Parkinson's disease: Alterations in iron and redox biology as a key to unlock therapeutic strategies.

A plethora of studies indicate that iron metabolism is dysregulated in Parkinson's disease (PD). The literature reveals well-documented alterations consistent with established dogma, but also intriguing paradoxical observations requiring mechanistic dissection. An important fact is the iron loading in dopaminergic neurons of the substantia nigra pars compacta (SNpc), which are the cells primarily affected in PD. Assessment of these changes reveal increased expression of proteins critical for iron uptake, namely transferrin receptor 1 and the divalent metal transporter 1 (DMT1), and decreased expression of the iron exporter, ferroportin-1 (FPN1). Consistent with this is the activation of iron regulator protein (IRP) RNA-binding activity, which is an important regulator of iron homeostasis, with its activation indicating cytosolic iron deficiency. In fact, IRPs bind to iron-responsive elements (IREs) in the 3ꞌ untranslated region (UTR) of certain mRNAs to stabilize their half-life, while binding to the 5ꞌ UTR prevents translation. Iron loading of dopaminergic neurons in PD may occur through these mechanisms, leading to increased neuronal iron and iron-mediated reactive oxygen species (ROS) generation. The "gold standard" histological marker of PD, Lewy bodies, are mainly composed of α-synuclein, the expression of which is markedly increased in PD. Of note, an atypical IRE exists in the α-synuclein 5ꞌ UTR that may explain its up-regulation by increased iron. This dysregulation could be impacted by the unique autonomous pacemaking of dopaminergic neurons of the SNpc that engages L-type Ca+2 channels, which imparts a bioenergetic energy deficit and mitochondrial redox stress. This dysfunction could then drive alterations in iron trafficking that attempt to rescue energy deficits such as the increased iron uptake to provide iron for key electron transport proteins. Considering the increased iron-loading in PD brains, therapies utilizing limited iron chelation have shown success. Greater therapeutic advancements should be possible once the exact molecular pathways of iron processing are dissected.

Increased SUMO-1 expression in the unilateral rotenone-lesioned mouse model of Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disease resulting from progressive loss of dopaminergic nigrostriatal neurons. α-Synuclein protein conformational changes, resulting in cytotoxic/aggregated proteins, have been linked to PD pathogenesis. We investigated a unilateral rotenone-lesioned mouse PD model. Unilateral lesion of the medial forebrain bundle for two groups of male C57 black mice (n=5); adult (6-12 months) group and aged (1.75-2 years) group, was via stereotactic rotenone injection. After 2 weeks post-lesion, phenotypic Parkinsonian symptoms, resting tremor, postural instability, left-handed bias, ipsiversive rotation and bradykinesia were observed and were more severe in the aged group. We investigated protein expression profiles of the post-translational modifier, SUMO-1, and α-synuclein between the treated and control hemisphere, and between adult and aged groups. Western analysis of the brain homogenates indicated that there were statistically significant (p<0.05) increases in several specific molecular weight species (ranging 12-190 kDa) of both SUMO-1 (0.75-4.3-fold increased) and α-synuclein (1.6-19-fold increase) in the lesioned compared to un-lesioned hemisphere, with the adult mice showing proportionately greater increases in SUMO-1 than the aged group.

Raised calcium and oxidative stress cooperatively promote alpha-synuclein aggregate formation.

