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Although disorders of the skeleton are individually rare, they are of clinical relevance because of their overall frequency. Many attempts have been made in the past to identify disease groups in order to facilitate diagnosis and to draw conclusions about possible underlying pathomechanisms. Traditionally, skeletal disorders have been subdivided into dysostoses, defined as malformations of individual bones or groups of bones, and osteochondrodysplasias, defined as developmental disorders of chondro-osseous tissue. In light of the recent advances in molecular genetics, however, many phenotypically similar skeletal diseases comprising the classical categories turned out not to be based on defects in common genes or physiological pathways. In this article, we present a classification based on a combination of molecular pathology and embryology, taking into account the importance of development for the understanding of bone diseases.

No abstract available.

Aggregation of huntingtin (htt) in neuronal inclusions is associated with the development of Huntington's disease (HD). Previously, we have shown that mutant htt fragments with polyglutamine (polyQ) tracts in the pathological range (>37 glutamines) form SDS-resistant aggregates with a fibrillar morphology, whereas wild-type htt fragments with normal polyQ domains do not aggregate. In this study we have investigated the co-aggregation of mutant and wild-type htt fragments. We found that mutant htt promotes the aggregation of wild-type htt, causing the formation of SDS-resistant co-aggregates with a fibrillar morphology. Conversely, mutant htt does not promote the fibrillogenesis of the polyQ-containing protein NOCT3 or the polyQ-binding protein PQBP1, although these proteins are recruited into inclusions containing mutant htt aggregates in mammalian cells. The formation of mixed htt fibrils is a highly selective process that not only depends on polyQ tract length but also on the surrounding amino acid sequence. Our data suggest that mutant and wild-type htt fragments may also co-aggregate in neurons of HD patients and that a loss of wild-type htt function may contribute to HD pathogenesis.

TBP functions in transcription initiation in all eukaryotes and in Archaebacteria. Although the 181-amino acid (aa) carboxyl (C-) terminal core of the protein is highly conserved, TBP proteins from different phyla exhibit diverse sequences in their amino (N-) terminal region. In mice, the TBP N-terminus plays a role in protecting the placenta from maternal rejection; however the presence of similar TBP N-termini in nontherian tetrapods suggests that this domain also has more primitive functions. To gain insights into the pretherian functions of the N-terminus, we investigated its phylogenetic distribution. TBP cDNAs were isolated from representative nontetrapod jawed vertebrates (zebrafish and shark), from more primitive jawless vertebrates (lamprey and hagfish), and from a prevertebrate cephalochordate (amphioxus). Results showed that the tetrapod N-terminus likely arose coincident with the earliest vertebrates. The primary structures of vertebrate N-termini indicates that, historically, this domain has undergone events involving intragenic duplication and modification of short oligopeptide-encoding DNA sequences, which might have provided a mechanism of de novo evolution of this polypeptide.

Branched polyethylenimine (PEI) chains with an average molecular mass of 2 kDa (PEI2) have been covalently attached to gold nanoparticles (GNPs), and the potency of the resulting PEI2-GNPs conjugates as vectors for the delivery of plasmid DNA into monkey kidney (COS-7) cells in the presence of serum in vitro has been systematically investigated. The transfection efficiencies vary as a function of the PEI/gold molar ratio in the conjugates, with the best one (PEI2-GNPII) being 12 times more potent than the unmodified polycation. This potency can be further doubled by adding amphiphilic N-dodecyl-PEI2 during complex formation with DNA. The resulting ternary complexes are at least 1 order of magnitude more efficient than the 25-kDa PEI, one of the premier polycationic gene-delivery vectors. Importantly, although unmodified PEI2 transfects just 4% of the cells, PEI2-GNPII transfects 25%, and the PEI2-GNPII/dodecyl-PEI2 ternary complex transfects 50% of the cells. The intracellular trafficking of the DNA complexes of these vectors, monitored by transmission electron microscopy, has detected the complexes in the nucleus <1 h after transfection.

Huntingtin protein is mutated in Huntington disease. We previously reported that wild-type but not mutant huntingtin stimulates transcription of the gene encoding brain-derived neurotrophic factor (BDNF; ref. 2). Here we show that the neuron restrictive silencer element (NRSE) is the target of wild-type huntingtin activity on BDNF promoter II. Wild-type huntingtin inhibits the silencing activity of NRSE, increasing transcription of BDNF. We show that this effect occurs through cytoplasmic sequestering of repressor element-1 transcription factor/neuron restrictive silencer factor (REST/NRSF), the transcription factor that binds to NRSE. In contrast, aberrant accumulation of REST/NRSF in the nucleus is present in Huntington disease. We show that wild-type huntingtin coimmunoprecipitates with REST/NRSF and that less immunoprecipitated material is found in brain tissue with Huntington disease. We also report that wild-type huntingtin acts as a positive transcriptional regulator for other NRSE-containing genes involved in the maintenance of the neuronal phenotype. Consistently, loss of expression of NRSE-controlled neuronal genes is shown in cells, mice and human brain with Huntington disease. We conclude that wild-type huntingtin acts in the cytoplasm of neurons to regulate the availability of REST/NRSF to its nuclear NRSE-binding site and that this control is lost in the pathology of Huntington disease. These data identify a new mechanism by which mutation of huntingtin causes loss of transcription of neuronal genes.

NeuroD (ND) is a basic helix-loop-helix transcription factor important for neuronal development and survival. By using a yeast two-hybrid screen, we identified two proteins that interact with ND, huntingtin-associated protein 1 (HAP1) and mixed-lineage kinase 2 (MLK2), both of which are known to interact with huntingtin (Htt). Htt is a ubiquitous protein important for neuronal transcription, development, and survival, and loss of its function has been implicated in the pathogenesis of Huntington's disease, a neurodegenerative disorder. However, the mechanism by which Htt exerts its neuron-specific function at the molecular level is unknown. Here we report that Htt interacts with ND via HAP1, and that MLK2 phosphorylates and stimulates the activity of ND. Furthermore, we show that Htt and HAP1 facilitate the activation of ND by MLK2. To our knowledge, ND is the first example of a neuron-specific transcription factor involved in neuronal development and survival whose activity is modulated by Htt. We propose that Htt, together with HAP1, may function as a scaffold for the activation of ND by MLK2.

Sox9 has essential roles in endochondral bone formation during axial and appendicular skeletogenesis. Sox9 is also expressed in neural crest cells, but its function in neural crest remains largely unknown. Because many craniofacial skeletal elements are derived from cranial neural crest (CNC) cells, we asked whether deletion of Sox9 in CNC cells by using the Cre recombinase/loxP recombination system would affect craniofacial development. Inactivation of Sox9 in neural crest resulted in a complete absence of cartilages and endochondral bones derived from the CNC. In contrast, all of the mesodermal skeletal elements and intramembranous bones were essentially conserved. The migration and the localization of Sox9-null mutant CNC cells were normal. Indeed, the size of branchial arches and the frontonasal mass of mutant embryos was comparable to that of WT embryos, and the pattern of expression of Ap2, a marker of migrating CNC cells, was normal. Moreover, in mouse embryo chimeras Sox9-null mutant cells migrated to their correct location in endochondral skeletal elements; however, Sox9-null CNC cells were unable to contribute chondrogenic mesenchymal condensations. In mutant embryos, ectopic expression of osteoblast marker genes, such as Runx2, Osterix, and Col1a1, was found in the locations where the nasal cartilages exist in WT embryos. These results indicate that inactivation of Sox9 causes CNC cells to lose their chondrogenic potential. We hypothesize that these cells change their cell fate and acquire the ability to differentiate into osteoblasts. We conclude that Sox9 is required for the determination of the chondrogenic lineage in CNC cells.

The mechanisms by which the hereditary hemochromatosis protein, HFE, decreases transferrin-mediated iron uptake were examined. Coimmunoprecipitation studies using solubilized cell extracts demonstrated that transferrin (Tf) competed with HFE for binding to the transferrin receptor (TfR) similar to previous in vitro studies using soluble truncated forms of HFE and the TfR. At concentrations of Tf approaching those found in the blood, no differences in Tf binding to cells were detected, which is consistent with the lower binding constant of HFE for TfR versus Tf. However, cells expressing HFE still showed a decrease in Tf-mediated iron uptake at concentrations of Tf sufficient to dissociate HFE from the TfR. These results indicate that the association of HFE with TfR is not essential for its ability to lower intracellular iron stores. To test the effect of HFE on lowering intracellular iron levels independently of its association with TfR, a mutated HFE (fW81AHFE) that shows greatly reduced affinity for the TfR was transfected into tetracycline-controlled transactivator HeLa cells. HeLa cells expressing fW81AHFE behaved in a similar manner to cells expressing wild-type HFE with respect to decreased intracellular iron levels measured by iron regulatory protein gel-shift assays and ferritin levels. The results indicate that HFE can lower intracellular iron levels independently of its interaction with the TfR.

