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<h4>Background</h4>Iron has been implicated in the pathogenesis of various disorders. Mutations in the HFE gene are associated with an increase in serum iron parameters. The aim of this study was to estimate the heritability in serum iron parameters explained by HFE.<h4>Methods</h4>Ninety families (980 subjects) were included in the present analysis. Heritability estimation was conducted using the variance component method. The likelihood ratio test was used to compare models. Phenotypic and genetic correlations between serum iron parameters were calculated.<h4>Results</h4>The heritability (h(2) +/- SE) estimates were 0.23 +/- 0.07 (p < 0.0001) for iron, 0.29 +/- 0.09 (p < 0.0001) for ferritin and 0.28 +/- 0.07 (p < 0.0001) for transferrin saturation while adjusting for age, age(2) and sex. The HFE genotypes explained between 2 to 6% of the sex and age-adjusted variance in serum iron, ferritin and transferrin saturation. There was a high genetic correlation between serum iron parameters, suggesting pleiotropy between these traits.<h4>Conclusion</h4>A substantial proportion of the variance of iron, ferritin and transferrin saturation can be explained by additive genetic effects, independent of sex and age. The HFE genotypes explained a considerable proportion of serum iron parameters and may be an important factor in the complex iron network.

Mitochondrial tRNAs (mtRNAs) often lack domains and posttranscriptional modifications that are found in cytoplasmic tRNAs. These structural and chemical elements normally stabilize the folding of cytoplasmic tRNAs into canonical structures that are competent for aminoacylation and translation. For example, the dihydrouridine (D) stem and loop domain is involved in the tertiary structure of cytoplasmic tRNAs through hydrogen bonds and a Mg2+ bridge to the ribothymidine (T) stem and loop domain. These interactions are often absent in mtRNA because the D-domain is truncated or missing. Using gel mobility shift analyses, UV, circular dichroism and NMR spectroscopies and aminoacylation assays, we have investigated the functional folding interactions of chemically synthesized and site-specifically modified mitochondrial and cytoplasmic tRNAs. We found that Mg2+ is critical for folding of the truncated D-domain of bovine mtRNAMet with the tRNA's T-domain. Contrary to the expectation that Mg2+ stabilizes RNA folding, the mtRNAMet D-domain structure was unfolded and relaxed, rather than stabilized in the presence of Mg2+. Because the D-domain is transcribed prior to the T-domain, we conclude that Mg2+ prevents misfolding of the 5'-half of bovine mtRNAMet facilitating its correct interaction with the T-domain. The interaction of the mtRNAMet D-domain with the T-domain was enhanced by a pseudouridine located in either the D or T-domains compared to that of the unmodified RNAs (Kd=25.3, 24.6 and 44.4 microM, respectively). Mg2+ also affected the folding interaction of a yeast mtRNALeu1, but had minimal effect on the folding of an Escherichia coli cytoplasmic tRNALeu. The D-domain modification, dihydrouridine, facilitated mtRNALeu folding. These data indicate that conserved modifications assist and stabilize the formation of the functional mtRNA tertiary structure.

The recently discovered repulsive guidance molecule c (RGMc or hemojuvelin) gene encodes a putative glycosylphosphatidylinositol (GPI)-anchored protein that is expressed in striated muscle and in liver. Mutations in this gene have been linked to the severe iron storage disease, juvenile hemochromatosis, although the mechanisms of action of RGMc in iron metabolism are unknown. As a first step toward understanding the molecular physiology of this protein, we studied its biosynthesis, processing and maturation. Production of RGMc occurs as an early and sustained event during skeletal muscle differentiation in culture and is secondary to RGMc gene activation. As assessed by pulse-chase studies and cell-surface labeling experiments, two classes of GPI-anchored and glycosylated RGMc molecules are targeted to the membrane and undergo distinct fates. Full-length RGMc is released from the cell surface and accumulates in extracellular fluid, where its half-life exceeds 24 hours. By contrast, the predominant membrane-associated isoform, a disulfide-linked heterodimer composed of N- and C-terminal fragments, is not found in the extracellular fluid, and is short-lived, as it disappears from the cell surface with a half-life of <3 hours after interruption of protein synthesis. A natural disease-associated RGMc mutant, with valine substituted for glycine at residue 320 (313 in mouse RGMc), does not undergo processing to generate the heterodimeric membrane-linked isoform of RGMc, and is found on the cell surface only as larger protein species. Our results define a series of biosynthetic steps leading to the normal production of different RGMc isoforms in cells, and provide a framework for understanding the biochemical basis of defects in the maturation of RGMc in juvenile hemochromatosis.

To understand the molecular basis for the rapid and robust memory T-cell responses, we examined gene expression and chromatin modification by histone H3 lysine 9 (H3K9) acetylation in resting and activated human naive and memory CD8(+) T cells. We found that, although overall gene expression patterns were similar, a number of genes are differentially expressed in either memory or naive cells in their resting and activated states. To further elucidate the basis for differential gene expression, we assessed the role of histone H3K9 acetylation in differential gene expression. Strikingly, higher H3K9 acetylation levels were detected in resting memory cells, prior to their activation, for those genes that were differentially expressed following activation, indicating that hyperacetylation of histone H3K9 may play a role in selective and rapid gene expression of memory CD8(+) T cells. Consistent with this model, we showed that inducing high levels of H3K9 acetylation resulted in an increased expression in naive cells of those genes that are normally expressed differentially in memory cells. Together, these findings suggest that differential gene expression mediated at least in part by histone H3K9 hyperacetylation may be responsible for the rapid and robust memory CD8(+) T-cell response.

A simple compartmental model is developed for investigating the mechanism of iron homeostasis. In contrast to previous mathematical models of iron metabolism, the liver is included as a key site of iron regulation. Compartments for free iron in blood, diferric transferrin (Tf) in blood, hepatocytes, red blood cells, and macrophages are included, and their roles in iron regulation are explored. The function of hepcidin in regulating iron absorption is modeled through an inverse relationship between hepatocyte transferrin receptor 2 (TfR2) levels and the rate of iron export processes mediated by ferroportin (Fpn). Simulations of anemia and erythropoiesis stimulation support the idea that the iron demands of the erythroid compartment can be communicated through diferric Tf. The iron-responsive element of Fpn is found to be important for stabilizing intracellular iron stores in response to changing iron demands and allowing proper iron regulation through diferric Tf. The contribution of iron dysregulation to the pathogenesis of iron overload disorders is also investigated. It is shown that the characteristics of HFE hemochromatosis can be reproduced by increasing the setpoint of iron absorption in the duodenum to a level where the system cannot downregulate iron absorption to meet the iron excretion rate.

A novel endogenous beta-1,4-endoglucanase (Ag-EGase III) gene belonging to the glycoside hydrolase family (GHF) 5 was cloned from the mulberry longicorn beetle, Apriona germari. The Ag-EGase III gene spans 1061 bp and consists of a single exon coding for 325 amino acid residues. The Ag-EGase III showed 89% protein sequence identity to another beetle, Psacothea hilaris, cellulase belonging to GHF 5. The Ag-EGase III has the potential proton donor and nucleophile amino acids conserved in GHF 5 and two putative N-glycosylation sites. Northern blot and Western blot analyses showed that Ag-EGases were expressed in the gut; Ag-EGase III and Ag-EGase I were expressed in three gut regions, and no Ag-EGase II was found in hindgut, indicating that the foregut and midgut are the prime sites for cellulase synthesis in A. germari larvae. The cDNA encoding Ag-EGase III was expressed as a 47-kDa polypeptide in baculovirus-infected insect Sf9 cells and the enzyme activity of the purified recombinant Ag-EGase III was approximately 1037 U per mg of recombinant Ag-EGase III. The enzymatic property of the purified recombinant Ag-EGase III showed the highest activity at 55 degrees C and pH 6.0, and was stable at 60 degrees C at least for 10 min. In addition, the N-glycosylation of Ag-EGase III was revealed by treatment with tunicamycin of recombinant virus-infected insect Sf9 cells and with endoglycosidase F of purified recombinant Ag-EGase III, demonstrating that the carbohydrate moieties are not necessary for enzyme activity.

