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The aim of this study was to assess and to compare the role of HFE polymorphisms and other genetic factors in variation in iron stores. Blood samples were obtained from 3,375 adult male and female twins (age range 29-82 years) recruited from the Australian Twin Registry. There were 1,233 complete pairs (562 monozygotic and 571 dizygotic twins). Serum iron, transferrin, transferrin saturation with iron, and ferritin were measured, and the HFE C282Y and H63D genotypes were determined. The frequency of the C282Y allele was.072, and that of the H63D allele was.141. Significant sources of variation in the indices of iron status included age, sex, age-sex interaction, body-mass index, and both the C282Y and H63D genotypes. The iron, transferrin, and saturation values of CC and CY subjects differed significantly, but the ferritin values did not. After correction for age and body-mass index, 23% and 31% of the variance in iron, 66% and 49% of the variance in transferrin, 33% and 47% of the variance in transferrin saturation, and 47% and 47% of the variance in ferritin could be explained by additive genetic factors, for men and women, respectively. HFE C282Y and H63D variation accounted for <5% of the corrected phenotypic variance, except for saturation (12% in women and 5% in men). We conclude that HFE CY and HD heterozygotes differ in iron status from the CC and HH homozygotes and that serum transferrin saturation is more affected than is serum ferritin. There are highly significant effects of other as-yet-unidentified genes on iron stores, in addition to HFE genotype.

Puromycin, an analog of the 3' end of aminoacyl-tRNA, causes premature termination of translation by being linked non-specifically to growing polypeptide chains. Here we report the interesting phenomenon that puromycin acting as a non-inhibitor at very low concentration (e.g. 0.04 microM) can bond only to full-length protein at the C-terminus. This was proved by using a carboxypeptidase digestion assay of the products obtained by Escherichia coli cell-free translation of human tau 4 repeat (tau4R) mRNA in the presence of low concentrations of puromycin or its derivatives. The tau4R mRNA was modified to code for three C-terminal methionines, which were radioactively labeled, followed by a stop codon. The translation products could not be digested by carboxy-peptidase if puromycin or a derivative was present at the C-terminus of full-length tau4R. Puromycin and its derivatives at 0. 04-1.0 microM bonded to 7-21% of full-length tau4R, depending on the ability to act as acceptor substrates. Furthermore, the bonding efficiency of a puromycin derivative to tau4R was decreased by addition of release factors. These results suggest that puromycin and its derivatives at concentrations lower than those able to compete effectively with aminoacyl-tRNA can bond specifically to full-length protein at a stop codon. This specific bonding of puromycin to full-length protein should be useful for in vitro selection of proteins and for in vitro and in vivo C-terminal end protein labeling.

Although the Drosophila melanogaster erect wing (ewg) gene is broadly transcribed in adults, an unusual posttranscriptional regulation involving alternative and inefficient splicing generates a 116-kDa EWG protein in neurons, while protein expression elsewhere or of other isoforms is below detection at this stage. This posttranscriptional control is important, as broad expression of EWG can be lethal. In this paper, we show that ELAV, a neuron-specific RNA binding protein, is necessary to regulate EWG protein expression in ELAV-null eye imaginal disc clones and that ELAV is sufficient for EWG expression in wing disc imaginal tissue after ectopic expression. Further, analysis of EWG expression elicited from intron-containing genomic transgenes and cDNA minitransgenes in ELAV-deficient eye discs shows that this regulation is dependent on the presence of ewg introns. Analyses of the ewg splicing patterns in wild-type and ELAV-deficient eye imaginal discs and in wild-type and ectopic ELAV-expressing wing imaginal discs, show that certain neuronal splice isoforms correspond to ELAV levels. The data presented in this paper are consistent with a mechanism in which ELAV increases the splicing efficiency of ewg transcripts in alternatively spliced regions rather than with a mechanism in which stability of specific splice forms is enhanced by ELAV. Additionally, we report that ELAV promotes a neuron-enriched splice isoform of Drosophila armadillo transcript. ELAV, however, is not involved in all neuron-enriched splice events.

<h4>Background</h4>Ulcerative colitis is associated with an increased risk of colorectal cancer above that of the normal population. The relative risk correlates with the extent and duration of the disease but the genetic basis of ulcerative colitis associated cancer risk is not known.<h4>Aims</h4>To assess the prevalence of microsatellite instability and mismatch repair gene abnormalities in ulcerative colitis associated colorectal cancer.<h4>Patients</h4>Forty six patients with colorectal cancer, with a previous histological diagnosis of ulcerative colitis.<h4>Methods</h4>The frequency of microsatellite instability and/or immunohistochemical expression of hMSH2 and hMLH1 was assessed. Thirty three cases were investigated using both approaches.<h4>Results</h4>Although 6/41 (14.6%) cases showed microsatellite instability at one or more markers, only one case (2. 4%) exhibited high level instability (at least two markers affected). Of 38 cases which were assessed using antibodies against hMSH2 and hMLH1, only one case (2.6%) showed loss of expression. This case, which showed loss of hMSH2 expression, was the same case which exhibited high level microsatellite instability. The 33 cases which were investigated using both approaches showed that loss of expression of either hMSH2 or hMLH1 was not seen in any case which exhibited microsatellite instability in no more than one marker.<h4>Conclusions</h4>This study suggests that both high level microsatellite instability and loss of expression of hMSH2/hMLH1 are infrequent events in ulcerative colitis associated colorectal cancers. Low level microsatellite instability was not associated with loss of expression of either hMSH2 or hMLH1.

<h4>Background</h4>Most patients with haemochromatosis have mutations of the HFE gene. However, the risk to people with HFE mutations of developing disease manifestations of haemochromatosis is not known.<h4>Aims</h4>To determine the risk of developing cirrhosis and liver cancer in individuals with HFE mutations in a population where few people were being treated for haemochromatosis.<h4>Methods</h4>215 archive biopsy specimens of liver cancer (n=34) and cirrhosis (n=190) were retrieved from histology archives. Blood samples from 1000 individuals from the normal population were also collected. DNA was extracted from the biopsy specimens and exons 2 and 4 of the HFE gene were amplified using polymerase chain reaction. The products were analysed for the C282Y (845A) and H63D (187G) mutations.<h4>Results</h4>Three (8.8%) patients from the liver cancer group were homozygous for the C282Y mutation. Five (2.6%) patients from the cirrhosis group were homozygous for the C282Y mutation. One case fell in both the liver cancer and cirrhosis groups. C282Y homozygosity was thus significantly more frequent in both groups than in the normal population. These 215 cases are representative of a population of about 250 000 over 20 years. During this period we estimate that about 260 births or deaths of C282Y homozygous individuals occurred within this population.<h4>Conclusions</h4>A diagnosis of liver cancer or cirrhosis is rare in the lifetime of individuals from this population who are homozygous for the C282Y mutation (2.5%; upper 95% confidence interval (CI) = 8%). Similarly liver disease is rare among C282Y/H63D compound heterozygotes (1%; upper 95% CI = 3.5%).