Haemochromatosis (HH) is a clinically and genetically heterogeneous disease caused by inappropriate iron absorption. Most HH patients are homozygous for the C282Y mutation in the HFE gene. However, penetrance of the C282Y mutation is incomplete, and other genetic factors may well affect the HH phenotype. Ferroportin and TFR2 mutations also cause HH, and two HAMP mutations have recently been reported that causes juvenile haemochromatosis (JH) in the homozygous state. Here, we report evidence for digenic inheritance of HH. We have detected two new HAMP mutations in two different families, in which there is concordance between severity of iron overload and heterozygosity for HAMP mutations when present with the HFE C282Y mutation. In family A, the proband has a JH phenotype and is heterozygous for C282Y and a novel HAMP mutation Met50del IVS2+1(-G). This is a four nucleotide ATGG deletion which causes a frameshift. The proband's unaffected mother is also heterozygous for Met50del IVS2+1(-G), but lacks the C282Y mutation and is heterozygous for the HFE H63D mutation. Met50del IVS2+1(-G) was absent from 642 control chromosomes. In family B, a second novel, less severe HAMP mutation, G71D, was identified. This was detected in the general population at an allele frequency of 0.3%. We propose that the phenotype of C282Y heterozygotes and homozygotes may be modified by heterozygosity for mutations which disrupt the function of hepcidin in iron homeostasis, with the severity of iron overload corresponding to the severity of the HAMP mutation.

<h4>Background</h4>EURIS (European Resistance Intervention Study) was launched as a multinational study in September of 2000 to identify the multitude of complex risk factors that contribute to the high carriage rate of drug resistant Streptococcus pneumoniae strains in children attending Day Care Centers in several European countries. Access to the very large number of data required the development of a web-based infrastructure - EURISWEB - that includes a relational online database, coupled with a query system for data retrieval, and allows integrative storage of demographic, clinical and molecular biology data generated in EURIS.<h4>Methods</h4>All components of the system were developed using open source programming tools: data storage management was supported by PostgreSQL, and the hypertext preprocessor to generate the web pages was implemented using PHP. The query system is based on a software agent running in the background specifically developed for EURIS.<h4>Results</h4>The website currently contains data related to 13,500 nasopharyngeal samples and over one million measures taken from 5,250 individual children, as well as over one thousand pre-made and user-made queries aggregated into several reports, approximately. It is presently in use by participating researchers from three countries (Iceland, Portugal and Sweden).<h4>Conclusion</h4>An operational model centered on a PHP engine builds the interface between the user and the database automatically, allowing an easy maintenance of the system. The query system is also sufficiently adaptable to allow the integration of several advanced data analysis procedures far more demanding than simple queries, eventually including artificial intelligence predictive models.

The presenilin-gamma-secretase complex plays a critical role in mediating intramembranous proteolysis of several type I membrane proteins, including beta-amyloid precursor protein (APP) and Notch. We now show that deleted in colorectal cancer (DCC) is subject to proteolysis within the ectodomain segment both in cultured cells and in vivo and that the residual membrane-tethered DCC "stub" is subsequently processed by gamma-secretase to generate a derivative termed DCC-intracellular domain (ICD). The production of DCC-ICD is inhibited by selective gamma-secretase inhibitors, and by the expression of the dominant negative PS1 D385A variant. Moreover, the membrane-tethered DCC "stubs" accumulate to high levels in PS1-deficient embryos. We also demonstrate that expression of a DCC-Gal4 chimera is capable of activating transcription in a luciferase-based reporter assay and this activity is dependent on gamma-secretase activity. Our findings offer the proposal that DCC performs dual roles both as a cell surface receptor that modulates intracellular signaling pathways and as a transcriptional coactivator that relies on gamma-secretase-dependent production and nuclear translocation of the cytoplasmic domain.

HFE, the protein mutated in hereditary haemochromatosis type 1, is known to interact with the transferrin receptor (TfR) on the cell surface and during endocytosis [Gross, Irrinki, Feder and Enns (1998) J. Biol. Chem. 273, 22068-22074; Roy, Penny, Feder and Enns (1999) J. Biol. Chem. 274, 9022-9028]. However, whether they are capable of interacting with each other once inside the cell is not known. In the present study we present several lines of evidence that they do interact in endosome compartments. Cells expressing a chimaera of HFE protein with the cytoplasmic domain of lysosomal-associated membrane protein 1 (LAMP1) in place of its own (HFE-LAMP) show a decrease in the half-life of the TfR. This implies that the interaction between HFE and TfR in endosomes targets the TfR to lysosomal compartments. The interaction between TfR and HFE-LAMP was confirmed by immunoprecipitation, in addition to immunofluorescence studies. Addition of transferrin (Tf) to HFE-LAMP-expressing cells competes with HFE for binding to the TfR, thereby increasing the half-life of TfR and confirming that the HFE-LAMP-TfR complex reaches the cell surface prior to entering the endosomal vesicles and trafficking to the lysosome. These results raise the possibility that interaction of HFE and TfR in intracellular vesicles may play an important role in determining the function of HFE in iron homoeostasis, which is still unknown. Analysis of endosomal pH and the iron content of internalized Tf indicated that HFE does not appear to alter the unloading of iron from Tf in the endosome.

We investigated the effects of several xenobiotics, including antimicrobial agents and general stress factors such as starvation, heat and osmotic shock, on the modulation of expression of Proteus mirabilis glutathione S-transferase B1-1 (PmGST B1-1). The level of expression of PmGST B1-1 was established by both Western- and Northern-blot experiments. Our results show that several compounds can modulate expression of PmGST B1-1. The level of PmGST B1-1 increased when bacterial cells were exposed to a variety of stresses such as 1-chloro-2,4-dinitrobenzene, H(2)O(2), fosfomycin or tetracycline. A knock-out gst B gene was also constructed using the suicide vector pKNOCKlox-Ap. Successful inactivation of the wild-type gene was confirmed by PCR, DNA sequence analysis and Western blotting. Under normal culture conditions, this mutant was viable and displayed no significant phenotypic differences compared with the wild-type. However, viability tests revealed that the null mutant was more sensitive to oxidative stress in the form of H(2)O(2) and to several antimicrobial drugs when compared with the wild-type. These results suggest that PmGST B1-1 has an active role in the protection against oxidative stress generated by H(2)O(2) and it appears to be involved in the detoxification of antimicrobial agents.

For two decades the glial sling has been hypothesized to act as a guidance substratum for developing callosal axons. However, neither the cellular nature of the sling nor its guidance properties have ever been clearly identified. Although originally thought to be glioblasts, we show here that the subventricular zone cells forming the sling are in fact neurons. Sling cells label with a number of neuronal markers and display electrophysiological properties characteristic of neurons and not glia. Furthermore, sling cells are continuously generated until early postnatal stages and do not appear to undergo widespread cell death. These data indicate that the sling may be a source of, or migratory pathway for, developing neurons in the rostral forebrain, suggesting additional functions for the sling independent of callosal axon guidance.

The cytolethal distending toxins (CDT) are responsible for the mitosis block at G2/M and the cycle arrest of cells in culture. Escherichia coli isolated from humans and animals with intestinal and extra-intestinal diseases can be positive for the production of a CDT-like cytopathic effect or for the presence of cdt-related genes. The purpose of this study was to compare the prevalence and the identity of cdt-related sequences in necrotoxigenic E. coli (NTEC). A collection of 98 bovine type 2 NTEC (NTEC2) and 45 bovine, 20 canine, 3 feline, 65 human and 129 porcine type 1 NTEC (NTEC1) isolates was studied by colony hybridisation and PCR assays specific for the cdtB genes encoding the B sub-unit of the CDT-I, CDT-II, CDT-III and CDT-IV toxins produced by E. coli. cdtB-III sequences were frequent amongst bovine NTEC2, since 83% of these isolates were positive by colony hybridisation and/or PCR, whereas cdtB-related sequences were rare amongst NTEC1, since only 2 bovine (4%), 3 canine (15%), 10 human (15%) and 13 porcine (10%) of these isolates were positive. The 28 probe-positive NTEC1 harboured cdtB-IV sequences (13 isolates), cdtB-I sequences (10 isolates), or still unidentified cdt-related sequences (5 isolates). After comparison with previously published and unpublished results of phenotypic assay on cell cultures, existence of other cdt-related sequences is suggested amongst NTEC1. The differences between NTEC1 and NTEC2 in their CDT profiles may have implication for the pathogenesis of those two classes of pathogenic E. coli.

Hereditary hemochromatosis has been recognized as a clinical disorder for more than 100 years. The common form of the disorder is caused by the Cys282Tyr mutation (C282Y) of the HFE gene. Hereditary hemochromatosis affects predominantly people of Northern European origin. The C282Y mutation probably occurred on a single chromosome carrying the ancestral hemochromatosis haplotype, which subsequently was spread by emigration and the founder effect. It has been estimated that the C282Y mutation appeared 60-70 generations ago. It was initially suggested that the ancestral C282Y mutation occurred within the Celtic group of peoples. However, we hypothesize that the distribution of the C282Y mutation in Europe is more consistent with an origin among the Germanic Iron Age population in Southern Scandinavia. From this area, the mutation could later be spread by the migratory activities of the Vikings. The aim of the present study was to evaluate the validity of these two hypotheses. Several arguments are in favor of the 'Viking hypothesis': first, the highest frequencies (5.1-9.7%) of the C282Y mutation are observed in populations in the Northern part of Europe, i.e. Denmark, Norway, Sweden, Faeroe Islands, Iceland, Eastern part of England (Danelaw) and the Dublin area, all Viking homelands and settlements. Second, the highest allele frequencies are reported among populations living along the coastlines. Third, the frequencies of the C282Y mutation decline from Northern to Southern Europe. Intermediate allele frequencies (3.1-4.8%) are seen in the populations in Central Europe, which is the original Celtic homeland. Low allele frequencies (0-3.1%) are recognized in populations in Southern Europe and the Mediterranean.

