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The first sequenced marsupial genome promises to reveal unparalleled insights into mammalian evolution. We have used the Monodelphis domestica (gray short-tailed opossum) sequence to construct the first map of a marsupial major histocompatibility complex (MHC). The MHC is the most gene-dense region of the mammalian genome and is critical to immunity and reproductive success. The marsupial MHC bridges the phylogenetic gap between the complex MHC of eutherian mammals and the minimal essential MHC of birds. Here we show that the opossum MHC is gene dense and complex, as in humans, but shares more organizational features with non-mammals. The Class I genes have amplified within the Class II region, resulting in a unique Class I/II region. We present a model of the organization of the MHC in ancestral mammals and its elaboration during mammalian evolution. The opossum genome, together with other extant genomes, reveals the existence of an ancestral "immune supercomplex" that contained genes of both types of natural killer receptors together with antigen processing genes and MHC genes.

The inner medullary collecting duct (IMCD) is an important site of vasopressin-regulated water and urea transport. Here we have used protein mass spectrometry to investigate the proteome of the IMCD cell and how it is altered in response to long-term vasopressin administration in rats. IMCDs were isolated from inner medullas of rats, and IMCD proteins were identified by liquid chromatography/tandem mass spectrometry (LC-MS/MS). We present a WWW-based "IMCD Proteome Database" containing all IMCD proteins identified in this study (n = 704) and prior MS-based identification studies (n = 301). We used the isotope-coded affinity tag (ICAT) technique to identify IMCD proteins that change in abundance in response to vasopressin. Vasopressin analog (dDAVP) or vehicle was infused subcutaneously in Brattleboro rats for 3 days, and IMCDs were isolated for proteomic analysis. dDAVP and control samples were labeled with different cleavable ICAT reagents (mass difference 9 amu) and mixed. This was followed by one-dimensional SDS-PAGE separation, in-gel trypsin digestion, biotin-avidin affinity purification, and LC-MS/MS identification and quantification. Responses to vasopressin for a total of 165 proteins were quantified. Quantification, based on semiquantitative immunoblotting of 16 proteins for which antibodies were available, showed a high degree of correlation with ICAT results. In addition to aquaporin-2 and gamma-epithelial Na channel (gamma-ENaC), five of the immunoblotted proteins were substantially altered in abundance in response to dDAVP, viz., syntaxin-7, Rap1, GAPDH, heat shock protein (HSP)70, and cathepsin D. A 28-protein vasopressin signaling network was constructed using literature-based network analysis software focusing on the newly identified proteins, providing several new hypotheses for future studies.

Mutations in aminolevulinate synthase 2 (ALAS2) are usually associated with sideroblastic anemia and iron overload. The objective of this study was to determine if "mild" mutations in ALAS2 might increase the severity of primary iron overload. Direct sequencing of the ALAS2 gene was performed on 24 subjects with primary hemochromatosis and one subject with sideroblastic anemia with severe iron overload. We identified a novel mutation P520L (c. 1559 C --> T) in ALAS2 in three subjects. Two had severe iron overload and no anemia: one also had HFE C282Y homozygosity, and the other was wildtype for HFE and other iron-related genes. The third subject had sideroblastic anemia with iron overload, and was hemizygous for both P520L and R560H (c. 1679 G --> A) mutations in ALAS2. The P520L mutation was found at a frequency of 0.0013 (741 alleles) in white control subjects, but was not found in 158 alleles from black control subjects. The proline in this position is highly conserved across species from humans to zebrafish. However, genotype/phenotype studies of the families demonstrate that the P520L mutation alone has no iron-associated phenotype, but it may act as a modifier of iron overload in the presence of mutations in HFE or other uncharacterized hemochromatosis genes. Thus, ALAS2 mutations might contribute to more severe iron loading in persons with primary hemochromatosis.

<h4>Background</h4>The successful use of serotonin reuptake inhibitors (SRIs) in obsessive-compulsive disorder (OCD) has led to the hypothesis that serotonin plays a pivotal role in the pathogenesis of OCD. The purpose of the present study was to investigate the role of the serotonin transporter (5-HTT) and serotonin 5-HT1B and 5-HT2A receptor genes in OCD.<h4>Method</h4>The distribution of polymorphic variants was analyzed in 156 OCD cases and 134 control individuals by means of case-control association studies. Potential relevant OCD phenotypes founded on age of onset, positive family history for OCD, clinical subtypes, comorbidity and symptom severity were stratified according to 5-HTT, 5-HT1B and 5-HT2A genotypes.<h4>Results</h4>Patients did not show significant differences in genotype distribution and allele frequency for polymorphisms investigated relative to controls. However, taking in account OCD phenotypes, we found indication towards an association of the 5-HTTLPR S-allele with female OCD patients, and the 5-HT2A G-allele and GG genotype with patients with a positive family history of OCD and an early onset of disease.<h4>Conclusions</h4>Our data yields interesting preliminary results as regards the genetic underpinnings of OCD phenotypes that warrant further discussion and investigation.

We report an experimental and theoretical study on type VIII beta-turn using a designed peptide of sequence GDNP. CD and NMR studies reveal that this peptide exists in equilibrium between type VIII beta-turn and extended conformations. Extensive MD simulations give a description of the free energy landscape of the peptide in which we retrieve the same two main conformations suggested by the experiments. The free energy difference between the two conformational states is very small and the transition between them occurs within a few kT at 300 K on a nanosecond timescale. The equilibrium is mainly driven by entropic contribution, which favors extended conformations over beta-turns. This confirms other theoretical studies showing that beta-turns are marginally stable in water solution because of the larger entropy of the extended state unless some stabilizing interactions exist. Our observations may be extended to any type of beta-turn and have important consequences for protein folding. A comparison of our MD and CD results also suggests a possible type VIII beta-turn CD signature indicated by one main band at 200 nm, close to that of random coil, and a fairly large shoulder at 220 nm. Last, our results clearly show that the XXXP motif can only fold into a type VIII beta-turn, which is consistent with its fairly strong propensity for this type of turn. This important finding may help for peptide design and is in line with recent studies on bioactive elastin peptides.

Mutant huntingtin lowered steady-state levels of DARPP-32 mRNA in the brain but not kidney of R6 transgenic HD mice by repressing transcription from one of two promoters. The activity of DARPP-32 promoter deletion constructs were lower in the presence of mutant huntingtin in immortalized striatal cell lines but no difference in transcription factor binding to the promoter was detected. The activity of CMV, TK and HPRT promoters was also affected by mutant huntingtin in these cell lines. Transient transfection experiments demonstrated that short-term expression of mutant huntingtin exerted a cell- and promoter-specific transcriptional repression. In in vitro experiments, transcription of the CMV promoter was reduced in the presence of striatal proteins and mutant huntingtin. It is likely that select combinations of trans-acting factors, co-activators and components of the Pol II holoenzyme acting in concert provide the basis for both the gene- and tissue-specific effects of mutant huntingtin.

In the developing nervous system, functional neural networks are constructed with intricate coordination of neuronal migrations and axonal projections. We have previously reported a ventral tangential migration of a special type of cortical neurons, lot cells, in the mouse embryo. These neurons originate from the ventricular zone of the entire neocortex, tangentially migrate in the surface layer of the neocortex into the ventral direction, align in the future pathway of the lateral olfactory tract (LOT) and eventually guide the projection of LOT axons. In this study, we developed an organotypic culture system to investigate the regulation of this cell migration in the developing telencephalon. Our data show that the neocortex contains the signals that direct lot cells ventrally, that the ganglionic eminence excludes lot cells by repelling the migration and that lot cells are attracted to netrin 1, an axon guidance factor. Furthermore, we demonstrate that mutations in the genes encoding netrin 1 and its functional receptor Dcc lead to inappropriate distribution of lot cells and subsequent partial disruption of LOT projection. These results suggest that netrin 1 regulates the migration of lot cells and LOT projections, possibly by ensuring the correct distribution of these guidepost neurons.

<h4>Background</h4>Alternative transcripts of protein kinases and protein phosphatases are known to encode peptides with altered substrate affinities, subcellular localizations, and activities. We undertook a systematic study to catalog the variant transcripts of every protein kinase-like and phosphatase-like locus of mouse http://variant.imb.uq.edu.au.<h4>Results</h4>By reviewing all available transcript evidence, we found that at least 75% of kinase and phosphatase loci in mouse generate alternative splice forms, and that 44% of these loci have well supported alternative 5' exons. In a further analysis of full-length cDNAs, we identified 69% of loci as generating more than one peptide isoform. The 1,469 peptide isoforms generated from these loci correspond to 1,080 unique Interpro domain combinations, many of which lack catalytic or interaction domains. We also report on the existence of likely dominant negative forms for many of the receptor kinases and phosphatases, including some 26 secreted decoys (seven known and 19 novel: Alk, Csf1r, Egfr, Epha1, 3, 5,7 and 10, Ephb1, Flt1, Flt3, Insr, Insrr, Kdr, Met, Ptk7, Ptprc, Ptprd, Ptprg, Ptprl, Ptprn, Ptprn2, Ptpro, Ptprr, Ptprs, and Ptprz1) and 13 transmembrane forms (four known and nine novel: Axl, Bmpr1a, Csf1r, Epha4, 5, 6 and 7, Ntrk2, Ntrk3, Pdgfra, Ptprk, Ptprm, Ptpru). Finally, by mining public gene expression data (MPSS and microarrays), we confirmed tissue-specific expression of ten of the novel isoforms.<h4>Conclusion</h4>These findings suggest that alternative transcripts of protein kinases and phosphatases are produced that encode different domain structures, and that these variants are likely to play important roles in phosphorylation-dependent signaling pathways.

Friedreich's ataxia (FRDA) is caused by reduction of frataxin levels to 5-35%. To better understand the biochemical sequelae of frataxin reduction, in absence of the confounding effects of neurodegeneration, we studied the gene expression profile of a mouse model expressing 25-36% of the normal frataxin levels, and not showing a detectable phenotype or neurodegenerative features. Despite having no overt phenotype, a clear microarray gene expression phenotype was observed. This phenotype followed the known regional susceptibility in this disease, most changes occurring in the spinal cord. Additionally, gene ontology analysis identified a clear mitochondrial component, consistent with previous findings. We were able to confirm a subset of changes in fibroblast cell lines from patients. The identification of a core set of genes changing early in the FRDA pathogenesis can be a useful tool in both clarifying the disease process and in evaluating new therapeutic strategies.

We report for the first time that morphine-6-glucuronide, a highly analgesic morphine-derived molecule, is present in adrenal chromaffin granules and secreted from chromaffin cells upon stimulation. We also demonstrate that phosphatidylethanolamine-binding protein (alternatively named Raf-1 kinase inhibitor protein or RKIP) acts as an endogenous morphine-6-glucuronide-binding protein. An UDP-glucuronosyltransferase 2B-like enzyme, described to transform morphine into morphine-6-glucuronide, has been immunodetected in the chromaffin granule matrix, and morphine-6-glucuronide de novo synthesis has been characterized, demonstrating the possible involvement of intragranular UDP-glucuronosyltransferase 2B-like enzyme in morphine-6-glucuronide metabolism. Once secreted into the circulation, morphine-6-glucuronide may mediate several systemic actions (e.g. on immune cells) based on its affinity for mu-opioid receptors. These activities could be facilitated by phosphatidylethanolamine-binding protein (PEBP), acting as a molecular shield and preventing morphine-6-glucuronide from rapid clearance. Taken together, our data represent an important observation on the role of morphine-6-glucuronide as a new endocrine factor.

Endotoxemia is often associated with extreme inflammatory responses and disseminated intravascular coagulation. Protease-activated receptors (PARs) mediate cellular responses to coagulation proteases, including platelet activation and endothelial cell reactions predicted to promote inflammation. These observations suggested that PAR activation by coagulation proteases generated in the setting of endotoxemia might promote platelet activation, leukocyte-mediated endothelial injury, tissue damage, and death. Toward testing these hypotheses, we examined the effect of PAR deficiencies that ablate platelet and endothelial activation by coagulation proteases in a mouse endotoxemia model. Although coagulation was activated as measured by thrombin-antithrombin (TAT) production and antithrombin III (ATIII) depletion, Par1(-/-), Par2(-/-), Par4(-/-), Par2(-/-):Par4(-/-), and Par1(-/-):Par2(-/-) mice all failed to show improved survival or decreased cytokine responses after endotoxin challenge compared with wild type. Thus, our results fail to support a necessary role for PARs in linking coagulation to inflammation or death in this model. Interestingly, endotoxin-induced thrombocytopenia was not diminished in Par4(-/-) mice. Thus, a mechanism independent of platelet activation by thrombin was sufficient to cause thrombocytopenia in our model. These results raise the possibility that decreases in platelet count in the setting of sepsis may not be caused by disseminated intravascular coagulation but instead report on a sometimes parallel but independent process.

Huntington's disease (HD) is a neurodegenerative disease caused by expansion of a polyglutamine tract within the huntingtin protein. Transcriptional dysregulation has been implicated in HD pathogenesis; recent evidence suggests a defect in Sp1-mediated transcription. We used chromatin immunoprecipitation (ChIP) assays followed by real-time PCR to quantify the association of Sp1 with individual genes. We find that, despite normal protein levels and normal to increased overall nuclear binding activity, Sp1 has decreased binding to specific promoters of susceptible genes in transgenic HD mouse brain, in striatal HD cells, and in human HD brain. Genes whose mRNA levels are decreased in HD have abnormal Sp1-DNA binding, whereas genes with unchanged mRNA levels have normal levels of Sp1 association. Moreover, the altered binding seen with Sp1 is not found with another transcription factor, NF-Y. These findings suggest that mutant huntingtin dissociates Sp1 from target promoters, inhibiting transcription of specific genes.

A rapidly growing body of data suggests that abnormalities in serotonergic function might be involved in the pathophysiology of schizophrenia and that serotonergic mechanisms play a role in the therapeutic effects of antipsychotics. The activity of the serotonin transporter (5-HTT), as determined by [(3)H]5-HT uptake to blood lymphocytes, was measured in 38 medicated schizophrenia patients (15 of them treated with typical antipsychotics and 23 treated with atypical antipsychotics) and 15 healthy control subjects. In addition, the pharmacodynamic characteristics of platelet 5-HTT were assessed by [(3)H]citalopram binding. There were no significant differences in the density (B(max)) of platelet [(3)H]citalopram binding sites between the three groups. Similarly, the dissociation constant (K(d)) values were indistinguishable. There were no significant differences in the maximal uptake velocity (V(max)) of [(3)H]5-HT to fresh lymphocytes between the three groups. The affinity (K(m)) values of 5-HT to the 5-HTT were significantly higher in schizophrenia patients treated with typical antipsychotics compared with control subjects. The K(m) values in schizophrenia patients treated with atypical antipsychotics were significantly lower compared with those observed in the group of schizophrenia patients treated with typical antipsychotics; however, they were comparable to values in the control group. The high values of K(m) associated with typical antipsychotic treatment may be relevant to the high risk of developing extrapyramidal side effects (EPS). The role of the various components of the serotonergic system in the etiopathology of schizophrenia and the mechanisms by which antipsychotics achieve their therapeutic effects need to be further evaluated.

SOX6 plays key functions in several developmental processes, including neurogenesis and skeleton formation. In this report, we show that SOX6 is modified in vitro and in vivo by small ubiquitin-related modifier (SUMO) on two distinct sites. Mutation of both sites abolished SOX6 sumoylation and increased SOX6 transcriptional activity. SUMO dependent repression of SOX6 transcription was promoted by UBC9 whereas siRNA to UBC9, cotransfection of inactive UBC9 or a SUMO protease increased SOX6 transcriptional activity. Furthermore, co-expression of SOX6 with SUMO2 results in the appearance of SOX6 in a punctate nuclear pattern that colocalized with promyelocytic leukemia protein, which was partially abolished by mutations in SOX6 sumoylation sites.

<h4>Background</h4>Genome-wide RNA interference (RNAi) screening is a very powerful tool for analyzing gene function in vivo in Caenorhabditis elegans. The effectiveness of RNAi varies from gene to gene, however, and neuronally expressed genes are largely refractive to RNAi in wild-type worms.<h4>Results</h4>We found that C. elegans strains carrying mutations in lin-35, the worm ortholog of the tumor suppressor gene p105Rb, or a subset of the genetically related synMuv B family of chromatin-modifying genes, show increased strength and penetrance for many germline, embryonic, and post-embryonic RNAi phenotypes, including neuronal RNAi phenotypes. Mutations in these same genes also enhance somatic transgene silencing via an RNAi-dependent mechanism. Two genes, mes-4 and zfp-1, are required both for the vulval lineage defects resulting from mutations in synMuv B genes and for RNAi, suggesting a common mechanism for the function of synMuv B genes in vulval development and in regulating RNAi. Enhanced RNAi in the germline of lin-35 worms suggests that misexpression of germline genes in somatic cells cannot alone account for the enhanced RNAi observed in this strain.<h4>Conclusion</h4>A worm strain with a null mutation in lin-35 is more sensitive to RNAi than any other previously described single mutant strain, and so will prove very useful for future genome-wide RNAi screens, particularly for identifying genes with neuronal functions. As lin-35 is the worm ortholog of the mammalian tumor suppressor gene p105Rb, misregulation of RNAi may be important during human oncogenesis.