Cell loss in Parkinson's and Parkinson's-plus diseases is linked to abnormal, aggregated forms of the cytoplasmic protein, α-synuclein (α-syn). The factors causing α-syn aggregation may include oxidative stress, changes in protein turnover and dysregulation of calcium homeostasis, resulting in cytotoxic aggregated α-syn species. Recently, we showed that raised calcium can promote α-syn aggregation. We have now investigated the effects of raised calcium combined with oxidation/oxidative stress on α-syn aggregation both in vitro and in vivo. We treated monomeric α-syn with calcium, hydrogen peroxide or calcium plus hydrogen peroxide in vitro and used size exclusion chromatography, fluorescence correlation spectroscopy, atomic force microscopy and scanning electron microscopy to investigate protein aggregation. Our in vitro data is consistent with a cooperative interaction between calcium and oxidation resulting in α-syn oligomers. In cell culture experiments, we used thapsigargin or ionophore A23187 to induce transient increases of intracellular free calcium in human 1321N1 cells expressing an α-syn-GFP construct both with and without co-treatment with hydrogen peroxide and observed α-syn aggregation by fluorescence microscopy. Our in vivo cell culture data shows that either transient increase in intracellular free calcium or hydrogen peroxide treatment individually were able to induce significantly (P=0.01) increased 1-4µm cytoplasmic α-syn aggregates after 12h in cells transiently transfected with α-syn-GFP. There was a greater proportion of cells positive for aggregates when both raised calcium and oxidative stress were combined, with a significantly increased proportion (P=0.001) of cells with multiple (3 or more) discrete α-syn focal accumulations per cell in the combined treatment compared to raised calcium only. Our data indicates that calcium and oxidation/oxidative stress can cooperatively promote α-syn aggregation both in vitro and in vivo and suggests that oxidative stress may play an important role in the calcium-dependent aggregation mechanism.

Raised calcium promotes α-synuclein aggregate formation.

Parkinson's and Parkinson's-plus diseases are associated with abnormal, aggregated forms of the protein, α-synuclein. We have investigated the effects of calcium on α-synuclein aggregation in vitro and in vivo. We treated monomeric α-synuclein with calcium in vitro and used fluorescence imaging, fluorescence correlation and scanning electron microscopy to investigate protein aggregation. Incubation of fluorescent-labelled monomeric α-synuclein (24h) at low concentration (10 µM) with calcium resulted in surface aggregates (1.5±0.7 µm(2)) detected by fluorescence microscopy saturating at a half-maximum calcium concentration of 80 µM, whilst incubations without calcium showed few protein aggregates. Scanning electron microscopy revealed that α-synuclein surface plaques (0.5-1 µm) form in the presence of calcium and comprise 10-20 nm globular particles. Incubation of α-synuclein at high concentration (75 µM; 6h) resulted in soluble oligomeric aggregates detected by fluorescence correlation spectroscopy in a calcium dependent process, saturating at a half maximum calcium concentration of 180 µM. In cell culture experiments, we used thapsigargin or calcium ionophore A23187 to induce transient increases of intracellular free calcium in human 1321N1 cells expressing an α-synuclein-GFP construct and observed calcium flux and α-synuclein aggregation by fluorescence microscopy. The cell culture data shows that a transient increase in intracellular free calcium significantly increased the proportion of cells bearing cytoplasmic α-synuclein aggregates 6 and 12h post-treatment (P, 0.01). Our data indicates that calcium accelerates α-synuclein aggregation on surfaces, in free solution and in cultured cells and suggests that surface adsorption may play an important role in the calcium-dependent aggregation mechanism.

Association of metallothionein-III with oligodendroglial cytoplasmic inclusions in multiple system atrophy.

Multiple system atrophy (MSA) is an adult-onset neurodegenerative disease characterised by Parkinsonian and autonomic symptoms and by widespread intracytoplasmic inclusion bodies in oligodendrocytes. These glial cytoplasmic inclusions (GCIs) are comprised of 9-10 nm filaments rich in the protein alpha-synuclein, also found in neuronal inclusion bodies associated with Parkinson's disease. Metallothioneins (MTs) are a class of low-molecular weight (6-7 kDa), cysteine-rich metal-binding proteins the expression of which is induced by heavy metals, glucocorticoids, cytokines and oxidative stress. Recent studies have shown a role for the ubiquitously expressed MT-I/II isoforms in the brain following a variety of stresses, whereas, the function of the brain-specific MT isoform, MT-III, is less clear. MT-III and MT-I/II immunostaining of post-mortem tissue in MSA and normal control human brains showed that the number of MT-III-positive cells is significantly increased in MSA in visual cortex, whereas MT-I/II isoforms showed no significant difference in the distribution of immunopositive cells in MSA compared to normal tissue. GCIs were immunopositive for MT-III, but were immunonegative for the MT-I/II isoforms. Immunofluorescence double labelling showed the co-localisation of alpha-synuclein and MT-III in GCIs in MSA tissue. In isolated GCIs, transmission electron microscopy demonstrated MT-III immunogold labelling of the amorphous material surrounding alpha-synuclein filaments in GCIs. High-molecular weight MT-III species in addition to MT-III monomer were detected in GCIs by Western analysis of the detergent-solubilised proteins of purified GCIs. These results show that MT-III, but not MT-I/II, is a specific component of GCIs, present in abnormal aggregated forms external to the alpha-synuclein filaments.