<h4>Background</h4>C282Y hereditary haemochromatosis is an appropriate condition for population screening. Transferrin saturation, the best screening test to date, is relatively expensive, labour intensive, and cannot be automated. Unsaturated iron binding capacity is a surrogate marker of transferrin saturation and its measurement can be automated.<h4>Aims</h4>To evaluate a screening strategy for C282Y hereditary haemochromatosis in a tertiary hospital environment based on unsaturated iron binding capacity as the initial screening test.<h4>Methods</h4>Measurement of unsaturated iron binding capacity was adapted to the main laboratory analyser. An unsaturated iron binding capacity of less than 30 micromol/l was identified as an appropriate decision point and 5182 consecutive subjects were screened over 28 consecutive days.<h4>Results</h4>Of those screened, 697 had an unsaturated iron binding capacity less than 30 micromol/l. Of these, transferrin saturation was greater than 40% in 294. A total of 227 were able to be genotyped for the C282Y mutation. Nine subjects homozygous for C282Y were identified. Based on full cost recovery, affected persons were identified at a cost of Aus$2268.77 per case (approximately US$1496).<h4>Conclusion</h4>Automated measurement of unsaturated iron binding capacity enables a cost effective, large scale population screening programme for C282Y hereditary haemochromatosis to be developed.

The serotonin transporter (5-HTT) regulates serotonergic neurotransmission by determining the magnitude and duration of serotonergic responses. We have recently described a polymorphism in the 5-HTT gene promoter (5-HTTLPR) which influences the function of the 5-HTT and is associated with several psychiatric disorders. Immortalized B lymphocytes express the 5-HTT, and a B lymphocyte line has been shown to express the receptor for brain-derived neurotrophic factor, trkB. Since brain-derived neurotrophic factor (BDNF) is a specific growth and differentiation factor for serotonergic neurons, we assessed whether BDNF is able to modulate 5-HTT function in B lymphoblasts. Nerve growth factor (NGF), another neurotrophin which acts via the trkA receptor, was also studied. Eight immortalized B lymphoblast lines were generated and genotyped for the 5-HTTLPR. After treatment with BDNF or NGF, 5-HT uptake and proliferation of the cell lines were assessed. Two of the B cell lines showed a dose-dependent reduction of 5-HT uptake after exposure to BDNF. Both of these cell lines were homozygous for the long allele of the 5-HTTLPR. NGF did not influence 5-HT uptake or cellular proliferation in any of the cell lines. Thus, BDNF but not NGF may influence 5-HT uptake in some B lymphocytes. The fact that regulation of the 5-HTT was observed preferentially in cells of the long/long genotype indicates that presence of a short allele confers reduced regulatory capacity on the 5-HTT. In conclusion, B lymphoblasts represent a practical model for functional regulation of the 5-HTT by neurotrophins in serotonergic neurons.

Optic nerve formation requires precise retinal ganglion cell (RGC) axon pathfinding within the retina to the optic disc, the molecular basis of which is not well understood. At CNS targets, interactions between Eph receptor tyrosine kinases on RGC axons and ephrin ligands on target cells have been implicated in formation of topographic maps. However, studies in chick and mouse have shown that both Eph receptors and ephrins are also expressed within the retina itself, raising the possibility that this receptor-ligand family mediates aspects of retinal development. Here, we more fully document the presence of specific EphB receptors and B-ephrins in embryonic mouse retina and provide evidence that EphB receptors are involved in RGC axon pathfinding to the optic disc. We find that as RGC axons begin this pathfinding process, EphB receptors are uniformly expressed along the dorsal-ventral retinal axis. This is in contrast to the previously reported high ventral-low dorsal gradient of EphB receptors later in development when RGC axons map to CNS targets. We show that mice lacking both EphB2 and EphB3 receptor tyrosine kinases, but not each alone, exhibit increased frequency of RGC axon guidance errors to the optic disc. In these animals, major aspects of retinal development and cellular organization appear normal, as do the expression of other RGC guidance cues netrin, DCC, and L1. Unexpectedly, errors occur in dorsal but not ventral retina despite early uniform or later high ventral expression of EphB2 and EphB3. Furthermore, embryos lacking EphB3 and the kinase domain of EphB2 do not show increased errors, consistent with a guidance role for the EphB2 extracellular domain. Thus, while Eph kinase function is involved in RGC axon mapping in the brain, RGC axon pathfinding within the retina is partially mediated by EphB receptors acting in a kinase-independent manner.

The light response of fly photoreceptor cells is modulated by changes in free Ca(2+) concentration. Fly phototransduction and most processes regulating it take place in or very close to the rhabdomere. We therefore measured the kinetics and the absolute values of the free Ca(2+) concentration in the rhabdomere of fly photoreceptor cells in vivo by making use of the natural optics of the fly's eye. We show that Ca(2+) flowing into the rhabdomere after light stimulation of dark-adapted cells causes fast Ca(2+) transients that reach peak values higher than 200 microM in <20 msec. Approximately 500 msec later, the free Ca(2+) concentration has declined again to approximately 20 microM. The duration of the Ca(2+) transients becomes still shorter, and their size reduced, when the photoreceptor cell is light-adapted. This reduction in duration and size of the Ca(2+) transients is graded with the intensity of the adapting light. The kinetics and absolute values of the free calcium concentration found to occur in the rhabdomere are suitable to mediate the fast feedback signals known to act on the fly phototransduction cascade.