Juvenile Huntington's disease (HD) becomes clinically manifest before 20 years of age. The diagnosis of HD is based on family history, characteristic clinical findings, and the detection of an expansion of a CAG polyglutamine tract in the Huntingtin gene. Juvenile HD is characterized by paternal anticipation and large CAG expansions that may be missed using routine molecular analysis. We have developed an easy, rapid, and reliable modified PCR method using XL (Extra Long) PCR that allowed us to diagnose one of the youngest children reported with juvenile HD. Without this innovation we would not have been able to demonstrate the large CAG expansion. This assay could become part of a standard protocol for HD testing in molecular diagnostic laboratories.

<h4>Background and aims</h4>In HFE associated hereditary haemochromatosis, the duodenal enterocyte behaves as if iron deficient and previous reports have shown increased duodenal expression of divalent metal transporter 1 (DMT1) and iron regulated gene 1 (Ireg1) in affected subjects. In those studies, many patients had undergone venesection, which is a potent stimulus of iron absorption. Our study investigated duodenal expression of DMT1 (IRE and non-IRE), Ireg1, hephaestin, and duodenal cytochrome-b (Dyctb) in untreated C282Y homozygous haemochromatosis patients, iron deficient patients, and iron replete subjects.<h4>Methods</h4>Total RNA was extracted from duodenal biopsies and expression of the iron transport genes was assessed by ribonuclease protection assay.<h4>Results</h4>Expression of DMT1 (IRE) and Ireg1 was increased 3-5-fold in iron deficient subjects compared with iron replete subjects. Duodenal expression of DMT1 (IRE) and Ireg1 was similar in haemochromatosis patients and iron replete subjects but in haemochromatosis patients with elevated serum ferritin concentrations, both DMT1 (IRE) and Ireg1 expression were inappropriately increased relative to serum ferritin concentration. Hephaestin and Dcytb levels were not upregulated in haemochromatosis. DMT1 (IRE) and Ireg1 levels showed significant inverse correlations with serum ferritin concentration in each group of patients.<h4>Conclusions</h4>These findings are consistent with DMT1 (IRE) and Ireg1 playing primary roles in the adaptive response to iron deficiency. Untreated haemochromatosis patients showed inappropriate increases in DMT1 (IRE) and Ireg1 expression for a given level of serum ferritin concentration, although the actual level of expression of these iron transport genes was not significantly different from that of normal subjects.

Clearance rates for serotonin (5-HT) in heterozygote (+/-) and homozygote (-/-) serotonin transporter (5-HTT) knockout (KO) mice have not been determined in vivo. Moreover, the effect of selective serotonin reuptake inhibitors (SSRIs) on 5-HT clearance in these mice has not been examined. In this study, the rate of clearance of exogenously applied 5-HT was measured in the CA3 region of the hippocampus of anesthetized mice using high-speed chronoamperometry. Compared with wild-type mice, the maximal rate of 5-HT clearance from extracellular fluid (ECF) was decreased in heterozygotes and more markedly so in KO mice. Heterozygote mice were more sensitive to the 5-HT uptake inhibitor, fluvoxamine, resulting in longer clearance times for 5-HT than in wild-type mice; as expected, the KO mice were completely unresponsive to fluvoxamine. There were no associated changes in norepinephrine transporter density, nor was there an effect of the norepinephrine uptake inhibitor, desipramine, on 5-HT clearance in any genotype. Thus, adaptive changes in the norepinephrine transport system do not occur in the CA3 region of hippocampus as a consequence of 5-HTT KO. These data highlight the potential of the heterozygote 5-HTT mutant mice to model the dynamic in vivo consequences of the human 5-HTT polymorphism.

Most eukaryotic cells contain nearly equimolar amounts of nucleosomes and H1 linker histones. Despite their abundance and the potential functional specialization of H1 subtypes in multicellular organisms, gene inactivation studies have failed to reveal essential functions for linker histones in vivo. Moreover, in vitro studies suggest that H1 subtypes may not be absolutely required for assembly of chromosomes or nuclei. By sequentially inactivating the genes for three mouse H1 subtypes (H1c, H1d, and H1e), we showed that linker histones are essential for mammalian development. Embryos lacking the three H1 subtypes die by mid-gestation with a broad range of defects. Triple-H1-null embryos have about 50% of the normal ratio of H1 to nucleosomes. Mice null for five of these six H1 alleles are viable but are underrepresented in litters and are much smaller than their littermates. Marked reductions in H1 content were found in certain tissues of these mice and in another compound H1 mutant. These results demonstrate that the total amount of H1 is crucial for proper embryonic development. Extensive reduction of H1 in certain tissues did not lead to changes in nuclear size, but it did result in global shortening of the spacing between nucleosomes.

Iron metabolism is dysregulated in critically ill patients. A mouse model of dysregulated iron metabolism was used to examine the consequence of iron loading upon sepsis. Mice deleted in the hfe gene (hfe-/-) abnormally accumulate iron in tissue; defects in the human hfe gene are clinically expressed as hemochromatosis. Hfe-/- mice and wild-type counterparts were randomized to receive either high- or low-iron diets for 2 weeks. After iron loading, mice were subjected to cecal ligation and puncture (CLP), a clinically relevant animal model of intra-abdominal sepsis. A preliminary (but underpowered) study suggested that iron-loaded hfe-/- mice had increased mortality as compared with hfe-/- mice fed a low-iron diet. There was no difference between wild-type and hfe-/- mice fed a low-iron diet or between wild-type mice fed hihg- and low-iron diets. A subsequent, appropriately powered study showed that iron-loaded hfe-/- mice had significantly higher mortality from intra-abdominal sepsis than hfe-/- mice fed a low-iron diet. Iron loading was confirmed through chemical assay of iron concentration in hepatic tissue. Animals with dysregulated iron handling, loaded with iron and subjected to CLP, had double the mortality of animals with normal iron levels. Critical care patients often have altered iron metabolism. In clinical practice, critically ill patients may receive iron through direct administration and the transfusion of blood products. Iron therapy may adversely affect the clinical outcome from sepsis.

Knock-out mice lacking the serotonin transporter [5-hydroxytryptamine transporter (5-HTT)] were used to assess the influence of 5-HT re-uptake on ethanol consumption. Under a free-choice paradigm, alcohol intake was lower in mutant than in wild-type mice, and pharmacological blockade of 5-HTT by fluoxetine reduced alcohol intake in wild-type mice only. These data confirm the inhibitory effect of 5-HTT inactivation on ethanol intake.

<h4>Background</h4>Two mutations in HFE, G845A (amino acid substitution C282Y) and C187G (H63D), are associated with hereditary hemochromatosis. We developed and validated a novel method, linked linear amplification (LLA), for detection of these two mutations.<h4>Methods</h4>Two segments of HFE were amplified by a multiplex LLA reaction that generated biotinylated LLA products. Aliquots of the multiplex LLA reaction were captured in microwells by hybridization to immobilized allele-specific oligonucleotides (ASOs). One wild-type and one mutant ASO represented the DNA sequence at each of the two mutation sites. Hybridization was detected by a streptavidin-horseradish peroxidase-based colorimetric method. Genotypes obtained by LLA and PCR-restriction fragment length polymorphism (PCR-RFLP) methods for 320 individuals were compared.<h4>Results</h4>The amplified samples included the following genotypes as determined by PCR-RFLP: wild-type 282 and 63 codons (n = 105), C282Y homozygous mutant (n = 54), C282Y heterozygous (n = 52), H63D homozygous mutant (n = 17), H63D heterozygous (n = 59), and compound H63D and C282Y heterozygous mutant (n = 33). There was complete concordance between the results obtained by LLA and those obtained by PCR-RFLP analysis. The presence of another HFE mutation, A193T (encoding S65C), did not interfere with genotyping at codon 63.<h4>Conclusions</h4>LLA provides a reliable method to detect the common mutations in HFE that cause hereditary hemochromatosis.

The African American Study of Kidney Disease and Hypertension (AASK) is a multicenter randomized clinical trial designed to test the effectiveness of three anti-hypertensive drug regimens and two levels of BP control on the progression of hypertensive kidney disease. Participants include African-American men and women aged 18 to 70 yr who have hypertensive kidney disease and GFR between 20 and 65 ml/min per 1.73 m(2). The three anti-hypertensive drug regimens include an angiotensin converting enzyme inhibitor (ramipril), a dihydropyridine calcium channel blocker (amlodipine) or a beta-blocker (metoprolol) as initial therapy. The BP control levels are a lower goal (mean arterial pressure, </=92 mmHg) and a usual goal (mean arterial pressure, 102 to 107 mmHg inclusive). The primary outcome is rate of change in renal function as measured by GFR, assessed by (125) I-iothalamate clearance. The main secondary patient outcome is a composite including the following events: (1) reduction in GFR by 50%, (2) end-stage renal disease, or (3) death.