Small interfering RNAs (siRNAs), the guides that direct RNA interference (RNAi), provide a powerful tool to reduce the expression of a single gene in human cells. Ideally, dominant, gain-of-function human diseases could be treated using siRNAs that specifically silence the mutant disease allele, while leaving expression of the wild-type allele unperturbed. Previous reports suggest that siRNAs can be designed with single nucleotide specificity, but no rational basis for the design of siRNAs with single nucleotide discrimination has been proposed. We systematically identified siRNAs that discriminate between the wild-type and mutant alleles of two disease genes: the human Cu, Zn superoxide dismutase (SOD1) gene, which contributes to the progression of hereditary amyotrophic lateral sclerosis through the gain of a toxic property, and the huntingtin (HTT) gene, which causes Huntington disease when its CAG-repeat region expands beyond approximately 35 repeats. Using cell-free RNAi reactions in Drosophila embryo lysate and reporter assays and microarray analysis of off-target effects in cultured human cells, we identified positions within an siRNA that are most sensitive to mismatches. We also show that purine:purine mismatches imbue an siRNA with greater discriminatory power than other types of base mismatches. siRNAs in which either a G:U wobble or a mismatch is located in the "seed" sequence, the specialized siRNA guide region responsible for target binding, displayed lower levels of selectivity than those in which the mismatch was located 3' to the seed; this region of an siRNA is critical for target cleavage but not siRNA binding. Our data suggest that siRNAs can be designed to discriminate between the wild-type and mutant alleles of many genes that differ by just a single nucleotide.

Huntington disease (HD) is an autosomal dominant neurodegenerative disorder characterised by chorea, cognitive impairment, dementia and personality changes, caused by the expansion of a CAG repeat in the HD gene. Often, patients with a similar clinical presentation do not carry expansions of the CAG repeat in this gene [Huntington disease-like (HDL) patients]. We report the genetic analysis of 107 Portuguese patients with an HDL phenotype. The HDL genes PRNP and JPH3, encoding the prion protein and junctophilin-3, respectively, were screened for repeat expansions in these patients. Given the partial clinical overlap of SCA17, DRPLA and neuroferritinopathy with HD, their causative genes (TBP, ATN1, and FTL, respectively) were also analysed. Finally, repeat expansions in two candidate genes, CREBBP and POU3F2, which encode the nuclear transcriptional coactivator CREB-binding protein and the CNS-specific transcription factor N-Oct-3, respectively, were also studied. Expansions of the repetitive tracts of the PRNP, JPH3, TBP, ATN1, CREBBP and POU3F2 genes were excluded in all patients, as were sequence alterations in the FTL gene. Since none of the genes already included in the differential diagnosis of HD was responsible for the disease in our sample, the genetic heterogeneity of the HDL phenotype is still open for investigation.

The enteric nervous system (ENS) forms from migrating neural crest-derived precursors that differentiate into neurons and glia, aggregate into ganglion cell clusters, and extend neuronal processes to form a complex interacting network that controls many aspects of intestinal function. Bone morphogenetic proteins (BMPs) have diverse roles in development and influence the differentiation, proliferation, and survival of ENS precursors. We hypothesized that BMP signaling might also be important for the ENS precursor migration, ganglion cell aggregation, and neurite fasciculation necessary to form the enteric nervous system. We now demonstrate that BMP signaling restricts murine ENS precursors to the outer bowel wall during migration. In addition, blocking BMP signaling causes faster colonization of the murine colon, reduces ganglion cell aggregation, and reduces neurite fasciculation. BMP signaling also influences patterns of neurite extension within the developing bowel wall. These effects on ENS precursor migration and neurite fasciculation appear to be mediated at least in part by increased polysialic acid addition to neural cell adhesion molecule (Ncam1) in response to BMP. Removing PSA enzymatically reverses the BMP effects on ENS precursor migration and neurite fasciculation. These studies demonstrate several novel roles for BMP signaling and highlight new functions for sialyltransferases in the developing ENS.

The transcription factor GATA2 plays an essential role in the establishment and maintenance of adult hematopoiesis. It is expressed in hematopoietic stem cells, as well as the cells that make up the aortic vasculature, namely aortic endothelial cells and smooth muscle cells. We have shown that GATA2 expression is predictive of location within the thoracic aorta; location is suggested to be a surrogate for disease susceptibility. The GATA2 gene maps beneath the Chromosome 3q linkage peak from our family-based sample set (GENECARD) study of early-onset coronary artery disease. Given these observations, we investigated the relationship of several known and novel polymorphisms within GATA2 to coronary artery disease. We identified five single nucleotide polymorphisms that were significantly associated with early-onset coronary artery disease in GENECARD. These results were validated by identifying significant association of two of these single nucleotide polymorphisms in an independent case-control sample set that was phenotypically similar to the GENECARD families. These observations identify GATA2 as a novel susceptibility gene for coronary artery disease and suggest that the study of this transcription factor and its downstream targets may uncover a regulatory network important for coronary artery disease inheritance.

<h4>Background</h4>Development of the enteric nervous system (ENS) requires interactions between migrating neural crest cells and the nascent gastrointestinal tract that are dependent upon genes expressed by both cell compartments. Hlx, a homeobox transcription factor gene that is expressed in mouse intestinal and hepatic mesenchyme, is required for normal embryonic growth of intestine and liver, and the Hlx-/- genotype is embryonic lethal. We hypothesized that Hlx is required for ENS development.<h4>Results</h4>Enteric neurons were identified in Hlx+/+ and Hlx-/- mouse embryos by immunostaining of embryo sections for the neural markers PGP9.5 and Phox2b, or by staining for beta-galactosidase in whole-mount embryos containing the dopamine beta-hydroxylase-nLacZ transgene. In Hlx+/+ embryos, neural crest cells/enteric neurons have moved from the stomach into the intestine by E10.5. By contrast, neural crest cells/enteric neurons remain largely restricted to the lateral stomach mesenchyme of Hlx-/- embryos, with only a few scattered neural crest cells/enteric neurons in the intestine between E10.5-16.5.<h4>Conclusion</h4>The Hlx homeobox transcription factor is required for early aspects of ENS development.

<h4>Background</h4>There are more than 50 genes for autosomal dominant and autosomal recessive nonsyndromic hereditary deafness that are yet to be cloned. The human genome sequence and expression profiles of transcripts in the inner ear have aided positional cloning approaches. The knowledge of protein interactions offers additional advantages in selecting candidate genes within a mapped region.<h4>Results</h4>We have employed a bioinformatic approach to assemble the genes encoded by genomic regions that harbor various deafness loci. The genes were then in silico analyzed for their candidacy by expression pattern and ability to interact with other proteins. Such analyses have narrowed a list of 2400 genes from suspected regions of the genome to a manageable number of about 140 for further analysis.<h4>Conclusion</h4>We have established a list of strong candidate genes encoded by the regions linked to various nonsyndromic hereditary hearing loss phenotypes by using a novel bioinformatic approach. The candidates presented here provide a starting point for mutational analysis in well-characterized families along with genetic linkage to refine the loci. The advantages and shortcomings of this bioinformatic approach are discussed.

Strategies for removal of high abundance proteins have been increasingly utilized in proteomic studies of serum/plasma and other body fluids to enhance the detection of low abundance proteins and achieve broader proteome coverage; however, both the reproducibility and specificity of the high abundance protein depletion process still represent common concerns. Here we report a detailed evaluation of immunoaffinity subtraction performed applying the ProteomeLab IgY-12 system that is commonly used in human serum/plasma proteome characterization in combination with high resolution LC-MS/MS. Plasma samples were repeatedly processed using this approach, and the resulting flow-through fractions and bound fractions were individually analyzed for comparison. The removal of target proteins by the immunoaffinity subtraction system and the overall process was highly reproducible. Non-target proteins, including one spiked protein standard (rabbit glyceraldehyde-3-phosphate dehydrogenase), were also observed to bind to the column at different levels but also in a reproducible manner. The results suggest that multiprotein immunoaffinity subtraction systems can be readily integrated into quantitative strategies to enhance detection of low abundance proteins in biomarker discovery studies.