N-terminal mutant huntingtin (N-mhtt) fragments form inclusions and cause cell death in vitro. Mutant htt expression stimulates autophagy and increases levels of lysosomal proteases. Here, we show that lysosomal proteases, cathepsins D, B and L, affected mhtt processing and levels of cleavage products (cp) known as A and B, which form inclusions. Adding inhibitors of cathepsin D, B and L to clonal striatal cells reduced mhtt, especially mhtt fragment cp A. Mutant htt fully degraded in cathepsin-L-treated lysates but formed stable N-mhtt fragments upon exposure to cathepsin D. Mutagenesis analysis of htt cDNA suggested that cathepsin D and the protease for cp A may cleave htt in the same region. Brain lysates from HD knock-in mice expressed N-mhtt fragments that accumulated with cathepsin D treatment and declined with aspartyl protease inhibition. Findings implicate lysosomal proteases in formation of N-mhtt fragments and clearance of mhtt.

The Olfactomedin family is a relatively new class of extracellular proteins. Two family members have been shown to play roles in the early development of ectodermal tissues: Noelin enhances neural crest generation in chick and Tiarin promotes dorsal neural specification in Xenopus. In this study, we introduce a novel member of the Olfactomedin family, ONT1. In the early chick embryo, ONT1 expression first appears at Hensen's node and subsequently in the axial and paraxial mesoderm. When the neural tube closes, strong expression of ONT1 is transiently found in the roof plate region from the rostral midbrain to the hindbrain. Overexpression of ONT1 in these regions prolongs the generation of neural crest cells in a manner similar to that of Noelin. Interestingly, ONT1 and Noelin have opposing effects on the expression of the migrating neural crest marker HNK-1 in the chick: they, respectively, cause suppression and ectopic induction of this marker. Differential activities among Olfactomedin-related factors are further examined in Xenopus. Microinjection of ONT1 mRNA into the Xenopus embryo expands the expression domain of the neural crest marker FoxD3 at the neurula stage whereas overexpression of Tiarin or Noelin suppresses FoxD3. ONT1 exhibits no dorsalizing effects on the Xenopus neural tube, which contrasts with the strong dorsalizing activity seen for Tiarin. Thus, distinct Olfactomedin-related factors evoke qualitatively different phenotypes even in the same experimental systems, suggesting that Olfactomedin family uses multiple response systems to mediate its signals in embryogenesis.

<h4>Background</h4>The serotonin transporter 5-HTT mediates responses to serotonin reuptake inhibitors (SSRIs), a mainstay treatment in mood disorders. The amygdala, a key emotional processing center, has functional abnormalities in mood disorders, which resolve following successful SSRI treatment. To better understand the effects of SSRIs in mood disorders, we examined the distribution of 5-HTT labeled fibers relative to specific nuclear groups in the amygdala.<h4>Methods</h4>Immunocytochemical techniques were used to chart 5-HTT labeled fibers in the amygdala in coronal sections through the brain of six adult Macaques. Nissl staining was used to define nuclear groups in the amygdala.<h4>Results</h4>The serotonin transporter 5-HTT is distributed heterogeneously in the primate amygdala, with the lateral subdivision of the central nucleus, intercalated cell islands, amygdalohippocampal area, and the paralaminar nucleus showing the heaviest concentrations.<h4>Conclusions</h4>5HTT-labeled fibers are very densely concentrated in output regions of the amygdala. High concentrations of 5-HTT-positive fibers in the central nucleus indicate that tight regulation of serotonin is critical in modulating fear responses mediated by this nucleus. High concentrations of 5-HTT-labeled fibers in the intercalated islands and parvicellular basal nucleus/paralaminar nucleus, which contain immature -appearing neurons, suggest a potential trophic role for serotonin in these subregions.

Maturation promoting factor (MPF), a complex of cyclin-dependent kinase 1 and cyclin B, drives oocyte maturation in all animals. Mechanisms to block MPF activation in developing oocytes must exist to prevent precocious cell cycle progression prior to oocyte maturation and fertilization. This study sought to determine the developmental consequences of precociously activating MPF in oocytes prior to fertilization. Whereas depletion of Myt1 in Xenopus oocytes causes nuclear envelope breakdown in vitro, we found that depletion of the Myt1 ortholog WEE-1.3 in C. elegans hermaphrodites causes precocious oocyte maturation in vivo. Although such oocytes are ovulated, they are fertilization incompetent. We have also observed novel phenotypes in these precociously maturing oocytes, such as chromosome coalescence, aberrant meiotic spindle organization, and the expression of a meiosis II post-fertilization marker. Furthermore, co-depletion studies of CDK-1 and WEE-1.3 demonstrate that WEE-1.3 is dispensable in the absence of CDK-1, suggesting that CDK-1 is a major target of WEE-1.3 in C. elegans oocytes.

Huntingtin is a caspase substrate, and loss of normal huntingtin function resulting from caspase-mediated proteolysis may play a role in the pathogenesis of Huntington disease. Here we tested the hypothesis that increasing huntingtin levels protect striatal neurons from NMDA receptor-mediated excitotoxicity. Cultured striatal neurons from yeast artificial chromosome (YAC)18 transgenic mice over-expressing full-length wild-type huntingtin were dramatically protected from apoptosis and caspase-3 activation compared with cultured striatal neurons from non-transgenic FVB/N littermates and YAC72 mice expressing mutant human huntingtin. NMDA receptor activation induced by intrastriatal injection of quinolinic acid initiated a form of apoptotic neurodegeneration within the striatum of mice that was associated with caspase-3 cleavage of huntingtin in neurons and astrocytes, decreased levels of full-length huntingtin, and the generation of a specific N-terminal caspase cleavage product of huntingtin. In vivo, over-expression of wild-type huntingtin in YAC18 transgenic mice conferred significant protection against NMDA receptor-mediated apoptotic neurodegeneration. These data provide in vitro and in vivo evidence that huntingtin may regulate the balance between neuronal survival and death following acute excitotoxic stress, and that the levels of huntingtin may modulate neuronal sensitivity to excitotoxic neurodegeneration. We suggest that further study of huntingtin's anti-apoptotic function will contribute to our understanding of the pathogenesis of Huntingdon's disease and provide insights into the selective vulnerability of striatal neurons to excitotoxic cell death.

We report carbon nanotube network field-effect transistors (NTNFETs) that function as selective detectors of DNA immobilization and hybridization. NTNFETs with immobilized synthetic oligonucleotides have been shown to specifically recognize target DNA sequences, including H63D single-nucleotide polymorphism (SNP) discrimination in the HFE gene, responsible for hereditary hemochromatosis. The electronic responses of NTNFETs upon single-stranded DNA immobilization and subsequent DNA hybridization events were confirmed by using fluorescence-labeled oligonucleotides and then were further explored for label-free DNA detection at picomolar to micromolar concentrations. We have also observed a strong effect of DNA counterions on the electronic response, thus suggesting a charge-based mechanism of DNA detection using NTNFET devices. Implementation of label-free electronic detection assays using NTNFETs constitutes an important step toward low-cost, low-complexity, highly sensitive and accurate molecular diagnostics.

A rapid throughput screening system involving gene expression analysis was developed in order to investigate the potential of bioactive chemicals contained in natural health products as effective drug therapy, in particular the ability of these chemicals to alleviate the inflammatory response in human airway epithelial cells. A number of databases were searched to retrieve the information needed to properly analyze the gene expression profiles obtained. The gene expression of human bronchial epithelial cells infected with rhinovirus and/or exposed to platelet activating factor was analyzed. Following analysis of the gene expression data the total number of expressed proteins that may potentially act as a marker for monitoring the modulation of airway inflammation was narrowed to 19. Further studies will involve selecting antibodies for these proteins, culturing airway epithelial cells in the presence of extracts of natural health products, extracting the proteins and identifying them by western blot analysis.

<h4>Background</h4>Unbalanced chromosomal translocations may present with a variety of clinical and laboratory findings and provide insight into the functions of genes on the involved chromosomal segments.<h4>Case presentation</h4>A 9 year-old boy presented to our clinic with Factor VII deficiency, microcephaly, a seizure disorder, multiple midline abnormalities (agenesis of the corpus callosum, imperforate anus, bilateral optic nerve hypoplasia), developmental delay, hypopigmented macules, short 5th fingers, and sleep apnea due to enlarged tonsils. Cytogenetic and fluorescence in situ hybridization analyses revealed an unbalanced translocation involving the segment distal to 16p13 replacing the segment distal to 13q33 [46, XY, der(13)t(13;16)(q33;p13.3)]. Specific BAC-probes were used to confirm the extent of the 13q deletion.<h4>Conclusion</h4>This unique unbalanced chromosomal translocation may provide insights into genes important in midline development and underscores the previously-reported phenotype of Factor VII deficiency in 13q deletions.

<h4>Background</h4>The extracellular matrix has a major effect upon the malignant properties of bladder cancer cells both in vitro in 3-dimensional culture and in vivo. Comparing gene expression of several bladder cancer cells lines grown under permissive and suppressive conditions in 3-dimensional growth on cancer-derived and normal-derived basement membrane gels respectively and on plastic in conventional tissue culture provides a model system for investigating the interaction of malignancy and extracellular matrix. Understanding how the extracellular matrix affects the phenotype of bladder cancer cells may provide important clues to identify new markers or targets for therapy.<h4>Methods</h4>Five bladder cancer cell lines and one immortalized, but non-tumorigenic, urothelial line were grown on Matrigel, a cancer-derived ECM, on SISgel, a normal-derived ECM, and on plastic, where the only ECM is derived from the cells themselves. The transcriptomes were analyzed on an array of 1186 well-annotated cancer derived cDNAs containing most of the major pathways for malignancy. Hypervariable genes expressing more variability across cell lines than a set expressing technical variability were analyzed further. Expression values were clustered, and to identify genes most likely to represent biological factors, statistically over-represented ontologies and transcriptional regulatory elements were identified.<h4>Results</h4>Approximately 400 of the 1186 total genes were expressed 2 SD above background. Approximately 100 genes were hypervariable in cells grown on each ECM, but the pattern was different in each case. A core of 20 were identified as hypervariable under all 3 growth conditions, and 33 were hypervariable on both SISgel and Matrigel, but not on plastic. Clustering of the hypervariable genes showed very different patterns for the same 6 cell types on the different ECM. Even when loss of cell cycle regulation was identified, different genes were involved, depending on the ECM. Under the most permissive conditions of growth where the malignant phenotype was fully expressed, activation of AKT was noted. TGFbeta1 signaling played a major role in the response of bladder cancer cells to ECM. Identification of TREs on genes that clustered together suggested some clustering was driven by specific transcription factors.<h4>Conclusion</h4>The extracellular matrix on which cancer cells are grown has a major effect on gene expression. A core of 20 malignancy-related genes were not affected by matrix, and 33 were differentially expressed on 3-dimensional culture as opposed to plastic. Other than these genes, the patterns of expression were very different in cells grown on SISgel than on Matrigel or even plastic, supporting the hypothesis that growth of bladder cancer cells on normal matrix suppresses some malignant functions. Unique underlying regulatory networks were driving gene expression and could be identified by the approach outlined here.

The human N-acetyltransferase genes NAT1 and NAT2 encode two phase-II enzymes that metabolize various drugs and carcinogens. Functional variability at these genes has been associated with adverse drug reactions and cancer susceptibility. Mutations in NAT2 leading to the so-called slow-acetylation phenotype reach high frequencies worldwide, which questions the significance of altered acetylation in human adaptation. To investigate the role of population history and natural selection in shaping NATs variation, we characterized genetic diversity through the resequencing and genotyping of NAT1, NAT2, and the pseudogene NATP in a collection of 13 different populations with distinct ethnic backgrounds and demographic pasts. This combined study design allowed us to define a detailed map of linkage disequilibrium of the NATs region as well as to perform a number of sequence-based neutrality tests and the long-range haplotype (LRH) test. Our data revealed distinctive patterns of variability for the two genes: the reduced diversity observed at NAT1 is consistent with the action of purifying selection, whereas NAT2 functional variation contributes to high levels of diversity. In addition, the LRH test identified a particular NAT2 haplotype (NAT2*5B) under recent positive selection in western/central Eurasians. This haplotype harbors the mutation 341T-->C and encodes the "slowest-acetylator" NAT2 enzyme, suggesting a general selective advantage for the slow-acetylator phenotype. Interestingly, the NAT2*5B haplotype, which seems to have conferred a selective advantage during the past approximately 6,500 years, exhibits today the strongest association with susceptibility to bladder cancer and adverse drug reactions. On the whole, the patterns observed for NAT2 well illustrate how geographically and temporally fluctuating xenobiotic environments may have influenced not only our genome variability but also our present-day susceptibility to disease.

The regulation of iron absorption is of considerable interest in mammals since excretion is minimal. Recent advances in iron metabolism have expounded the molecular mechanisms by which iron absorption is attuned to the physiological demands of the body. The pinnacle was the discovery and identification of hepcidin, a hepatic antimicrobial peptide that regulates absorption to maintain iron homeostasis. While the intricacies of its expression and regulation by HFE, transferrin receptor 2 and hemojuvelin are still speculative, hepcidin responsiveness has correlated negatively with iron absorption in different models and disorders of iron metabolism. Consequently, hepcidin expression is repressed to enhance iron absorption during stimulated erythropoiesis even in situations of elevated iron stores. Animal models have been crucial to the advances in understanding iron metabolism and the present review focuses on phenylhydrazine treated and hypotransferrinaemic rodents. These, respectively, experimental and genetic models of enhanced erythropoiesis highlight the shifting focus of iron absorption regulation from the marrow to the liver.

Membrane-mimetic thin films containing thrombomodulin (TM) and/or heparin were produced and their capacity to inhibit thrombin generation evaluated in a continuous flow system. Tissue factor (TF) along with TM and heparin were immobilized in spatially restricted zones as components of a membrane-mimetic film. Specifically, TF was positioned as an upstream trigger for thrombin generation and TM and/or heparin positioned over the remaining downstream portion of test films. Peak and steady-state levels of thrombin were decreased by antithrombin III (ATIII), as well as by surface bound heparin and TM. Although physiologic concentrations of ATIII have the capacity to significantly inhibit thrombin activity, surface bound TM and heparin nearly abolished steady-state thrombin responses. In particular, surface bound TM appears to be superior to heparin in reducing local thrombin concentrations. These studies are the first to demonstrate the additive effect of surface bound heparin and TM as a combined interactive strategy to limit TF-induced thrombin formation.

The interaction between integrins and extracellular matrix proteins play an important role in the regulation of hematopoiesis. Human hematopoietic progenitor cells express very late antigen-4 (VLA-4) and VLA-5, which mediate their interaction with fibronectin by recognizing the connecting segment-1 (CS-1 and RGD motifs, respectively. In this study, we investigated the ex vivo expansion of human umbilical cord blood (UCB) CD34+ cells on synthetic substrates surface-immobilized with peptides containing the CS-1 binding motif (EILDVPST) and the RGD motif (GRGDSPC). These peptides were covalently conjugated to poly(ethylene terephthalate) (PET) film at a surface density of 2.0-2.3 nmol/cm2. UCB CD34+ cells were cultured for 10 days in serum-free medium supplemented with recombinant human thrombopoietin, stem cell factor, flt3-ligand and interleukin 3. The highest cell expansion fold was observed on the CS-1 peptide-modified surface, where total nucleated cells, total colony forming unit, and long-term culture initiating cells were expanded by 589.6+/-58.6 (mean+/-s.d.), 76.5+/-8.8, and 3.2+/-0.9-fold, respectively, compared to unexpanded cells. All substrates surface-immobilized with peptides, including the control peptides, were more efficient in supporting the expansion of CD34+, CFU-GEMM and LTC-ICs than tissue culture polystyrene surface. Nevertheless, after 10-days of ex vivo expansion from 600 CD34+ cells, only cells cultured on CS-1-immobilized surface yielded positive engraftment, even though the frequency was low. PET surface immobilized with RGD peptide was less efficient than that with CS-1 peptide. Our results suggest that covalently immobilized adhesion peptides can significantly influence the proliferation characteristics of cultured UCB CD34+ cells.

Huntingtin (htt), the protein encoded by the Huntington's disease (HD) gene, contains a polymorphic stretch of glutamines (polyQ) near its N-terminus. When the polyQ stretch is expanded beyond 37Q, HD results. However, the role of the normal polyQ stretch in the function of htt is still unknown. To determine the contribution of the polyQ stretch to normal htt function, we have generated mice with a precise deletion of the short CAG triplet repeat encoding 7Q in the mouse HD gene (Hdh(DeltaQ)). Hdh(DeltaQ/DeltaQ) mice are born with normal Mendelian frequency and exhibit no gross phenotypic differences in comparison to control littermates, suggesting that the polyQ stretch is not essential for htt's functions during embryonic development. Adult mice, however, commit more errors initially in the Barnes circular maze learning and memory test and perform slightly better than wild-type controls in the accelerating rotarod test for motor coordination. To determine whether these phenotypes may reflect an altered cellular physiology in the Hdh(DeltaQ) mice, we characterized the growth and energy status of primary embryonic and adult Hdh(DeltaQ/DeltaQ) fibroblasts in culture. The Hdh(DeltaQ) fibroblasts exhibited elevated levels of ATP, but senesced prematurely in comparison with wild-type fibroblasts. Taken altogether, these results suggest that htt's polyQ stretch is required for modulating longevity in culture and support the hypothesis that the polyQ stretch may also modulate a htt function involved in regulating energy homeostasis.