Neuroactive steroids are synthesized de novo in brain, yet their physiological significance remains elusive. We provide biochemical, electrophysiological, and behavioral evidence that several specific actions of alcohol (ethanol) are mediated by the neurosteroid 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-THP; allopregnanolone). Systemic alcohol administration elevates 3alpha, 5alpha-THP levels in the cerebral cortex to pharmacologically relevant concentrations. The elevation of 3alpha,5alpha-THP is dose- and time-dependent. Furthermore, there is a significant correlation between 3alpha,5alpha-THP levels in cerebral cortex and the hypnotic effect of ethanol. Blockade of de novo biosynthesis of 5alpha-reduced steroids using the 5alpha-reductase inhibitor finasteride prevents several effects of ethanol. Pretreatment with finasteride causes no changes in baseline bicuculline-induced seizure threshold but reverses the anticonvulsant effect of ethanol. Finasteride pretreatment also reverses ethanol inhibition of spontaneous neural activity in medial septal/diagonal band of Broca neurons while having no direct effect on spontaneous firing rates. Thus, elevation of 3alpha,5alpha-THP levels by acute ethanol administration represents a novel mechanism of ethanol action as well as an important modulatory role for neurosteroids in the CNS.

SOX proteins form a large family of transcription factors related by a DNA-binding domain known as the HMG box. Some 30 Sox genes have been identified in mammals and orthologues have been found in a wide range of other metazoans. Sox genes are highly conserved and are known to play important roles in embryonic development, including roles in gonadal, central nervous system, neural crest and skeletal development. Several SOX genes have been implicated in human congenital diseases. We report here the isolation of Sox8 and its characterisation in mice and humans. This gene has a remarkably similar primary structure and genomic organisation to the campomelic dysplasia gene SOX9 and the Waardenburg-Shah syndrome gene SOX10. SOX8 protein is able to bind to canonical SOX target DNA sequences and activate transcription in vitro through two separate trans -activation regions. Further, Sox8 is expressed in the central nervous system, limbs, kidneys, gonads and craniofacial structures during mouse embryo development. Sox8 maps to the t complex on mouse chromosome 17 and to human chromosome 16p13.3, a region associated with the microphthalmia-cataract syndrome CATM and the alpha-thalassemia/mental retardation syndrome ATR-16.

H1 histones bind to linker DNA and nucleosome core particles and facilitate the folding of chromatin into a more compact structure. Mammals contain seven nonallelic subtypes of H1, including testis-specific subtype H1t, which varies considerably in primary sequence from the other H1 subtypes. H1t is found only in pachytene spermatocytes and early, haploid spermatids, constituting as much as 55% of the linker histone associated with chromatin in these cell types. To investigate the role of H1t in spermatogenesis, we disrupted the H1t gene by homologous recombination in mouse embryonic stem cells. Mice homozygous for the mutation and completely lacking H1t protein in their germ cells were fertile and showed no detectable defect in spermatogenesis. Chromatin from H1t-deficient germ cells had a normal ratio of H1 to nucleosomes, indicating that other H1 subtypes are deposited in chromatin in place of H1t and presumably compensate for most or all H1t functions. The results indicate that despite the unique primary structure and regulated synthesis of H1t, it is not essential for proper development of mature, functional sperm.

Inherited and acquired factors have been implicated in the pathogenesis of porphyria cutanea tarda (PCT), a disorder characterized by a photosensitive dermatosis and hepatic siderosis. This study, comprising 108 patients with PCT, was intended to define the role of hemochromatosis gene (HFE) mutations in the expression of PCT and to determine the contribution of acquired factors including alcohol, hepatitis C virus (HCV), and estrogen. The 2 known HFE mutations, cysteine 282 tyrosine (Cys282Tyr) and histidine 63 asparagine (His63Asp), were detected by polymerase chain reaction, and anti-HCV immunoglobulin G was detected serologically. Liver biopsies were graded for iron content, inflammation, and fibrosis. Estimates of alcohol and estrogen use were based on a questionnaire. Of the PCT patients tested, 19% were homozygous for the Cys282Tyr mutation; controls were equal to 0.5%. The compound heterozygous genotype was detected in 7% of the PCT patients; controls were less than 1%. The transferrin saturation, serum ferritin, and liver iron burden of all PCT patients were higher than those of nonporphyric controls. The highest values were found in PCT patients homozygous for the Cys282Tyr mutation. Of the patients studied, 59% were HCV positive (compared with 1.8% of the population), and 46% consumed more than 70 g of alcohol daily. Of the female patients, 63% were ingesting estrogens. Hepatic damage was most marked in patients with the Cys282Tyr/Cys282Tyr genotype who had HCV and drank heavily. Homozygosity for the Cys282Tyr mutation and HCV are the greatest risk factors for expression of PCT, and in most patients, more than 1 risk factor was identified. It was common for patients with HCV to consume alcohol. Patients with PCT should be screened for HFE mutations and for HCV. (Blood. 2000;95:1565-1571)

An increased sensitivity to glucose was observed in islets pre-exposed for 1 h to glibenclamide (0.1 micromol 1(-1)), but not to tolbutamide (100 micromol l(-1)), as indicated by a shift to the left of the dose-response curve (EC(50) at 5.8+/-0.3 mmol l(-1) glucose vs 10.6+/-0.8 in control islets; n=11, P<0.005). According to this secretory pattern also glucose utilization at 2.5 and 5.0 mmol l(-1) glucose was higher in islets exposed to glibenclamide. Since binding to mitochondria results in an increased enzyme activity, we measured hexokinase (HK) and glucokinase (GK) activity both in a cytosolic and in a mitochondrion-enriched fractions. Cytosolic hexokinase activity was similar in islets exposed to glibenclamide and in control islets but mitochondrial hexokinase activity was significantly increased after exposure to glibenclamide (124+/-7 vs 51+/-9 nmol microgram prot(-1) 90 min(-1), P<0.01), with no change in the enzyme protein content. In contrast, glucokinase activity in the two groups of islets was similar. When in islets < exposed to glibenclamide hexokinase binding to mitochondria was inhibited by the addition of 20 nmol l(-1) dicyclohexylcarbodiimide (DCC), no increase of glucose sensitivity was observed (EC(50) 10.9+/-1.3 mmol l(-1) glucose, n=3, similar to that of control islets). These data indicate that a 1 h exposure to glibenclamide causes the beta cell to become more sensitive to glucose. This increased sensitivity is associated with (and may be due to) an increased hexokinase activity, in particular the mitochondrial-bound, more active, form. This mechanism may contribute to the hypoglycemic action of this drug.