Ca(2+) influx through voltage-gated R-type (Ca(V)2.3) Ca(2+) channels is important for hormone and neurotransmitter secretion and other cellular events. Previous studies have shown that Ca(V)2.3 is both inhibited and stimulated through signaling mechanisms coupled to muscarinic ACh receptors. We previously demonstrated that muscarinic stimulation of Ca(V)2.3 is blocked by regulator of G protein signaling (RGS) 2. Here we investigated whether muscarinic inhibition of Ca(V)2.3 is antagonized by RGS3. RGS3 is particularly interesting because it contains a lengthy ( approximately 380 residue) amino-terminal domain of uncertain physiological function. Ca(V)2.3, M(2) muscarinic ACh receptors (M(2)R), and various deletion mutants of RGS3, including its native isoform RGS3T, were expressed in HEK293 cells, and agonist-dependent inhibition of Ca(V)2.3 was quantified using whole cell patch-clamp recordings. Full-length RGS3, RGS3T, and the core domain of RGS3 were equally effective in antagonizing inhibition of Ca(V)2.3 through M(2)R. These results identify RGS3 and RGS3T as potential physiological regulators of R-type Ca(2+) channels. Furthermore, they suggest that the signaling activity of RGS3 is unaffected by its extended amino-terminal domain. Confocal microscopy was used to examine the intracellular locations of four RGS3-enhanced green fluorescent protein fusion proteins. The RGS3 core domain was uniformly distributed throughout both cytoplasm and nucleus. By contrast, full-length RGS3, RGS3T, and the amino-terminal domain of RGS3 were restricted to the cytoplasm. These observations suggest that the amino terminus of RGS3 may serve to confine it to the cytoplasmic compartment where it can interact with cell surface receptors, heterotrimeric G proteins, and other signaling proteins.

<h4>Rationale</h4>Determining whether, under what circumstances, and to what extent 3,4-methylenedioxymethamphetamine (MDMA) exposure produces chronic changes in human brain function is a critical public health issue. MDMA is a widely used recreational drug commonly sold as "Ecstasy". Because findings from the animal literature have indicated that specific dosage regimens of MDMA can produce long-lasting alterations in serotonergic function, existing studies of MDMA effects in humans have examined brain serotonin (5-HT) transporters (5-HTT) and receptors or have examined brain structures or functions potentially affected by MDMA.<h4>Objectives</h4>The objectives of this review are to provide a background for interpreting human MDMA neuroimaging research, to examine existing neuroimaging data regarding the rationale for and limitations to human MDMA research, and to provide suggestions for improving the design and interpretation of future neuroimaging approaches.<h4>Results</h4>Of the existing neuroimaging studies in human MDMA users, few experimental designs have been replicated across different research groups. Only investigations employing nuclear imaging methods to assay brain 5-HTT levels have been replicated across methods and research laboratories. These studies have found reduced levels of the 5-HTT in recently abstinent MDMA users with some evidence for normalization of 5-HTT levels with prolonged abstinence. However, the sensitivity of these methods is unknown.<h4>Conclusions</h4>The current state of neuroimaging in human MDMA users does not permit conclusions regarding the long-term effects of MDMA exposure. Future study designs might benefit from improved sample homogeneity, increased length of MDMA abstinence, longitudinal study design, test-retest measures, serotonergic specificity, and multimodal approaches.

The expansion of a polymorphic CAG repeat in the HD gene encoding huntingtin has been identified as the major cause of Huntington's disease (HD) and determines 42-73% of the variance in the age-at-onset of the disease. Polymorphisms in huntingtin interacting or associated genes are thought to modify the course of the disease. To identify genetic modifiers influencing the age at disease onset, we searched for polymorphic markers in the GRIK2, TBP, BDNF, HIP1 and ZDHHC17 genes and analysed seven of them by association studies in 980 independent European HD patients. Screening for unknown sequence variations we found besides several silent variations three polymorphisms in the ZDHHC17 gene. These and polymorphisms in the GRIK2, TBP and BDNF genes were analysed with respect to their association with the HD age-at-onset. Although some of the factors have been defined as genetic modifier factors in previous studies, none of the genes encoding GRIK2, TBP, BDNF and ZDHHC17 could be identified as a genetic modifier for HD.

Protein interaction networks are useful resources for the functional annotation of proteins. Recently, we have generated a highly connected protein-protein interaction network for Huntington's disease (HD) by automated yeast two-hybrid (Y2H) screening (Goehler et al., 2004). The network included several novel direct interaction partners for the disease protein huntingtin (htt). Some of these interactions, however, have not been validated by independent methods. Here we describe the verification of the interaction between htt and GASP2 (G protein-coupled receptor associated sorting protein 2), a protein involved in membrane receptor degradation. Using membrane-based and classical coimmunoprecipitation assays we demonstrate that htt and GASP2 form a complex in cotransfected mammalian cells. Moreover, we show that the two proteins colocalize in SH-SY5Y cells, raising the possibility that htt and GASP2 interact in neurons. As the GASP protein family plays a role in G protein-coupled receptor sorting, our data suggest that htt might influence receptor trafficking via the interaction with GASP2.

Dilated cardiomyopathy (DCM) is a heart muscle disease characterized by ventricular dilatation and impaired systolic function. Patients with DCM suffer from heart failure, arrhythmia, and are at risk of premature death. DCM has a prevalence of one case out of 2500 individuals with an incidence of 7/100,000/year (but may be under diagnosed). In many cases the disease is inherited and is termed familial DCM (FDC). FDC may account for 20-48% of DCM. FDC is principally caused by genetic mutations in FDC genes that encode for cytoskeletal and sarcomeric proteins in the cardiac myocyte. Family history analysis is an important tool for identifying families affected by FDC. Standard criteria for evaluating FDC families have been published and the use of such criteria is increasing. Clinical genetic testing has been developed for some FDC genes and will be increasingly utilized for evaluating FDC families. Through the use of family screening by pedigree analysis and/or genetic testing, it is possible to identify patients at earlier, or even presymptomatic stages of their disease. This presents an opportunity to invoke lifestyle changes and to provide pharmacological therapy earlier in the course of disease. Genetic counseling is used to identify additional asymptomatic family members who are at risk of developing symptoms, allowing for regular screening of these individuals. The management of FDC focuses on limiting the progression of heart failure and controlling arrhythmia, and is based on currently accepted treatment guidelines for DCM. It includes general measures (salt and fluid restriction, treatment of hypertension, limitation of alcohol intake, control of body weight, moderate exercise) and pharmacotherapy. Cardiac resynchronization, implantable cardioverter defibrillators and left ventricular assist devices have progressively expanding usage. Patients with severe heart failure, severe reduction of the functional capacity and depressed left ventricular ejection fraction have a low survival rate and may require heart transplant.

The physical restoration of dopamine circuits damaged or lost in Parkinson disease by implanting embryonic stem (ES)-derived cells may become a treatment. It is critical to understand responses of ES-derived dopamine (DA) neurons to guidance signals that determine axonal path and targeting. Using a collagen gel culture system, we examined effects of secreted molecules Netrin-1 and Slits on neurite outgrowth of fetal DA neurons and murine ES-differentiated DA neurons. We have previously shown that fetal DA neurons express DCC and Robo1/2 receptors and that Netrin-1 and Slit2 function as an attractant and a repellent for DA neurite outgrowth. In the present study, we observe that both Slit1 and Slit3 repel and inhibit neurite growth of fetal DA neurons. Here, we also demonstrate that ES-differentiated neurons including DA neurons express the Netrin receptor DCC and Slit receptor Robo proteins. In the gel culture system of ES cells, Netrin-1 promoted neurite outgrowth mediated by DCC receptor, and Slit1 and Slit3 were inhibitory for neurite outgrowth through Robo receptors. Slit2 appeared to exert inhibitory as well as repulsive effects in the coculture assay. However, unlike fetal DA neurites, no directed neurite outgrowth was observed in the cocultures of ES-derived DA neurons with Netrin-1-, Slit1-, and Slit3-producing cells. The findings suggest that ES-derived DA neurons generated by current protocols can respond to guidance cues in vitro in a similar manner to fetal cells but also exhibit distinct responses. This may result from developmental differences generated by present in vitro methods of cell patterning or conditioning during ES cell differentiation.