To identify the genetic modifier(s) that might alter the age at onset in Huntington's disease (HD) we have analyzed variations in GluR6 kainate receptor (GluR6), CA150 gene, Delta2642 and polymorphic CCG repeat variation in huntingtin (htt) gene in 77 HD patients and normal individuals. In addition, variation in the RAI1 gene was analyzed in 30 spinocerebellar ataxia (SCA2) patients and normal individuals to show the possible influence on the age at onset. Multiple regression analysis indicated that variation in GluR6 and CCG repeat genotype might explain 6.2% and 3.1%, respectively, of the variability in the age at onset in HD. Similar analysis with SCA2 patients indicated that RAI1 might explain about 13% of the variability in the age at onset. Specific alleles in GluR6 and CA150 locus were only observed in HD patients.

The goal of this study was to determine whether a panel of tumor suppressor gene markers of allelic loss could serve as a representative indicator of gene damage and thereby provide further discriminative power over current staging systems for recurrence-free prognostication in patients undergoing liver transplantation in the presence of hepatocellular carcinoma. The paraffin blocks from 103 cases of hepatocellular carcinoma were obtained, and cellular targets were selected for tissue microdissection genotyping. Tumor suppressor gene loss was based on loss of heterozygosity situated within or adjacent to specific genes of interest (APC, CDKN2A, DCC, MET, MYC1, OGG1, p34, p53, PTEN). Microdissected tissue was amplified using polymerase chain reaction (PCR) with flanking oligonucleotides bearing fluorescent labels designed for GeneScan fragment analysis; PCR products were separated by capillary electrophoresis. Normal microdissected tissue samples for each case were evaluated for informative status with respect to individual alleles for 18 microsatellites at 10 genomic loci-1p, 3p, 5q, 7q, 8q, 9p, 10q, 17p, 17q, 18q. The measure of allelic loss of heterozygosity combined with tumor number, tumor size, vascular invasion, lobar distribution, and patient gender provide a highly discriminatory model for predicting cancer recurrence after liver transplantation. Using our previously developed artificial neural network model in combination with the genotyping results, unambiguous predictions were made for 91 of the103 patients (88.3%). Of these, 1 was lost to follow-up, and 9 died recurrence-free less than 3 years posttransplantation. For the remaining 81, the combined models predicted tumor recurrence outcomes with complete accuracy. Microdissection genotyping provides powerful supplementary discriminative information for tumor-free survival.

RARRES3 is a retinoid-inducible class II tumour-suppressor gene. This study analysed the expression of RARRES3 protein in normal, adenoma and carcinoma tissues of the colorectum and its correlation with tumour differentiation. The expression of RARRES3 protein in 151 paraffin-embedded colorectal tissues (11 distal normal mucosa, 20 adenoma and 120 colorectal adenocarcinoma) was determined by immunohistochemistry. RARRES3 protein was expressed in all 11 distal normal, 120 adjacent normal and 20 adenoma tissues. In distal normal tissues, RARRES3 protein was expressed at the highest levels in differentiated mucosal epithelial cells. Among 120 carcinoma tissues, RARRES3 protein was detected in 97.6% (40 out of 41), 79.4% (54 out of 68) and 17.3% (three out of 11) of well-, moderately and poorly differentiated tumours, respectively. The expression of RARRES3 protein was positively correlated to tumour differentiation (test for trend, P<0.0001). Also, levels of RARRES3 protein were found to be higher in the normal tissues adjacent to 14.6% (six out of 41), 51.5% (35 out of 68), and 90.1% (10 out of 11) of well-, moderately and poorly differentiated tumours, respectively. The decreases in tumour differentiation and RARRES3 expression were significantly correlated compared to the adjacent normal tissues (test for trend, P<0.0001). The prognostic implication of RARRES3 protein expression was studied in 107 tumour, and no statistical difference in survival was observed. The expression of RARRES3 protein was positively correlated to cellular differentiation of normal and adenocarcinoma tissues of the colorectum, which supports the role of RARRES3 in normal and malignant epithelial differentiation of colorectum. RARRES3 expression was decreased only in carcinoma tissue, which suggests that altered RARRES3 expression occurs late in colorectal carcinogenesis.

Cell motility is an important cellular function closely related to the processes of tumour progression and metastasis. Several members of transmembrane 4 superfamily (TM4SF) have been reported to be associated with cell motility and metastatic potential of solid tumour. The aim of this study is to clarify the clinical significance of the member of TM4SF (MRP-1/CD9, KAI1/CD82 and CD151) in human colon cancer. We studied 146 colon cancer patients who underwent curative surgery and studied the expression of MRP-1/CD9, KAI1/CD82 and CD151 using reverse transcriptase - polymerase chain reaction and immunohistochemistry. We found that 64 patients (43.8%) had MRP-1/CD9-positive tumours and that the overall survival rate of patients with MRP-1/CD9-positive tumours was much higher than that of patients with MRP-1/CD9-negative tumours (89.8 vs 50.8%, P<0.001). In contrast, 63 patients (43.2%) had KAI1/CD82-positive tumours and the overall survival rate of patients with KAI1/CD82-positive tumours was also higher than that of patients with KAI1/CD82-negative tumours (84.8 vs 54.9%, P=0.002). On the other hand, positive CD151 expression had a bad effect on the overall survival rate of patients with colon cancer (61.2 vs 74.9%, P=0.022). In a multivariate analysis, MRP-1/CD9 status was a good indicator of the overall survival (P=0.007). We have shown that the reduction of MRP-1/CD9 and KAI1/CD82 expression, and the increasing CD151 expression are indicators for a poor prognosis in patients with colon cancer. This is a first report describing about the relation between CD151 and colon cancer.

Two related protein complexes, cohesin and condensin, are essential for separating identical copies of the genome into daughter cells during cell division. Cohesin glues replicated sister chromatids together until they split at anaphase, whereas condensin reorganizes chromosomes into their highly compact mitotic structure. Unexpectedly, mutations in the subunits of these complexes have been uncovered in genetic screens that target completely different processes. Exciting new evidence is emerging that cohesin and condensin influence crucial processes during interphase, and unforeseen aspects of mitosis. Each complex can perform several roles, and individual subunits can associate with different sets of proteins to achieve diverse functions, including the regulation of gene expression, DNA repair, cell-cycle checkpoints and centromere organization.

A newly developed oligonucleotide array suited for multilocus sequence typing (MLST) of Staphylococcus aureus strains was analyzed with two strain collections in a two-center study. MLST allele identification for the first strain collection fully agreed with conventional strain typing. Analysis of strains from the second collection revealed that chip-defined MLST was concordant with conventional MLST. Array-mediated MLST data were reproducible, exchangeable, and epidemiologically concordant.

We describe a patient with a history of asthma and remote use of steroids the development of necrotizing fascitis due to Vibrio alginolyticus after an injury from a coral reef during bathing in the Caribbean Sea off Colombia. The patient recovered with aggressive surgical debridement and antibiotics.

Staufen1, the mammalian homolog of Drosophila Staufen, assembles into ribonucleoprotein particles (RNPs), which are thought to transport and localize RNA into dendrites of mature hippocampal neurons. We therefore investigated whether additional components of the RNA localization complex besides Staufen are conserved. One candidate is the mammalian homolog of Drosophila Barentsz (Btz), which is essential for the localization of oskar mRNA to the posterior pole of the Drosophila oocyte and is a component of the oskar RNA localization complex along with Staufen. In this study, we report the characterization of mammalian Btz, which behaves like a nucleocytoplasmic shuttling protein. When expressed in the Drosophila egg chamber, mammalian Btz is still able to interact with Drosophila Staufen and reach the posterior pole in the wild-type oocyte, but does not rescue the btz mutant phenotype. Most interestingly, we show by immunoprecipitation assays that Btz interacts with mammalian Staufen in an RNA-dependent manner through a conserved domain, which encompasses the region of homology to the Drosophila Btz protein and contains a novel conserved motif. One candidate for an RNA that mediates this interaction is the dendritically localized brain cytoplasmic 1 transcript. In addition, Btz and Staufen1 colocalize within particles in the cell body and, to a more variable extent, in dendrites of mature hippocampal neurons. Together, our data suggest that the mRNA transport machinery is conserved during evolution, and that mammalian Btz is an additional component of the dendritic RNPs in hippocampal neurons.

During forward swimming, motor neurons that innervate each crayfish swimmeret fire periodic coordinated bursts of impulses. These bursts occur simultaneously in neurons that are functional synergists but alternate with bursts in their antagonists. These impulses ride on periodic oscillations of membrane potential that occur simultaneously in neurons of each type. A model of the local circuit that generates this motor pattern has been proposed. In this model, each motor neuron is driven alternately by excitatory and inhibitory synaptic currents from nonspiking local interneurons. I tested this model by perturbing individual interneurons and recording synaptic currents and changes in input resistance from each class of motor neuron. I also simulated the synaptic currents that would be observed in a cell subject to different patterns of presynaptic input. When the CNS was actively expressing the swimming motor pattern, changes in the membrane potential of individual local interneurons controlled firing of whole sets of motor neurons. Membrane currents in these motor neurons oscillated in phase with the motor output from their own local circuit. The phases of these oscillations differed in different functional classes of motor neurons. In neurons that could be clamped at the reversal potential of their outward currents, the model predicted that large periodic inward currents would be recorded. I observed no signs of periodic inward currents, even when the outward currents clearly had reversed. These results permit a simplification of the cellular model. They are discussed in the context of neural control of locomotion in crustacea and insects.