Protein aggregates are a neuropathological feature of Huntington's disease and Parkinson's disease. Mutant huntingtin exon 1 with 72 CAG repeats fused to enhanced green fluorescent protein (EGFP) forms hyperfluorescent inclusions in PC12 cells. Inclusion formation is enhanced in cells co-transfected with EGFP-huntingtin-(CAG)(72) and alpha-synuclein, a major component of Parkinson's disease aggregates. However, alpha-synuclein does not form aggregates by itself, nor does it appear in huntingtin inclusions in vitro.

A set of radioiodinatable phosphatidylcholines (PCs) derivatized with the Bolton-Hunter reagent (BHPCs) was synthesized to probe the substrate recognition and activity of phospholipases. A common feature of this series is the presence of a bulky 4-hydroxyphenyl group at the end of the fatty acyl chain attached to position sn-2. The distance between the end group and the glycerol backbone was varied by changing the length of the intervening fatty acyl chain (3-25 atoms). Except for the shortest, this chain includes at least one amide linkage. The usefulness of this series of substrates as a molecular ruler was tested by measuring the hydrolytic activities of Naja naja naja phospholipase A(2) (PLA(2)) and Bacillus cereus phospholipase C (PLC) in Triton X-100 micelles. The activity of PLA(2) proved to be highly dependent on the length of the fatty acyl chain linker, the shorter compounds (3-10 atoms) being very poor substrates. In contrast, the PLC activity profile exhibited much less discrimination. In both cases, PCs with 16-21 atom chains at position sn-2 yielded optimal activity. We interpret these findings in terms of fatty acyl chain length-related steric hindrance caused by the terminal aromatic group, affecting the activity of PLA(2) and, to a smaller extent, that of PLC. This notion agrees with the more extended recognition of aliphatic chains inside the narrow channel leading to the catalytic site in the former case. Molecular models of these substrates bound to PLA(2) were built on the basis of the crystallographic structure of Naja naja atra PLA(2) complexed with a phospholipid analogue. Docking of these substrates necessarily requires the intrusion of the bulky 4-hydroxyphenyl group inside the binding pocket and also the failure of the amide group to form hydrogen bonds inside the hydrophobic substrate channel.

Major strides have been taken in the regulation of lead intoxication in the general population, but studies using genetic markers of susceptibility to environmental toxicants raise the question of whether genes can make certain individuals more vulnerable to environmental toxins such as lead. At least three polymorphic genes have been identified that potentially can influence the bioaccumulation and toxicokinetics of lead in humans. The first gene to be discussed in this review is the gene coding for delta-aminolevulinic acid dehydratase (ALAD), an enzyme of heme biosynthesis, that exists in two polymorphic forms. The resulting isozymes have been shown to affect the blood and bone lead levels in human populations. The effects of ALAD in lead intoxication have also been studied in laboratory mice that differ in the genetic dose for this enzyme. The second gene reviewed here is the vitamin D receptor (VDR) gene. The VDR is involved in calcium absorption through the gut and into calcium-rich tissues such as bone. Recent findings suggest that VDR polymorphism may influence the accumulation of lead in bone. Finally, the third gene to be discussed here that may influence the absorption of lead is the hemochromatosis gene coding for the HFE protein. The presence of mutations in the HFE gene leads to hemochromatosis in homozygotic individuals. Because of the associations between iron and lead transport, it is possible that polymorphisms in the HFE gene may also influence the absorption of lead, but this has not yet been studied. More studies will be needed to define the role of these genes in lead intoxication.

Members of the family Picornaviridae are the most common viruses infecting humans, and species in several genera also infect a wide variety of other mammals. Picornaviruses have traditionally been classified by antigenic type, based on a serum neutralization assay. However, this method is time-consuming and labor-intensive, is sensitive to virus aggregation and antigenic variation, and requires a large number of antisera to identify all serotypes, even when antiserum pools are used. We developed generic reverse transcription (RT)-PCR primers that will amplify all human enterovirus serotypes, as well as many rhinoviruses and other picornaviruses, and used RT-PCR amplification of the VP1 gene and amplicon sequencing to identify enteroviruses that were refractory to typing by neutralization with pooled antisera. Enterovirus serotypes determined by sequencing were confirmed by neutralization with monospecific antisera. Of 55 isolates tested, 49 were of known enterovirus serotypes, two were rhinoviruses, and four were clearly picornaviruses but did not match any known picornavirus sequence. All four untyped picornaviruses were closely related to one another in sequence, suggesting that they are of the same serotype. RT-PCR, coupled with amplicon sequencing, is a simple and rapid method for the typing and classification of picornaviruses and may lead to the identification of many new picornavirus serotypes.

Telomerase plays a crucial role in telomere maintenance in vivo. To understand telomerase regulation, we have been characterizing components of the enzyme. To date several components of the mammalian telomerase holoenzyme have been identified: the essential RNA component (human telomerase RNA [hTR]), the catalytic subunit human telomerase reverse transcriptase (hTERT), and telomerase-associated protein 1. Here we describe the identification of two new proteins that interact with hTR: hStau and L22. Antisera against both proteins immunoprecipitated hTR, hTERT, and telomerase activity from cell extracts, suggesting that the proteins are associated with telomerase. Both proteins localized to the nucleolus and cytoplasm. Although these proteins are associated with telomerase, we found no evidence of their association with each other or with telomerase-associated protein 1. Both hStau and L22 are more abundant than TERT. This, together with their localization, suggests that they may be associated with other ribonucleoprotein complexes in cells. We propose that these two hTR-associated proteins may play a role in hTR processing, telomerase assembly, or localization in vivo.

The netrin-1 protein elicits spinal commissural axon outgrowth and turning in vitro and has been shown to be required for commissural axon guidance in vivo in the developing spinal cord. Biochemical observations made during the purification of netrin-1 suggest that this ligand and its receptor, DCC, may not function alone in directing commissural axon guidance. Recombinant netrin-1 protein is approximately 10 times more active in eliciting axon outgrowth from embryonic day (E) 13 rat dorsal spinal cord explants than from E11 rat dorsal spinal cord explants (Serafini, T., Kennedy, T. E., Galko, M. J., Mirzayan, C., Jessell, T. M., and Tessier-Lavigne, M. (1994) Cell 78, 409-424) even though the starting material for the netrin purification, a high salt extract of E10 chicken brain membranes, is equally active on E13 and E11 explants. We previously reported an activity termed netrin-synergizing activity (NSA) that can potentiate the outgrowth-promoting activity of netrin-1 on E11 explants (Serafini et al.). Here we report a biochemical characterization of NSA in netrin-depleted high salt extracts of E10 chicken brain membranes. We provide evidence that NSA is composed of a denaturation-resistant basic protein(s) in the 25-35-kDa size range. We also provide evidence that the activity may be heterogeneous, splitting into three species that may be distinct or related. The results reported here should facilitate purification of this activity from a more abundant source or identification of the activity based on similarity to known proteins that share its distinctive biochemical properties.