Iron homeostasis is maintained through meticulous regulation of circulating hepcidin levels. Hepcidin levels that are inappropriately low or high result in iron overload or iron deficiency, respectively. Although hypoxia, erythroid demand, iron, and inflammation are all known to influence hepcidin expression, the mechanisms responsible are not well defined. In this report we show that the inflammatory cytokine interleukin-6 (IL-6) directly regulates hepcidin through induction and subsequent promoter binding of signal transducer and activator of transcription 3 (STAT3). STAT3 is necessary and sufficient for the IL-6 responsiveness of the hepcidin promoter. Our findings provide a mechanism by which hepcidin can be regulated by inflammation or, in the absence of inflammatory stimuli, by alternative mechanisms leading to STAT3 activation.

SOX13 is a member of the SOX family of transcription factors. SOX proteins play essential roles in development, and some are associated with human genetic diseases. SOX13 maps to a multi-disease locus on chromosome 1q31-32, yet its function is unknown. Here we describe the temporal and spatial expression of SOX13 protein during mouse organogenesis. SOX13 is expressed in the three embryonic cell lineages, suggesting that it may direct various developmental processes. SOX13 is expressed in the developing central nervous system including the neural tube and the developing brain. Expression is also detected in the condensing mesenchyme and cartilage progenitor cells during endochondral bone formation in the limb as well as the somite sclerotome and its derivatives. SOX13 is also detected in the developing kidney, pancreas, and liver as well as in the visceral mesoderm of the extra-embryonic yolk sac and spongiotrophoblast layer of the placenta.

A 24-year-old female patient presented to her community hospital with mild elevations of serum transaminase and bilirubin levels. Because of multiple sclerosis, she was treated with interferon beta-1a for 6 weeks. After exclusion of viral hepatitis due to hepatitis A-E, interferon beta-1a was withdrawn under the suspicion of drug-induced hepatitis. One week later, she was admitted again to her community hospital with severe icterus. The transaminase and bilirubin levels were highly elevated, and a beginning impairment of the liver synthesis was expressed by a reduced prothrombin time. The confinement to our department occurred with a fulminant hepatitis and the suspicion of beginning acute liver failure. There was no evidence for hepatitis due to potentially hepatotoxic viruses, alcoholic hepatitis, Budd-Chiari syndrome, hemochromatosis, and Wilson's disease. In her serum there were high titers of liver-kidney microsomal type 1 autoantibody; the serum gamma globulin levels were in the normal range. Fine-needle aspiration biopsy of the liver ruled out an autoimmune hepatitis but showed signs of drug-induced toxicity. During the interview, she admitted that for 'general immune system stimulation' she had been drinking Noni juice, a Polynesian herbal remedy made from a tropical fruit (Morinda citrifolia), during the past 4 weeks. After cessation of the Noni juice ingestion, her transaminase levels normalized quickly and were in the normal range within 1 month.

Several neurodegenerative diseases are associated with the toxicity of misfolded proteins. This toxicity must arise from a combination of the amino acid sequences of the misfolded proteins and their interactions with other factors in their environment. A particularly compelling example of how profoundly these intramolecular and intermolecular factors can modulate the toxicity of a misfolded protein is provided by the polyglutamine (polyQ) diseases. All of these disorders are caused by glutamine expansions in proteins that are broadly expressed, yet the nature of the proteins that harbor the glutamine expansions and the particular pathologies they produce are very different. We find, using a yeast model, that amino acid sequences that modulate polyQ toxicity in cis can also do so in trans. Furthermore, the prion conformation of the yeast protein Rnq1 and the level of expression of a suite of other glutamine-rich proteins profoundly affect polyQ toxicity. They can convert polyQ expansion proteins from toxic to benign and vice versa. Our work presents a paradigm for how a complex, dynamic interplay between intramolecular features of polyQ proteins and intermolecular factors in the cellular environment might determine the unique pathobiologies of polyQ expansion proteins.

Protein misfolding is the molecular basis for several human diseases. How the primary amino acid sequence triggers misfolding and determines the benign or toxic character of the misfolded protein remains largely obscure. Among proteins that misfold, polyglutamine (polyQ) expansion proteins provide an interesting case: Each causes a distinct neurodegenerative disease that selectively affects different neurons. However, all are broadly expressed and most become toxic when the glutamine expansion exceeds approximately 39 glutamine residues. The disease-causing polyQ expansion proteins differ profoundly in the amino acids flanking the polyQ region. We therefore hypothesized that these flanking sequences influence the specific toxic character of each polyQ expansion protein. Using a yeast model, we find that sequences flanking the polyQ region of human huntingtin exon I can convert a benign protein to a toxic species and vice versa. Further, we observe that flanking sequences can direct polyQ misfolding to at least two morphologically distinct types of polyQ aggregates. Very tight aggregates always are benign, whereas amorphous aggregates can be toxic. We thereby establish a previously undescribed systematic characterization of the influence of flanking amino acid sequences on polyQ toxicity.

The goal of this work was to assess the effect of the controlled delivery of neurotrophin-3 (NT-3) from an affinity-based delivery system in fibrin scaffolds on regeneration following spinal cord injury (SCI). A heparin-based delivery system (HBDS) was used to immobilize NT-3 within fibrin scaffolds via non-covalent interactions. The fibrin scaffolds were implanted in lesions immediately after injury in an adult rat model of SCI (complete ablation of a 2 mm segment of the cord at T9). Delivery of NT-3 was controlled by an affinity-based delivery system that limits drug loss by diffusion and releases the drug via cell-mediated processes. Twelve weeks after injury and treatment, animals treated with fibrin scaffolds and NT-3, with or without the delivery system, did not show functional improvement over saline controls. Substantial cavitation at edges of the lesion was present, and while neuronal fibers were present inside the lesion, traced corticospinal and dorsal sensory tracts did not regenerate into the lesion. Therefore, while previous studies indicate that the controlled delivery of NT-3 from fibrin scaffolds may increase the short term regenerative response, the continued degeneration of the cord, indicative of the severity of the injury, limits the long term regeneration stimulated by this treatment. Chronic or repeated treatments or a less severe injury model may prove useful in assessing the utility of controlled delivery systems for the treatment of spinal cord injury.

An enkephalin analogue coupled to 'aminofentanyl' has been synthesized and tested for biological activities at the mu and delta opioid receptors. Aminofentanyl which represents a structural derivative of fentanyl has been synthesized by acylation of 1-(2-phenethyl)-4-(N-anilino)piperidine with phthaloyl protected beta-alaninyl chloride in the presence of DIPEA, followed by deprotection with hydrazine hydrate. Aminofentanyl has also been successfully acylated with ethyl isocyanate, various acid anhydrides, to further investigate structure-activity relationships of these new fentanyl derivatives. Among the new derivatives compound 7 which carries a Tyr-D-Ala-Gly-Phe opioid message sequence showed good opioid affinity (1 nM at both delta and mu opioid receptors) and bioactivity (34.9 nM in MVD and 42 nM in GPI/LMMP bioassays).

There is no information available on left ventricular (LV) systolic function and the response to stress echocardiography in asymptomatic subjects with hereditary hemochromatosis (HH). To evaluate this topic, 43 asymptomatic subjects with HH homozygous for the C282Y HFE gene mutation (22 untreated subjects [group A] and 21 long-term treated subjects [group B]) were compared with 21 age- and gender-matched normal volunteers negative for HFE mutations. Contractile reserve, as a measure of LV systolic function, was assessed using continuous echocardiographic imaging and electrocardiography during supine bicycle exercise. Nineteen subjects in group A had repeat tests after 6 months of induction phlebotomy therapy to assess the effect of iron removal. Exercise performance and hemodynamic variables of supine bicycle exercise were comparable between subjects with HH and controls. LV contractile reserve of asymptomatic subjects with HH was not impaired at either a 75-W submaximal exercise level (mean +/- SD difference in ejection fraction from baseline 13.8 +/- 6.2%, 11.5 +/- 6.8%, and 13.4 +/- 7.8% in groups A, B, and C, respectively; p = NS for all by analysis of variance) or at peak exercise (difference in ejection fraction from baseline 18.9 +/- 6.9%, 18.4 +/- 7.8%, and 20.3 +/- 8.1% in groups A, B, and C, respectively; p = NS for all by analysis of variance). However, the incidence of abnormal ischemic stress electrocardiographic responses was more frequent in subjects with HH as a whole (33%) compared with normal subjects (10%). Stress imaging revealed no regional wall motion abnormalities, suggesting that these were false-positive results. Iron removal by induction phlebotomy did not affect stress echocardiographic performance. In conclusion, LV systolic function during exercise in asymptomatic subjects with HH is preserved, and 6-month induction phlebotomy does not affect stress echocardiographic performance.