In the UK, 90% of patients with hereditary haemochromatosis (HH) are homozygous for HFE C282Y, as are one in 150 people in the general population. However, only a minority of these will develop clinical haemochromatosis. Iron loss modifies iron accumulation but so may other genetic factors. Haptoglobin (Hp) exists as three major types (Hp 1-1, Hp 2-1 or Hp 2-2) and binds free plasma haemoglobin. In men, Hp 2-2 has been shown to be associated with increased macrophage iron accumulation and serum ferritin concentration. Furthermore, the frequency of Hp 2-2 was shown to be increased in patients with HH. We determined Hp types by phenotyping and genotyping 265 blood donor control subjects and 173 subjects who were homozygous for HFE C282Y. The latter group included 66 blood donors lacking clinical features suggestive of haemochromatosis and without a known family history, and 68 patients presenting clinically with haemochromatosis. Hp 2-2 frequencies did not differ in control subjects and C282Y homozygotes. Hp 2-2 was not a risk factor for disease development in HH. To investigate the relationship between iron accumulation and haptoglobin type, we determined transferrin saturation and serum ferritin concentration in 192 male, first-time blood donors aged 20-40 years who lacked both HFE C282Y and H63D. Transferrin saturation and serum ferritin concentrations did not vary with Hp type.

Defective handling of proteins is a central feature of major neurodegenerative diseases. The discovery that neuronal dysfunction or degeneration can be caused by mutations in single cellular proteins has given new opportunities to model the underlying disease processes by genetic modification of cells in vitro or by generation of transgenic animals carrying the disease-causing gene. Recent developments in recombinant viral-vector technology have opened up an interesting alternative possibility, based on direct gene transfer to selected subregions or subsets of neurons in the brain. Using the highly efficient adeno-associated virus or lentivirus vectors, recent reports have shown that overexpression of mutated human huntingtin or alpha-synuclein in neurons in the striatum or substantia nigra induces progressive neuropathology and neurodegeneration, similar to that seen in Huntington's and Parkinson's diseases. Targeted overexpression of disease-causing genes by recombinant viral vectors provides a new and highly flexible approach for in vivo modeling of neurodegenerative diseases, not only in mice and rats but also in primates.

There is interest in the role of iron in age-related diseases such as atherosclerosis. Tissue iron deposition could be harmful, because Fe(2+) can react with H(2)O(2) to form OH(-) radicals and Fe(2+) can react with O(2) to form reactive oxygen species. Free radicals react with cell membranes and cell organelles and could lead to the development of atherosclerosis by initiating lipid peroxidation. Hereditary hemochromatosis provides an opportunity for studying the effects of iron on cardiovascular disease. Some studies have shown that individuals who carried HFE mutations may be at greater risk of developing coronary heart disease than those without the mutations. In contrast, a large number of studies have reported no association between HFE mutations and coronary heart disease. These studies have possible confounding factors, such as the homogeneity of the population in term of geographical origin among others. We studied the relation between HFE mutations and acute myocardial infarction in two case-control studies involving two sets of subjects representing different age groups from different geographic regions in Italy. The first one was composed of 172 older patients (139 males and 33 females; mean age 67) and 207 healthy controls (91 males and 116 females; mean age 46) from Emilia-Romagna. The second one was composed of younger 77 patients (75 males and 2 females; mean age 41) and 172 healthy controls (75 males and 97 females, mean age: 38) from Sicily. All patients were genotyped for ApoE alleles, since the ApoE- epsilon 4 allele is considered a risk factor for cardiovascular diseases and can interfere with other genetic and environmental factors by modifying susceptibility to this disease. DNA typing for C282Y and H63D HFE alleles was performed also. There were no significant differences in frequencies of the different HFE alleles between acute myocardial infarction patients and controls in cohorts of both old and young patients. Also taking into account the presence or absence of the ApoE- epsilon 4 allele, no significant differences in H63D allele frequencies were observed. Thus, our study, performed in two samples of genetically homogeneous patients and controls, does not support the suggestion that HFE mutations may be associated with acute myocardial infarction in susceptible individuals.

Eight hemochromatosis probands with HFE C282Y homozygosity had frequent, severe, or unusual infections and common variable immunodeficiency (CVID) or immunoglobulin (Ig) G subclass deficiency (IgGSD). Thus, we performed serum Ig isotyping and other characterization of 43 additional unselected probands, 5 human leukocyte antigen (HLA)-identical siblings, and 240 consecutive CVID or IgGSD index patients. C282Y allele frequencies were estimated in 58 CVID or IgGSD index patients without hemochromatosis phenotypes and in 341 controls. HLA-A and -B haplotypes and frequencies were determined in all 51 probands, 186 CVID or IgGSD index patients without hemochromatosis phenotypes, and 751 controls. Thirteen unselected probands (30%) had CVID or IgGSD. Among all 21 hemochromatosis probands with CVID (n = 4) or IgGSD (n = 17), Ig subclass deficiency patterns were IgG(1) (n = 5), IgG(1) and IgG(3) (n = 6), IgG(3) (n = 9), and IgG(1), IgG(3), and IgG(4) (n = 1). IgG(2) or IgA deficiency was not detected; one proband had IgM deficiency. Mean values of total IgG, IgG(1), and IgG(3) were significantly lower in probands with CVID or IgGSD. Mean values of age, transferrin saturation, and ferritin at diagnosis and phlebotomy units required to induce iron depletion were similar in probands with or without CVID or IgGSD; phlebotomy had no apparent effect on IgG levels. C282Y frequencies were similar in CVID or IgGSD index cases without hemochromatosis phenotypes and in controls. There was concordance of Ig and hemochromatosis phenotypes in probands and respective HLA-identical siblings. Eight of 240 CVID or IgGSD index patients had hemochromatosis phenotypes and C282Y homozygosity (3 vs 0.7% and 0.2% controls; P < 0.0001, respectively). The frequency of A*03-B*07 was greater in CVID and IgGSD index cases without hemochromatosis phenotypes than in controls (0.0968 vs 0.0546, respectively; P = 0.0032). HLA-A*03-B*07 was the predominant haplotype in probands grouped by presence or absence of CVID or IgGSD. Some probands in each group were A*03-B*07 homozygotes; group A*03-B*07 frequencies were similar. We conclude that serum IgG abnormalities characteristic of CVID or IgGSD are common in hemochromatosis probands, and that the prevalence of hemochromatosis is increased in CVID and IgGSD index cases. These observations could be explained by the increased frequencies of HLA-A*03-B*07 in C282Y homozygotes and in CVID and IgGSD, and by the common occurrence of putative CVID or IgGSD allele(s) on haplotypes bearing C282Y.

Hereditary hemochromatosis is a common inherited disorder characterized by iron overload. A single mutation (C282Y) in the HFE gene is present in 80-95% of cases in populations of northern European extraction. The disorder presents a large phenotypic heterogeneity, and its expression can be influenced by environmental factors. This 1977-2002 study aimed to identify the influence of alcohol consumption on expression of the disease. The authors retrospectively registered 378 C282Y-homozygous patients treated in a blood center of western Brittany, France. In this cohort, 33 patients reported excessive alcohol consumption (8.7%). Those subjects presented significantly increased iron parameters (serum ferritin: 1745.2 vs. 968.7 microg/liter, p< 0.0001; serum iron: 39.9 vs. 36.0 micromol/liter, p = 0.0040; transferrin saturation: 87.1 vs. 80.1%, p = 0.0071) and elevated liver enzymes (alanine aminotransferase: 66.3 vs. 41.1 IU/liter, p = 0.0003; aspartate aminotransferase: 56.2 vs. 34.9 IU/liter, p = 0.0002). Their risk of skin pigmentation was also higher (odds ratio = 3.4, p = 0.0006). Results remained unchanged after adjustment. This study provides precise quantitative data about the impact of alcohol on expression of hereditary hemochromatosis in C282Y-homozygous patients. Excessive alcohol consumption accentuates disease expression and therefore the risk of cirrhosis and cancer. Consequently, these patients should be encouraged to consume very moderate quantities of alcohol.

The benefit of postoperative adjuvant chemotherapy in patients with Dukes' B colorectal cancer is still uncertain and its routine use is not recommended. Prognostic biomarkers may be useful for identifying high-risk patients with resected, node-negative disease, and this stratification may represent an innovative strategy for designing adjuvant chemotherapy trials. Featured prognostic molecular markers can be divided into the following categories: cell proliferation indices (Ki-67, Mib-1, proliferating cell nuclear antigen); oncogenes/tumor suppressor genes [p53, K-ras, Deleted in Colorectal Cancer (DCC), Bcl-2, c-erbB2]; DNA repair (microsatellite instability); markers of angiogenesis (vascular count, vascular endothelial growth factor); markers of invasion/metastasis (plasminogen-related molecules, matrix metalloproteinases); and biochemical markers (thymidylate synthase). Studies that have investigated their prognostic role in Dukes' B colorectal cancer patients are reviewed here. Current data do not provide sufficient evidence for the incorporation of available prognostic biomarkers into clinical practice. However, a biomarker-based approach could be an effective strategy for improving results of postoperative adjuvant treatments in high-risk Dukes' B colorectal cancer patients. Markers of altered DCC function have shown promising prognostic role and sufficient prevalence in retrospective investigations and they deserve further assessment in prospective studies.