The present study examines the frequency of the two main HFE mutations (C282Y and H63D) in a randomly selected population of 346 individuals including 201 DNA samples from women with cervical neoplasia (including high-grade squamous intraepithelial lesions and invasive squamous cell carcinoma) and a control population of 146 women from the same geographical area. We found a significantly lower risk of development of cervical neoplasia in H63D carriers (OR = 0.56; 95% CI 0.35-0.92; p = 0.01). Multivariate logistic regression analysis confirms this observation (OR = 0.55; 95% CI 0.35-0.88, p = 0.01). Regarding the C282Y mutation no association was found (OR = 1.32; 95% CI 0.53-3.33; p = 0.52). In addition, a significant difference between H63D carrier and non-carrier women on the time-to-onset of cervical lesions was observed (log-rank test: p = 0.0012). These results indicate that HFE could be considered a candidate modifier gene of viral-related neoplasia such as cervical carcinoma possibly by a dual role on iron metabolism and immunological system.

Peroxiredoxin 6 (Prd x 6) is a novel peroxidase enzyme that is expressed at a high level in the lung. We tested the hypothesis that transgenic (Tg) mice overexpressing Prd x 6 would exhibit increased resistance to hyperoxia-induced lung injury. Wild-type and Tg mice were exposed to 100% O(2) and evaluated for survival, lung histopathology, total protein, and nucleated cells in bronchoalveolar lavage fluid (BALF), and oxidation of lung protein and lipids. Prd x 6 protein expression and enzyme activity were approximately 3-fold higher in Tg lungs compared with wild-type. Tg mice survived longer during exposure to 100% O(2) (LT(50) 104+/-2.8 h in Tg versus 88.9+/-1.1 h for wild-type). Lung wet/dry weight ratio and total protein and nucleated cell count in lung lavage fluid were significantly greater in wild-type mice at 72 and 96 h of hyperoxia compared with Tg mice. At 96 h of hyperoxia, Tg mice had less epithelial cell necrosis, perivascular edema, and inflammatory cell recruitment by light microscopy, and lower TBARS and protein carbonyls in lung homogenate (P<0.05). These results show that Tg mice have increased defense against lung injury in hyperoxia, providing evidence that Prd x 6 functions as a lung antioxidant enzyme.

<h4>Introduction</h4>In hemolytic diseases such as sickle cell disease and beta-thalassemia, the mechanisms of thrombosis are poorly understood, however erythrocyte/endothelium interactions are thought to play an important role. Appropriate animal models would increase our understanding of the pathophysiology of thrombosis and aid in the development of new therapeutic strategies. We previously reported that rats exposed to 2-butoxyethanol (2-BE) develop hemolysis and enhanced adherence of erythrocytes to the extracellular matrix, possibly secondary to the recruitment of cellular adhesion molecules at the erythrocyte/endothelium interface.<h4>Methods</h4>We exposed rats to 250 mg/kg/day of 2-BE for 4 days, and collected blood for coagulation markers on each day.<h4>Results</h4>As previously observed, erythrocytes dropped precipitously (8.0 to 1.8x10(6)/microl in 48 h), and diffuse microvascular thrombosis developed in the heart, lungs, liver, bones and eyes. Prothrombin times, activated partial thromboplastin times, fibrinogen, and antithrombin-III were unchanged between treated and control rats, indicating that hemostasis is largely unperturbed. However the thrombin-antithrombin III levels in the 2-BE treated rats for all days were 3-7 times greater than the control rats. The plasma intercellular adhesion molecule-1 (ICAM-1) levels of 2-BE treated animals were approximately twice that of the controls on days 2 and 3 and 1.5 times the controls on day 4 (P<0.05).<h4>Conclusion</h4>Our findings are consistent with the observations of increased erythrocyte aggregation, increased erythrocyte/endothelium interaction, and increased plasma ICAM-1 levels observed in sickle cell disease and beta-thalassemia patients. This model may be useful for studying therapeutic agents that disrupt erythrocyte/endothelium interactions.

In view of the limited success of available treatment modalities for breast cancer, alternative and complementary strategies need to be developed. The delineation of the molecular basis of breast cancer provides the possibility of specific intervention by gene therapy through the introduction of genetic material for therapeutic purposes. In this regard, several gene therapy approaches for carcinoma of the breast have been developed. These approaches can be divided into six broad categories: (1) mutation compensation, (2) molecular chemotherapy, (3) proapoptotic gene therapy, (4) antiangiogenic gene therapy, (5) genetic immunopotentiation, and (6) genetic modulation of resistance/sensitivity. Clinical trials for breast cancer have been initiated to evaluate safety, toxicity, and efficacy. Combined modality therapy with gene therapy and chemotherapy or radiation therapy has shown promising results. It is expected that as new therapeutic targets and approaches are identified and advances in vector design are realized, gene therapy will play an increasing role in clinical breast cancer treatment.

Embryos of avian eggs and mammals are highly sensitive to oxidative stress and hence maintaining a steady reducing environment during the embryonic development is known to confer protection. Although information is completely lacking, proteins of avian egg albumin which have been suggested to play various biological functions, are the major targets for such reducing state during embryogenesis. In this study, we found that ovotransferrin (OTf), the second major protein in egg albumin, undergoes autocleavage at distinct sites upon reduction with thiol-reducing agent or thioredoxin-reducing system. Mass spectral and microsequencing analysis indicated that OTf is able to cleave itself through the unique chemical reactivity of four tripeptides motifs, HTT (residues 209-211), HST (residues 542-544) and two CHT (residues 115-117 and 454-456). Intriguingly, these self-cleavage sites were uniquely located upstream and downstream of the two disulfide kringle domains (residues 115-211 and 454-544) of OTf. These reduction-scissile sequences, His/Cys-X-Thr, are evolutionary conserved self-cleavage motifs found in several autoprocessing proteins including hedgehog proteins. Interestingly, reduction of other two members of transferrin family induced autocleavage patterns, similar to that of OTf, in bovine lactoferrin (bLf) while human lactoferrin (hLf) showed much less self-cleaving activity. This finding is the first to describe that transferrins are a new subset in the class of proteins able to carry out autoprocessing, providing insight into this unusual biochemical process that appears to be a molecular switch involved in triggering a yet unidentified function(s) of OTf as well as bLf.

Hepcidin is an antimicrobial peptide secreted by the liver during inflammation that plays a central role in mammalian iron homeostasis. Here we demonstrate the endogenous expression of hepcidin by macrophages and neutrophils in vitro and in vivo. These myeloid cell types produced hepcidin in response to bacterial pathogens in a toll-like receptor 4 (TLR4)-dependent fashion. Conversely, bacterial stimulation of macrophages triggered a TLR4-dependent reduction in the iron exporter ferroportin. In vivo, intraperitoneal challenge with Pseudomonas aeruginosa induced TLR4-dependent hepcidin expression and iron deposition in splenic macrophages, findings mirrored in subcutaneous infection with group A Streptococcus where hepcidin induction was further observed in neutrophils migrating to the tissue site of infection. Hepcidin expression in cultured hepatocytes or in the livers of mice infected with bacteria was independent of TLR4, suggesting the TLR4-hepcidin pathway is restricted to myeloid cell types. Our findings identify endogenous myeloid cell hepcidin production as a previously unrecognized component of the host response to bacterial pathogens.

Hereditary hemochromatosis (HH), the most common genetic disease in northern Europeans, is an autosomal recessive disorder of iron metabolism. The association between hepatocellular carcinoma and HFE homozygosity is well documented, but recently HFE hetero- and homozygosity has also been linked to nonhepatocellular malignancies, including female breast cancer. We hypothesized that C282Y and H63D mutations in the HFE gene could contribute to male breast cancer (MBC) and prostate cancer (PC) susceptibility at the population level in Finland. We screened the 2 major HFE mutations, H63D and C282Y, from 116 MBC cases diagnosed in Finland between 1967 and 1996, 843 consecutive unselected PC cases diagnosed at the Pirkanmaa Hospital District between 1999 and 2001 and 480 anonymous blood donor controls by minisequencing. Our results indicate that the frequencies of the HFE mutations do not significantly differ between MBC and PC patients and the population-based controls. No significantly altered risks for MBC or PC among carriers of the 2 variants were observed. However, HFE mutations were seen twice as often among carriers of a common BRCA2 mutation 9346(-2)A-->G compared with the rest of the MBC cases, indicating that HFE may be an MBC risk modifier gene among BRCA2 mutation carriers. In conclusion, our results indicate a minor role for the HFE mutations C282Y and H63D in the causation of MBC and PC, but carriers of both BRCA2 9346(-2)A-->G and an HFE mutation may be at an increased risk.

Barrett's esophagus (BE) is a major predisposing factor for the development of esophageal adenocarcinoma. Current strategies for treatment of BE, both dysplastic and nondysplastic, include photodynamic therapy (PDT) and argon plasma coagulation (APC). However, the effect of ablative therapy at the genetic level is unclear. We performed loss of heterozygosity (LOH) analysis of BE in baseline and follow-up biopsy specimens from 21 patients with BE (17 male, 4 female) treated with PDT and/or APC. At baseline, 14 patients had intestinal metaplasia without dysplasia (MET), 4 low-grade dysplasia (LGD) and 3 high-grade dysplasia (HGD). LOH was assessed using a panel of 9 polymorphic markers for evaluation of the P53 gene on 17p, P16 on 9p, DCC and SMAD4 on 18q and the APC gene on 5q. The tissue specimens obtained at baseline (t = 0) were analysed, as well as the first (t = 1; mean interval: 4 months) and last (t = 2; mean interval: 8 months) available biopsy with residual or recurrent BE after ablation. At t = 0, allelic loss was detected of 5q in 27%, 9p in 56%, 17p in 31% and 18q in 6% of informative cases. At t = 1 (18 patients with persistent MET and 3 with LGD) and at t = 2 (8 MET, 2 LGD), the LOH patterns were not statistically different from t = 0. Further, multiple genetic lineages before and after therapy were detected in 15 cases illustrating the multiclonal nature of BE. We conclude that recurrent and/or persistent BE after ablative therapy still contains genetic alterations associated with malignant progression to cancer. Therefore, the goal of treatment should be the complete elimination of Barrett's mucosa.

MCJ (DNAJD1) is a recently discovered member of the DNAJ protein family whose expression is controlled epigenetically by methylation of a CpG island located within the 5' transcribed region of its gene. Methylation-dependent transcriptional silencing of MCJ has been observed in ovarian cancers and associated with increased resistance to chemotherapeutic agents; however, its role in other cancer types has not been widely investigated. We examined the status of MCJ in intracranial primitive neuroectodermal tumours [PNETs, comprising cerebellar PNETs (medulloblastomas) and supratentorial PNETs (stPNETs)] and ependymomas, together representing the most common malignant brain tumours of childhood. Evidence of MCJ hypermethylation was found in all 3 tumour types [medulloblastomas, 3/9 (33%) cell lines, 2/28 (7%) primary tumours; stPNETs, 2/2 (100%) cell lines, 3/10 (30%) primary tumours; and ependymomas, 2/20 (10%) primary tumours] but not in nonneoplastic brain tissues (n = 11), indicating that MCJ methylation is a tumour-specific event. In methylated cases, the distribution of methylated CpG sites across the CpG island could be broadly divided into 2 patterns: (i) extensive methylation of the majority of CpG sites across the island or (ii) limited methylation of individual CpG sites concentrated towards the 5' end of the island. Extensive methylation patterns were associated with the methylation-dependent transcriptional silencing of MCJ in medulloblastoma and stPNET cell lines. Further investigations of the mechanism of MCJ inactivation revealed that its loss could occur either through biallelic epigenetic methylation or by methylation in association with genetic loss of its second allele. These data indicate that epigenetic inactivation of MCJ may play a role in the development of a range of paediatric brain tumour types, and its role in disease pathogenesis and chemotherapeutic resistance should now be investigated further.

Mammalian Stau1 (Staufen1), a modular protein composed of several dsRBDs (double-stranded RNA-binding domains), is probably involved in mRNA localization. Although Stau1 is mostly described in association with the rough endoplasmic reticulum and ribosomes in the cytoplasm, recent studies suggest that it may transit through the nucleus/nucleolus. Using a sensitive yeast import assay, we show that Stau1 is actively imported into the nucleus through a newly identified bipartite nuclear localization signal. As in yeast, the bipartite nuclear localization signal is necessary for Stau1 nuclear import in mammalian cells. It is also required for Stau1 nucleolar trafficking. However, Stau1 nuclear transit seems to be regulated by mechanisms that involve cytoplasmic retention and/or facilitated nuclear export. Cytoplasmic retention is mainly achieved through the action of dsRBD3, with dsRBD2 playing a supporting role in this function. Similarly, dsRBD3, but not its RNA-binding activity, is critical for Stau1 nucleolar trafficking. The function of dsRBD3 is strengthened or stabilized by the presence of dsRBD4 but prevented by the interdomain between dsRBD2 and dsRBD3. Altogether, these results suggest that Stau1 nuclear trafficking is a highly regulated process involving several determinants. The presence of Stau1 in the nucleus/nucleolus suggests that it may be involved in ribonucleoprotein formation in the nucleus and/or in other nuclear functions not necessarily related to mRNA transport.

The CRT (creatine transporter) is a member of the Na+- and Cl--dependent neurotransmitter transporter family and is responsible for the import of creatine into cells, and thus is important for cellular energy metabolism. We established for CRT an expression system in HEK-293 cells that allowed biochemical, immunological and functional analysis of CRT wild-type and glycosylation-deficient mutants. Analysis of HA (haemagglutinin)-tagged CRT-NN (wild-type rat CRT with an HA-tag at the C-terminus) revealed several monomeric immunoreactive species with apparent molecular masses of 58, 48 and 43 kDa. The 58 kDa species was shown to be plasma-membrane-resident by EndoHf (endoglycosidase Hf) and PNGase F (peptide N-glycosidase F) treatments and represents fully glycosylated CRT, whereas the 48 kDa and 43 kDa species were glycosylation intermediates and non-glycosylated CRT respectively. Glycosylation-deficient mutants (Asn192Asp, Asn197Asp and Asn192Asp/Asn197Asp) showed altered electrophoretic mobility, indicating that CRT is indeed N-glycosylated. In addition, a prominent CRT band in the range of 75-91 kDa was also detected. Pharmacological inhibition of N-linked glycosylation by tunicamycin in CRT-NN-expressing cells gave a similar reduction in molecular mass, corroborating the finding that Asn192 and Asn197 are major N-glycosylation sites in CRT. Although the apparent Km was not significantly affected in glycosylation-deficient mutants compared with CRT-NN, we measured reduced Vmax values for all mutants (21-28% residual activity), and 51% residual activity after enzymatic deglycosylation of surface proteins in intact CRT-NN cells by PNGase F. Moreover, immunocytochemical analysis of CRT-NN- and CRT-DD-expressing cells (where CRT-DD represents a non-glycosylated double mutant of CRT, i.e. Asn192Asp/Asn197Asp) showed a lower abundance of CRT-DD in the plasma membrane. Taken together, our results suggest that plasma-membrane CRT is glycosylated and has an apparent monomer molecular mass of 58 kDa. Furthermore, N-linked glycosylation is neither exclusively important for the function of CRT nor for surface trafficking, but affects both processes. These findings may have relevance for closely related neurotransmitter transporter family members.

A well known strategy to prepare high affinity ligands for a biological receptor is to link together low affinity ligands. DCC (dynamic combinatorial chemistry) was used to select bifunctional protein ligands with high affinity relative to the corresponding monofunctional ligands. Thiol to disulfide linkage generated a small dynamic library of bifunctional ligands in the presence of calmodulin, a protein with two independently mobile domains. The binding constant of the bifunctional ligand (disulfide) most amplified by the presence of calmodulin is nearly two orders of magnitude higher than that of the corresponding monofunctional ligand (thiol).

The p53 protein is a transcription factor that integrates various cellular stress signals. The accumulation of the mutant huntingtin protein with an expanded polyglutamine tract plays a central role in the pathology of human Huntington's disease. We found that the huntingtin gene contains multiple putative p53-responsive elements and p53 binds to these elements both in vivo and in vitro. p53 activation in cultured human cells, either by a temperature-sensitive mutant p53 protein or by gamma-irradiation (gamma-irradiation), increases huntingtin mRNA and protein expression. Similarly, murine huntingtin also contains multiple putative p53-responsive elements and its expression is induced by p53 activation in cultured cells. Moreover, gamma-irradiation, which activates p53, increases huntingtin gene expression in the striatum and cortex of mouse brain, the major pathological sites for Huntington's disease, in p53+/+ but not the isogenic p53-/- mice. These results demonstrate that p53 protein can regulate huntingtin expression at transcriptional level, and suggest that a p53 stress response could be a modulator of the process of Huntington's disease.