<h4>Background</h4>Pathological brain iron deposition has been implicated as a source of neurotoxic reactive oxygen species in Alzheimer (AD) and Parkinson diseases (PD). Iron metabolism is associated with the gene hemochromatosis (HFE Human genome nomenclature committee ID:4886), and mutations in HFE are a cause of the iron mismetabolism disease, hemochromatosis. Several reports have tested the association of HFE variants with neurodegenerative diseases, such as AD and PD with conflicting results.<h4>Methods</h4>Genotypes were analysed for the two most common variants of HFE in a series of 130 AD, 55 Mild Cognitive Impairment (MCI) and 132 PD patients. Additionally, a series of 115 healthy age-matched controls was also screened.<h4>Results</h4>A statistically significant association was found in the PD group when compared to controls, showing that the presence of the C282Y variant allele may confer higher risk for developing the disease.<h4>Conclusion</h4>Taken together these results suggest that the common variants in HFE may be a risk factor for PD, but not for AD in the Portuguese population.

We performed a network-based analysis of DNA microarray data from allergen-challenged CD4(+) T cells from patients with seasonal allergic rhinitis. Differentially expressed genes were organized into a functionally annotated network using the Ingenuity Knowledge Database, which is based on manual review of more than 200,000 publications. The main function of this network is the regulation of lymphocyte apoptosis, a role associated with several genes of the tuber necrosis factor superfamily. The expression of TNFRSF4, one of the genes in this family, was found to be 48 times higher in allergen-challenged cells than in diluent-challenged cells. TNFRSF4 is known to inhibit apoptosis and to enhance Th2 proliferation. Examination of a different material of allergen-stimulated peripheral blood mononuclear cells showed a higher number of interleukin-4(+) type 2 CD4(+) T (Th2) cells in patients than in controls (P<0.01), as well as a higher number of non-apoptotic Th2 cells in patients (P<0.01). The number of Th2 cells expressing TNFRSF4, TNFSF7 and TNFRSF1B was also significantly higher in patients. Treatment with anti-TNFSF4 resulted in a significantly decreased number of Th2 cells (P<0.05). A logical inference from all this is that the proliferation of allergen-challenged Th2 cells is associated with a decreased apoptosis of Th2 cells and an increase in TNFRSF4 signalling.

Photodynamic therapy (PDT) involves a two-stage process. A light-absorbing photosensitiser (Ps) is endocytosed and then stimulated by light, inducing transfer of energy to a cytoplasmic acceptor molecule and the generation of reactive oxygen species that initiate damage to cellular membrane components and cytolysis. The expanded use of PDT in the clinic is hindered by the lack of Ps target-cell specificity and the limited tissue penetration by external light radiation. This study demonstrates that bioconjugates composed of transferrin and haematoporphyrin (Tf-Hp), significantly improve the specificity and efficiency of PDT for erythroleukemic cells by a factor of almost seven-fold. Fluorescence microscopy showed that the conjugates accumulate in intracellular vesicles whereas free Hp was mostly membrane bound. Experiments with cells deliberately exposed to Tf-Hp at <LD(100) doses showed that surviving cells did not develop resistance to subsequent treatments with the conjugate. Furthermore, we show that the compound luminol induces intracellular chemiluminescence. This strategy was then used to obviate the use of external radiation for Ps activation by incubating the cells with luminol either before or together with Tf-Hp. This novel chemical means of PDT activation induced cytotoxicity in 95% of cells. These combined approaches provide an opportunity to develop broader and more effective applications of PDT.

Early onset of alcohol and tobacco use during adolescence increases the risk for establishing a substance use disorder in adulthood. Both alcohol and nicotine stimulate the dopamine (DA) and the serotonin (5-HT) systems. The DA system has been implicated in the mediation of the rewarding effects of self-administered drugs of abuse. A possible role of an interaction between these neurotransmitter systems in substance use behavior has been suggested but is as yet unknown. The present study was designed to examine the influence of the DA D4 receptor (DRD4) and the serotonin transporter (5-HTT) genotype and their interaction on adolescent alcohol and tobacco experimentation. Participants were from a longitudinal study of a birth cohort consisting initially of 384 children from a high-risk community sample. At the age of 15 years, adolescents completed a self-report questionnaire measuring tobacco and alcohol consumption. DNA was taken from 305 participants (146 boys, 159 girls) and genotyped for the DRD4 exon III and the 5-HTTLPR polymorphisms. The DRD4 7-repeat allele was associated with greater smoking and drinking involvement in boys. In girls, a significant DRD4 x 5-HTT interaction was detected. Girls without the DRD4 7-repeat allele and who were homozygous for the long allele of 5-HTTLPR displayed the highest smoking and drinking activity. The genetic and potential molecular background underlying adolescent vulnerability to substance abuse is discussed.

Thymic stromal lymphopoietin, a four helix-bundle cytokine, is expressed mainly by barrier epithelial cells and is a potent activator of several cell types, particularly myeloid dendritic cells. TSLP influences the outcome of interactions between dendritic cells and CD4+ thymocytes and T cells in many situations, such as the regulation of the positive selection of regulatory T cells, maintenance of peripheral CD4+ T cell homeostasis and induction of CD4+ T cell-mediated allergic inflammation.

<h4>Purpose</h4>Cell adhesion molecule integrin alpha v beta 3 is an excellent target for tumor interventions because of its unique expression on the surface of several types of solid tumor cells and on almost all sprouting tumor vasculatures. Here, we describe the development of near-infrared (NIR) fluorochrome Cy7-labeled RGD peptides for tumor integrin targeting.<h4>Procedures</h4>Mono-, di-, and tetrameric RGD peptides were synthesized and conjugated with Cy7. The integrin specificity of these fluorescent probes was tested in vitro for receptor binding assay and fluorescence microscopy and in vivo for subcutaneous U87MG tumor targeting.<h4>Results</h4>The tetrameric RGD peptide probe with the highest integrin affinity showed the highest tumor activity accumulation and strongest tumor-to-normal tissue contrast. This uptake is integrin-specific as the signal accumulated in the tumor can be effectively blocked by unconjugated RGD peptide antagonist of integrin alpha v beta 3.<h4>Conclusions</h4>Noninvasive NIR fluorescence imaging is able to detect and semiquantify tumor integrin expression based upon the highly potent tetrameric RGD peptide probe.

<h4>Background and aims</h4>The Hemochromatosis and Iron Overload Screening (HEIRS) Study screened 101,168 primary care participants for iron overload with serum transferrin saturation (TS), ferritin, and C282Y and H63D mutations of the HFE gene.<h4>Methods</h4>All C282Y homozygotes and participants with an increased TS (>45% women, >50% men) and serum ferritin level (> 200 microg/L women, >300 microg/L men) were recalled for a clinical history and physical examination, and blood tests including alanine transaminase (ALT) and aspartate transaminase levels. Hepatitis B surface antigen and anti-hepatitis C virus were measured if the ALT level was increased (>31 IU/L in women, >40 IU/L in men).<h4>Results</h4>In the group of participants selected to return for clinical examination because of increased TS and ferritin levels, ALT increases and anti-hepatitis C virus were found in 95 of 284 (33%) African Americans, 50 of 466 (11%) Asian and Pacific Islanders, 21 of 120 (18%) Hispanics, and 40 of 477 (8.4%) Caucasians. ALT increases and hepatitis B surface antigen were detected in 24 of 466 (5%) Asian and Pacific Islanders, 10 of 284 (3.5%) African Americans, 3 of 120 (2.5%) Hispanics, and 2 of 477 (.42%) Caucasians. Of 86 liver biopsy specimens obtained for clinical purposes, 53 were reviewed by a single study pathologist. Liver fibrosis (stage 3 or 4) was present in 2 of 11 (18.2%) C282Y homozygotes that underwent central review and 2 of 302 (.66%) C282Y homozygotes attending the clinical examination.<h4>Conclusions</h4>Screening for iron overload with ferritin and TS detects persons with viral hepatitis and other types of liver disease. A minimum of .66% C282Y homozygotes have liver fibrosis.

