importance to a number of diseases such as Alzheimer's disease and multiple sclerosis. Iron, copper, and zinc are metals of special interest in the pathogenesis of these disorders. This review will focus primarily on iron.

Alzheimer Disease↗
PubMed2003

Myelination and motor coordination are increased in transferrin transgenic mice.

Myelin deficiency in the central nervous system (CNS) can cause severe disabling conditions. Most of the transgenic mice models overexpressing myelin components have limitations for investigators of myelin deficiency and myelin therapy as they severely alter CNS architecture. It has been postulated that transferrin (Tf) is involved in oligodendrocyte (OL) maturation and myelinogenesis. Because Tf is not an intrinsic myelin constituent, we decided to investigate if its overexpression could have an impact on the myelination process without affecting myelin integrity. We generated transgenic mice containing the complete human Tf gene specifically overexpressed in OLs. This overexpression leads to more than a 30% increase in myelin components, such as galactolipids, phospholipids, and proteins. Electron microscopy showed that myelin is structurally normal in terms of thickness and compaction. Behavior analysis showed that mice do not display significant modifications in their locomotion and cognitive and emotional abilities. Furthermore, in one of the genetic background, animals presented a significant increase in motor coordination. We did not find any modification in OL number during early postnatal development, suggesting that Tf does not act on OL proliferation. In addition, the levels of iron and ferritin remained unchanged in the brain of transgenic mice compared to control mice. Our findings indicate that, besides its known iron transport function, Tf is able to influence myelination process and induce behavioral improvements in mice.

Animals↗
PubMed2003

Iron status and neural functioning.

Iron deficiency in early life is associated with delayed development as assessed by a number of clinical trials using similar global scales of development; this poor development during infancy persists in most cases after iron therapy has corrected iron status. If iron deficiency occurs in preschool and older children, the consequences appear reversible with treatment. The biologic understanding of this relationship between development, brain iron status, and functioning is sparse though animal studies repeatedly demonstrate alterations in dopamine metabolism and in the myelination process. Dietary iron deficiency can rapidly deplete brain iron concentrations and repletion is able to normalize them. Residual alterations in striatal dopamine metabolism and myelin production persist if neonatal animals are used. Future studies with more specific measures of neurodevelopment in iron-deficient human infants, and animal models, will allow investigators to more clearly define causal roles of brain iron in neural development and functioning.

Adolescent↗
PubMed2003

Iron deficiency alters iron regulatory protein and iron transport protein expression in the perinatal rat brain.

Iron plays an important role in numerous vital enzyme systems in the perinatal brain. The membrane proteins that mediate iron transport [transferrin receptor (TfR) and divalent metal transporter 1 (DMT-1)] and the iron regulatory proteins (IRP-1 and IRP-2) that stabilize their mRNAs undergo regional developmental changes in the iron-sufficient rat brain between postnatal day (P) 5 and 15. Perinatal iron deficiency (ID) affects developing brain regions nonhomogeneously, suggesting potential differences in regional iron transporter and regulatory protein expression. The objective of the study was to determine the effect of perinatal ID on regional expression of IRP-1, IRP-2, TfR, and DMT-1 in the developing rat brain. Gestationally iron-deficient Sprague Dawley rat pups were compared with iron-sufficient control pups at P10. Serial 12-mu coronal sections of fixed frozen brain from pups on P10 were assessed by light microscopy for IRP-1, IRP-2, DMT-1, and TfR localization. ID did not change the percentage of cells with positive staining for the four proteins in the choroid epithelium, ependyma, vascular endothelium, or neurons of the striatum. ID increased the percentage of neurons expressing the four proteins in the hippocampus and the cerebral cortex. Increased numbers of TfR- and DMT-1-positive cells were always associated with increased IRP-positive cells. The P10 rat responds to perinatal ID by selectively increasing the number of neurons expressing IRP-regulated transporters in brain regions that are rapidly developing, without any change at transport surfaces or in regions that are quiescent. Brain iron distribution during ID seems to be locally rather than globally regulated.

Anemia, Iron-Deficiency↗
PubMed2003

Brain iron uptake and homeostatic mechanisms: an overview.