We evaluated a short, prepubertal 13.9 year-old boy with Blackfan-Diamond anemia and significant liver iron stores due to multiple blood transfusions and found him to have several endocrine abnormalities, including hypothyroidism, hypoparathyroidism, primary and secondary hypogonadism and IGF-I insufficiency. Growth velocity was poor despite treatment with levothyroxine, calcitriol, calcium and aggressive therapy with chelating agents. After 25 months of treatment with rhGH his growth velocity, height for age and PAH increased significantly, suggesting a degree of sensitivity to GH despite his liver damage.

Herein we describe a recombinant factor X (fX) with a single substitution at position 347 (fXR347N). Activated fXR347N had a reduced affinity for factor Va (fVa), although the catalytic impact of fVa binding remained intact. The mutation was selective as demonstrated by normal activation and inhibition, except in the presence of subsaturating heparin where the rate of inhibition by antithrombin III (ATIII) was 15% of normal. The reactivity of fXaR347N toward prothrombin was equivalent to wild-type fXa (fXaWT) in the absence of fVa and phospholipid. Addition (without phospholipid) of fVa dramatically increased the catalytic efficiency of fXaWT toward prothrombin but had a negligible effect on fXaR347N. On addition of phosphatidylcholine:phosphatidylserine (PC:PS, 3:1) vesicles, fXaR347Ndisplayed an increased catalytic activity in response to fVa, but the apparent affinity for fVa on the phospholipid surface was 5-20-fold lower than that of fXaWT. On an activated platelet surface, however, fXaWT and fXaR347N activated prothrombin similarly. In a competitive binding assay that measures the displacement of radiolabeled fXa from fVa on a phospholipid surface, fXaR347N was approximately 10-fold less effective than fXaWT. Substitution of fXa at position 347 selectively attenuates the interaction between fXa and fVa without affecting its catalytic activity.

<h4>Background</h4>Numerous findings indicate alterations in brain serotonin systems in seasonal affective disorder (SAD). [(123)I]-2-beta-carbomethoxy-3-beta-(4-iodophenyl)-tropane ([(123)I]-beta-CIT) labels serotonin transporters (5-HTTs) in the midbrain. We performed a [(123)I]-beta-CIT single photon emission computer tomography (SPECT) study under the hypothesis of lower [(123)I]-beta-CIT binding reflecting reduced central 5-HTT availability in depressed SAD patients.<h4>Methods</h4>Depressed SAD patients and healthy control subjects were investigated using [(123)I]-beta-CIT SPECT 4 hours and again 24 hours after tracer injection. Subjects had either never used psychotropic medication or had been drug-free for at least 6 months prior to the investigation. Specific-to-nondisplaceable partition coefficient (V(3)") was calculated for the thalamus-hypothalamus and the midbrain-pons; the cerebellum served as a reference region.<h4>Results</h4>Patients showed a reduction in V(3)" in thalamus-hypothalamus (2.41+/-0.3 vs. 2.84+/-0.4; p = .026) 24 hours post tracer injection (p.i.). No difference between patients and control subjects was found in midbrain-pons (1.31+/-0.2 vs. 1.42+/-0.2; p = .39). No differences were detected in the SPECT acquisitions 4 hours p.i.<h4>Conclusions</h4>Depressed SAD patients showed lower specific-to-nondisplaceable [(123)I]-beta-CIT binding in the region of interest (ROI) thalamus-hypothalamus. The small size of the midbrain-pons ROI may have contributed to the failure to show a difference in this ROI as well. Similar to reduced midbrain 5-HTT availability in nonseasonal depression, depression in SAD seems to be associated with reduced 5-HTT availability to the thalamus-hypothalamus.

Low temperature is an important environmental factor that has effects on all living organisms. Various low-temperature-inducible genes encode products that are essential for acclimation to low temperature, but low-temperature sensors and signal transducers have not been identified. However, systematic disruption of putative genes for histidine kinases and random mutagenesis of almost all the genes in the genome of the cyanobacterium Synechocystis sp. PCC 6803 have allowed us to identify two histidine kinases and a response regulator as components of the pathway for perception and transduction of low-temperature signals. Inactivation, by targeted mutagenesis, of the gene for each of the two histidine kinases and inactivation of the gene for the response regulator depressed the transcription of several lowtemperature-inducible genes.

Two brefeldin A (BFA)-inhibited guanine nucleotide-exchange proteins for ADP-ribosylation factors, 200-kDa BIG1 and 190-kDa BIG2, were copurified from bovine brain cytosol associated with >670-kDa macromolecular complexes. When observed by immunofluorescence in HeLa S3 and HepG2 cells, endogenous BIG1 and coexpressed BIG2 were distributed in a punctate pattern throughout the cytosol, and also concentrated in the perinuclear region, where endogenous BIG1 and BIG2 each partially colocalized with Golgi-specific 58K protein and gamma-adaptin. On Western blot analysis, both BIG1 and BIG2 were clearly more abundant in the cytosol than in the microsomal fractions. After density gradient centrifugation of a microsomal fraction, BIG1 and BIG2 were recovered in the same fraction as beta-COP, a marker for Golgi membranes. When cytosol from HeLa S3 cells was subjected to gel filtration and fractions were analyzed by Western blotting, the largest percentages of both BIG1 and BIG2 were detected in fractions containing proteins with a molecular mass of >670 kDa. Western blotting using anti-peptide antibodies specific for BIG1 or BIG2 demonstrated that approximately 70% of BIG2 was immunoprecipitated along with 100% of BIG1 by the anti-BIG1 IgG, and approximately 75% of BIG1 was coprecipitated with 100% of BIG2 by the anti-BIG2 IgG. All observations were consistent with the conclusion that significant fractions of BIG1 and BIG2 exist as components of the same macromolecular complexes in bovine brain cytosol and are similarly localized in cultured cells.