Polymorphisms of genes linked to iron metabolism may account for individual variability in hemochromatosis and iron status connected with liver and cardiovascular diseases, cancers, toxicity, and infection. Mouse strains exhibit marked differences in levels of non-heme iron, with C57BL/6J and SWR showing low and high levels, respectively. The genetic basis for this variability was examined using quantitative trait loci (QTL) analysis together with expression profiling and chromosomal positions of known iron-related genes. Non-heme iron levels in liver and spleen of C57BL/6J x SWR F2 mice were poorly correlated, indicating independent regulation. Highly significant (P < .01) polymorphic loci were found on chromosomes 2 and 16 for liver and on chromosomes 8 and 9 for spleen. With sex as a covariate, additional significant or suggestive (P < 0.1) QTL were detected on chromosomes 7, 8, 11, and 19 for liver and on chromosome 2 for spleen. A gene array showed no clear association between most loci and differential iron-related gene expression. The gene for transferrin and a transferrin-like gene map close to the QTL on chromosome 9. Transferrin saturation was significantly lower in C57BL/6J mice than in SWR mice, but there was no significant difference in the serum level of transferrin, hepatic expression, or functional change in cDNA sequence. beta2-Microglobulin, which, unlike other loci, was associated with C57BL/6J alleles, is a candidate for the chromosome 2 QTL for higher iron. In conclusion, the findings show the location of polymorphic genes that determine basal iron status in wild-type mice. Human equivalents may be pertinent in predisposition to hepatic and other disorders.

<h4>Background</h4>The histopathologic heterogeneity of lung cancer remains a significant confounding factor in its diagnosis and prognosis-spurring numerous recent efforts to find a molecular classification of the disease that has clinical relevance.<h4>Methods and findings</h4>Molecular profiles of tumors from 186 patients representing four different lung cancer subtypes (and 17 normal lung tissue samples) were compared with a mouse lung development model using principal component analysis in both temporal and genomic domains. An algorithm for the classification of lung cancers using a multi-scale developmental framework was developed. Kaplan-Meier survival analysis was conducted for lung adenocarcinoma patient subgroups identified via their developmental association. We found multi-scale genomic similarities between four human lung cancer subtypes and the developing mouse lung that are prognostically meaningful. Significant association was observed between the localization of human lung cancer cases along the principal mouse lung development trajectory and the corresponding patient survival rate at three distinct levels of classical histopathologic resolution: among different lung cancer subtypes, among patients within the adenocarcinoma subtype, and within the stage I adenocarcinoma subclass. The earlier the genomic association between a human tumor profile and the mouse lung development sequence, the poorer the patient's prognosis. Furthermore, decomposing this principal lung development trajectory identified a gene set that was significantly enriched for pyrimidine metabolism and cell-adhesion functions specific to lung development and oncogenesis.<h4>Conclusions</h4>From a multi-scale disease modeling perspective, the molecular dynamics of murine lung development provide an effective framework that is not only data driven but also informed by the biology of development for elucidating the mechanisms of human lung cancer biology and its clinical outcome.

Protein-directed dynamic combinatorial chemistry (DCC) relies on reversible chemical reactions that can function under the near-physiological conditions required by the biological target. Few classes of reaction have so far proven effective at generating dynamic combinatorial libraries (DCLs) under such constraints. In this study, we establish the conjugate addition of thiols to enones as a reaction well-suited for the synthesis of dynamic combinatorial libraries (DCLs) directed by the active site of the enzyme glutathione S-transferase (GST). The reaction is fast, freely reversible at basic pH, and easily interfaced with the protein, which is a target for the design of inhibitors in cancer therapy and the treatment of parasitic diseases such as schistosomiasis. We have synthesized DCLs based on glutathione (GSH, 1) and the enone ethacrynic acid, 2a. By varying either set of components, we can choose to probe either the GSH binding region ("G site") or the adjacent hydrophobic acceptor binding region ("H site") of the GST active site. In both cases the strongest binding DCL components are identified due to molecular amplification by GST which, in the latter system, leads to the identification of two new inhibitors for the GST enzyme.

Neuronal differentiation requires exquisitely timed cell cycle arrest for progenitors to acquire an appropriate neuronal cell fate and is achieved by communication between soluble signals, such as growth factors and extracellular matrix molecules. Here we report that the expression of TIMP-2, a matrix metalloproteinase inhibitor, is up-regulated by signals that control proliferation (bFGF and EGF) and differentiation (retinoic acid and NGF) in neural progenitor and neuroblastoma cell lines. TIMP-2 expression coincides with the appearance of neurofilament-positive neurons, indicating that TIMP-2 may play a role in neurogenesis. The up-regulation of TIMP-2 expression by proliferate signals suggests a role in the transition from proliferation to neuronal differentiation. Live labeling experiments demonstrate TIMP-2 expression only on alpha(3) integrin-positive cells. Thus, TIMP-2 function may be mediated via interaction with integrin receptor(s). We propose that TIMP-2 represents a component of the neurogenic signaling cascade induced by mitogenic stimuli that may withdraw progenitor cells from the cell cycle permitting their terminal neuronal differentiation.

Synthetic analogues were designed to highlight the effect of the glycine moiety of bacillibactin on the overall stability of the ferric complex as compared to synthetic analogues of enterobactin. Insertion of a variety of amino acids to catecholamide analogues based on a Tren (tris(2-aminoethyl)amine) backbone increased the overall acidity of the ligands, causing an enhancement of the stability of the resulting ferric complex as compared to TRENCAM. Solution thermodynamic behavior of these siderophores and their synthetic analogues was investigated through potentiometric and spectrophotometric titrations. X-ray crystallography, circular dichroism, and molecular modeling were used to determine the chirality and geometry of the ferric complexes of bacillibactin and its analogues. In contrast to the Tren scaffold, addition of a glycine to the catechol chelating arms causes an inversion of the trilactone backbone, resulting in opposite chiralities of the two siderophores and a destabilization of the ferric complex of bacillibactin compared to ferric enterobactin.

We recently reported that the transient expression of polyglutamine tracts of various size in exon 1 of the huntingtin polypeptide (httEx1) generated abnormally high levels of intracellular reactive oxygen species that directly contributed to cell death. Here, we compared the protection generated by heat shock proteins to that provided by the antioxidant agent N-acetyl-L-cysteine. In cells expressing httEx1 with 72 glutamine repeats (httEx1-72Q), the overexpression of Hsp27 or Hsp70 plus Hdj-1(Hsp40) or treatment of the cells with N-acetyl-L-cysteine inhibited not only mitochondrial membrane potential disruption but also the increase in reactive oxygen species, nitric oxide and protein oxidation. However, only heat shock proteins and not N-acetyl-L-cysteine reduced the size of the inclusion bodies formed by httEx1-72Q. In cells expressing httEx1 polypeptide with 103 glutamine repeats (httEx1-103Q), heat shock proteins neither decreased oxidative damage nor reduced the size of the inclusions. In contrast, N-acetyl-L-cysteine still efficiently decreased the oxidative damage induced by httEx1-103Q polypeptide without altering the inclusions. N-Acetyl-L-cysteine was inactive with regard to proteasome inhibition, whereas heat shock proteins partially restored the caspase-like activity of this protease. These observations suggest some relationships between the presence of inclusion bodies and the oxidative damage induced by httEx1-polyQ.