The presence of growth hormone (GH) and GH receptors (GHRs) in the lung suggests it is an autocrine/paracrine target site for pulmonary GH action and/or an endocrine site of pituitary GH action. Roles for GH in lung growth or pulmonary function are, however, uncertain. The possibility that pituitary and/or pulmonary GH have physiological roles in lung development has therefore been investigated in GHR knockout (KO or -/-) mice, using a proteomics approach to determine if an absence of GH-signaling affects the proteome of the developing lung. More than 600 proteins were detected by 2-DE in the lungs of control [GHR (+/+)] and GHR (-/-) mice at the end of the alveolarization period (at day 14 postnatally). Of these, 39 differed significantly in protein content at the p>0.05 level [6 were of higher abundance in the GHR (-/-) group, 33 were of lower abundance] and 17 differed at the p>0.02 level [5 of higher abundance in the GHR (-/-) group, 12 of lower abundance] and 7 were definitively identified by MS. Vimentin, a protein involved in cellular proliferation, was reduced in content by approximately 75% in the lungs of the GHR (-/-) mice. Three proteins involved in oxidative protection [SH3 domain-binding glutamic acid-rich-like protein, peroxiredoxin 6 (Prdx6), and isocitrate dehydrogenase 1] were also of lower content in the GHR (-/-) lungs (by approximately 88%, 81% and 70%, respectively). Prdx6 is also involved in lipid and surfactant metabolism, as is apolipoprotein A-IV, the lung content of which was reduced by approximately 73% in these mice. Proteasome 26S ATPase subunit 4, a protein involved in the non-lysosomal degradation of intracellular proteins, and electron flavoprotein alpha subunit , involved in intracellular metabolism, were also reduced in content in the lungs of the GHR (-/-) mice (by approximately 70% and 49%, respectively). These results therefore suggest that these proteins are normally dependent upon GH signaling, and that GH is normally involved in early lung growth, oxidative protection, lipid and energy metabolism and in proteasomal activity. These roles may reflect endocrine actions of pituitary GH and/or local autocrine/paracrine actions of GH produced within the lung.

<h4>Background</h4>In some Caucasian populations it has been found that the C282Y hemochromatosis (HFE) gene mutation is a risk factor for the development of leukemia and other malignancies.<h4>Methods</h4>In a group of 50 Mexican mestizo patients and 153 normal controls, the HFE gene mutations H63D and C282Y were studied by means of ARMS-PCR.<h4>Results</h4>In the group of patients with leukemia we found a heterozygote for the C282Y mutation, seven heterozygotes for the H63D mutation, a double heterozygote for the H63D / C282Y mutation and 41 normal homozygotes. These data are not different from those observed in normal controls, where the allele frequencies were 0.062 and 0.013 for the H63D and C282Y HFE gene mutations, respectively.<h4>Conclusions</h4>These HFE gene mutations are not risk factors for the development of leukemia in Mexican mestizos.

The chronic phase of pulmonary arterial hypertension (PAH) is associated with vascular remodeling, especially thickening of the smooth muscle layer of large pulmonary arteries and muscularization of small pulmonary vessels, which normally have no associated smooth muscle. Serotonin (5-hydroxytryptamine, 5-HT) has been shown to induce proliferation and hypertrophy of pulmonary artery smooth muscle cells (PASMC), and may be important for in vivo pulmonary vascular remodeling. Here, we show that 5-HT stimulates migration of pulmonary artery PASMC. Treatment with 5-HT for 16h increased migration of PASMC up to four-fold as monitored in a modified Boyden chamber assay. Increased migratory responses were associated with cellular morphological changes and reorganization of the actin cytoskeleton. 5-HT-induced alterations in morphology were previously shown in our laboratory to require cAMP [Lee SL, Fanburg BL. Serotonin produces a configurational change of cultured smooth muscle cells that is associated with elevation of intracellular cAMP. J Cell Phys 1992;150(2):396-405], and the 5-HT4 receptor was pharmacologically determined to be the primary activator of cAMP in bovine PASMC [Becker BN, Gettys TW, Middleton JP, Olsen CL, Albers FJ, Lee SL, et al. 8-Hydroxy-2-(di-n-propylamino)tetralin-responsive 5-hydroxytryptamine4-like receptor expressed in bovine pulmonary artery smooth muscle cells. Mol Pharmacol 1992;42(5):817-25]. We examined the role of the 5-HT4 receptor and cAMP in 5-HT-induced bovine PASMC migration. PASMC express 5-HT4 receptor mRNA, and a 5-HT4 receptor antagonist and a cAMP antagonist completely blocked 5-HT-induced cellular migration. Consistent with our previous report that a cAMP-dependent Cl(-) channel is required for 5-HT-induced morphological changes in PASMC, phenylanthranilic acid, a Cl(-) channel blocker, inhibited actin cytoskeletal reorganization and migration produced by 5-HT. We conclude that 5-HT stimulates PASMC migration and associated cytoskeletal reorganization through the 5-HT4 receptor and cAMP activation of a chloride channel.

The knowledge of estrogen receptor (ER) status is important in the management of breast cancer patients. More precisely, analytical methods for ER determination have changed over the last two decades from ligand binding assay (LBA) dextran-coated charcoal (DCC) to enzyme immuno-assay (EIA) and more recently immunohistochemistry (IHC). We examined the respective clinical impact of ER determination according to these 3 methods over the period 1983-1999 within a group of 1940 patients, all operated and followed in the single institution Centre Antoine Lacassagne. Validated cut off values were 10 and 15 fmol/mg protein for both LBA-DCC and EIA, respectively and 10% of stained cells for IHC. During the years it was noted that the initial size of the tumor decreased and that the proportion of positive axillary nodes and negative ER tumors was different according to the ER method. ER negativity was 20, 13 and 10% in LBA-DCC, EIA, IHC, respectively. ER was a strong predictor of overall survival in the whole population (Mantel-Cox, p < 0.00001); however when stratifying the analysis on ER method groups, ER was still a prognostic indicator in the EIA, LBA-DCC group but not in the IHC group (the follow-up was too short). It is important to keep these data in mind when conducting large retrospective studies evaluating prognostic markers in breast cancer patients.

Periventricular heterotopia (PH) is a malformation of cortical development characterized by nodules of neurons, ectopically located along the lateral ventricles of the brain. Mutations in the vesicle transport ADP-ribosylation factor guanine exchange factor 2 gene (ARFGEF2) or the actin-binding Filamin A (FLNA) gene cause PH. Previous studies have shown that FLNA expression is developmentally regulated, with strongest expression observed along the ventricular zone (VZ) and to a lesser degree in postmitotic neurons in the cortex. Here we characterize the expression patterns for ARFGEF2 within the central nervous systems of human and mouse in order to better understand their potential roles in causing PH. ARFGEF2 mRNA was widely expressed in all cortical layers, especially in the neural precursors of the ventricular and subventricular zones (SVZ) during development, with persistent but diminished expression in adulthood. ARFGEF2 encodes for the protein brefeldin-inhibited guanine exchange factor 2 (BIG2). BIG2 protein immunoreactivity was most strongly localized to the neural progenitors along the neuroependymal lining of the VZ during development, with decreased expression in adulthood. Furthermore, overlapping BIG2 and FLNA expression was greatest in these same neuroependymal cells of human embryonic brain and was co-expressed in progenitors by Western blot. Finally, transfection of a dominant-negative construct of ARFGEF2 in SHSY5Y neuroblastoma cells partially blocked FLNA transport from the Golgi apparatus to the cell membrane. These results suggest that mutations in ARFGEF2 may impair targeted transport of FLNA to the cell surface within neural progenitors along the neuroependyma and that disruption of these cells could contribute to PH formation.

This phase I study investigated the maximum tolerated dose and pharmacokinetics of a 3-weekly administration of BMS-188797, a paclitaxel derivate, at three dose levels (DLs) (80, 110 and 150 mg m(-2) DL), combined with cisplatin (standard dose 75 mg m(-2)). In 16 patients with advanced malignancies treated, one patient experienced dose-limiting febrile neutropenia, sepsis and severe colitis at the 150 mg m(-2) DL; at the 110 mg m(-2) DL one episode of dose-limiting grade 3 diarrhoea/nausea occurred. Grade 3/4 haematological toxicities were leucopenia/neutropenia; grade 3 nonhaematological toxicities were neuropathy, nausea, diarrhoea and stomatits. Objective response was seen in four patients, with three complete remissions in ovarian and cervical cancer patients. Pharmacokinetics of BMS-188797 appeared linear through the 110 mg m(-2), but not through the 150 mg m(-2) DL. The mean+/-SD values for clearance, distribution volume at steady state and terminal half-life during cycle 1 were 317+/-60 ml min(-1) m(-2), 258+/-96 l m(-2) and 30.8+/-7.7 h, respectively. The maximum tolerated and recommended phase II dose for BMS-188797 was 110 mg m(-2) (1-h infusion, every 3 weeks) combined with cisplatin 75 mg m(-2).

<h4>Background</h4>Although statin-treatment during the acute phase of unstable coronary syndromes improve the outcome their effects on thrombosis/fibrinolysis system in normocholesterolemic patients admitted with unstable angina remain obscure. We assessed the effects of short-term atorvastatin treatment on thrombotic/fibrinolysis markers in normocholesterolemic in patients with unstable angina.<h4>Methods</h4>Forty-five patients with unstable angina were allocated into two groups to receive atorvastatin 10 mg/day (n = 24) or no statin (n = 21) for 6 weeks. Circulating levels of von Willebrand Factor (vWF), factor V (fV), protein C (prC), tissue plasminogen activator (tPA) and antithrombin III (ATIII) were measured by enzyme linked immunosorbent assay, by the patients admission and at the 1st and 6th week of the study.<h4>Results</h4>After 1 week of treatment, a significant increase of ATIII (p < 0.05), fV (p < 0.01) and vWF (p < 0.05) was found in the control group, but not in atorvastatin-treated group. Similarly, at 6 weeks after admission, plasma levels of ATIII were still significantly higher than at baseline in controls (p < 0.05), but not in atorvastatin-treated group. Plasma levels of PrtC were significantly increased in both controls (p < 0.01) and atorvastatin-treated patients (p < 0.05) at 1 week, while remained unaffected in atorvastatin-treated group at 6th week. There was no significant difference in the variations of plasma levels of tPA, PrtS and fVII between the two groups at 1 and 6 weeks after admission.<h4>Conclusions</h4>In normocholesterolemic patients admitted with unstable angina the early administration of atorvastatin, significantly affects von Willebrand factor levels and the expression of liver-derived components of both thrombosis and fibrinolysis system.

Transfer of iron from the mucosa is a critical step in dietary iron assimilation that is tightly regulated to ensure the appropriate amount of iron is absorbed to meet the body's demands. Too much iron is highly toxic, and failure to properly control intestinal iron export causes iron overload associated with hereditary forms of hemochromatosis. One form of genetic iron overload, ferroportin disease, originates due to defects in ferroportin, the membrane iron exporter. Ferroportin acts in conjunction with the intestinal ferroxidase hephaestin to mediate release of iron from the enterocyte. How iron is then acquired by transferrin and released into circulation remains an unknown step in this process.

We report the coexistence of Wilson's disease and genetic haemochromatosis in one family. The diagnosis of genetic haemochromatosis was established in a 52-year-old man. Among his siblings, one 57-year-old sister and one 55-year-old brother had decreased copper and ceruloplasmin levels in serum and increased urinary copper excretion. The sister shared the same human leucocyte antigen haplotypes and was homozygous for the HFE mutation C282Y, like the propositus. However, she had normal liver iron content and increased liver copper content. Her dietary copper intake was probably excessive. The association of Wilson's disease and genetic haemochromatosis is rare and has only been described twice. The onset of Wilson's disease after 50 years of age is rare; Wilson's disease should be considered in any patient with unexplained chronic liver disease; an excess in liver copper content might be induced by excessive dietary input in a susceptible individual.

A field demonstration of reduction of lead availability in a soil and cabbage (Brassica Chinensis L.) contaminated by mining tailings, located in Shaoxing, China was carried out to evaluate the effects of applications of phosphorus fertilizers on Pb fractionation and Pb phyto-availability in the soil. It was found that the addition of all three P fertilizers including single super phosphate (SSP), phosphate rock (PR), and calcium magnesium phosphate (CMP) significantly decreased the percentage of water-soluble and exchangeable (WE) soil Pb and then reduced the uptake of Pb, Cd, and Zn by the cabbage compared to the control (CK). The results showed that the level of 300 g P/m(2) soil was the most cost-effective application rate of P fertilizers for reducing Pb availability at the first stage of remediation, and that at this P level, the effect of WE fraction of Pb in the soil decreased by three phosphorus fertilizers followed the order: CMP (79%)>SSP (41%)>PR (23%); Effectiveness on the reduction of Pb uptake by cabbage was in the order: CMP (53%)>SSP (41%)>PR (30%). Therefore our field trial demonstrated that it was effective and feasible to reduce Pb availability in soil and cabbage contaminated by mining tailings using P fertilizers in China and PR would be a most cost-effective amendment.

During neuronal development, netrin and its receptors UNC5 and DCC (deleted in colorectal cancer) guide axonal growth cones in navigating to their targets. Netrin also plays important roles in the regulation of cell migration, tissue morphogenesis and tumor growth. Here, we show that netrin induces UNC5 tyrosine phosphorylation and that this effect of netrin is dependent on its co-receptor DCC. UNC5 tyrosine phosphorylation is known to be important for netrin to induce cell migration and axonal repulsion. Src tyrosine kinase activity is required for netrin to stimulate UNC5 tyrosine phosphorylation in neurons and transfected cells. The SH2 domain of Src kinase directly interacts with the cytosolic domain of UNC5 in a tyrosine-phosphorylation-dependent manner. Furthermore, the tyrosine kinase focal adhesion kinase (FAK) is also involved in netrin-induced UNC5 tyrosine phosphorylation. Both Src and FAK can phosphorylate UNC5. Our data suggest a model in which netrin stimulates UNC5 tyrosine phosphorylation and signaling in a manner dependent on the co-receptor DCC, through the recruitment of Src and FAK kinases.

The current studies of apoptosis in rheumatoid arthritis (RA) suggest that the TNF ligand-receptor superfamily (TNFRsF) molecules, downstream pathways (activation of proapoptosis or anti-apoptosis pathway), cell types (lymphocytes and synovial fibroblast), and the mechanism that triggers apoptosis (tolerance induction-related, downmodulation of inflammation-related, or DNA damage-related) all exhibit a capability to determine the induction or prevention of RA. This series of defects at different levels and in different cells have been shown to lead to T cell and synovial hyperproliferation, defective apoptosis, excessive apoptosis, or bone erosion. In this chapter, we summarize the available knowledge of the regulation of TNFRsF and their likely pathogenic roles in RA to help identify candidate target cells and target molecules for delivery of gene constructs to modulate apoptosis to prevent the development of RA in both humans and mice.

Differentiation of embryonic stem (ES) cells from a pluripotent to a committed state involves global changes in genome expression patterns. Gene activity is critically determined by chromatin structure and interactions of chromatin binding proteins. Here, we show that major architectural chromatin proteins are hyperdynamic and bind loosely to chromatin in ES cells. Upon differentiation, the hyperdynamic proteins become immobilized on chromatin. Hyperdynamic binding is a property of pluripotent cells, but not of undifferentiated cells that are already lineage committed. ES cells lacking the nucleosome assembly factor HirA exhibit elevated levels of unbound histones, and formation of embryoid bodies is accelerated. In contrast, ES cells, in which the dynamic exchange of H1 is restricted, display differentiation arrest. We suggest that hyperdynamic binding of structural chromatin proteins is a functionally important hallmark of pluripotent ES cells that contributes to the maintenance of plasticity in undifferentiated ES cells and to establishing higher-order chromatin structure.

<h4>Background and aim</h4>Hereditary hemochromatosis (HH) is a disorder characterized by inappropriately high intestinal iron absorption. In populations of Northern European descent, HH is most commonly caused by mutations (C282Y/H63D) in the HFE gene.<h4>Methods and results</h4>We investigated the effects of dietary heme iron intake and HFE mutations on serum ferritin concentrations in a population-based random sample of 1611 women aged >50 years using analysis of covariance (ANCOVA). Higher heme iron intake was associated with significantly higher serum ferritin concentrations (P(trend) < 0.001). Also, women with the compound or C282Y homozygous genotype had significantly higher serum ferritin concentrations (geometric mean 115.2 microg/L (95% CI 81.4-162.9 microg/L) than women carrying normal alleles (geometric mean 76.6 microg/L (95% CI 72.5-80.9 microg/L). We observed the highest serum ferritin concentrations among postmenopausal women who are compound heterozygous or C282Y homozygous, and who consume relatively high amounts of heme iron (geometric mean 183.9 microg/L (95% CI 97.2-347.8 microg/L).<h4>Conclusions</h4>Even when there are currently no clinical signs, women with the compound or C282Y homozygous genotype may still be at risk for developing iron overload sometime after menopause.

Bladder cancer is a common malignancy requiring a high degree of surveillance because of the frequent recurrences and the poor clinical outcome of invasive disease. To date, serum biomarkers for bladder cancer lack optimal sensitivity and specificity to assist in diagnosis and disease categorization. Here, we designed antibody arrays for bladder cancer by selecting antibodies against targets differentially expressed in bladder tumors. Serum protein profiles measured by an antibody array containing 254 antibodies discriminated bladder cancer patients from controls (n = 95) with a correct classification rate of 93.7%. A second independent antibody array containing 144 antibodies revealed that protein profiles provide predictive information by stratifying patients with bladder tumors (n = 37) based on their overall survival (P = 0.0479). In addition, serum proteins, such as c-met, that were top ranked at identifying bladder cancer patients were associated with pathological stage, tumor grade, and survival when validated by immunohistochemistry of tissue microarrays containing bladder tumors (n = 173). This study provides experimental evidence for the use of several integrated technologies strengthening the process of biomarker discovery. Serum protein profiles obtained by antibody arrays represent comprehensive means for bladder cancer diagnosis and clinical outcome stratification, which could potentially assist in selection of cancer patients who would benefit from early, individualized therapeutic intervention.