Alpha-synuclein is upregulated in neurones in response to chronic oxidative stress and is associated with neuroprotection.

Chronic oxidative stress has been linked to the neurodegenerative changes characteristic of Parkinson's disease, particularly alpha-synuclein accumulation and aggregation. However, it remains contentious whether these alpha-synuclein changes are cytotoxic or neuroprotective. The current study utilised long-term primary neural culture techniques with antioxidant free media to study the cellular response to chronic oxidative stress. Cells maintained in antioxidant free media were exquisitely more vulnerable to acute exposure to hydrogen peroxide, yet exposure of up to 10 days in antioxidant free media did not lead to morphological alterations in neurones or glia. However, a subpopulation of neurones demonstrated a significant increase in the level of alpha-synuclein expressed within the cell body and at synaptic sites. This subset of neurones was also more resistant to apoptotic changes following exposure to antioxidant free media relative to other neurones. These data indicate that increased alpha-synuclein content is associated with neuroprotection from relatively low levels of oxidative stress.

SUMO-1 marks subdomains within glial cytoplasmic inclusions of multiple system atrophy.

Conjugation of the small ubiquitin-like modifier, SUMO-1, to target proteins is linked to the regulation of multiple cellular pathways, including nucleocytoplasmic trafficking, cell cycle progression, the ubiquitin-proteasome system and apoptosis. Recently, the accumulation of SUMOylated proteins in pathological neuronal intranuclear aggregates has been found in several neurodegenerative diseases. The aim of our study was to examine SUMO-1 in the alpha-synucleinopathy diseases, Multiple System Atrophy (MSA) and Dementia with Lewy Bodies (DLB). We conducted anti-SUMO-1 immunostaining of fixed brain tissue sections and smears of unfixed brain tissue homogenates of DLB and MSA cases. We found that oligodendroglial cytoplasmic inclusions, the alpha-synuclein-positive cytoplasmic aggregates that characterize MSA, exhibit robust punctate SUMO-1 immunostaining, marking discrete submicron-sized subdomains within the inclusion bodies. Lewy bodies in smears of DLB tissue homogenates showed similar SUMO-1-positive structures, although these were not detected in fixed tissue. In cell culture experiments, we found that the nuclear and perinuclear accumulation of SUMO-1 aggregates could be induced in glioma cells by chemical inhibition of proteasomal protein degradation.

Alpha B-crystallin is a major component of glial cytoplasmic inclusions in multiple system atrophy.

Multiple system atrophy (MSA) is characterized by the formation of oligodendroglial cytoplasmic inclusions (GCIs) consisting of alpha-synuclein filaments. AlphaB-crystallin, a small chaperone protein that binds to unfolded proteins and inhibits aggregation, has been documented in GCIs. We investigated the relative abundance and speciation of alphaB-crystallin in GCIs in MSA brains. We also examined the influence of alphaB-crystallin on the formation of cytoplasmic inclusions in cultured glial cells. Immunohistochemistry and confocal microscopy revealed alphaB-crystallin is a prominent component of GCIs, more abundant than in Lewy bodies in Lewy body dementia. One- and two-dimensional gel electrophoresis and mass spectrometric analysis of GCIs immunopurified from MSA brains indicated that alphaB-crystallin is a major protein component with multiple post-translationally modified species. In cultured C6 glioma cells treated with the proteasomal inhibitor, lactacystin, to induce accumulation of ubiquitinated proteins, a subset of cells showed increased cytoplasmic staining for alphaB-crystallin. Proteasome-inhibited cells transfected with GFP-tagged alpha-synuclein resulted in ubiquitin- and alphaB-crystallin-positive aggregates resembling GCIs in MSA brains. Our results indicate that alphaB-crystallin is a major chaperone in MSA, and suggest a role of the protein in the formation of inclusion bodies in glial cells.