Timely and adequate iron acquisition by the brain is essential to normal neurological function. Despite the numerous cognitive and neurological impairments that are associated with disruptions in brain iron acquisition, including both too much and too little iron, the mechanism and regulation of the mechanisms by which the brain acquires iron are poorly understood. In this article, we review the current state of knowledge regarding expression of iron transport proteins in the brain, brain iron uptake and discuss why a model for brain iron uptake must take into consideration the potentially competing influences on the endothelial cell between the status of iron in the brain versus the systemic iron status.

Animals↗
PubMed2003

Gene expression of transferrin and transferrin receptor in brains of control vs. iron-deficient rats.

The mechanism of the regulation of transferrin (Tf) and transferrin receptor (TfR) levels in rat brain by dietary iron status is not fully elucidated. We examined Tf and TfR protein and mRNA contents in various brain regions affected by dietary iron deficiency, and analyzed the relationships between protein and mRNA contents in brains of control vs. iron-deficient rats. In a region-specific fashion, iron-deficient diet decreased significantly brain iron concentration by 22-63%, and increased Tf level by 22-130% and TfR level by 74% in thalamus and 40% in cortex. Tf mRNA content decreased by 20-50% in most brain regions demonstrating inverse correlation of Tf and its mRNA in response to iron deficiency. TfR mRNA levels remained unaffected by iron status. The corpus callosum, white matter of the cerebellum and lateral ventricles expressed highest levels of Tf mRNA, whereas TfR mRNA levels was lowest in these regions, but highest in cortex, hippocampus, and the gray matter of the cerebellum. The data demonstrate that the cells in brain have the capacity to maintain minimum iron levels during iron deficiency. This capacity may be associated with increased iron-Tf uptake from plasma, stabilization of TfR mRNA, or increased Tf mRNA translation efficiency in specific cell types within the brain.

Animals↗
PubMed2002

Developmental changes in the expression of iron regulatory proteins and iron transport proteins in the perinatal rat brain.

The perinatal brain requires a tightly regulated iron transport system. Iron regulatory proteins (IRPs) 1 and 2 are cytosolic proteins that regulate the stability of mRNA for the two major cellular iron transporters, transferrin receptor (TfR) and divalent metal transporter-1 (DMT-1). We studied the localization of IRPs, their change in expression during perinatal development, and their relationship to TfR and DMT-1 in rat brain between postnatal days (PND) 5 and 15. Twelve-micron frozen coronal sections of fixed brain tissue were obtained from iron-sufficient Sprague-Dawley rat pups on PND 5, 10, and 15, and were visualized at 20 to 1,000x light microscopy for diaminobenzidine activity after incubation with specific primary IRP-1, IRP-2, DMT-1, and TfR antibodies and a universal biotinylated secondary and tertiary antibody system. IRP and transport protein expression increased in parallel over time. IRP1, IRP2, and DMT-1 were partially expressed in the choroid plexus epithelial cells at PND 5 and 10, and fully expressed at PND 15. The cerebral blood vessels and ependymal cells strongly expressed IRP1, IRP2, and DMT-1 as early as PND 5. Substantive TfR staining was not seen in the choroid plexus or ependyma until PND 15. Glial and neuronal expression of IRP1, IRP2, DMT-1, and TfR in cortex, hippocampal subareas and striatum increased over time, but showed variability in cell number and intensity of expression based on brain region, cell type, and age. These developmental changes in IRP and transporter expression suggest potentially different time periods of brain structure vulnerability to iron deficiency or iron overload.

Animals↗
PubMed2002

Regulation, mechanisms and proposed function of ferritin translocation to cell nuclei.