We previously demonstrated that chromosome 18 is frequently deleted in neuroblastoma. To further elucidate the role of chromosome 18 deletions in the development of neuroblastomas we examined 82 cases of neuroblastomas for allelic imbalance (AI) at 17 loci on chromosome 18 to define the common region of AI in neuroblastoma. AI at one or more loci on chromosome 18 was detected in 18/82 (22%) cases. AI on 18q was detected in 17/82 (21%) cases, whereas AI on 18p was detected in 4/82 (5%) cases. There was a distinct common region of AI at 18q21.1 between the D18S363 and D18S858 loci. In addition, cases 16 and 53, which did not show AI at 18q21.1, showed AI at 18pter-q12.3 between the D18S52 and D18S36 loci, indicating that another common region of AI may exist on chromosome 18. AI on chromosome 18 did not significantly correlate with any clinicopathological findings of patients with neuroblastoma. The common region of AI at 18q21.1 includes the DCC gene but not the Smad2 and Smad4 genes. However, our previous studies together with the present study indicated that the incidence of DCC mutation is much less than that of AI at 18q21.1 in neuroblastoma. These results indicate that novel tumour suppressor genes involved in the development of neuroblastoma are present at 18q21.1, and possibly at 18pter-q12.3.

Although iron can catalyze the production of free radicals involved in LDL lipid peroxidation, the contribution of iron overload to atherosclerosis remains controversial. The description of two mutations in the HFE gene (Cys282Tyr and His63Asp) related to hereditary hemochromatosis provides an opportunity to address the question of the association between iron overload and atherosclerosis. We investigated the prevalence of HFE mutations in 160 survivors of myocardial infarction with angiographically demonstrated severe coronary atherosclerotic disease, and in 160 age-, gender- and race-matched healthy control subjects. PCR amplification of genomic DNA followed by RsaI and BclI restriction enzyme digestion was used to determine the genotypes. The frequency of the mutant Cys282Tyr allele was identical among patients and controls (0.022; carrier frequency, 4.4%), whereas the mutant His63Asp allele had a frequency of 0.143 (carrier frequency, 27.5%) in controls and of 0.134 (carrier frequency, 24.5%) in patients. Compound heterozygotes were found in 2 of 160 (1.2%) controls and in 1 of 160 (0.6%) patients. The finding of a similar prevalence of Cys282Tyr and His63Asp mutations in the HFE gene among controls and patients with coronary atherothrombotic disease, indirectly questions the possibility of an association between hereditary hemochromatosis and atherosclerosis.

No abstract available.

The FHIT gene is located at a chromosomal site (3p14.2) which is commonly affected by translocations and deletions in human neoplasia. Although FHIT alterations at the DNA and RNA level are frequent in many types of tumours, the biological and clinical significance of these changes is not clear. In this study we aimed at correlating loss of Fhit protein expression with a large number of molecular genetic and clinical parameters in a well-characterized cohort of non-small-cell lung cancers (NSCLCs). Paraffin sections of 99 non-small-cell carcinomas were reacted with an anti-Fhit polyclonal antibody in a standard immunohistochemical reaction. Abnormal cases were characterized by complete loss of cytoplasmic Fhit staining. The Fhit staining results were then correlated with previously obtained clinical and molecular data. Fifty-two of 99 tumours lacked cytoplasmic Fhit staining, with preserved reactivity in adjacent normal cells. Lack of Fhit staining correlated with: loss of heterozygosity (LOH) at the FHIT 3p14.2 locus, but not at other loci on 3p; squamous histology; LOH at 17p13 and 5q but not with LOH at multiple other suspected tumour suppressor gene loci; and was inversely correlated with codon 12 mutations in K-ras. Fhit expression was not correlated overall with a variety of clinical parameters including survival and was not associated with abnormalities of immunohistochemical expression of p53, RB, and p16. All of these findings are consistent with loss of Fhit protein expression being as frequent an abnormality in lung cancer pathogenesis as are p53 and p16 protein abnormalities and that such loss occurs independently of the commitment to the metastatic state and of most other molecular abnormalities.

The synthesis and monoamine transporter activity of additional members of a series of 3,4-disubstituted piperidines (truncated analogues of the WIN series) are described. All members of this series were prepared from arecoline hydrobromide in optically pure form and were evaluated for their ability to inhibit high affinity uptake of dopamine (DA), serotonin (5-HT) and norepinephrine (NE) into rat brain nerve endings (synaptosomes). Most of the compounds prepared in this series are reasonably potent DAT inhibitors (K(i) values of 4-400 nM) and have selectivity for the 5-HT transporter relative to both the NE transporter (3-9-fold) and to the DAT ( approximately 25-fold). In the present series, (-)-methyl 1-methyl-4beta-(2-naphthyl)piperidine-3beta-carboxylate (6) was found to be the most potent piperidine-based ligand, exhibiting K(i)'s of 21 nM and 7.6 nM at the DAT and 5-HTT, respectively. While the 5-HTT activity of compound 6 is comparable to that of the antidepressant medication fluoxetine, it is less selective. As is apparent from the data presented, the naphthyl substituted piperidines 6-9, which differ in their stereochemistry, show different degrees of selectivity for the three transporters. Consistent with results reported in the literature for the tropane analogues, removal of the methyl group from the nitrogen atom of 9 leads to a further enhancement in 5-HTT activity. To examine the in vivo effects of these piperidines, preliminary behavioral screening was carried out on piperidine 14. Despite its 2.5-fold greater DAT activity compared to cocaine, piperidine 14 was found to be about 2. 5-fold less potent in increasing distance traveled in mice. However, consistent with its DAT activity, piperidine 14 was found to be about 2.5-fold more potent than cocaine in enhancing stereotypic movements. Further studies of these piperidine-based ligands may provide valuable insights into the pharmacological mechanisms underlying the enhancement in distance traveled versus stereotypic movements. The present results have important implications for better understanding the structural motifs required in the design of agents with specific potency and selectivity at monoamine transporters.

We herein summarize the reports on genetic changes in precancerous lesions in the gastrointestinal tract. It has been reported that with esophageal lesions such as dysplasia and Barrett's esophagus there is a high frequency of p53 mutations. Among gastric lesions, some cases of chronic atrophic gastritis have been shown to harbor K-ras mutations. p53 and APC mutations in intestinal metaplasia have also been demonstrated, as have APC mutations in flat adenomas. With colorectal lesions, it has been reported that K-ras, DCC, p53 mutations commonly occur while APC mutations are also seen in cases of adenoma-carcinoma. p53 and K-ras mutations have been demonstrated with serrated adenoma, and K-ras mutations with hyperplastic polyps APC mutations in familial polyposis coli, LKB1 mutations in Peutz-Jeghers syndrome, and SMAD4/DPC4 mutations in juvenile polyposis syndrome have been found. Besides these genes, other genetic changes likely occur in carcinogenesis among those with hereditary diseases. K-ras mutations in aberrant crypt foci and hMSH2 mutations in ulcerative colitis have been found. Research into the genetic changes associated with cancerous lesions should lead to the development of early diagnosis and treatment methods for gastrointestinal cancer as well as the improved comprehension of carcinogenesis.