SUMO-1 marks the nuclear inclusions in familial neuronal intranuclear inclusion disease.

Neuronal intranuclear inclusion disease (NIID) is a rare neurodegenerative disorder characterized by progressive ataxia and neuronal nuclear inclusions (NIs), similar to the inclusions found in expanded CAG repeat diseases. NIID may be familial or sporadic. The cause of familial NIID is poorly understood, as no CAG expansion has been detected. We examined three cases, from two unrelated families, who had autosomal dominant NIID but normal CAG repeats in genes involved in polyglutamine neurodegenerative diseases. We found that NIs in all three cases were intensely immunopositive for SUMO-1, a protein which covalently conjugates to other proteins and targets them to the nuclear regions (nuclear bodies) responsible for nuclear proteasomal degradation. Electron microscopy demonstrated that SUMO-1 was located on the 10-nm fibrils of NIs. In cultured PC12 cells, we found that inhibition of proteasome function by specific inhibitors resulted in the appearance of SUMO-1-immunopositive nuclear inclusions. Our study suggests that recruitment of SUMO-1 modified proteins into insoluble nuclear inclusions and proteasomal dysfunction may be involved in the pathogenesis of NIs in familial NIID cases.

Localization of alpha-, beta-, and gamma-synuclein during neuronal development and alterations associated with the neuronal response to axonal trauma.

Genetic and protein studies have indicated abnormalities in alpha-synuclein in neurodegenerative diseases. However, the developmental localization and cellular role of synuclein isoforms is contentious. We investigated the cellular localization of alpha-, beta-, and gamma-synuclein in developing cultured rat neurons and following axonal transection of relatively mature neurons, a model that disrupts the axonal cytoskeleton and results in regenerative sprouting. Cortical neurons were grown up to 21 days in vitro (DIV). Axon bundles at 21 DIV were transected and cellular changes examined at 4 and 24 h post-injury. Immunohistochemistry demonstrated that alpha- and beta-synuclein were localized to cellular cytosol and growth cones at 3DIV, with accumulating puncta-like labeling within axons and growth cones by 10-21DIV. In contrast, gamma-synuclein immunoreactivity was limited at all time points. By 21DIV, alpha- and beta-synuclein were present in the same neurons but largely in separate subregions, only 26% of puncta contained both alpha- and beta-synuclein immunoreactivity. Less than 20% of alpha-, beta-, and pan-synuclein immunoreactive puncta directly colocalized to synaptophysin profiles at 10DIV, decreasing to 10% at 21DIV. Both alpha- and beta-synuclein accumulated substantially within damaged axons at 21DIV and were localized to cytoskeletal abnormalities. At latter time points post-injury, alpha- and beta-synuclein immunoreactive puncta were localized to growth cone-like structures in regenerating neurites. This study shows that alpha- and beta-synuclein have a precise localization within cortical neurons and are generally nonoverlapping in their distribution within individual neurons. In addition, synuclein proteins accumulate rapidly in damaged axons and may have a role in regenerative sprouting.

alpha-Synuclein fibrils constitute the central core of oligodendroglial inclusion filaments in multiple system atrophy.

Multiple system atrophy (MSA) belongs to synucleinopathies and is characterized pathologically by oligodendroglial inclusions (GCIs) composed of 20- to 30-nm tubular filaments. alpha-Synuclein fibrils formed in vitro, however, range between 10 and 12 nm in diameter. To understand the relationship between alpha-synuclein and GCI filaments, we conducted structural analyses of GCIs in fixed brain sections and isolated from fresh-frozen MSA brains. In fixed brain sections, GCIs were composed of amorphous material-coated filaments up to 30 nm in size. The filaments were often organized in parallel bundles extending into oligodendroglial processes. In freshly isolated GCIs, progressive buffer washes removed amorphous material and revealed that GCI filaments consisted of 10-nm-sized central core fibrils that were strongly alpha-synuclein immunoreactive. Image analysis revealed that each core fibril was made of two subfibrils, and each subfibril was made of a string of 3- to 6-nm-sized particles probably alpha-synuclein oligomers. Immunogold labeling demonstrated that epitopes encompassing entire alpha-synuclein molecule were represented in the core fibrils, with the N-terminal 11-26 and C-terminal 108-131 amino acid residues most accessible to antibodies, probably exposed on the surface of the fibril. Our study indicates that GCI filaments are multilayered in structure, with alpha-synuclein oligomers forming the central core fibrils of the filaments.