Nerve growth factor (NGF) and its apoptosis-promoting low-affinity receptor (p75NTR) regulate murine hair cycling. However, it is unknown whether human hair growth is also controlled through p75NTR, its high-affinity ligand pro-NGF, and/or the growth-promoting high-affinity NGF receptor tyrosine kinase A (TrkA). In microdissected human scalp anagen hair bulbs, mRNA for NGF, pro-NGF, p75NTR, and TrkA was transcribed. Immunohistomorphometry and in situ hybridization detected strong NGF and pro-NGF expression in terminally differentiating inner root sheath keratinocytes, whereas TrkA was co-expressed with p75NTR in basal and suprabasal outer root sheath keratinocytes. During spontaneous catagen development of organ-cultured human anagen hair follicles, p75NTR mRNA levels rose, and p75NTR and pro-NGF immunoreactivity increased dramatically in involuting compartments primarily devoid of TrkA expression. Here, TUNEL(+) apoptotic cells showed prominent p75NTR expression. Joint pro-NGF/NGF administration inhibited hair shaft elongation and accelerated catagen development in culture, which was antagonized by co-administration of p75NTR-blocking antibodies. In addition, mRNA and protein expression of transforming growth factor-beta2 increased early during spontaneous catagen development, and its neutralization blocked pro-NGF/NGF-dependent hair growth inhibition. Our findings suggest that pro-NGF/NGF interacts with transforming growth factor-beta2 and p75NTR to terminate anagen in human hair follicles, implying that p75NTR blockade may alleviate hair growth disorders characterized by excessive catagen development.

Glutathione S-transferase pi (GST pi) has been shown to reactivate oxidized 1-cysteine peroxiredoxin (1-Cys Prx, Prx VI, Prdx6, and AOP2). We now demonstrate that a heterodimer complex is formed between 1-Cys Prx with a C-terminal His6 tag and GST pi upon incubation of the two proteins at pH 8.0 in buffer containing 20% 1,6-hexanediol to dissociate the homodimers, followed by dialysis against buffer containing 2.5 mM glutathione (GSH) but lacking 1,6-hexanediol. The heterodimer can be purified by chromatography on nickel-nitriloacetic acid agarose in the presence of GSH. N-Terminal sequencing showed that equimolar amounts of the two proteins are present in the isolated complex. In the heterodimer, 1-Cys Prx is fully active toward either H2O2 or phospholipid hydroperoxide, while the GST pi activity is approximately 25% of that of the GST pi homodimer. In contrast, the 1-Cys Prx homodimer lacks peroxidase activity even in the presence of free GSH. The heterodimer is also formed in the presence of S-methylglutathione, but no 1-Cys Prx activity is found under these conditions. The yield of heterodimer is decreased in the absence of 1,6-hexanediol or GSH. Rapid glutathionylation of 1-Cys Prx in the heterodimer is detected by immunoblotting. Subsequently, a disulfide-linked dimer is observed on SDS-PAGE, and the free cysteine content is decreased by 2 per heterodimer. The involvement of particular binding sites in heterodimer formation was tested by site-directed mutagenesis of the two proteins. For 1-Cys Prx, neither Cys47 nor Ser32 is required for heterodimer formation but Cys47 is essential for 1-Cys Prx activation. For GST pi, Cys47 and Tyr7 (at or near the GSH-binding site) are needed for heterodimer formation but three other cysteines are not. We conclude that reactivation of oxidized 1-Cys Prx by GST pi occurs by heterodimerization of 1-Cys Prx and GST pi harboring bound GSH, followed by glutathionylation of 1-Cys Prx and then formation of an intersubunit disulfide. Finally, the GSH-mediated reduction of the disulfide regenerates the reduced active-site sulfhydryl of 1-Cys Prx.

The N-terminal domain of thrombospondin-1 (TSPN-1) mediates the protein's interaction with (1) glycosaminoglycans, calreticulin, and integrins during cellular adhesion, (2) low-density lipoprotein receptor-related protein during uptake and clearance, and (3) fibrinogen during platelet aggregation. The crystal structure of TSPN-1 to 1.8 A resolution is a beta sandwich with 13 antiparallel beta strands and 1 irregular strand-like segment. Unique structural features of the N- and C-terminal regions, and the disulfide bond location, distinguish TSPN-1 from the laminin G domain and other concanavalin A-like lectins/glucanases superfamily members. The crystal structure of the complex of TSPN-1 with heparin indicates that residues R29, R42, and R77 in an extensive positively charged patch at the bottom of the domain specifically associate with the sulfate groups of heparin. The TSPN-1 structure and identified adjacent linker region provide a structural framework for the analysis of the TSPN domain of various molecules, including TSPs, NELLs, many collagens, TSPEAR, and kielin.

[reaction: see text] Lysophosphatidic acids bearing a benzophenone group in either the sn-1 or sn-2 chain of an oleoyl-type ester or oleyl-type ether chain and (32)P in the phosphate group were synthesized. The benzophenone moiety was introduced by selective hydroboration of the double bond of enyne 11 at low temperature, followed by a Suzuki reaction with 4-bromobenzophenone. The key intermediates for the preparation of ester-linked lysophosphatidic acid (LPA) 1 and 3 were obtained in one pot by a modified DIBAL-H reduction of orthoformate intermediate 22. These probes were shown to covalently modify a single protein target in rat plasma containing albumin and several protein targets in rat plasma containing a low level of albumin.

A number of genetic disorders can result in the accumulation of excess iron in the body. These causes of hereditary hemochromatosis include defects in genes encoding HFE, transferrin receptor 2, ferroportin, hepcidin, and hemojuvelin. Hepcidin, with its cognate receptor, ferroportin, has emerged as a central regulator of iron homeostasis; all of the known causes of hemochromatosis appear to prevent this system from functioning normally. The most common form of primary hemochromatosis is that caused by C282Y mutation of the HFE gene. This mutation is most prevalent among Northern Europeans. Although the frequency of the homozygous genotype is approximately 5 per 1000, the disease itself is quite rare because the clinical penetrance of the genotype is very low.

Subunits located near the cardiolipin binding sites of bovine heart cytochrome c oxidase (CcO) were identified by photolabeling with arylazido-cardiolipin analogues and detecting labeled subunits by reversed-phase HPLC and HPLC-electrospray ionization mass spectrometry. Two arylazido-containing cardiolipin analogues were synthesized: (1) 2-SAND-gly-CL with a nitrophenylazido group attached to the polar headgroup of cardiolipin (CL) via a linker containing a cleavable disulfide; (2) 2',2''-bis-(AzC12)-CL with two of the four fatty acid tails of cardiolipin replaced by 12-(N-4-azido-2-nitrophenyl) aminododecanoic acid. Both arylazido-CL derivatives were used to map the cardiolipin binding sites within two types of detergent-solubilized CcO: (1) intact 13-subunit CL-containing CcO (three to four molecules of endogenous CL remain bound per CcO monomer); (2) 11-subunit CL-free CcO (subunits VIa and VIb are missing because they dissociate during CL removal). Upon the basis of these photolabeling studies, we conclude that (1) subunits VIIa, VIIc, and possibly VIII are located near the two high-affinity cardiolipin binding sites, which are present in either form of CcO, and (2) subunit VIa is located adjacent to the lower affinity cardiolipin binding site, which is only present in the 13-subunit form of CcO. These data are consistent with the recent CcO crystal structure in which one cardiolipin is located near subunit VIIa and a second is located near subunit VIa (PDB ID code referenced in Tomitake, T. et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 15304-15309). However, we propose that a third cardiolipin is bound between subunits VIIa and VIIc near the entrance to the D-channel. Cardiolipin bound at this location could potentially function as a proton antenna to facilitate proton entry into the D-channel. If true, it would explain the CcO requirement of bound cardiolipin for full electron transport activity.

Synthetic polymer surfaces require surface modification to improve biocompatibility. A generic route to biocompatible silicone elastomers is described involving high yield surface functionalization of standard silicones with hydrosilanes, hydrosilylation using asymmetric, allyl-, NSC-terminated PEO of narrow molecular weight, and covalent modification in one step with amine-containing biological molecules including oligopeptides (YIGSR, RGDS), proteins (EGF, albumin, fibrinogen, mucin), and glycosaminoglycans (heparin). Efficient, high-density binding (e.g., 0.2 EGF molecules/nm2) was demonstrated using radiolabeling studies. The resulting surfaces were demonstrated to be biocompatible by further reaction with biomolecules, for example, thrombosis suppression on surfaces modified by heparin + ATIII, and the formation of confluent corneal epithelial cell layers on EGF, RGDS, or YIGSR surfaces.

The remodeling of phosphatidylinositol polyphosphates in cellular membranes by phosphatases and kinases orchestrates the signaling by these lipids in space and time. To provide chemical tools to study the changes in cell physiology mediated by these lipids, three new metabolically stabilized (ms) analogues of phosphatidylinositol-3-phosphate (PtdIns(3)P) were synthesized. We describe herein the total asymmetric synthesis of 3-methylphosphonate, 3-(monofluoromethyl)phosphonate and 3-phosphorothioate analogues of PtdIns(3)P. From differentially protected D-myo-inositol key intermediates, a versatile phosphoramidite reagent was employed in the synthesis of PtdIns(3)P analogues with diacylglyceryl moieties containing dioleoyl, dipalmitoyl, and dibutyryl chains. In addition, we introduce a new phosphorylation reagent, (monofluoromethyl)phosphonyl chloride, which has general applications for the preparation of "pKa-matched" monofluorophosphonates. These ms-PtdIns(3)P analogues exhibited reduced binding activities with 15N-labeled FYVE and PX domains, as significant 1H and 15N chemical shift changes in the FYVE domain were induced by titrating ms-PtdIns(3)P analogues into membrane-mimetic dodecylphosphocholine micelles. In addition, the PtdIns(3)P analogues with dioleoyl and dipalmitoyl chains were substrates for the 5-kinase enzyme PIKfyve; the corresponding phosphorylated ms-PI(3,5)P2 products were detected by radio-TLC analysis.

Factors that mediate p53 tumor suppressor activity remain largely unknown. In this study we describe a systematic approach to identify downstream mediators of tumor suppressor function of p53, consisting of global gene expression profiling, focused short hairpin RNA (shRNA) library creation, and functional selection of genetic elements cooperating with oncogenic Ras in cell transformation. This approach is based on our finding that repression of gene expression is a major event, occurring in response to p53 inactivation during transformation and immortalization of primary cells. Functional analysis of the subset of genes universally down-regulated in the cells that lacked functional p53 revealed BTG2 as a major downstream effector of p53-dependent proliferation arrest of mouse and human fibroblasts transduced with oncogenic Ras. shRNA-mediated knockdown of BTG2 cooperates with oncogenic Ras to transform primary mouse fibroblasts containing wild-type transcriptionally active p53. Repression of BTG2 results in up-regulation of cyclins D1 and E1 and phosphorylation of Rb and, in cooperation with other oncogenic elements, induces neoplastic transformation of primary human fibroblasts. BTG2 expression was found to be significantly reduced in a large proportion of human kidney and breast carcinomas, suggesting that BTG2 is a tumor suppressor that links p53 and Rb pathways in human tumorigenesis.

Mammalian Staufen2 (Stau2) is a member of the double-stranded RNA-binding protein family. Its expression is largely restricted to the brain. It is thought to play a role in the delivery of RNA to dendrites of polarized neurons. To investigate the function of Stau2 in mature neurons, we interfered with Stau2 expression by RNA interference (RNAi). Mature neurons lacking Stau2 displayed a significant reduction in the number of dendritic spines and an increase in filopodia-like structures. The number of PSD95-positive synapses and miniature excitatory postsynaptic currents were markedly reduced in Stau2 down-regulated neurons. Akin effects were caused by overexpression of dominant-negative Stau2. The observed phenotype could be rescued by overexpression of two RNAi cleavage-resistant Stau2 isoforms. In situ hybridization revealed reduced expression levels of beta-actin mRNA and fewer dendritic beta-actin mRNPs in Stau2 down-regulated neurons. Thus, our data suggest an important role for Stau2 in the formation and maintenance of dendritic spines of hippocampal neurons.

The immune expulsion of gastrointestinal nematode parasites is usually associated with T helper type 2 (Th2) responses, but the effector mechanisms directly responsible for parasite loss have not been elucidated. The intestinal inflammatory response accompanying infection with gastrointestinal helminths is thought to be a contributory factor leading to the expulsion of the parasite. However, we have shown that the intestinal inflammation, which is controlled by interleukin (IL)-4, is not required for parasite expulsion. OX40-OX40 ligand (L) signals have been shown to be important for the development of Th2 immune responses but are also involved in a number of inflammatory diseases including those of the intestine. Here, we have investigated the effect of OX40 and OX40L fusion protein treatment on the induction of protective Th2 responses and enteropathy following infection with the gastrointestinal nematode Trichinella spiralis. Treatment with an OX40-immunoglobulin (Ig) blocking fusion protein resulted in enhanced expulsion of the parasite and an increase in the accompanying mastocytosis, despite unaltered levels of Th2 cytokines. Furthermore, there was a delay in the villus atrophy and crypt hyperplasia usually associated with this infection. In contrast, levels of Th2 cytokines were greatly up-regulated in mice treated with an OX40L-Ig activating fusion protein, yet the expulsion of the parasite and the enteropathy were unaffected. Therefore, OX40 ligation potentiates the Th2 response without enhancing host protective immune responses, whereas blocking the OX40-OX40L interaction enhances host protection without promoting Th2 cytokine responses during Trichinella spiralis infection.

Slits mediate multiple axon guidance decisions, but the mechanisms underlying the responses of growth cones to these cues remain poorly defined. We show here that collapse induced by Slit2-conditioned medium (Slit2-CM) in Xenopus retinal growth cones requires local protein synthesis (PS) and endocytosis. Slit2-CM elicits rapid activation of translation regulators and MAP kinases in growth cones, and inhibition of MAPKs or disruption of heparan sulfate blocks Slit2-CM-induced PS and repulsion. Interestingly, Slit2-CM causes a fast PS-dependent decrease in cytoskeletal F-actin concomitant with a PS-dependent increase in the actin-depolymerizing protein cofilin. Our findings reveal an unexpected link between Slit2 and cofilin in growth cones and suggest that local translation of actin regulatory proteins contributes to repulsion.

We report three mutations of transferrin receptor 2 (TFR2)--R396X (exon 9; nt 1186C-->T), R455Q (exon 10; nt 1364G-->A) and G792R (exon 18; nt 2374G-->A)--in a man of Scottish descent with hemochromatosis and severe iron overload. He was also heterozygous for the common HFE H63D polymorphism. The patient did not have coding region mutations in HAMP, FPN1, HJV or ALAS2. We conclude that this patient represents another example of hemochromatosis due to mutations of the gene encoding transferrin receptor 2.

<h4>Background</h4>A 25-year-old woman of English/Irish background was diagnosed with hemochromatosis. She manifested hypogonadotrophic hypogonadism and congestive heart failure. Although there were abnormal liver function tests, no cirrhosis was present. The patient has been treated intermittently by phlebotomy for 24 years. The aim of this study was to investigate the genetic basis of the patient's iron overload disease.<h4>Methods</h4>Genetic analysis was performed by direct sequencing of the genes for hemojuvelin, HFE, hepcidin, ferroportin and transferrin receptor 2.<h4>Results and conclusions</h4>Molecular analysis showed that the patient was homozygous for a previously undescribed mutation of HJV, the gene encoding hemojuvelin. This mutation, nt 81G deletion, causes a frameshift encoding 23 additional irrelevant amino acids and premature termination. No mutations were found in the other hemochromatosis genes, hepcidin, HFE, ferroportin or transferrin receptor 2, which might have contributed to her iron overload.

Iron overload disease due to mutations in ferroportin has a dominant inheritance and a variable clinical phenotype, such that some patients show early Küpffer cell iron loading and low transferrin saturation, while others show hepatocyte iron loading and high transferrin saturation. Studies expressing ferroportin mutant proteins in cultured cells have shown that mutant proteins fall into two main classes; proteins that do not localize to the cell surface and are unable to export iron, and those that localize to the cell surface but are unable to respond to the antimicrobial peptide hepcidin. Patients with mutant ferroportin proteins that do not localize to the cell surface show typical ferroportin disease with low transferrin saturation and early Küpffer cell iron loading, while patients with mutant proteins unable to respond to hepcidin show high transferrin saturation and early hepatocyte iron loading similar to classic hereditary hemochromatosis. The dominant genetic transmission of ferroportin-linked disorders is explained by the in vitro data, which suggest that ferroportin is a multimer and that the behavior of the mutant protein can affect the behavior of the wild type protein.

This review highlights the role of a CD4(+)CD3(-) accessory cell in the development of organized lymphoid infrastructures as well as in the development of high-affinity antibody responses and T-cell memory. These 2 functions are linked in the development of the vertebrate immune system and are effected by the constitutive expression of 2 sets of tumor necrosis factor (TNF) family members. The expression of lymphotoxin 3 (LT3), LT3, and TNF-3, which are closely linked genetically, affects the organization of lymphoid structures into B-cell and T-cell areas; the dual expression of OX40 ligand (TNFSF4) and CD30 ligand (TNFSF8) influences both the survival of T-cells within germinal centers and T-cell memory.