Recent research in iron metabolism has revealed the existence of iron-responding elements in the 5'UTR of the mRNA of ferritin. Binding of these structures with iron-regulatory proteins regulates ferritin synthesis within the cell, according to the intracellular iron level. Several mutations of the iron-responding elements located at the 5'UTR of the L-ferritin subunit, which lead to the hereditary hyperferritinaemia cataract syndrome, an autosomal dominant hereditary disease, have been described. Patients with congenital bilateral nuclear cataract present high serum ferritin (360-2264 micrograms/l) in the absence of iron overload. The purpose of our study was to look for this syndrome in Switzerland and in particular in the Geneva population. About 3000 cases of cataract operated on during a 4-year period (1995-1998) in the University Clinic of Ophthalmology were screened. We found 135 patients operated on before the age of 51 years. However, only 19 had bilateral nuclear cataract. 15 patients agreed to undergo iron screening. In 2 of them, a slight elevation of ferritin (267 micrograms/l in a female, 416 micrograms/l in a male) was found in the absence of iron overload. In both cases there is a positive family history of cataract. DNA sequencing analysis in these patients showed a normal nucleotide sequence of the whole iron-responding elements region. One of them (male) was found to present the codon 63 mutation at HFE gene in the heterozygous state. Our local study indicates that hereditary hyperferritinaemia cataract syndrome is extremely rare in Switzerland. However, similar studies should be carried out in other regions of the country. Iron status evaluation and ferritin level monitoring should become routine examinations in all new cases presenting with bilateral nuclear cataract before the age of 50 years.

Patho-epidemiological studies showed that thyroid lymphoma (TL) arises in inflammatory lesions of chronic lymphocytic thyroiditis (CLTH). Replication error (RER) is found in inflammatory lesions and associated cancer, suggesting that chronic inflammation could be a risk factor for neoplastic development through causing RER. To clarify whether RER is involved in the pathogenesis of TL, we examined the microsatellite instability (MSI) in 9 cases with CLTH and 19 with TL, including 10 diffuse large B-cell lymphoma (DLBL), 4 follicle center cell lymphoma, 3 marginal zone B-cell lymphoma of extranodal (MALT) type, and 2 lymphoplasmacytic type. Sixteen distinct microsatellite repeats were analyzed. Mutations of p53 and k-ras genes were also examined. When alterations at 2 or more microsatellite loci were judged as positive, only 5 DLBL cases exhibited MSI. The frequency of MSI in DLBL was significantly higher than that in other types of TL and CLTH (P < 0.05). Four of 19 cases (21.1%) showed point mutation of the k-ras gene. The k-ras mutations occurred in the cases with DLBL with RER, and four of five cases with RER had a k-ras mutation, indicating a close association between RER and k-ras mutation. p53 mutations were not found in the CLTH. Two of 19 TL cases showed mutations of p53 gene. There was no significant association between RER and p53 mutation. These findings indicate that genomic instability contributes to the progression of TL from low grade to high grade, but not to the development of low grade lymphoma in CLTH lesions.

Huntington disease (HD) is a progressive neurodegenerative disorder with autosomal dominant inheritance, characterized by choreiform movements and cognitive impairment. Onset of symptoms is around 40 years of age and progression to death occurs in approximately 10 to 15 years from the time of disease onset. HD is associated with an unstable CAG repeat expansion at the 5' and of the IT15 gene. We have genotyped the CAG repeat in the IT15 gene in 44 Brazilian individuals (42 patients and 2 unaffected family members) belonging to 34 unrelated families thought to segregate HD. We found one expanded CAG allele in 32 individuals (76%) belonging to 25 unrelated families. In these HD patients, expanded alleles varied from 43 to 73 CAG units and normal alleles varied from 18 to 26 CAGs. A significant negative correlation between age at onset of symptoms and size of the expanded CAG allele was found (r=0.6; p=0.0001); however, the size of the expanded CAG repeat could explain only about 40% of the variability in age at onset (r2=0.4). In addition, we genotyped 25 unrelated control individuals (total of 50 alleles) and found normal CAG repeats varying from 16 to 33 units. The percentage of heterozigocity of the normal allele in the control population was 88%. In conclusion, our results showed that not all patients with the "HD" phenotype carried the expansion at the IT15 gene. Furthermore, molecular diagnosis was possible in all individuals, since no alleles of intermediate size were found. Therefore, molecular confirmation of the clinical diagnosis in HD should be sought in all suspected patients, making it possible for adequate genetic counseling.

Neurodegenerative disorders like Huntington's disease (HD) are characterized by progressive and putative irreversible clinical and neuropathological symptoms, including neuronal protein aggregates. Conditional transgenic models of neurodegenerative diseases therefore could be a powerful means to explore the relationship between mutant protein expression and progression of the disease. We have created a conditional model of HD by using the tet-regulatable system. Mice expressing a mutated huntingtin fragment demonstrate neuronal inclusions, characteristic neuropathology, and progressive motor dysfunction. Blockade of expression in symptomatic mice leads to a disappearance of inclusions and an amelioration of the behavioral phenotype. We thus demonstrate that a continuous influx of the mutant protein is required to maintain inclusions and symptoms, raising the possibility that HD may be reversible.

A mutation analysis of the HFE gene followed, when applicable, by sequencing was performed on 47 patients with hereditary haemochromatosis (HH) living in Saguenay-Lac-Saint-Jean. The C282Y and H63D mutations were present on 50% and 20.3% of the HH chromosomes respectively. These frequencies were very different from those found in other populations and could be, at least partially, the result of a founder effect. No new mutation was identified among the remaining 28.1% of the HH chromosomes. Five of the eight probands with no mutation in the HFE gene had a severe and early onset suggestive of juvenile haemochromatosis.