The pathogenesis of spinocerebellar ataxia type 7 and other neurodegenerative polyglutamine (polyQ) disorders correlates with the aberrant accumulation of toxic polyQ-expanded proteins in the nucleus. Promyelocytic leukemia protein (PML) nuclear bodies are often present in polyQ aggregates, but their relation to pathogenesis is unclear. We show that expression of PML isoform IV leads to the formation of distinct nuclear bodies enriched in components of the ubiquitin-proteasome system. These bodies recruit soluble mutant ataxin-7 and promote its degradation by proteasome-dependent proteolysis, thus preventing the aggregate formation. Inversely, disruption of the endogenous nuclear bodies with cadmium increases the nuclear accumulation and aggregation of mutant ataxin-7, demonstrating their role in ataxin-7 turnover. Interestingly, beta-interferon treatment, which induces the expression of endogenous PML IV, prevents the accumulation of transiently expressed mutant ataxin-7 without affecting the level of the endogenous wild-type protein. Therefore, clastosomes represent a potential therapeutic target for preventing polyQ disorders.

We report the computer-aided design, chemical synthesis, and biological evaluation of a novel family of delta opioid receptor (DOR) antagonists containing a 1,2,4-triazole core structure that are structurally distinct from other known opioid receptor active ligands. Among those delta antagonists sharing this core structure, 8 exhibited strong binding affinity (K(i) = 50 nM) for the DOR and appreciable selectivity for delta over mu and kappa opioid receptors (delta/mu = 80; delta/kappa > 200).

In Escherichia coli, Fe-S clusters are assembled by gene products encoded from the isc and suf operons. Both the iscRSUA and sufABCDSE operons are induced highly by oxidants, reflecting an increased need for providing and maintaining Fe-S clusters under oxidative stress conditions. Three cis-acting oxidant-responsive elements (ORE-I, II, III) in the upstream of the sufA promoter serve as the binding sites for OxyR, IHF and an uncharacterized factor respectively. Using DNA affinity fractionation, we isolated an ORE-III-binding factor that positively regulates the suf operon in response to various oxidants. MALDI-TOF mass analysis identified it with IscR, known to serve as a repressor of the iscRSUA gene expression under anaerobic condition as a [2Fe-2S]-bound form. The iscR null mutation abolished ORE-III-binding activity in cell extracts, and caused a significant decrease in the oxidant induction of sufA in vivo. OxyR and IscR contributed almost equally to activate the sufA operon in response to oxidants. Purified IscR that lacked Fe-S cluster bound to the ORE-III site and activated transcription from the sufA promoter in vitro. Mutations in Fe-S-binding sites of IscR enabled sufA activation in vivo and in vitro. These results support a model that IscR in its demetallated form directly activates sufA transcription, while it de-represses isc operon, under oxidative stress condition.

<h4>Background & aims</h4>Aceruloplasminemia is a novel hereditary iron overload disease caused by a mutation in the ceruloplasmin gene and characterized by a complete deficiency of serum ceruloplasmin and iron accumulation in the liver and brain.<h4>Methods</h4>We herein studied a novel clinical type of aceruloplasminemia in which a low amount of ceruloplasmin was detected in the serum of a patient. The patient presented with an asymptomatic hepatic iron overload, retinal degeneration, and diabetes mellitus. Magnetic resonance imaging of the liver and basal ganglia showed T2-hypointensity signals associated with parenchymal iron accumulation because of an absence of the ferroxidase activity in ceruloplasmin.<h4>Results</h4>A gene analysis showed a novel G969S mutation in the ceruloplasmin gene. A biochemical analysis of the patients' serum and a biogenesis study of G969S mutant ceruloplasmin using mammalian cell culture system resulted in the synthesis and secretion of only apoceruloplasmin without any ferroxidase activity.<h4>Conclusions</h4>This novel clinical type of aceruloplasminemia should therefore be considered in the differential diagnosis of unexplained hemochromatosis, which is associated with a decrease in the serum ceruloplasmin level.

<h4>Objective</h4>To assess the practical usefulness of magnetic resonance imaging (MRI) in establishing a positive diagnosis of rheumatoid arthritis (RA) in a cohort of patients with early inflammatory polyarthralgia, in the absence of anti-cyclic citrullinated peptide (anti-CCP) antibodies.<h4>Methods</h4>We prospectively followed 30 outpatients with inflammatory polyarthralgia and/or synovitis of at least one joint. Patients were disease modifying antirheumatic drug-naive and received no corticosteroids. At the initial visit a clinical examination, radiographs of hands, wrists and feet, and MRI of hands were performed. Rheumatoid factor and anti-CCP antibodies were assessed. The MRI procedure was T1 fat saturation with gadolinium injection [scores were established on the basis of the axial view of the carpal and metacarpal joints, using the RA MRI scoring system (RAMRIS) defined in the OMERACT study]. In all patients, radiographs at baseline were normal and anti-CCP antibodies were negative.<h4>Results</h4>At one-year followup, the final diagnosis was: 16 RA; the non-RA group was composed of 4 cases of spondyloarthropathy, 2 cases of fibromyalgia, 4 cases of undifferentiated arthritis (3 of which were self-limiting), 1 sicca syndrome, 1 hemochromatosis, 1 polymyositis, and 1 paraneoplastic syndrome. No statistical difference was found between patients with and without RA for carpal erosion, synovitis, and tenosynovitis. However, a statistical difference was observed between the RA and non-RA group where metacarpophalangeal (MCP) erosion scores were concerned (p = 0.024). This difference persisted when we compared erosions of the second and third MCP in the 2 groups (p = 0.044). ROC curve analysis revealed a positive MCP score at 15, with a specificity of 70% and a sensitivity of 64%.<h4>Conclusion</h4>In our population of 30 anti-CPP negative patients with normal radiographs, MRI of hands, showing MCP erosions, can be helpful for the diagnosis of RA.

<h4>Background</h4>The INTERCEPT Blood System, a photochemical treatment (PCT) process, has been developed to inactivate pathogens in platelet concentrates. These studies evaluated the efficacy of PCT to inactivate pathogens in plasma and the effect of PCT on plasma function.<h4>Study design and methods</h4>Jumbo (600 mL) plasma units were inoculated with high titers of test pathogens and treated with 150 micromol per L amotosalen and 3 J per cm(2) long-wavelength ultraviolet light. The viability of each pathogen before and after treatment was measured with biological assays. Plasma function was evaluated through measurement of coagulation factors and antithrombotic protein activities.<h4>Results</h4>The levels of inactivation expressed as log-reduction were as follows: cell-free human immunodeficiency virus-1 (HIV-1), greater than 6.8; cell-associated HIV-1, greater than 6.4; human T-lymphotropic virus-I (HTLV-I), 4.5; HTLV-II, greater than 5.7; hepatitis B virus (HBV) and hepatitis C virus, greater than 4.5; duck HBV, 4.4 to 4.5; bovine viral diarrhea virus, 6.0; severe acute respiratory syndrome coronavirus, 5.5; West Nile virus, 6.8; bluetongue virus, 5.1; human adenovirus 5, 6.8; Klebsiella pneumoniae, greater than 7.4; Staphylococcus epidermidis and Yersinia enterocolitica, greater than 7.3; Treponema pallidum, greater than 5.9; Borrelia burgdorferi, greater than 10.6; Plasmodium falciparum, 6.9; Trypanosoma cruzi, greater than 5.0; and Babesia microti, greater than 5.3. Retention of coagulation factor activity after PCT was expressed as the proportion of pretreatment (baseline) activity. Retention was 72 to 73 percent of baseline fibrinogen and Factor (F)VIII activity and 78 to 98 percent for FII, FV, FVII, F IX, FX, FXI, FXIII, protein C, protein S, antithrombin, and alpha2-antiplasmin.<h4>Conclusion</h4>PCT of plasma inactivated high levels of a wide range of pathogens while maintaining adequate coagulation function. PCT has the potential to reduce the risk of transfusion-transmitted diseases in patients requiring plasma transfusion support.

Mitotic cell death (MCD) is a prominent but poorly defined form of death that stems from aberrant mitosis. One of the early steps in MCD is premature mitosis and uneven chromatin condensation (UCC). The mechanism underlying this phenomenon is currently unknown. In this study, we show that DNA damage in cells with a compromised p53-mediated G2/M checkpoint triggers the unscheduled activation of cyclin-dependent kinase 1 (Cdk1), activation and chromatin loading of the condensin I complex, and UCC followed by the appearance of multimicronucleated cells, which is evidence of MCD. We demonstrate that these processes engage some of the players of normal mitotic chromatin packaging but not those that drive the apoptotic chromatin condensation. Our findings establish a link between the induction of DNA damage and mitotic abnormalities (UCC) through the unscheduled activation of Cdk1 and recruitment of condensin I. These results demonstrate a clear distinction between the mechanisms that drive MCD-associated and apoptosis-related chromatin condensation and provide mechanistic insights and new readouts for a major cell death process in treated tumors.