OX40 belongs to the tumor necrosis factor receptor superfamily, and its expression is restricted to activated T-cells. Ligation of OX40 during T-cell-dendritic cell interaction is crucial for clonal expansion of antigen-specific T-cells and generation of T-cell memory. The ligand of OX40 (OX40L) is expressed not only on dendritic cells but also on other cell types, such as B-cells, vascular endothelial cells, natural killer cells, and mast cells. The pathophysiological relevance of this broad distribution needs further investigation. In particular, OX40L on vascular endothelial cells may play a role in inflammatory vasculitis as well as in atherosclerotic change. Recent studies with animal models have indicated the critical involvement of OX40 in the pathogenesis of a variety of immunologic abnormalities of inflammatory, autoimmune, infectious, allergic, and allotransplantation-related diseases. Blockade of OX40-OX40L interaction has been shown to prevent, cure, or ameliorate these diseases. In contrast, activation of OX40 is known to break an existing state of tolerance in malignancies, leading to a reactivation of antitumor immunity. These findings clearly suggest that the OX40/OX40L system is one of the most promising targets of immune intervention for treatment of these diseases.

Thirty-five patients diagnosed to have ovarian cancer (early FIGO stage I/II n = 11, advanced FIGO stage III/IV, n = 24) were evaluated for hemostatic parameters relating to survival outcome by 36 months of disease. Systemic plasminogen activators and inhibitors were evaluated and we found no significant association with survival outcome and eventually only fibrinogen, von Willebrand Factor (vWF), antithrombin III (ATIII), and D-dimer levels were determined for their association with disease outcome by 12 months, 24 months, and 36 months. Twenty-four patients succumbed to the disease by 36 months (early n = 2, advanced n = 22). The 11 surviving patients (advanced n = 3, including one deceased at 52 months) is still living past 36 months and 82 months at the time of analysis. Elevated fibrinogen, vWF, and D-dimer together with reduced ATIII levels were found to be associated with poor survival outcome by 12 months of disease. Moreover, elevated vWF and D-dimer with reduced ATIII levels was strongly implicated with poor survival outcome by 36 months from ovarian cancer. The overall survival rate at 36 months from ovarian cancer was 31.4%. It is therefore suggested that fibrinogen, vWF, ATIII, and D-dimer levels be used together as prognostic markers for disease outcome especially in patients with advanced ovarian cancer within 36 months of disease. An expanded study is required to confirm these findings.

Heart failure remains a common diagnosis, especially in older individuals. It continues to be associated with significant morbidity and mortality, but major advances in both diagnosis and management have occurred and will continue to improve symptoms and other outcomes in patients. The Canadian Cardiovascular Society published its first consensus conference recommendations on the diagnosis and management of heart failure in 1994, followed by two brief updates, and reconvened this consensus conference to provide a comprehensive review of current knowledge and management strategies. New clinical trial evidence and meta-analyses were critically reviewed by a multidisciplinary primary panel who developed both recommendations and practical tips, which were reviewed by a secondary panel. The resulting document is intended to provide practical advice for specialists, family physicians, nurses, pharmacists and others who are involved in the care of heart failure patients. Management of heart failure begins with an accurate diagnosis, and requires rational combination drug therapy, individualization of care for each patient (based on their symptoms, clinical presentation and disease severity), appropriate mechanical interventions including revascularization and devices, collaborative efforts among health care professionals, and education and cooperation of the patient and their immediate caregivers. The goal is to translate best evidence-based therapies into clinical practice with a measureable impact on the health of heart failure patients in Canada.

A central role of iron in the pathogenesis of Parkinson's disease (PD) has been discussed for many years. So far, however, a biomarker indicating increased iron levels in the substantia nigra (SN) in PD patients has been missing. Performing transcranial ultrasound we detected an increased area of SN echogenicity as a typical echofeature in PD, visible already in the early stages of the disease and in subjects with subclinical impairment of the nigrostriatal system. Animal studies and post mortem analyses of human brain tissue revealed that this echofeature is associated with increased iron levels of the substantia nigra as well as a reduced neuromelanin content. The apparently autosomal dominant inheritance of this echofeature in relatives of patients with idiopathic PD indicates a primary role of disturbances of iron metabolism in PD. Consequently performed mutation analyses in genes involved in brain iron metabolism lead to the discovery of specific mutations in the ferritin-H, IRP2 and HFE gene in single PD patients. Moreover, variations in the ceruloplasmin gene were found to be associated with PD or SN hyperechogenicity. Functional relevance of some of these mutations for iron metabolism could be proven. Therefore, SN hyperechogenicity can be regarded as biomarker for both: impairment of the nigrostriatal system and increased iron levels of the SN. Future studies aim at substantiating the hypothesis that healthy subjects with SN hyperechogenicity indeed represent a population at risk for nigrostriatal degeneration, which would have a significant impact on therapeutical options.

The development and progression of gastric cancer involves a number of genetic and epigenetic alterations of tumor suppressor and tumor-related genes. The majority of differentiated carcinomas arise from intestinal metaplastic mucosa and exhibit structurally altered tumor suppressor genes, typified by p53, which is inactivated via the classic two-hit mechanism, i.e. loss of heterozygosity (LOH) and mutation of the remaining allele. LOH at certain chromosomal loci accumulates during tumor progression. Approximately 20% of differentiated carcinomas show evidence of mutator pathway tumorigenesis due to hMLH1 inactivation via hypermethylation of promoter CpG islands, and exhibit high-frequency microsatellite instability. In contrast, undifferentiated carcinomas rarely exhibit structurally altered tumor suppressor genes. For instance, while methylation of E-cadherin is often observed in undifferentiated carcinomas, mutation of this gene is generally associated with the progression from differentiated to undifferentiated carcinomas. Hypermethylation of tumor suppressor and tumor-related genes, including APC, CHFR, DAP-kinase, DCC, E-cadherin, GSTP1, hMLH1, p16, PTEN, RASSF1A, RUNX3, and TSLC1, can be detected in both differentiated and undifferentiated carcinomas at varying frequencies. However, the significance of the hypermethylation varies according to the analyzed genomic region, and hypermethylation of these genes can also be present in non-neoplastic gastric epithelia. Promoter demethylation of specific genes, such as MAGE and synuclein Y, can occur during the progressive stages of both histological types, and is associated with patient prognosis. Thus, while the molecular pathways of gastric carcinogenesis are dependent on histological background, specific genetic alterations can still be used for risk assessment, diagnosis, and prognosis.

<h4>Aim</h4>To assess the effect of iron reduction after phlebotomy in rats with "normal" hepatic iron concentration (HIC) on the progression of hepatic fibrosis, as a result of bile duct ligation (BDL).<h4>Methods</h4>Rats underwent phlebotomy before or after sham operation or BDL. Animals undergone only BDL or sham operation served as controls. Two weeks after surgery, indices of hepatic damage and fibrosis were evaluated.<h4>Results</h4>Phlebotomy lowered HIC. Phlebotomy after BDL was associated with body weight increase, lower hepatic weight, less portal hypertension, less periportal necrosis, less portal inflammation, lower hepatic activity index score and higher albumin levels. On the other hand, phlebotomy before BDL was associated with body weight decrease and hepatic activity index score increase. Phlebotomy after sham operation was not associated with any hepatic or systemic adverse effects.<h4>Conclusion</h4>Reduction of HIC after induction of liver damage may have beneficial effects in BDL rats. However, iron deficiency could induce impairment of liver function and may make the liver more susceptible to insults like BDL.

<h4>Aim</h4>There is strong evidence that interleukin-11 (IL-11) is involved in the regulation of tumor progression, cellular growth and differentiation. Recently, interleukin-11 receptor (IL-11R) has been detected on some cancer cells. In this study, we investigated the expression of IL-11 and IL-11R in colorectal adenocarcinoma.<h4>Methods</h4>To elucidate the involvement of IL-11 and IL-11Ra in human intestinal adenocarcinomas, we examined 115 cases of surgically resected human colonic adenocarcinoma and 11 cases of adenoma by immunohistochemistry and Western blotting.<h4>Results</h4>Among 115 cases of adenocarcinoma, 100 cases (87.0%) showed positive staining in the cytoplasm of carcinoma cells for the IL-11, and 87 cases (75.6%) were positive for the IL-11Ra. Six cases (54.5%) and four cases (36.4%) of 11 adenomas were positive for IL-11 and IL-11Ra, respectively. The expression of IL-11Ra correlated with the histological differentiation (P = 0.033503), the depth of tumor invasion (P = 0.006395), Dukes'classification (P = 0.015648) and lymphatic invasion (P = 0.003865). However, the expression of IL-11Ra was not correlated with the venous invasion and the presence of lymph node metastasis. The expression of IL-11 was not correlated with any clinicopathological factors. In Western blot analysis, two human colorectal carcinoma cell lines and four tissues of surgically resected human carcinoma expressed both IL-11 and IL-11Ra proteins.<h4>Conclusion</h4>IL-11 and IL-11Ra are highly expressed in human colorectal adenocarcinoma and the IL-11Ra expression is correlated with clinicopathological factors. These findings suggest that the expression of IL-11Ra is an important factor for the invasion of human colorectal adenocarcinoma.

Mice with knock-out of peroxiredoxin 6 (Prdx6), a recently described antioxidant enzyme, were evaluated for susceptibility to lung injury with paraquat (PQ) administration. With high dose PQ (30 mg/kg i.p.), all Prdx6-/- mice died (LT50 54 +/- 2.05 h, mean +/- SE) by 4 days, whereas 86% of the wild-type (WT) mice (C57BL/6) survived (n = 14). At 2 days after PQ, lung wet/dry weight ratio increased significantly (p < 0.05) to 7.57 +/- 0.37 in Prdx6-/- mice vs. 5.42 +/- 0.25 in WT mice. Total protein and nucleated cells in bronchoalveolar lavage fluid and TBARS and protein carbonyls in lung homogenate also showed more marked increases in Prdx6-/- mice. At 2.5 days after PQ, light microscopy of WT lungs showed mild injury while Prdx6-/- lungs showed epithelial cell necrosis, perivascular edema, and inflammatory cells. With low dose PQ (12.5 mg/kg), mortality and lung injury were less marked but were significantly greater with Prdx6-/- compared to WT mice. These results show that Prdx6-/- mice have increased susceptibility to lung injury with PQ administration. Thus, Prdx6 protects lungs against PQ toxicity as shown previously for hyperoxia, indicating that it functions as an important lung antioxidant enzyme.

Non-alcoholic fatty liver disease (NAFLD) encompasses a wide spectrum of fat-induced liver injury, ranging from relatively benign steatosis to cirrhosis and liver failure. The presence of obesity and insulin resistance is strongly associated with non-alcoholic fatty liver and confers on it a greater risk of histologically advanced disease. There is a growing concern in the medical profession as the prevalence of this disease continues to rise in parallel with the rise in obesity and the metabolic syndrome. Treatment options are limited and dietary weight loss is often advised. Low fat diets are difficult to adhere to and recent studies have shown the potential of low carbohydrate diets for weight loss and improving insulin resistance. Thus far, no study has evaluated the effect of low carbohydrate diets on NAFLD. Future studies will be required to address this question and others with regards to the nutritional adequacy and long-term side effects of these diets.

<h4>Aim</h4>To determine whether body weight and/or serum leptin were independent predictors of response to antiviral treatment in patients with chronic hepatitis C.<h4>Methods</h4>A retrospective evaluation was performed in 139 patients with chronic hepatitis C treated with interferon (IFN) from 1996 to 2000. Sustained response was defined as negative by hepatitis C virus (HCV) RNA analysis using PCR and normal transaminase at 24 wk after cessation of IFN therapy. Patients who remained positive for HCV RNA at the end of IFN treatment were defined as resistant to IFN therapy. Sex, age, body mass index (BMI) (> or =25 vs <25), complication of diabetes mellitus, serum leptin level (> or =8.0 microg/L vs < 8.0 microg/L), and the stage of liver fibrosis by needle biopsy (F1/F2 vs F3/F4) were examined.<h4>Results</h4>Sustained response was achieved in 33 patients (23.7%), while others failed to show a response to IFN therapy. Overall, the factors associated with sustained antiviral effects were HCV-RNA load, HCV genotype, serum leptin level, and stage of liver fibrosis evaluated by univariate analysis. BMI was not associated with any therapeutic effect of IFN. Multivariate analysis indicated that HCV-RNA load was a significant risk factor, but among the patients with low viremia (HCV-RNA <100 MU/L), leptin level was an independent risk factor for IFN resistance. Namely, a high level of serum leptin attenuated the effect of IFN on both male and female patients with low viremia.<h4>Conclusion</h4>High serum leptin level is a negative predictor of response to antiviral treatment in chronic hepatitis C with low viremia.

We have reported a case of hepatic adenomatosis associated with hormone replacement therapy (estrogen and progesterone) and hemosiderosis caused by excessive blood transfusion for the treatment of chronic myeloid leukemia. A 34-year-old woman was found to have several hepatic tumors on a routine medical examination. The general condition was good. Laboratory studies showed iron overload. Abdominal computed tomography and selective hepatic angiography showed several hypervascular tumors in the right lobe of the liver (up to 20 mm in diameter). Since hepatocellular carcinoma could not be ruled out, subsegmental hepatectomy was performed. Histopathological examination of the surgical specimen showed hepatic adenomatosis with hemosiderosis. Both hormone replacement therapy and iron overload could be the cause of hepatic adenomatosis.

Our current psychopharmacological treatments for anxiety disorders evince a number of shortcomings, including troublesome side effects and lack of primary effects. Whereas many new drugs have been developed in the past few decades, most are based on outmoded theories of the pathogenesis of these disorders (i.e., monoamine hypotheses), thus frustrating our ability to create more specific and effective interventions. Recently, however, the neurobiological literature has shown a convergence of findings focusing on the glutamatergic system in anxiety disorders, and the growth of pharmacological tools targeting these receptors has led to the development of novel treatments having anxiolytic effects in humans and animals alike. Additionally, as this system is showing promise as a final common pathway in the pathogenesis of anxiety disorders, we may be able to employ glutamate-specific neuroimaging techniques (e.g., N-acetyl-aspartate, GLX) to both guide treatment decisions and present reliable objective biomarkers for treatment efficacy.

It is now widely recognized that the packaging of genomic DNA together with core histones, linker histones, and other functional proteins into chromatin profoundly influences nuclear processes such as transcription, replication, repair and recombination. How chromatin structure modulates the expression and maintenance of knowledge encoded in eukaryotic genomes, and how these processes take place within the context of a highly complex and compacted genomic chromatin environment remains a major unresolved question in biology. Here we review recent advances in our understanding of how nucleosome and chromatin structure may have to adapt to promote these vital functions.

The effects of a combined oral contraceptive (COC) containing 20 microg ethinyl estradiol (EE) and 75 microg gestodene (GSD) on prothrombin activity (PA), activated partial thromboplastin time (APTT), thrombin time (TT), platelet number, fibrinogen, antithrombin III (ATIII), protein C, protein S and D-dimer were evaluated over 6 months in 23 young, healthy women. Laboratory assessments were performed prior to initiation of COC use (pretreatment) and after 3 and 6 months of use. Results showed no significant changes in fibrinogen, protein C, ATIII or D-dimer during COC use, compared with pretreatment values. The increase in platelet count, decreases in protein S level, PA and APTT, and the prolongation of TT were significant. In conclusion, the use of a COC containing 20 microg EE and 75 microg GSD did not cause any significant changes in the hemostatic parameters studied that could be suggestive of a higher prothrombotic risk. Further studies with a larger sample size are necessary in order to obtain conclusive data.

Hepatocellular carcinoma is a common malignancy affecting approximately one million people around the world every year. The incidence is low in the occidental world and high in locations such as Southeast Asia and sub-Saharan Africa. Hepatocellular carcinoma primarily affects old people, reaching its highest prevalence among those aged 65 to 69 years old. Chronic infection by the hepatitis B virus is the most common cause of this disease. Other important causes are cirrhosis, chronic viral hepatitis (hepatitis C virus, and hepatitis B plus D viruses), alcohol abuse, obesity, hemochromatosis, alfa1-antitripsin deficiency, and toxins similar to aflatoxin. In most cases, hepatocellular carcinoma is asymptomatic and has a low life expectancy. This article presents a review of the most important epidemiological, diagnostic and treatment data about this disease.

One of the fundamental questions of modern biology is to unravel how genes are switched on and off at the right time and in the correct tissues. It is well recognized that gene regulation depends on a dynamic balance between activating and repressing forces, and multiple mechanisms are involved in both gene silencing and activation. Work over the last decade has revealed that in some cases transcriptional silencing of specific genes is mediated by RNAs that specifically recruit repressing complexes to homologous DNA sequences. Examples of both cis and trans RNA driven transcriptional silencing have been reported. This review focuses on those examples of transcriptional gene silencing in which the RNA component seems to act uniquely in cis. Speculative models of how such cis acting transcripts may trigger transcriptional silencing are proposed. Future experimental testing of these and other mechanisms is important to gain a fuller understanding of how genes are regulated and to identify instances in which such mechanisms are defective, leading to disease. Understanding the basic molecular basis of these phenomena will provide us with invaluable tools for the future development of targeted therapies and drugs for those diseases in which they are faulty.

It is now widely recognized that the packaging of genomic DNA, together with core histones, linker histones, and other functional proteins into chromatin profoundly influences nuclear processes such as transcription, replication, DNA repair, and recombination. How chromatin structure modulates the expression of knowledge encoded in eukaryotic genomes, and how these processes take place within the context of a highly complex and compacted genomic chromatin environment remains a major unresolved question in biology. Here we review recent advances in nucleosome structure and dynamics.