Ferritin is traditionally considered a cytoplasmic iron-storage protein, but recent reports indicate that it is also found in cell nuclei. Nuclear ferritin has been proposed to be involved in both the protection of DNA and the exacerbation of iron-induced oxidative damage to DNA. We demonstrate that H-rich ferritin is present in the nucleus of human astrocytoma tumor cells. To study the mechanism and regulation of ferritin translocation to the nucleus, we developed a cell culture model using SW1088 human astrocytoma cells. Changes in cellular iron levels, cytokine treatments and hydrogen peroxide exposure affected the distribution of ferritin between the cytosol and the nucleus. Ferritin enters the nucleus via active transport through the nuclear pore and does not require NLS-bearing cytosolic factors for transport. Furthermore, H-rich ferritin is preferred over L-rich ferritin for uptake into the nucleus. Whole cell crosslinking studies revealed that ferritin is associated with DNA. Ferritin protected DNA from iron-induced oxidative damage in both in vitro and in cell culture models. These results strongly suggest a novel role for ferritin in nuclear protection. This work should lead to novel characterization of ferritin functions in the context of genomic stability and may have unparalleled biological significance in terms of the accessibility of metals to DNA. The knowledge generated as a result of these studies will also improve our understanding of iron-induced damage of nuclear constituents.

Active Transport, Cell Nucleus↗
PubMed2002

Microscopic R2* mapping of reduced brain iron in the Belgrade rat.

R2* mapping has recently been used to detect iron overload in patients with movement disorders. We demonstrate here that this technique can also be used to detect reduced brain iron, as in the case of a missense mutation in the iron-transporting protein divalent metal transporter 1. Surprisingly, we found that the same brain regions are affected (ie, the globus pallidus, substantia nigra, and cerebellar dentate nucleus); this suggests a much more extensive role for these structures in regulating overall brain iron homeostasis. Therefore, for the clinical monitoring of movement disorders for which normal brain iron homeostasis (either overload or reduction) may be implicated, R2* mapping appears to be well-suited.

Animals↗
PubMed2002

A light and electron microscopic study of divalent metal transporter-1 distribution in the rat hippocampus, after kainate-induced neuronal injury.

An accumulation of iron occurs in the hippocampus of rats injected with kainate over time, but thus far whether this accumulation is associated with any changes in expression of iron transporters is not known. The present study was therefore carried out using an antibody to the divalent metal transporter-1 (DMT-1) and immunoblot and immunocytochemical analyses to elucidate possible changes in expression of the transporter in the rat hippocampus after kainate injections. A significant increase in density ratios of DMT-1/beta-actin bands was observed in Western blots in the 1-week, 1-month, and 2-months post-kainate-injected hippocampus, compared to uninjected and 1-day post-kainate-injected hippocampus. The increase in DMT-1 protein was paralleled by an increase in DMT-1 immunoreactivity in astrocytes. Light staining for DMT-1 was observed in the uninjected, saline-injected, and 1-day post-kainate-injected rat hippocampus. In contrast, an upregulation of DMT-1 was observed in reactive glial cells at 1 week, 1 month, and 2 months post-kainate injection. Electron microscopy confirmed that the glial cells had morphological features of astrocytes. DMT-1 is a cellular iron transporter responsible for transport of metal ions from the plasma membrane to endosomes. The observation that DMT-1 is present on astrocytic end feet in contact with blood vessels suggests that these cells may be involved in uptake of iron from endothelial cells.

Animals↗
PubMed2002

Abnormal iron delivery to the bone marrow in neonatal hypotransferrinemic mice.

Hypotransferrinemic (HP) mice have a splicing defect in the transferrin gene, resulting in < 1% of the normal plasma levels of transferrin. They have severe anemia, suggesting that transferrin is essential for iron uptake by erythroid cells in the bone barrow. To clarify the significance of transferrin on iron delivery to the bone marrow, iron concentration and 59Fe distribution were determined in 7-day-old HP mice. Iron concentration in the femur, bone containing the bone marrow, of HP mice was approximately twice higher than in wild type mice. Twenty-four h after injection of 59FeCl3, 59Fe concentration in the bone and bone marrow of HP mice was also twice higher than in wild type mice. The present findings indicate that iron is abnormally delivered to the bone marrow of HP mice. However, the iron seems to be unavailable for the production of hemoglobin. These results suggest that transferrin-dependent iron uptake by erythroid cells in the bone marrow is essential for the development of erythrocytes.

Anemia↗