<h4>Objective</h4>Peripheral neuropathy (PN) often complicates nucleoside reverse transcriptase inhibitor (NRTI) therapy of HIV infection and may involve mitochondrial dysfunction. Since iron deficiency is associated with some types of PN, and iron is essential for mitochondrial function, we tested the hypothesis that hemochromatosis (HFE) gene mutations influence susceptibility to NRTI-induced PN.<h4>Design</h4>Case-control study involving multicenter, AIDS Clinical Trials Group (ACTG) protocol 384 and ACTG Human DNA Repository specimens.<h4>Methods</h4>Study participants were randomized to receive three- or four-drug antiretroviral therapy with didanosine (ddI) plus stavudine (d4T) or zidovudine plus lamivudine, given with efavirenz, nelfinavir, or both, with up to three years of follow-up. PN was ascertained based on signs and symptoms. HFE C282Y and H63D genotypes were determined, and associations with PN were assessed using logistic regression.<h4>Results</h4>: Of 509 participants, 147 (29%) developed PN, 73% of whom had been randomized to receive ddI plus d4T. Among ddI/d4T-ever-treated individuals, HFE C282Y heterozygotes developed PN on ddI/d4T significantly less often than C282Y non-carriers, adjusting for age, CD4 lymphocyte count and viral load at baseline, and concomitant antiretroviral drugs [6% vs. 35%, respectively, in whites; adjusted odds ratio (OR), 0.17; 95% confidence interval (CI) 0.03-0.83; P = 0.021]. Regardless of race/ethnicity, ddI/d4T-associated PN was uncommon in C282Y heterozygotes [race-adjusted OR, 0.30; 95% CI 0.09-0.96); P = 0.042].<h4>Conclusions</h4>Iron-loading HFE mutations such as C282Y are associated with a decreased risk of PN during antiretroviral therapy. This finding has potential implications for the prediction and prevention of NRTI-associated PN, particularly in populations at risk of iron deficiency.

A general method for the non-oxidative functionalization of single-crystal silicon(111) surfaces is described. The silicon surface is fully acetylenylated using two-step chlorination/alkylation chemistry. A benzoquinone-masked primary amine is attached to this surface via Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition ("click" chemistry). The benzoquinone is electrochemically reduced, resulting in quantitative cleavage of the molecule and exposing the amine terminus. Molecules presenting a carboxylic acid have been immobilized to the exposed amine sites. X-ray photoelectron spectroscopy (XPS), Fourier transform infrared spectroscopy (FTIR), cyclic voltammetry (CV), and contact angle goniometry were utilized to characterize and quantitate each step in the functionalization process. This work represents a strategy for providing a general platform that can incorporate organic and biological molecules on Si(111) with minimal oxidation of the silicon surface.

A series of cyclo-3beta-(4-aminophenyl)-2beta-tropanemethanol analogues (5a-m) possessing varying linker groups between the 2- and 3-position on the tropane ring were synthesized and evaluated for their monoamine transporter binding properties. The results show that binding to the dopamine and serotonin transporters (DAT and 5-HTT) is highly dependent on the specific linker used. Cyclo-3beta-(4-aminophenyl)-2beta-tropanemethanol pimelic acid ester/amide (5b) had an IC50 of 3.8 nM at the DAT. Cyclo-3beta-(4-aminophenyl)-2beta-tropanemethanol sebacic acid ester/amide (5e) had a Ki of 1.9 nM at the 5-HTT and was 68- and 737-fold selective for the 5-HTT relative to the DAT and NET. Small changes to the size as well as the electrostatic and hydrophobic properties of the 2,3-linker in 5b or 5e led to much less potent analogues at all three transporters. These results suggest that the high affinity for 5b and 5e at the DAT and 5-HTT may be due to their specific conformational properties.

<h4>Background</h4>The aim of the present study was to examine the diversity of liver diseases in outpatients referred because of elevated serum ferritin.<h4>Methods</h4>A retrospective review was performed of outpatient referrals for serum ferritin elevations made to a tertiary care centre liver clinic between 1999 and 2005. Information regarding serum ferritin, transferrin saturation, liver biopsy, liver iron concentration and final diagnosis was extracted. Patients were stratified into two groups based on ferritin concentration: ferritin concentration 300 microg/L to 1000 microg/L, and ferritin concentration greater than 1000 microg/L.<h4>Results</h4>A total of 482 charts were reviewed, of which 119 (25%) had ferritin concentrations greater than 1000 microg/L. HFE-linked hemochromatosis, nonalcoholic steatohepatitis and alcohol-related liver disease were the top three diagnoses. HFE-linked hemochromatosis accounted for 28% to 42% of the diagnoses in all subgroups. The percentage of patients diagnosed with HFE-linked hemochromatosis was similar in the ferritin 300 microg/L to 1000 microg/L and the ferritin greater than 1000 microg/L groups (P = 0.067). Among patients with ferritin greater than 1000 microg/L, 63% underwent a liver biopsy. Of those with an elevated liver iron concentration (greater than 35 micromol/g dry weight), 71% had a transferrin saturation greater than 50% (88% of C282Y homozygotes and 43% of non-C282Y homozygotes). In non-C282Y homozygotes with an elevated serum ferritin concentration greater than 1000 microg/L, 64% did not have iron overload on liver biopsy.<h4>Conclusion</h4>HFE-linked hemochromatosis accounted for less than one-half of the diagnoses in an outpatient population referred for elevated ferritin, suggesting a need to search further for an alternate cause.

The gastrointestinal tract is lined by a simple epithelium that undergoes constant renewal involving cell division, differentiation and cell death. In addition, the epithelial lining separates the hostile processes of digestion and absorption that occur in the intestinal lumen from the aseptic environment of the internal milieu by defensive mechanisms that protect the epithelium from being breached. Central to these defensive processes is the synthesis of heme and its catabolism by heme oxygenase (HO). Dietary heme is also an important source of iron for the body which is taken up intact by the enterocyte. This review describes the recent literature on the diverse properties of heme/HO in the intestine tract. The roles of heme/HO in the regulation of the cell cycle/apoptosis, detoxification of xenobiotics, oxidative stress, inflammation, development of colon cancer, heme-iron absorption and intestinal motility are specifically examined.

<h4>Background</h4>Nonalcoholic steatohepatitis is a chronic liver disease with a high prevalence in the general population and a potential to evolve into cirrhosis. It is speculated that iron overload could be associated with liver injury and unfavorable progress in affected patients.<h4>Aims</h4>To evaluate the prevalence of mutation of the hemochromatosis gene (HFE) in patients with nonalcoholic steatohepatitis and to correlate it with histological findings in liver specimens.<h4>Patients and methods</h4>Twenty-nine patients with nonalcoholic steatohepatitis were evaluated. The presence of mutation in the hemochromatosis gene (C282Y and H63D) was tested in all patients and its result was evaluated in relation to hepatic inflammatory activity, presence of fibrosis, and iron overload in the liver. The control group was composed of 20 patients with normal liver function tests and 20 patients infected with the hepatitis C virus, with elevated serum levels of aminotransferases and with chronic hepatitis as shown by biopsy.<h4>Results</h4>Mutation of the hemochromatosis gene (C282Y and/or H63D) was diagnosed in 16 (55.2%) patients with nonalcoholic steatohepatitis, in 12 (60%) patients with hepatitis C and in 8 (40%) patients with no liver disease. No association was found between the presence of mutation and inflammatory activity, nor with the presence of fibrosis in patients with nonalcoholic steatohepatitis. An association was found between the presence of mutation and the occurrence of iron overload in liver, but there was no association between liver iron and the occurrence of fibrosis.<h4>Conclusions</h4>The findings suggest that iron does not play a major role in the pathogenesis and progression of nonalcoholic steatohepatitis, and routine tests of the hemochromatosis gene mutation in these patients should not be recommended.