<h4>Background</h4>Primary haemochromatosis is characterized by iron overload in the body tissues. It is common in populations of northern European descent. In such populations, 85%-90% of patients with this disease have a C282Y mutation in the HFE gene. In India, the disease is uncommon and the genetic defects associated with it are unknown. We therefore looked for mutations in the HFE and other genes involved in iron metabolism in Indian patients with primary haemochromatosis.<h4>Methods</h4>Five patients (including a brother-sister pair) with primary haemochromatosis diagnosed on clinical, biochemical and histological findings were studied. Genomic DNA was analysed by sequencing for the presence of mutations in all the 6 exons of the HFE gene and for previously described mutations in genes encoding hepcidin antimicrobial peptide and ferroportin.<h4>Results</h4>No patient had the C282Y mutation. One had homozygous H63D mutation. No other mutation was found in any HFE exon. Two previously reported splice site mutations in the HFE gene (IVS3 + 1 G/T and IVS5+1 G/A) were not detected. Four of the 5 patients had an HFE splice site mutation (IVS2 + 4 T/C; homozygous 2, heterozygous 2); however, this change was as frequent in 29 healthy subjects (homozygous 9, heterozygous 7), and was present in only 1 of the sibling pair patients, indicating that this represented a polymorphism. No patient had any of the previously described mutations in the genes for hepcidin and ferroportin.<h4>Conclusion</h4>Our patients with primary haemochromatosis lacked mutations in the HFE, hepcidin and ferroportin genes. Further genetic analysis may help identify novel mutations responsible for primary haemochromatosis in these patients.

Molecular chaperones play crucial roles in various aspects of the biogenesis and maintenance of proteins in the cell. The heat shock protein 70 (HSP70) chaperone system, in which HSP70 proteins act as chaperones, is one of the major molecular chaperone systems conserved among a variety of organisms. To shed light on the evolutionary history of the constituents of the chordate HSP70 chaperone system and to identify all of the components of the HSP70 chaperone system in ascidians, we carried out a comprehensive survey for HSP70s and their cochaperones in the genome of Ciona intestinalis. We characterized all members of the Ciona HSP70 superfamily, J-proteins, BAG family, and some other types of cochaperones. The Ciona genome contains 8 members of the HSP70 superfamily, all of which have human and protostome counterparts. Members of the STCH subfamily of the HSP70 family and members of the HSPA14 subfamily of the HSP110 family are conserved between humans and protostomes but were not found in Ciona. The Ciona genome encodes 36 J-proteins, 32 of which belong to groups conserved in humans and protostomes. Three proteins seem to be unique to Ciona. J-proteins of the RBJ group are conserved between humans and Ciona but were not found in protostomes, whereas J-proteins of the DNAJC14, ZCSL3, FLJ13236, and C21orf55 groups are conserved between humans and protostomes but were not found in Ciona. J-proteins of the sacsin group seem to be specific to vertebrates. There is also a J-like protein without a conserved HPD tripeptide motif in the Ciona genome. The Ciona genome encodes 3 types of BAG family proteins, all of which have human and protostome counterparts (BAG1, BAG3, and BAT3). BAG2 group is conserved between humans and protostomes but was not found in Ciona, and BAG4 and BAG5 groups seem to be specific to vertebrates. Members for SIL1, UBQLN, UBADC1, TIMM44, GRPEL, and Magmas groups, which are conserved between humans and protostomes, were also found in Ciona. No Ciona member was retrieved for HSPBP1 group, which is conserved between humans and protostomes. For several groups of the HSP70 superfamily, J-proteins, and other types of cochaperones, multiple members in humans are represented by a single counterpart in Ciona. These results show that genes of the HSP70 chaperone system can be distinguished into groups that are shared by vertebrates, Ciona, and protostomes, ones shared by vertebrates and protostomes, ones shared by vertebrates and Ciona, and ones specific to vertebrates, Ciona, or protostomes. These results also demonstrate that the components of the HSP70 chaperone system in Ciona are similar to but simpler than those in humans and suggest that changes of the genome in the lineage leading to humans after the separation from that leading to Ciona increased the number and diversity of members of the HSP70 chaperone system. Changes of the genome in the lineage leading to Ciona also seem to have made the HSP70 chaperone system in this species slightly simpler than that in the common ancestor of humans and Ciona.

Anxiety and stress response/resiliency are heritable traits central to the etiology of multiple psychiatric diseases, but efforts to identify genetic variation influencing this broad domain of neurobiological function are hampered by the coarseness of the phenotypic measures and the effects of environmental factors. Neuroimaging offers a powerful approach for assessing functional neuronal activity. Neurophysiological measures can serve as intermediate phenotypes more directly linked to small gene effects, compared with behavioral end points of neural dysfunction. Imaging genomics is a relatively new research area that is concerned with linking functional gene variants and brain information processing. Here, we will focus on processes affected by anxiety and stress. Neuroimaging has been combined with genetic analysis to reveal genetic effects of functional variants of the serotonin transporter (5-HTT) and catechol-O-methyltransferase (COMT) genes on brain response to stressful stimuli. The low-expressing allele of the 5-HTT promoter polymorphism (HTTLPR) is associated with anxiety and with greater amygdala and other regional responses to emotional. The COMT Met158 allele leads to lower COMT activity and has also been associated with anxiety, and the effect of this gene is apparently additive with HTTLPR. Individuals with Met158 genotypes are more sensitive to pain stress and, as shown by C11 Carfentanil imaging, have diminished ability to upregulate opioid release after pain/stress. These results suggest that functional variants of 5-HTT and COMT impact brain functions involved in stress and anxiety.

The aim of this study was to examine the influence of different fat diets on serotonin receptor and transporter binding. Male Sprague-Dawley rats were fed a diet of either high saturated fat, omega-6 polyunsaturated fatty acid, omega-3 polyunsaturated fatty acid or low fat (control) for eight weeks. Using Beta-Imager quantification techniques, [(3)H]ketanserin, [(3)H]mesulergine and [(3)H]paroxetine binding to serotonin (5-HT)(2A), 5-HT(2C) receptors and 5-HT transporters (5-HTT) was measured throughout the brain in all four groups. All three high fatty acid diets influenced serotonin receptor binding, however the most pronounced effects were that compared with the low fat control group, i) 5-HT(2A) receptor binding was increased in the caudate putamen, but reduced in the mammillary nucleus in high saturated fat and high omega-6 polyunsaturated fatty acid diet groups; ii) 5-HT(2C) receptor binding was reduced in the mamillary nucleus of saturated fat group and reduced in prefrontal cortex of the omega-6 polyunsaturated fatty acid and omega-3 polyunsaturated fatty acid groups; and iii) 5-HTT binding was reduced in the hippocampus in the omega-6 polyunsaturated fatty acid group. Overall, the omega-6 polyunsaturated fatty acid diet exerted the most influence on serotonin receptor and transporter binding. These results may be of importance in relation to neuropsychiatric diseases such as schizophrenia, where associations between altered fatty acid levels and the serotonergic system have been made.

<h4>Background</h4>Hereditary Haemochromatosis (HH) and Coeliac disease (CD) are common disorders in Northern European populations, particularly the Irish population.<h4>Aims</h4>To investigate whether there was increased frequency of the two common HFE gene mutations, C282Y and H63D, associated with HH amongst a cohort of CD patients, and to determine the penetrance of the HH associated genotypes in this cohort.<h4>Methods</h4>HFE genotypes of a cohort of CD patients were determined using standard PCR techniques. HFE allele frequencies were compared to those of a previously reported, ethnically similar, cohort of 800 neonates, using Fishers exact test. Patients with HH-associated genotypes were subsequently evaluated.<h4>Results</h4>The C282Y and H63D allele frequencies, 24/222 (11%) and 28/222 (13%) respectively, in the CD patients were similar to those of the neonatal group, 171/1600 (11%) and 242/1600 (15%). Eight patients had HH-associated genotypes, of which two demonstrated biochemical evidence of iron overload.<h4>Conclusion</h4>The HFE mutations associated with Hereditary Haemochromatosis are not more common in Irish CD patients.

<h4>Context</h4>Theoretically, the risk of compartment syndrome is increased during creatine monohydrate (CrM) supplementation because of intracellular fluid retention in muscle cells and the overall increased size of the muscle tissue. Whether this change in intracellular fluid is associated with an increase in anterior compartment pressure in the lower leg when subjects are under thermal stress is unknown.<h4>Objective</h4>To assess the influence of CrM on the resting and postexercise anterior compartment pressure of the lower leg in mildly to moderately dehydrated males exercising in the heat.<h4>Design</h4>Double-blind, randomized, crossover design.<h4>Setting</h4>Human Performance Laboratory.<h4>Patients or other participants</h4>Eleven well-trained, non- heat-acclimated, healthy males (age = 22 +/- 2 years, height = 181.1 +/- 7 cm, mass = 78.4 +/- 4.2 kg, V(O2)max = 50.5 +/- 3.4 mL.kg(-1).min(-1)).<h4>Intervention(s)</h4>Subjects were supplemented with 21.6 g/d of CrM or placebo for 7 days. On day 7, they performed 2 hours of submaximal exercise, alternating 30 minutes of walking with 30 minutes of cycling in the heat, resulting in approximately 2% dehydration. This was followed by an 80-minute heat tolerance test (temperature = 33.5 +/- 0.5 degrees C, humidity = 41.0 +/- 12%), which included 12 repetitions of a 3-minute walk (pace = 4.0 +/- 0.1 miles/h, intensity = 37.1 +/- 6.1% V(O2)max) alternating with a 1-minute, high-intensity run (pace = 11.8 +/- 0.4 miles/h, intensity = 115.0 +/- 5.6% V(O2)max), resulting in an additional 2% decrease in body weight.<h4>Main outcome measures</h4>Before supplementation and on day 7 of supplementation, anterior compartment pressure was measured at rest, after dehydration, and at 1, 3, 5, 10, 15, and 60 minutes after the heat tolerance test. Analysis of variance with repeated measures was calculated to compare differences within the trials and time points and to identify any interaction between trial and time.<h4>Results</h4>The CrM intake was associated with an increase in body weight (P < .05). A moderate effect size was noted for compartment pressures between the trials for the differences between predehydration and postdehydration (eta2 = 0.414). This effect diminished substantially by 3 minutes after the heat tolerance test. Compared with the placebo trial, the change in anterior compartment pressure from rest to dehydration was greater, as was the change from rest to 1 minute after the heat tolerance test (P < .05) during the CrM trial.<h4>Conclusions</h4>A 7-day loading dose of CrM increased anterior compartment pressures after dehydration and immediately after the heat tolerance tests, but the changes did not induce symptoms and the pressure changes were transient.

<h4>Objective</h4>To investigate the effects of different doses of hydrocortisone (HC) on the disorder of coagulation in rats at early stage of septic shock.<h4>Methods</h4>The model of early septic shock accompanied by the disorder of coagulation in rats was set up by bolus injection of lipopolysaccharide (LPS) at 25 mg/kg through right femoral vein. Forty male Wistar rats were randomly divided into the following 5 groups: normal control (LPS and HC were substituted by same volume of normal saline solution), shock group (HC was substituted by same volume of normal saline), high-dose HC (HD group, HC 100 mg/kg), medium-dose HC (MD group, HC 50 mg/kg) and low-dose HC (LD group, HC 5 mg/kg). Four hours after the HC treatment, the blood specimens were collected for the count of blood platelet (PLT), the activity of antithrombin III (ATIII), the content of D-dimer and von Willebrand factor (vWF).<h4>Results</h4>The count of PLT (x 10(9)/L) and the activities of ATIII (%) in shock group (586.00 +/- 71.179 and 50.600 +/- 19.248) were significantly lower than that in normal control group (1012.600 +/- 20.852 and 89.200 +/- 12.109), and the level of D-dimer (microg/dl) and vWF (%) in LPS group (1.680 +/- 0.999 and 7.288 +/- 0.652) as significantly higher than that in normal control group (0.66 +/- 0.772 and 3.656 +/- 1.407), P < 0.05. After HC treatment, the disorder of coagulation was attenuated to different degree. However, significant difference was found between HD, MD groups and shock group only in the count of PLT (P = 0.000), and there were no significant differences between HD, MD groups and shock group in the other three indices. Significant improvements were found in LD group (P < 0.05) in all the four indices and there were no significant differences between LD group and normal group in D-dimer and vWF. The effects of medium-dose HC was between LD and HD groups, and there were no significant differences between HD and MD group, P > 0.05.<h4>Conclusions</h4>The results indicated that different doses of HC had different effects on coagulation in early stage of septic shock in rats. Low-dose HC may ameliorate the disorder of coagulation in early septic shock in rats. High-dose and medium-dose of HC had no significant improving effects on the disorder of coagulation.

Previous studies revealed novel genetic changes in the duodenal mucosa of iron-deprived rats during postnatal development. These observations are now extended to compare the genetic response to iron deficiency in the duodenum versus jejunum of 12-wk-old rats. cRNA samples were prepared from the duodenal and jejunal mucosa of three groups each of control and iron-deficient rats and hybridized with RAE 230A and 230B gene chips (Affymetrix). Stringent data reduction strategies were employed. Results showed that several genes were similarly induced in both gut segments, including DMT1, Dcytb, transferrin receptor 1, heme oxygenase 1, metallothionein, the Menkes copper ATPase (ATP7A), tripartitie motif protein 27, and the sodium-dependent vitamin C transporter. However, a subset of genes showed regulation in only one or the other gut segment. In duodenum only, gastrokine 1, trefoil factor 1 and claudin 2 were induced by iron-deficiency. Other genes previously identified were only regulated in the duodenum. Overall, these studies demonstrate similarities and distinct differences in the genetic response to iron deprivation in the duodenum versus jejunum and provide evidence that more distal gut segments also may play a role in increasing iron absorption in iron-deficiency anemia.

Hereditary hemochromatosis (HH) is the most common inherited disorder in people of Northern European descent. Over 83% of the cases of HH result from a single mutation of a Cys to Tyr in the HH protein. HFE. This mutation causes a recessive disease resulting in an accumulation of iron in selected tissues. Iron overload damages these organs leading to cirrhosis of the liver, diabetes, cardiomyopathy, and arthritis. The mechanism by which HFE influences iron homeostasis in cells and in the body remains elusive. Lack of functional HFE in humans produces the opposite effects in different cell types in the body. In the early stages of the disease. Kupffer cells in the liver and enterocytes in the intestine cells are iron depleted and have low intracellular ferritin levels, whereas hepatocytes in the liver are iron overloaded and have high intracellular iron levels. This review gives the background and a model as to possible mechanisms of how HFE could exert different effects on iron homeostasis in different cell types.

Levels of body iron should be tightly controlled to prevent the formation of oxygen radicals, lipoperoxidation, genotoxicity, and the production of cytotoxic cytokines, which result in damage to a number of organs. Enterocytes in the intestinal villae are involved in the apical uptake of iron from the intestinal lumen: iron is further exported from the cells into the circulation. The apical divalent metal transporter-1 (DMT1) transports ferrous iron from the lumen into the cells, while the basolateral transporter ferroportin extrudes iron from the enterocytes into the circulation. Patients with hereditary hemochromatosis display an accelerated transepithelial uptake of iron, which leads to body iron accumulation that results in cirrhosis, hepatocellular carcinoma, pancreatitis, and cardiomyopathy. Hereditary hemochromatosis, a recessive genetic condition, is the most prevalent genetic disease in Caucasians, with a prevalence of one in 300 subjects. The majority of patients with hereditary hemochromatosis display mutations in the gene coding for HFE, a protein that normally acts as an inhibitor of transepithelial iron transport. We discuss the different control points in the homeostasis of iron and the different mutations that exist in patients with hereditary hemochromatosis. These control sites may be influenced by gene therapeutic approaches; one general therapy for hemochromatosis of different etiologies is the inhibition of DMT1 synthesis by antisense-generating genes, which has been shown to markedly inhibit apical iron uptake by intestinal epithelial cells. We further discuss the most promising strategies to develop gene vectors and deliver them into enterocytes.

<h4>Purpose</h4>Hereditary hemochromatosis is commonly associated with iron overload and hepatitis C virus (HCV). Association between hemochromatosis C282Y or H63D mutation has been observed, although not uniformly, and iron overload is also commonly found in chronic HCV hepatitis. This study explored the contribution of genetic hemochromatosis to iron accumulation in hepatitis C.<h4>Design</h4>Review of current literature.<h4>Results</h4>The prevalence of increased serum iron stores in patients with HCV infection is 28% (patients having an elevated ferritin or transferrin saturation). Patients with elevated serum iron markers have more active chronic hepatitis with more liver fibrosis. In the opinion of the experts HFE mutations are not associated with a high hepatic iron content. No relation was detected between hepatic iron stores and HFE gene mutation. Significant iron deposition in the liver was uncommon and overall the quantity of iron that was detectable histologically and biochemically was unrelated to the grade and stage of HCV related liver injury. The mechanism by which liver iron accumulates in patients is unclear.<h4>Conclusions</h4>Carriage of HFE mutations does not have a role in the accumulation of iron or the liver disease in HCV. These findings do not support a role for iron depletion in patients with chronic HCV infection, including these with elevated serum studies.