<h4>Aims</h4>To assess the expression of SIX5 (a homeobox gene) mRNA in surface coelomic epithelium, endocervical epithelium, Fallopian tube epithelium, and benign, borderline, and malignant epithelial ovarian tumours.<h4>Methods</h4>10 normal premenopausal ovaries, 10 normal Fallopian tubes, 10 normal cervices, 10 normal postmenopausal ovaries, 10 benign epithelial ovarian tumours, 10 malignant epithelial ovarian tumours, and 40 borderline epithelial ovarian tumours were studied retrospectively. The tissues had been fixed in formalin and embedded in paraffin wax. The tumours had previously been typed into mucinous, serous, or mixed tumours and assigned to the borderline category according to the FIGO/WHO criteria. Expression was assessed by in situ binding of SIX5 specific sense and antisense riboprobes. Hybridization of the riboprobes was detected using a standard immunohistochemical technique and the results correlated with expression in the normal epithelium of the endocervix, Fallopian tube, surface coelomic epithelium, and ovarian tumours.<h4>Results</h4>Expression of SIX5 mRNA was demonstrated in normal Fallopian tube epithelium and normal endocervical epithelium. SIX5 mRNA was not detected in normal ovarian epithelial tissue at any of the times studied during the menstrual cycle. Expression of SIX5 was not shown in benign epithelial ovarian tumours or in any of the malignant epithelial ovarian tumours. In 31 of 37 borderline epithelial ovarian tumours (84%), SIX5 expression was found in the epithelial cells.<h4>Conclusions</h4>SIX5 expression is present in the normal epithelium throughout most of the female reproductive tract, suggesting it may have a role in maintaining epithelial differentiation in these tissues. SIX5 expression appears to be restricted to borderline epithelial ovarian tumours and may be a marker of epithelial differentiation in these tumours; thus borderline ovarian tumours may not be part of a continuum of disease between benign and malignant epithelial ovarian tumours. Further investigation of expression of SIX5 may clarify the molecular processes that promote differentiation of the ovarian surface epithelium.

Occasional reports have suggested an unfavorable effect of high estrogen receptor (ER) concentrations in primary breast cancer. In a population-based study we identified a subgroup explicitly exhibiting this seemingly paradoxical effect. ER concentrations were prospectively measured in a single laboratory by multipoint DCC assay. The relative risk of death in relation to the concentration of the interval-scaled variables ER and PgR was continually estimated by serial Cox regression analyses. Thus we circumvented loss of information due to primary categorization and avoided assumptions about relations between factor and risk. Based on 2,735 consecutively accrued primary breast cancer cases (median follow-up 56 months) we identified node-negative patients up to 60 years of age as the relevant subpopulation. High (>/=300 fmol/mg protein) ER concentrations exhibited an even more unfavorable impact (p < 0.03) on overall survival than ER concentrations of less than 10 fmol/mg protein. The well-known association of age and ER concentration was definitely excluded as an underlying biological cause for the increased risk. Differences in the distribution of other prognostic factors (HER-2/neu, Ki-67, DNA ploidy) were also excluded. As we observed a preponderance of pT2 tumors in the high ER group, we repeated the analysis, selectively focusing on pT2 tumors in the relevant subgroup, but the effect remained unchanged. In contrast, node-positive patients adjusted for age significantly (p = 0.02) profited from high ER concentrations as compared to the ER-negative group. As the phenomenon did not occur in node-positive patients, receptor defects in the high-ER group seem unlikely. To the contrary, we suspect that ER overexpressing cells are hypersensitive even to low levels of estrogens. Once they have sneaked past local barriers prior to primary surgery, they may cause early death in the absence of appropriate adjuvant endocrine therapy.

This study was conducted to immunohistochemical and biochemical measurements of estrogen receptor (ER) and progesterone receptor (PR) in endometrial carcinoma and elucidate the relationship between ER/PR contents and the clinico-pathologic features. Specific monoclonal antibodies to human ER and PR were used and the immunohistochemical (IHC) assay was performed on 36 specimens of endometrial carcinomas. Twenty-eight out of 36 specimens' ER and PR contents were also measured by dextran-coated charcoal analysis (DCC). ER and PR positive rates were 44.4% and 52.8% by IHC, and 50% and 57.1% by DCC, respectively. The two methods correlated significantly, the correlation coefficient between DCC and IHC being 0.565 for ER and 0.563 for PR (P < 0.05). ER and PR contents were much lower in grade 3 than in grades 1 and 2 endometrial carcinomas (P < 0.01). The authors conclude that the levels of ER and PR are associated with the differentiation of endometrial carcinoma. The immunohistochemical and biochemical methods correlate well. A combination of the two methods may be more useful in predicting the prognosis and choosing the treatment.

The physical alignment of the entire region of the pig major histocompatibility complex (MHC) has been almost completed. In swine, the MHC is called the SLA (swine leukocyte antigen) and most of its class I region has been sequenced. Over one hundred genes have been characterised, including the classical class I and class I-related genes, as well as the class II gene families. These results in swine provide new evidence for the striking conservation during the evolution of a general MHC framework, and are consistent with the location of the class I genes on segments referred to as permissive places within the MHC class I region. Recent results confirm the involvement of the SLA region in numerous quantitative traits.

Some key advances occurred last year in understanding mechanisms involved in nutrient absorption. A novel "prechylomicron transport vesicle" was identified; its movement to the Golgi is the rate-limiting step for triacylglycerol absorption. A scavenger receptor (type BI) in the brush border membrane appears to facilitate cholesterol uptake. Several studies define mechanisms for gastrointestinal peptide hormone stimulation of glucose uptake. An oligopeptide transporter, PepT1, is transcriptionally upregulated by certain dietary amino acids and dipeptides. Surprisingly, both insulin and fasting double the maximum velocity for dipeptide uptake (via PepT1), but they act by different mechanisms. Three transporters, SMVT (sodium-dependent multivitamin transporter for biotin and pantothenate), SVCT (for vitamin C), and CaT1 (for Ca uptake from the lumen) have been cloned and are active when expressed in various cells. Additional studies provide insights on Ca absorption and vitamin D action in aging, estrogen deficiency, and adaptation to a low Ca diet. Nramp2, also called DMT1 (divalent metal ion transporter), seems to be a major regulator of transferrin-independent, nonheme iron uptake. Finally, the protein HFE associates with the transferrin receptor and is part of an iron-sensing mechanism that regulates iron absorption. It is defective in hereditary hemochromatosis. HFE and Nramp2 (DMT1) genes are reciprocally regulated.