In an effort to understand the role of the linker histone in chromatin folding, its structure and location in the nucleosome has been studied by molecular modeling methods. The structure of the globular domain of the rat histone H1d, a highly conserved part of the linker histone, built by homology modeling methods, revealed a three-helical bundle fold that could be described as a helix-turn-helix variant with its characteristic properties of binding to DNA at the major groove. Using the information of its preferential binding to four-way Holliday junction (HJ) DNA, a model of the domain complexed to HJ was built, which was subsequently used to position the globular domain onto the nucleosome. The model revealed that the primary binding site of the domain interacts with the extra 20 bp of DNA of the entering duplex at the major groove while the secondary binding site interacts with the minor groove of the central gyre of the DNA superhelix of the nucleosomal core. The positioning of the globular domain served as an anchor to locate the C-terminal domain onto the nucleosome to obtain the structure of the chromatosome particle. The resulting structure had a stem-like appearance, resembling that observed by electron microscopic studies. The C-terminal domain which adopts a high mobility group (HMG)-box-like fold, has the ability to bend DNA, causing DNA condensation or compaction. It was observed that the three S/TPKK motifs in the C-terminal domain interact with the exiting duplex, thus defining the path of linker DNA in the chromatin fiber. This study has provided an insight into the probable individual roles of globular and the C-terminal domains of histone H1 in chromatin organization.

<h4>Aim</h4>To explore the prevalence of autoimmune gastritis in chronic hepatitis C virus (HCV) patients and the influence of alpha-interferon (IFN) treatment on autoimmune gastritis.<h4>Methods</h4>We performed a prospective study on 189 patients with positive anti-HCV and viral RNA enrolled in a 12-month IFN protocol. We evaluated: a) the baseline prevalence of autoimmune gastritis, b) the impact of IFN treatment on development of biochemical signs of autoimmune gastritis (at 3, 6 and 12 months), c) the evolution after IFN withdrawal (12 months) in terms of anti-gastric-parietal-cell antibodies (APCA), gastrin, anti-thyroid, and anti-non-organ-specific antibodies.<h4>Results</h4>APCA positivity and 3-fold gastrin levels were detected in 3 (1.6 %) and 9 (5 %) patients, respectively, at baseline, in 25 (13 %) and 31 (16 %) patients at the end of treatment (both P<0.001, vs baseline), and in 7 (4 %) and 14 (7 %) patients 12 months after withdrawal (P=0.002 and P=0.01 respectively, vs baseline; P=not significant vs end of treatment). The development of autoimmune gastritis was strictly associated with the presence of autoimmune thyroiditis (P =0.0001), no relationship was found with other markers of autoimmunity.<h4>Conclusion</h4>In HCV patients, IFN frequently precipitates latent autoimmune gastritis, particularly in females. Following our 12-month protocol, the phenomenon generally regressed. Since APCA positivity and high gastrin levels are associated with the presence of antithyroid antibodies, development of autoimmune thyroiditis during IFN treatment may provide a surrogate preliminary indicator of possible autoimmune gastritis to limit the need for invasive examinations.

<h4>Aim</h4>To investigate the safety and efficacy of long-term combination therapy with alpha interferon and lamivudine in non-responsive patients with anti-HBe-positive chronic hepatitis B.<h4>Methods</h4>34 patients received combination treatment (1 month lamivudine, 12 month lamivudine+interferon, 6 month lamivudine), 24 received lamivudine (12 months), 24 received interferon (12 months). Interferon was administered at 6 MU tiw and lamivudine at 100 mg orally once daily. Patients were followed up for 6 months after treatment.<h4>Results</h4>At the end of treatment, HBV DNA negativity rates were 88 % with lamivudine+interferon, 99 % with lamivudine and 55 % with interferon, (P=0.004, combination therapy vs. interferon, and P=0.001 lamivudine vs. interferon), and serum transaminase normalization rates were 84 %, 91 % and 53 % (P=0.01 combination therapy vs. interferon, and P=0.012 lamivudine vs. interferon). Six months later, HBV DNA negativity rates were 44 % with lamivudine+interferon, 33 % with lamivudine and 25 % with interferon, and serum transaminase normalization rates were 61 %, 42 % and 45 %, respectively, without statistical significance. No YMDD variants were observed with lamivudine+interferon (vs. 12 % with lamivudine). The combination therapy appeared to be safe.<h4>Conclusion</h4>Although viral clearance and transaminase normalization are slower with long-term lamivudine+interferon than that with lamivudine alone, the combination regimen seems to provide more lasting benefits and to protect against the appearance of YMDD variants. Studies with other regimens regarding sequence and duration are needed.

<h4>Introduction</h4>It has been suggested that a proopiomelanocortin peptide beta-endorphin may play an important role in temperature regulation and therefore have specific heat stroke response patterns.<h4>Methods</h4>The purpose of this study was to examine the response patterns of plasma beta-endorphin at baseline, during a 7-d exercise-heat acclimation period (HA; 90 min x d(-1), 40 degrees C), and during a 6-h exercise-heat tolerance test (HTT; 6 h, 40 degrees C) performed before and after HA. Subjects were nine previously diagnosed heatstroke patients (P) and eight matched controls (C). No differences between the two groups were observed at rest, prior to HTT.<h4>Results</h4>Plasma beta-endorphin concentration in P significantly increased above resting values at 2, 4, and 6 h of HTT and were significantly greater than C responses. During HA tests, a significantly higher beta-endorphin concentration was observed in P before exercise on day 1 and day 7, but not on day 4. A significant increase occurred after exercise in both groups, on all days of HA; no differences were observed between the groups on days 4 and 7.<h4>Discussion</h4>These results demonstrate that the opioid peptide responses in individuals with recent exertional heatstroke were greater than control subjects during the initial 6-h HTT. Decreased physical training among P may have contributed to these results. However, 7 d of HA abolished these between-group differences by reducing the magnitude of beta-endorphin responses in P, indicative of an adaptation of pituitary function.

<h4>Purpose</h4>Examine the penetrance (defined by high transferrin saturation [TS]) of C282Y and H63D in the U.S. population.<h4>Methods</h4>5171 participants from the Third National Health and Nutrition Examination Survey, 1992 to 1994.<h4>Results</h4>77.1% (95% confidence interval [CI], 2.3, 95.1) of men and 51.9% (95% CI, 0, 84.2) of women with C282Y homozygosity had high TS. The associations of H63D homozygosity with high TS were stronger in people aged 50 years or older than in younger persons. Among Mexican-Americans, simple H63D heterozygosity was associated with high TS.<h4>Conclusions</h4>The associations between HFE genotype and high TS may vary by sex, age, and ethnic group.

PURPOSE The increasing use of the Internet to obtain genetics information and to order medical services without a prescription, combined with a rise in direct-to-consumer marketing for genetic testing, suggests the potential for the Internet to be used to sell genetic services. METHODS A systematic World Wide Web search was conducted in May 2002 to assess the availability of genetic services sold directly to consumers on the Internet. RESULTS Out of 105 sites that offered genetic services directly, most offered non-health-related services, including parentage confirmation testing (83%), identity testing (56%), and DNA banking (24%); however, health-related genetic tests were offered through 14 sites (13%). The health-related genetic tests available ranged from standard tests, such as hemochromatosis and cystic fibrosis, to more unconventional tests related to nutrition, behavior, and aging. Of these 14 sites, 5 described risks associated with the genetic services and 6 described the availability of counseling. CONCLUSIONS The availability of direct sales of health-related genetic tests creates the potential for inadequate pretest decision making, misunderstanding test results, and access to tests of questionable clinical value.

No abstract available.

In a prospective-longitudinal study of a representative birth cohort, we tested why stressful experiences lead to depression in some people but not in others. A functional polymorphism in the promoter region of the serotonin transporter (5-HT T) gene was found to moderate the influence of stressful life events on depression. Individuals with one or two copies of the short allele of the 5-HT T promoter polymorphism exhibited more depressive symptoms, diagnosable depression, and suicidality in relation to stressful life events than individuals homozygous for the long allele. This epidemiological study thus provides evidence of a gene-by-environment interaction, in which an individual's response to environmental insults is moderated by his or her genetic makeup.

<h4>Background</h4>In the Italian general population, prevalence of C282Y is lower than in Northern European countries. We hypothesised a higher prevalence of C282Y in Northern than in Central and Southern Italy. We previously identified a nonsense mutation (W169X) in haemochromatosis probands originating from a Northern Italian region (Brianza).<h4>Aim</h4>To define the prevalence of HFE mutations in that region. Subjects and methods. A total of 1132 unrelated blood donors from the Blood Banks of Monza and Merate were investigated for C282Y, H63D, S65C and W169X mutations by PCR-restriction assays. A total of 300 were also tested for rare HFE and TFR2 mutations by reverse-hybridization test strips.<h4>Results</h4>Two C282Y homozygotes, eight C282Y/H63D compound heterozygotes, 27 H63D homozygotes and one W169X heterozygote were found. The allele frequencies of C282Y, H63D, S65C, and W169X were 3.2, 13.4, 1.3, and 0.04%, respectively.<h4>Conclusions</h4>Our results confirm the existence of a decreasing frequency of C282Y allele from upper to lower Northern Italy. This difference is probably related to the larger Celtic component of upper Northern Italian populations in which screening studies for haemochromatosis may even be cost effective. W169X, due to its severity, should be looked for in all haemochromatosis patients of Northern ancestry with an incomplete HFE genotype.