Differential subcellular localization of hZip1 in adherent and non-adherent cells.

Two human divalent cation transporters of the ZIP family, hZip1 and hZip2, homologous to Irt1 (Arabidopsis thaliana), the first identified member, have been described. They were shown by transfection into K562 cells to be localized at the plasma membrane and to mediate zinc uptake. Here we report a differential subcellular localization of hZip1 according to cell type. By transient expressions of EGFP-hZip1, FLAG-tagged or native hZip1, we observed that hZip1 has a vesicular localization in COS-7 cells or in several epithelial cell lines, corresponding partially to the endoplasmic reticulum. Using anti-hZip1 antibodies, we confirmed the intracellular localization of the endogenous protein in PC-3, a prostate cancer cell line.

A role for frequenin, a Ca2+-binding protein, as a regulator of Kv4 K+-currents.

Frequenin, a Ca(2+)-binding protein, has previously been implicated in the regulation of neurotransmission, possibly by affecting ion channel function. Here, we provide direct evidence that frequenin is a potent and specific modulator of Kv4 channels, the principal molecular components of subthreshold activating A-type K(+) currents. Frequenin increases Kv4.2 current amplitudes (partly by enhancing surface expression of Kv4.2 proteins) and it slows the inactivation time course in a Ca(2+)-dependent manner. It also accelerates recovery from inactivation. Closely related Ca(2+)-binding proteins, such as neurocalcin and visinin-like protein (VILIP)-1 have no such effects. Specificity for Kv4 currents is suggested because frequenin does not modulate Kv1.4 or Kv3.4 currents. Frequenin has negligible effects on Kv4.1 current inactivation time course. By using chimeras made from Kv4.2 and Kv4.1 subunits, we determined that the differential effects of frequenin are mediated by means of the Kv4 N terminus. Immunohistochemical analysis demonstrates that frequenin and Kv4.2 channel proteins are coexpressed in similar neuronal populations and have overlapping subcellular localizations in brain. Coimmunoprecipitation experiments demonstrate that a physical interaction occurs between these two proteins in brain membranes. Together, our data provide strong support for the concept that frequenin may be an important Ca(2+)-sensitive regulatory component of native A-type K(+) currents.

Is the molecular composition of K(ATP) channels more complex than originally thought?

ATP-sensitive K+ (K(ATP)) channels are abundantly expressed in the heart and may be involved in the pathogenesis of myocardial ischemia. These channels are heteromultimeric, consisting of four pore-forming subunits (Kir6.1, Kir6.2) and four sulfonylurea receptor (SUR) subunits in an octameric assembly. Conventionally, the molecular composition of K(ATP) channels in cardiomyocytes and pancreatic beta -cells is thought to include the Kir6.2 subunit and either the SUR2A or SUR1 subunits, respectively. However, Kir6.1 mRNA is abundantly expressed in the heart, suggesting that Kir6.1 and Kir6.2 subunits may co-assemble to form functional heteromeric channel complexes. Here we provide two independent lines of evidence that heteromultimerization between Kir6.1 and Kir6.2 subunits is possible in the presence of SUR2A. We generated dominant negative Kir6 subunits by mutating the GFG residues in the channel pore to a series of alanine residues. The Kir6.1-AAA pore mutant subunit suppressed both wt-Kir6.1/SUR2A and wt-Kir6.2/SUR2A currents in transfected HEK293 cells. Similarly, the dominant negative action of Kir6.2-AAA does not discriminate between either of the wild-type subunits, suggesting an interaction between Kir6.1 and Kir6.2 subunits within the same channel complex. Biochemical data support this concept: immunoprecipitation with Kir6.1 antibodies also co-precipitates Kir6.2 subunits and conversely, immunoprecipitation with Kir6.2 antibodies co-precipitates Kir6.1 subunits. Collectively, our data provide direct electrophysiological and biochemical evidence for heteromultimeric assembly between Kir6.1 and Kir6.2. This paradigm has profound implications for understanding the properties of native K(ATP)channels in the heart and other tissues.