Sleep deprivation exerts transient antidepressant efficacy. As a potential mechanism of action an enhancement of serotonergic and dopaminergic neurotransmission within the CNS is discussed. Because genetic variations influencing neurotransmission could have an impact on therapeutic outcome and stability of improvement, we investigated the functional polymorphism of the serotonin transporter (5-HTT) gene, the 5-HTT-linked polymorphic region (5-HTTLPR), to examine the serotonergic pathway. We included 56 patients with major depression (DSM-IV). Psychiatric ratings including the HAM-D21 and HAM-D6 scale were assessed on the day prior to partial sleep deprivation (PSD) and on day 1 and 2 after PSD and related to the different genotypes. The 5-HTTLPR variants were determined following PCR amplification using genomic DNA. 58.1% of the patients were responders to PSD. A significant overall reduction in depression scores could be observed on day 1. Subdivision according 5-HTTLPR gene variants showed no differences in clinical outcome on day 1. As expected the therapeutical effect of PSD was only transient and most patients experienced an exacerbation of depressive symptoms on day 2. 5-HTTLPR variants had no influence on reduction of depressive symptoms on day 2 or relapse on day 3. Thus, the previously reported influence of the serotonin transporter gene on PSD outcome in bipolar depression could not be confirmed in unipolar depressed patients.

Huntingtin, a protein altered by polyglutamine expansion in Huntington's disease (Httexp), forms a signaling complex with the InsP3R, an intracellular calcium channel, and Htt-associated protein 1A (HAP1A). The addition of Httexp increases the InsP3R sensitivity to InsP3, which subsequently makes neurons hyperresponsive to stimulation and presumably more prone to neurodegenerative processes.

Huntington's disease (HD) is caused by polyglutamine expansion (exp) in huntingtin (Htt). The type 1 inositol (1,4,5)-triphosphate receptor (InsP3R1) is an intracellular calcium (Ca2+) release channel that plays an important role in neuronal function. In a yeast two-hybrid screen with the InsP3R1 carboxy terminus, we isolated Htt-associated protein-1A (HAP1A). We show that an InsP3R1-HAP1A-Htt ternary complex is formed in vitro and in vivo. In planar lipid bilayer reconstitution experiments, InsP3R1 activation by InsP3 is sensitized by Httexp, but not by normal Htt. Transfection of full-length Httexp or caspase-resistant Httexp, but not normal Htt, into medium spiny striatal neurons faciliates Ca2+ release in response to threshold concentrations of the selective mGluR1/5 agonist 3,5-DHPG. Our findings identify a novel molecular link between Htt and InsP3R1-mediated neuronal Ca2+ signaling and provide an explanation for the derangement of cytosolic Ca2+ signaling in HD patients and mouse models.

The different serotonin (5-HT) receptors, including the serotonin transporter (5-HTT), are excellent candidate genes for suicide and suicidal behavior, and thus, they have been investigated in a large number of allelic association studies. The individual results of these studies have been inconsistent and definite conclusions are difficult to establish. A reliable method for assessing individual studies and generating combined results is provided by systematic reviews using meta-analytical techniques. In this study, we carried out a systematic review of studies investigating 5-HT receptors and the 5-HTT in suicidal behavior. Studies were identified by means of MEDLINE database searches and by scanning reference lists. More than 190 articles were reviewed and 26 met the inclusion criteria. In all, 14 studies investigated six different 5-HT receptor loci and 12 studies investigated the 5-HTT promoter 44 bp insertion/deletion polymorphism. Two specific meta-analyses were carried out, pooling studies investigating the 5-HT2A 102 T/C and the 5-HTT promoter loci that included, respectively, a total of 1599 and 2539 subjects. The combined evidence was significant for association with the 5-HTT locus (Mantel-Haenszel weighted odds ratio (M-H(w) OR)=1.17 CI : 1.04-1.32, P=0.009), but not for the 5-HT2A 102 T/C variant (M-H(w) OR)=1.09 CI : 0.93-1.27, P=0.319). The 5-HTT result was robust and remained significant following sensitivity analysis, suggesting that 5-HTT may play a role in the predisposition to suicide.

Densities of serotonin transporters (5-HTT) in the postmortem neocortex of behaviorally assessed Alzheimer's disease (AD) patients and aged controls were measured by radioligand binding with [3H]citalopram. It was found that 5-HTT sites in the temporal cortex of AD patients with prominent antemortem anxiety were unaltered compared with controls, but were reduced in non-anxious AD subjects. Furthermore, homozygosity for the high activity allele of a functional polymorphism in the 5-HTT gene promoter region (5-HTTLPR) was associated with both increased [3H]citalopram binding and occurrence of anxiety in the AD subjects. Since serotonin-synthesizing neurons are known to be lost in the AD cortex, this study suggests that the preservation of 5-HTT may exacerbate serotonergic deficits and underlie anxiety symptoms in AD.

<h4>Background</h4>There is a heritable component to suicidal behaviour, encouraging the search for the associated risk alleles. Given the putative role of the 5-HT (5-hydroxytryptamine; serotonin) system in suicidal behaviour, serotonergic genes are leading candidates. In particular, several studies have reported an association with variants in the tryptophan hydroxylase (TPH) gene.<h4>Method</h4>We studied six serotonergic gene polymorphisms in a well-characterized sample of 129 deliberate self-harm subjects and 329 comparison subjects. The polymorphisms were TPH (A779C), 5-HT transporter (5-HTT, LPR S/L), monoamine oxidase A (MAOA G941T), 5-HT1B receptor (HTR1B G861C), 5-HT2A receptor (HTR2A T102C), and 5-HT2C receptor (HTR2C Cys23Ser). Genotyping was done using polymerase chain reaction (PCR)-based assays. The primary analyses compared allele and genotype frequencies between cases and controls. There were a limited number of planned secondary analyses within the deliberate self-harm group.<h4>Results</h4>The TPH A779 allele was more common in deliberate self-harm subjects than in controls (OR 1.38, 95% CI 1.02-1.88; P = 0.03). None of the other polymorphisms was associated with deliberate self-harm. Within the deliberate self-harm group there were no associations with impulsivity, suicide risk, lifetime history of depression, or family history of deliberate self-harm.<h4>Conclusions</h4>Our data extend the evidence that allelic variation in the TPH gene is a risk factor for deliberate self-harm. No evidence was found to implicate the other polymorphisms.

AII amacrine cells play a critical role in the high-fidelity signal transmission pathways involved with nighttime vision. The temporal properties of the light responses strongly depend on the transfer function at different synaptic stages and consequently on presynaptic calcium influx. AII light responses are complex waveforms generated by graded input, they comprise Na+-based spikes as well as a sustained component, and they are transferred to graded cone bipolar cells. It is, therefore, of interest to determine the properties of AII voltage-dependent calcium channels (VDCCs) to establish whether these cells express N-type and/or P/Q-type VDCCs, characteristic of spiking neurons, or whether they are more like graded neurons, which mostly use L-type VDCCs. We combined electrophysiological, molecular biological, and imaging techniques to characterize calcium currents and their sites of origin in mouse AII amacrine cells. Calcium currents activated at potentials more positive than -60 mV (maximally between -50 and -20 mV) and inactivated slowly. These currents were blocked by dihydropyridine (DHP) antagonists and were enhanced by the DHP agonist BayK 8644. Single-cell RT-PCR analysis of mRNA encoding for different calcium channel alpha subunits in AIIs revealed a consistent expression of the alpha1-D subunit. Calcium imaging of AII cells showed that the greatest change in intracellular calcium occurred in the lobular appendages, with minor changes being observed in the arboreal dendrites. Depolarization-induced calcium rises were also modulated by DHPs, suggesting that a particular kind of L-type VDCC, mainly localized to the lobular appendages, enables these spiking-capable neurons to release neurotransmitter in a sustained manner onto OFF-cone bipolar cells.

Huntington's disease (HD) is caused by a polyglutamine expansion in the disease protein huntingtin. The polyglutamine expansion causes huntingtin to interact abnormally with a number of proteins. However, it is unclear whether, and how, huntingtin-associated proteins are involved in the neurodegeneration in HD. Here, we show that huntingtin-associated protein-1 (HAP1), which is involved in intracellular trafficking of epidermal growth factor receptor (EGFR), is highly expressed in the hypothalamus. Mice lacking HAP1 die after birth because of depressed feeding activity. Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling staining and electron microscopic examination revealed the degeneration in hypothalamic regions that control feeding behavior. Hypothalamic degeneration was also observed in HD transgenic mice that have a significant loss of body weight. Inhibition of HAP1 expression decreases EGFR signaling and cell viability, whereas overexpression of HAP1 enhances this signaling activity and inhibits mutant huntingtin-mediated cytotoxicity. These results suggest that the effect of mutant huntingtin on HAP1 and EGFR signaling may contribute to the hypothalamic neurodegeneration and loss of body weight in HD.

<h4>Background</h4>The deleted in colorectal cancer (DCC) gene has been shown to be frequently deleted or its expression reduced or absent in glioblastomas, colorectal, gastro-intestinal, pancreatic and prostatic tumors. In the present study, we investigated the expression of DCC in surgical specimen from 75 patients with primary breast cancer.<h4>Methods</h4>The expression of the DCC, estrogen receptors (ER), and progesterone receptors (PR) was studied in 75 surgical specimens of primary breast cancer using an immunohistochemical method. To evaluate the outcomes of the breast cancer patients, we followed up the patients during minimum of 10 years.<h4>Results</h4>Reduced or loss of expression of DCC was identified in 45 out of 75 samples. There were significant differences between cases without metastasis or local recurrences versus these with metastasis or local recurrences (p = 0.006), and between patients alive with no evidence of malignancy versus those with recurrence or dead of disease (p = 0.005). There were no significant differences between the DCC status and age, sex, tumor location, stage, grade, or proportion of patients who received adjuvant therapy.<h4>Conclusions</h4>These findings suggest that a decrease in DCC expression may influence the prognosis of breast carcinoma patients.

Molecular epidemiologic studies suggest genetic factors in the etiology of smoking behavior. Dopamine receptor genes, transporter genes (serotonin and dopamine), and other genes related to metabolism of nicotine are plausible functional candidate genes. Research examining the role of allelic variation in those genes is being actively pursued with respect to nicotine dependence as well as personality characteristics and mental disorders. Some of these genes were reported to be connected with schizophrenia. Although nicotine dependence itself is one of the mental disorders according to the fourth edition of Diagnostic and Statistical Manual of Mental Disorders and the World Health Organization's International Statistical Classification of Diseases and Related Health Problems-10 nomenclature, the high comorbidity between nicotine dependence and other mental disorders such as schizophrenia or affective disorders has been noted. Therefore, the relationship between those mental disorders and tobacco addiction should be cleared up considering the interactive effect of genetic and environmental factors.

Hereditary hemochromatosis (HH) is a frequent recessive disorder of iron metabolism characterised by systemic iron overload. In Northern Europe, more than 90% of HH patients are homozygous for a mis-sense mutation (C282Y) in the HFE1 gene product. The HFE protein is the heavy chain of a MHC class I-related molecule and associates with beta2 microglobulin and the transferrin receptor. Its precise roles in iron metabolism and in the pathophysiology of HH are still unclear. In order to identify the cellular processing of HFE, an important step towards the understanding of the function of the protein, we stably over-expressed the wild type and mutated forms fused to the Green Fluorescent Protein in a melanocytic MHC class I expressing cell line, the Mel Juso cell line. In wild type and mutant clones, the fusion proteins were not detected at the cell surface but only in the cytoplasm. Their sub-cellular localisation was determined by co-labelling of cells with organite-specific antibodies and confocal microscopy. HFE-GFP followed initially HLA class I intracellular processing but co-localised with transferrin in early endosomes without recycling at the cell surface. The C282Y-GFP fusion protein followed a different folding pathway to exit endoplasmic reticulum. Over-expression of the wild-type protein lead to a decrease in diferric transferrin uptake. Our model will be of use in the elucidation of the functional interaction between intracellular HFE and iron transporters transferrin/transferrin receptor complexes and Slc11A2 (also named N-Ramp2 or DMT1) in different endosomal compartments.

Expression of hereditary hemochromatosis as well as predisposition to iron overload syndrome and sporadic porphyria cutanea tarda are currently believed to be associated with the inheritance of certain allelic variants of the HFE gene. Allele frequencies of the C282Y (845A) and H63D (187G) mutations in the HFE gene in human populations of different races are remarkably different, and the prevalence of the S65C (193T) mutation is still poorly studied. In the present study we estimated allele frequencies of HFE mutations in Russians and in a number of Siberian ethnic indigenous populations. In Russians, allele frequencies of the C282Y, H63D and S65C mutations were 3.7, 13.3 and 1.7%, respectively. These values were similar to those observed in populations of Europe. The C282Y mutation was not detected in the population samples of Siberian ethnic groups, including Mansis, Khantys (Finno-Ugric group), Altaians, and Nivkhs (Mongoloids), suggesting that the frequency of this allele in the populations examined was lower than 1%. The frequency of the C282Y allele in the Tuvinian and Chukchi samples (Mongoloids) constituted 0.45 and 0.8%, respectively. Furthermore, pedigree analysis of both Chukchi carriers discovered showed that some of their ancestors were from other ethnic groups. Low frequencies of this allelic variant is typical of many Eastern Asian populations, which are also characterized by rather low frequencies of the H63D variant. In contrast, in some ethnic groups of Western Siberia allelic frequency of the H63D mutation is rather high, constituting 8.7% in Altaians, 15.5% in Mansis, and 11.3% in Khantys. The frequency of this allele in Tuvinians, Nivkhs, and Chukchis constituted 5, 4.7, and 0.8%, respectively. These findings make it possible to estimate the proportion of individuals predisposed to iron overload syndrome in different Russian ethnic groups. The HFE allele frequency distribution patterns observed in the populations examined pointed to pre-Celtic appearance of the CY82 allele. It also provides elucidation of the evolutionary genetic relationships between Siberian ethnic groups and the contemporary populations of Eastern and Western Europe.

<h4>Background</h4>Ensuring the appropriate use of migraine therapies is an important consideration for care providers, patients, employers, and managed care organizations (MCOs) because of the high cost of treatment for this fairly prevalent disabling disease. A review of utilization of serotonin 5-HT1 receptor agonists (triptans) in an MCO determined that about 24% of the patients who received triptan therapy exceeded the manufacturers. recommendations regarding the maximum daily dose and safe treatment guidelines in a 30-day period. An initiative was designed to manage the coverage of migraine abortive therapies with the anticipated outcome of decreasing potential misuse or overuse of the medications.<h4>Objective</h4>The objective of this retrospective, observational study was to determine the impact of a monthly drug-specific milligram coverage maximum (quantity limit) on serotonin 5-HT1 receptor agonists (triptans) and dihydroergotamine (DHE) nasal spray on the utilization and costs of migraine care in an MCO with approximately 600000 covered members.<h4>Methods</h4>A longitudinal, retrospective cohort analysis was conducted. All migraine-related services were analyzed, including outpatient medical visits, emergency department utilization, inpatient hospitalizations, and outpatient prescription drug use. The analysis was conducted using medical and pharmacy administrative claims. Analysis of data was performed for the period 12 months prior (October 1999 to September 2000) and 18 months postimplementation of the monthly drug-specific milligram coverage maximum (October 2000 through March 2002).<h4>Results</h4>Imposition of a monthly coverage maximum for migraine-abortive therapies was associated with a 26.1% reduction in overall per- patient-per-month (PPPM) medical costs for migraine care, from US dollars 55.52 PPPM to US dollars 41.02 PPPM (P<0.01). Utilization of serotonin 5-HT1 receptor agonists and DHE nasal spray declined by 16.7%, from 0.18 prescriptions PPPM to 0.15 prescriptions PPPM (P=0.039), and direct drug costs declined by 28.8%, from US dollars 29.18 PPPM to US dollars 20.78 PPPM (P<0.001). Utilization and costs of outpatient and inpatient migraine-related medical services declined by 40% from US dollars 16.58 PPPM in the preperiod to US dollars 9.94 PPPM in the postperiod (P<0.001).<h4>Conclusion</h4>A monthly drug-specific milligram coverage maximum was associated with significant reduction in drug costs and utilization of serotonin 5-HT1 receptor agonists (triptans) and DHE nasal spray. Utilization and costs of migraine-related medical services also declined after implementation of the coverage maximum for triptans and DHE nasal spray. The monthly drug-specific milligram coverage maximum appeared to have been successful in managing utilization of triptans and DHE nasal spray, including reduction of overall costs of migraine-related medical services and direct drug costs.

<h4>Background & aims</h4>Iron overload is observed frequently in chronic liver disease, and some studies have suggested that chronic iron overload may contribute to the pathogenesis of hepatocellular carcinoma (HCC). Heterozygosity for hereditary hemochromatosis (HH) is associated with increased body iron stores. The discovery of the HH gene HFE has enabled identification of the heterozygote status. The aim of this study was to evaluate if heterozygosity for HH is a risk factor for HCC.<h4>Methods</h4>The C282Y and the H63D mutation of the HFE gene were analyzed in 137 patients with HCC and no history of HH, 107 patients with cirrhosis without HCC and 126 healthy controls. Hepatic iron content was measured by using a semiquantitative histologic score.<h4>Results</h4>Seventeen of 137 HCC patients (12.4%) were C282Y heterozygote, compared with only 4 of 107 (3.7%) cirrhotic patients without HCC and 6 of 126 (4.8%) healthy controls (P < 0.05). The frequency of the H63D mutation showed no significant differences. C282Y heterozygote HCC patients had significantly higher levels of serum ferritin and transferrin saturation than C282Y wild-type patients (793 +/- 122 vs. 355 +/- 23 ng/mL, and 42.3% +/- 7.3% vs. 29.2% +/- 1.7%, respectively), and significantly higher iron deposition in HCC as well as in nontumorous liver tissue.<h4>Conclusions</h4>The C282Y heterozygous genotype is significantly more common in HCC patients and is associated with significantly increased intrahepatic iron deposition and systemic iron stores. These results suggest that C282Y heterozygosity plays a role in liver iron deposition and could contribute to hepatocarcinogenesis via the accumulation of potentially carcinogenic iron. These findings may have implications for HCC screening and prevention strategies.