KT3.2 and KT3.3, two novel human two-pore K(+) channels closely related to TASK-1.

We report the cloning of human KT3.2 and KT3.3 new members of the two-pore K(+) channel (KT) family. Based on amino acid sequence and phylogenetic analysis, KT3.2, KT3.3, and TASK-1 constitute a subfamily within the KT channel mammalian family. When Xenopus oocytes were injected with KT3.2 cRNA, the resting membrane potential was brought close to the potassium equilibrium potential. At low extracellular K(+) concentrations, two-electrode voltage-clamp recordings revealed the expression of predominantly outward currents. With high extracellular K(+) (98 mM), the current-voltage relationship exhibited weak outward rectification. Measurement of reversal potentials at different [K(+)](o) revealed a slope of 48 mV per 10-fold change in K(+) concentration as expected for a K(+)-selective channel. Unlike TASK-1, which is highly sensitive to changes of pH in the physiological range, KT3.2 currents were relatively insensitive to changes in intracellular or extracellular pH within this range due to a shift in the pH dependency of KT3.2 of 1 pH unit in the acidic direction. On the other hand, the phorbol ester phorbol 12-myristate 13-acetate (PMA), which does not affect TASK-1, produces strong inhibition of KT3.2 currents. Human KT3.2 mRNA expression was most prevalent in the cerebellum. In rat, KT3.2 is exclusively expressed in the brain, but it has a wide distribution within this organ. High levels of expression were found in the cerebellum, medulla, and thalamic nuclei. The hippocampus has a nonhomogeneous distribution, expressing at highest levels in the lateral posterior and inferior portions. Medium expression levels were found in neocortex. The KT3.2 gene is located at chromosome 8q24 1-3, and the KT3.3 gene maps to chromosome 20q13.1.

Different effects of the Ca(2+)-binding protein, KChIP1, on two Kv4 subfamily members, Kv4.1 and Kv4.2.

The Ca(2+)-binding protein, K(+) channel-interacting protein 1 (KChIP1), modulates Kv4 channels. We show here that KChIP1 affects Kv4.1 and Kv4.2 currents differently. KChIP1 slows Kv4.2 inactivation but accelerates the Kv4.1 inactivation time course. Kv4.2 activation is shifted in a hyperpolarizing direction, whereas a depolarizing shift occurs for Kv4.1. On the other hand, KChIP1 increases the current amplitudes and accelerates recovery from inactivation of both currents. An involvement of the Kv4 N-terminus in these differential effects is demonstrated using chimeras of Kv4.2 and Kv4.1. These results reveal a novel interaction of KChIP1 with these two Kv4 members. This represents a mechanism to further increase the functional diversity of K(+) channels.

Importance of anemia and transferrin levels in the regulation of intestinal iron absorption in hypotransferrinemic mice.

The hypotransferrinemic mouse (trf (hpx)) is a mutant strain exhibiting transferrin deficiency, marked anemia, hyperabsorption of iron, and elevated hepatic iron stores. We set out to investigate the relative roles of anemia and of transferrin in the malregulation of intestinal iron absorption in these animals. Transfusion of erythrocytes obtained from littermate controls increased hemoglobin levels and reduced reticulocyte counts in recipient animals. Although mucosal to carcass (59)Fe transfer was reduced, total duodenal iron uptake was not significantly affected. Iron absorption in homozygotes, in contrast to littermate controls, was not reduced by hyperoxia. Mouse transferrin injections, in the short term, increased delivery of iron to the marrow and raised hemoglobin levels. Although mucosal transfer and total iron uptake were reduced at the higher transferrin doses, total uptake was still higher than in controls. Daily injections of mouse/human transferrin for 3 weeks from weaning, normalized hemoglobin values, and markedly reduced liver iron and intestinal iron absorption values in trf (hpx) animals. When such daily-injected mice were left for a week to allow transferrin clearance, iron absorption values were significantly enhanced; hemoglobin or hepatic iron levels were, however, not significantly altered. These data indicate that hyperabsorption of iron in trf (hpx) mice is not solely because of the anemia; transferrin levels per se do affect iron absorption, possibly via a direct effect on the intestinal mucosa.

Iron proteins of duodenal enterocytes isolated from mice with genetically and experimentally altered iron metabolism.

The molecular basis for the control of iron absorption by the duodenum remains unknown: however, ferritin (Ft) and the iron status of enterocytes have been suggested as regulatory factors. We determined the iron and Ft status of duodenal enterocytes from mice with hypotransferrinaemia, a genetic defect leading to greatly enhanced iron absorption, and for comparison we also investigated mice with experimentally-altered iron absorption. Duodenal enterocytes were isolated and analysed for Ft and non-haem iron content and for transferrin binding (as a measure of transferrin receptor activity). RNA was extracted from the duodenal mucosa and examined for transferrin receptor and H- and L-Ft mRNA levels by Northern hybridization analysis. Ft levels were elevated in enterocytes of hypotransferrinaemic mice, similar to that seen in iron dextran-injected mice of the CD1-strain. Enterocyte Ft levels were reduced in mice fed a diet diminished in iron, but unchanged in hypoxic mice enterocytes. Enterocytes of hypotransferrinaemic mice had normal non-haem iron levels and transferrin binding; however, enterocytes from CD-1 mice fed a low iron diet had increased transferrin binding and a decreased non-haem iron content. Duodenal mRNA levels for transferrin receptor and H-Ft were unchanged in hypotransferrinaemic mice, whereas L-Ft was increased. We conclude from the Ft and non-haem iron contents and transferrin binding that duodenal enterocytes from hypotransferrinaemic mice are not simply iron deficient, leading to increased expression of iron carriers proteins. Duodenal iron absorption can be enhanced in mice even when enterocyte Ft levels are raised or unchanged, suggesting that iron absorption is regulated by developmentally programmed expression of iron transporters by enterocytes.

Molecular diversity of K+ channels.

K+ channel principal subunits are by far the largest and most diverse of the ion channels. This diversity originates partly from the large number of genes coding for K+ channel principal subunits, but also from other processes such as alternative splicing, generating multiple mRNA transcripts from a single gene, heteromeric assembly of different principal subunits, as well as possible RNA editing and posttranslational modifications. In this chapter, we attempt to give an overview (mostly in tabular format) of the different genes coding for K+ channel principal and accessory subunits and their genealogical relationships. We discuss the possible correlation of different principal subunits with native K+ channels, the biophysical and pharmacological properties of channels formed when principal subunits are expressed in heterologous expression systems, and their patterns of tissue expression. In addition, we devote a section to describing how diversity of K+ channels can be conferred by heteromultimer formation, accessory subunits, alternative splicing, RNA editing and posttranslational modifications. We trust that this collection of facts will be of use to those attempting to compare the properties of new subunits to the properties of others already known or to those interested in a comparison between native channels and cloned candidates.

The ferric-reducing activity of duodenal brush-border membrane vesicles is associated with a b-type haem.

Rabbit brush-border membrane vesicles possess ferricyanide reducing activity. This activity is preferentially dependent on NADH as reductant, and can be stimulated by the addition of FMN. The latency of activity observed following vesicle solubilisation suggests that the responsible component is transmembranous, and partially sequestered on the inner-face of the vesicles prior to full solubilisation. Subsequent increases in detergent concentration (> 0.3% w/v lauryl maltoside) were found to be inhibitory. Ferricyanide reducing activity was effectively inhibited by the sulphydryl modifying reagents N-ethyl malemide and p-chloromercuribenzoate, but not by the flavin analogue diphenylene iodonium. The ferric-reducing activity co-purified with a b-type haem when applied to Sephacryl S-200 columns. The putative cytochrome was found to be immunologically distinct from neutrophil cytochrome b558.