This study focused on the synthesis and characterization of poly(ethylene glycol)-poly(D,L-lactide-co-glycolide)-poly(ethylene glycol) tri-block co-polymer (PEG-PDLLG-PEG), and its modification with type-I collagen. To this aim, a PEG-PDLLG-PEG tri-block co-polymer was synthesized in two steps by reacting poly(ethylene glycol)bis(carboxymethyl)ether with thionyl chloride to obtain an acyl-halide-terminated poly(ethylene glycol) and subsequently coupling this compound to hydroxyl-terminated poly(D,L-lactide-co-glycolide) (PDLLG). The new carboxyl endgroups of PEG-PDLLG-PEG were subsequently reacted with N-hydroxysuccinimide (NHS) in the presence of the hetero-bifunctional cross-linking agent dicyclohexylcarbodiimide (DCC) in order to activate the co-polymer for coupling with collagen. PEG-PDLLG-PEG and its activated form PEG-PDLLG-NHS were characterized by Fourier transform infrared (FT-IR) and 1H-NMR spectroscopy. Molecular weights of the polymeric products were determined by SEC. Type-I collagen in phosphate buffer was reacted with PEG-PDLLG-NHS. The resultant product, PEG-PDLLG-Col, was characterized by FT-IR. This biopolymer was used for preparation of a suitable surface for cell growth experiment. To measure the degree of cell proliferation, the films prepared with PDLLG, PEG-PDLLG-NHS and PEG-PDLLG-Col were seeded with L929 mouse fibroblasts. Cell growth was followed by SEM photography and quantitated by the neutral red uptake assay. It was shown that the attachment of collagen significantly increased the number of cells on the co-polymers.

<h4>Unlabelled</h4>It is not known whether FABP suggested as an early marker of myocardial necrosis increases after direct current cardioversion (DCC). Moreover there are no data on FABP levels in patients with atrial fibrillation (AF) and flutter.<h4>Aim</h4>To find out whether DCC induces acute changes of FABP levels in patients with AF or flutter without acute coronary syndrome.<h4>Methods</h4>Serum FABP and troponin I (TnI) were measured in 63 pts treated with DCC (monophasic shocks) because of AF (n=45, 71.4%) or atrial flutter (n=18, 28.6%). Initial energy was 50J for atrial flutter and 200J for AF. Median total energy was 200J, max 660J. Blood was sampled before DCC and in 30, 60 min, 3, 6 h after first shock. TnI and FABP were measured with single-step sandwich method ("Hytest" Finland). Upper limit of normal range (ULN) for TnI was 0.4 ng/ml (recommended by manufacturer). Serum FABP was measured in healthy volunteers and 95th percentile was used as ULN (4.67 ng/ml).<h4>Results</h4>Most pts (n=40, 63.5%) had baseline FABP > ULN (median for all pts--5.55 ng/ml). In 11 pts (17.4%) baseline FABP was > 2 ULN. No relationship was found between baseline FABP and age, sex, duration of arrhythmia, concomitant ECG changes, and presence of overt coronary heart disease or clinical signs of heart failure. Median FABP level increased after DCC (p for trend 0.00014). FABP levels after DCC correlated with total delivered energy. Delivery of > or = 2 shocks compared with 1 shock was associated with higher FABP (median 60 min--9.65 and 5.24 ng/ml, p=0.009; 3 h--12.41 and 5.84 ng/ml, p=0.01, respectively). Median TnI levels were below ULN at each study point. After DCC TnI did not exceed ULN in 61 pts and remained unchanged in 2 pts with baseline TnI above ULN.<h4>Conclusion</h4>Unexpectedly most pts with AF and atrial flutter had elevated FABP at baseline. After DCC FABP increased in proportion with total delivered energy. Elevations of FABP levels were not associated with rapid increases of TnI so skeletal muscle damage can be a likely cause of elevated FABP. AF, atrial flutter and DCC for these arrhythmias should be considered as sources of false positive results when FABP is used for diagnosis of acute myocardial infarction.

The Raf kinase inhibitor protein (RKIP) belongs to an evolutionarily conserved family of phosphatidylethanolamine-binding proteins (PEBPs), which have important functions as inhibitors of kinase signaling pathways and metastasis. Most notably, RKIP can interrupt signaling through the Ras-Raf-MEK-ERK pathway by dissociating the interaction between Raf-1 and its substrate MEK, highlighting the importance of protein interactions as regulatory interfaces. Furthermore, RKIP was shown to inhibit IkappaB kinases (IKKs) interfering with the activation of nuclear factor kappa B (NFkappaB), and G-protein coupled receptor-kinase 2 (GRK2), impeding receptor downregulation and prolonging signaling. More recently, RKIP has emerged as an important suppressor of metastasis. Here, we review the functions of RKIP and present methods to detect and measure RKIP expression and activity in cells and tissues.

Liver cirrhosis is a frequent phenomenon in chronic liver diseases such as hepatitis B, hepatitis C, alcohol-related liver damage, autoimmune hepatitis and hemochromatosis. Ascites is the most frequent complication of cirrhosis. We discuss pathogenesis, diagnosis and state-of-the-art clinical management of ascites with emphasis on recent promising developments, such as covered transjugular intrahepatic portosystemic shunt (TIPS). Spontaneous bacterial peritonitis occurs in up to 10% of patients with ascites because of bacterial overgrowth with translocation through the increased permeable small intestinal wall and impaired defence mechanisms. The addition of albumin to standard antibiotic therapy may decrease mortality of spontaneous bacterial peritonitis by decreasing the incidence of renal insufficiency. Patients with coexistent marked hyperbilirubinaemia or pre-existent renal impairment could benefit from adjuvant albumin. Probiotics (bacterial food supplements) have been claimed to improve the state of underlying liver disease and may be useful in the primary and secondary prevention of spontaneous bacterial peritonitis.

Human neurodegenerative and neuromuscular disorders are associated with a class of gene mutations represented by expansion of trinucleotide repeats. DNA testing is important for the diagnosis of these diseases because clinical discrimination is complicated by their late onset and frequently overlapping symptomatology. However, detection of pathologic alleles expanded up to several thousand trinucleotides poses a challenge for the introduction of rapid, fully automatic, and simple DNA diagnostic procedures. Here we propose a simple two-step polymerase chain reaction (PCR) protocol for rapid molecular diagnostics of myotonic dystrophy, Huntington's disease, and possibly also other triplet expansion diseases. Standard PCR amplification with target repeat flanking primers is used for the detection of alleles of up to 100 repeats; next, triplet-primed PCR is applied for detection of larger expansions. Automated capillary electrophoresis of amplicons allows rapid discrimination between normal, premutated and expanded (CTG/CAG)(n) alleles. Using the suggested protocol, the expanded allele was successfully detected in all test DNA samples with known genotypes. Our experience demonstrates that the suggested two-step PCR protocol provides high sensitivity, specificity, and reproducibility; is significantly less time-consuming; is easier to perform; and provides a better basis for automation than previous methods requiring Southern analysis. Therefore, it can be used for confirmation of uncertain clinical diagnoses, for prenatal testing in at-risk families, and, generally in research on these diseases.

In recent years, the number of proteins implicated in iron homeostasis has increased dramatically, and genetic causes have apparently been identified for the major disorders associated with tissue iron overload. These dramatic steps forward have transformed the way we look at iron-related disorders, particularly hemochromatosis. This review presents a concept of this disease that is based on this new knowledge and stems from the idea that, beyond their genetic diversities, all known hemochromatoses originate from the same metabolic error, the genetic disruption of human tendency for circulatory iron constancy. Hepcidin, the iron hormone, seems to hold a central pathogenic place in hemochromatosis, similar to insulin in diabetes: Genetically determined lack of hepcidin synthesis or activity may cause the disease.

Hepcidin, a peptide hormone made in the liver, is the principal regulator of systemic iron homeostasis. Hepcidin controls plasma iron concentration and tissue distribution of iron by inhibiting intestinal iron absorption, iron recycling by macrophages, and iron mobilization from hepatic stores. Hepcidin acts by inhibiting cellular iron efflux through binding to and inducing the degradation of ferroportin, the sole known cellular iron exporter. Synthesis of hepcidin is homeostatically increased by iron loading and decreased by anemia and hypoxia. Hepcidin is also elevated during infections and inflammation, causing a decrease in serum iron levels and contributing to the development of anemia of inflammation, probably as a host defense mechanism to limit the availability of iron to invading microorganisms. At the opposite side of the spectrum, hepcidin deficiency appears to be the ultimate cause of most forms of hemochromatosis, either due to mutations in the hepcidin gene itself or due to mutations in the regulators of hepcidin synthesis. The emergence of hepcidin as the pathogenic factor in most systemic iron disorders should provide important opportunities for improving their diagnosis and treatment.

Despite its potent ability to inhibit proinflammatory cytokine synthesis, interleukin (IL)-10 has a marginal clinical effect in rheumatoid arthritis (RA) patients. Recent evidence suggests that IL-10 induces monocyte/macrophage maturation in cooperation with macrophage-colony stimulating factor (M-CSF). In the present study, we found that the inducible subunit of the IL-10 receptor (IL-10R), type 1 IL-10R (IL-10R1), was expressed at higher levels on monocytes in RA than in healthy controls, in association with disease activity, while their expression of both type 1 and 2 tumour necrosis factor receptors (TNFR1/2) was not increased. The expression of IL-10R1 but not IL-10R2 was augmented on monocytes cultured in the presence of RA synovial tissue (ST) cell culture supernatants. Cell surface expression of TNFR1/2 expression on monocytes was induced by IL-10, and more efficiently in combination with M-CSF. Two-color immunofluorescence labeling of RA ST samples showed an intensive coexpression of IL-10R1, TNFR1/2, and M-CSF receptor in CD68+ lining macrophages. Adhered monocytes, after 3-day preincubation with IL-10 and M-CSF, could produce more IL-1beta and IL-6 in response to TNF-alpha in the presence of dibutyryl cAMP, as compared with the cells preincubated with or without IL-10 or M-CSF alone. Microarray analysis of gene expression revealed that IL-10 activated various genes essential for macrophage functions, including other members of the TNFR superfamily, receptors for chemokines and growth factors, Toll-like receptors, and TNFR-associated signaling molecules. These results suggest that IL-10 may contribute to the inflammatory process by facilitating monocyte differentiation into TNF-alpha-responsive macrophages in the presence of M-CSF in RA.

LINE-1 (L1) elements are the most abundant autonomous non-LTR retrotransposons in the human genome. Having recently performed a meta-analysis of L1 endonuclease-mediated retrotranspositional events causing human genetic disease, we have extended this study by focusing on two key issues, namely, mutation detection bias and the multiplicity of mechanisms of target gene disruption. Our analysis suggests that whereas an ascertainment bias may have generally militated against the detection of autosomal L1-mediated insertions, autosomal L1 direct insertions could have been disproportionately overlooked owing to their unusually large size. Our analysis has also indicated that the mechanisms underlying the functional disruption of target genes by L1-mediated retrotranspositional events are likely to be dependent on several different factors such as the type of insertion (L1 direct, L1 trans-driven Alu, or SVA), the precise locations of the inserted sequences within the target gene regions, the length of the inserted sequences, and possibly also their orientation.

Genetic and environmental factors may play a role in the etiopathology of fibromyalgia syndrome (FMS) and other related syndromes. There is a high aggregation of FMS in families of FMS patients. The mode of inheritance is unknown but it is most probably polygenic. There is evidence that polymorphisms of genes in the serotoninergic, dopaminergic and catecholaminergic systems play a role in the etiology of FMS. These polymorphisms are not specific for FMS and are associated with other functional somatic disorders and depression. Future genetic studies in the field of FMS and related conditions should be conducted in larger cohorts of patients and ethnically matched control groups.

Major depression is believed to be a multifactorial disorder involving predisposing temperament and personality traits, exposure to traumatic and stressful life events, and biological susceptibility. Depression, both unipolar and bipolar, is a "phasic" disease. Stressful life events are known to trigger depressive episodes, while their influence seems to decrease over the course of the illness. This suggests that depression is associated with progressive stress response abnormalities, possibly linked to impairments of structural plasticity and cellular resilience. It therefore appears crucial to adequately treat depression in the early stages of the illness, in order to prevent morphological and functional abnormalities. While evidence suggests that a severely depressed patient needs antidepressant drug therapy and that a non-severely depressed patient may benefit from other approaches (ie, "nonbiological"), little research has been done on the effectiveness of different treatments for depression. The assertion that the clinical efficacy of antidepressants is comparable between the classes and within the classes of those medications may be true from a statistical viewpoint, but is of limited value in practice. The antidepressant drugs may produce differences in therapeutic response and tolerability. Among the possible predictors of outcome in depression treatment, those derived from clinical assessment, neuroendocrine investigations, polysomnographic sleep parameters, genetic variables, and brain imaging techniques have been extensively studied. This article also reviews therapeutic strategies used when initial treatment fails, and describes briefly new concepts in antidepressant therapies such as the regulation of disturbances in circadian rhythms. The treatment of depressive illness does not stop with treatment of acute episodes, and has to be envisaged as a continuous therapeutic intervention, of which we are still not able to determine the optimal duration of treatment and the moment that it should be ceased.

<h4>Objective</h4>To detect hereditary hemochromatosis (HH) in low-income residents of a medically underserved region through free screening and confirmatory laboratory testing and to raise awareness of HH in the general population.<h4>Design</h4>Two-tiered reflexive laboratory testing was used to screen for HH. Participants evaluated the project by written survey upon its conclusion. Data were analyzed by descriptive techniques.<h4>Setting</h4>Local public health departments in 18 counties in western North Carolina (WNC). PARTICIPANTS/SUBJECTS: Phase 1: adult volunteers age > or = 20 without previous diagnosis of HH; Phase 2: Phase 1 participants with elevated screening results and adult family members of Phase 2 participants found to have HFE mutations; Phase 3: randomly-selected Phase 1 participants (Survey A) and all Phase 2 participants with HFE mutations (Survey B).<h4>Interventions</h4>Phase 1 (initial screening): non-fasting blood collection by venipuncture with testing for transferrin saturation (TS). Phase 2 (confirmatory testing): fasting blood collection by venipuncture for TS, serum ferritin (SF), and cheek swab for DNA analysis for two HFE mutations (C282Y and H63D). Phase 3: written Surveys A and B.<h4>Main outcome measures</h4>Total number participants screened for HH; prevalence of elevated TS in Phase 1 participants; prevalence of HFE genotypes consistent with HH (C282Y/C282Y and C282Y/H63D) in Phase 2 participants; prevalence of elevated SF in subjects with HFE mutations; number of family members tested; number of participants being treated for HH as a result of screening; increase in awareness of HH among Phase 1 participants.<h4>Results</h4>2,034 total subjects participated in screening events and/or family member testing. Of the 1,976 Phase 1 participants, 130 (6.6%) had elevated TS (> or = 45%). Twenty of 130 (15.4%) Phase 2 subjects were homozygotes for C282Y. The prevalence of the C282Y/C282Y genotype among the Phase 1 participants who were tested in Phase 2 was 20/1976 (1.0%). Fourteen of 20 (70%) C282Y homozygotes had elevated SE Eleven of 130 (8.5%) Phase 2 subjects were compound heterozygotes for C282Y and H63D, and none had elevated SE Of 58 family members tested, two (3.4%) were homozygotes for C282Y and eight (13.8%) were compound heterozygotes for C282Y and H63D. One of two (50%) family members homozygous for C282Y had an elevated SE No compound heterozygotes had elevated SE Sixty-four of 120 (54.2%) Phase 1 subjects responded to Survey A. 53.1% of respondents were unaware of HH prior to the screening event. 92.1% of respondents told their family and friends about HH after participating. 73.4% discussed their laboratory results with their healthcare provider. Twenty of 41 participants (48.8%) found to have HFE mutations associated with HH responded to Survey B. Eleven of 20 (55.0%) stated that they were being treated for HH.<h4>Conclusion</h4>The prevalence of the major genetic mutation, C282Y/C282Y, associated with HH among Phase 1 study participants in WNC was 1%, more than three times the national prevalence of approximately 0.33%. Results suggest that free screening using laboratory tests in a two-tiered reflexive approach may be an effective means of detecting HH, especially in high-risk populations. Early detection through free laboratory screening tests may reduce morbidity and, ultimately, healthcare costs for low-income individuals. Awareness of HH as a health concern may increase as a result of publicity generated by screening events.

Review of diagnostic services offered by molecular genetic laboratories classified according the number of laboratories dealing with individual diagnoses is presented. Three different categories were distinguished: frequently, medium and rarely offered diagnostic service. Services that concerned trombophilic factors F2 a F5 and methylentetrahydrofolatreductase, cystic fibrosis, paternity testing, gender determination, molecular testing for hemochromatose--HFE, detection of BCR/ABL fused gene, and Y-chromosomal loci DAZ and AYF examination were most frequently offered.

One of the challenges in the coming years will be to better understand the mechanisms of neuronal cell death with the objective of developing adequate drugs for the treatment of neurodegenerative disorders. Caspases and calpains are among the best-characterized cysteine proteases activated in brain disorders. Likewise, during the last decade, extensive research revealed that the deregulation of calpains activity is a key cytotoxic event in a variety of neurodegenerative disorders. Moreover, interest in the role of calpain in neurodegenerative processes is growing due to implication of the involvement of cdk5 in neurodegenerative diseases. Since calpain inhibitors appear to not only protect brain tissue from ischemia, but also to prevent neurotoxicity caused by such neurotoxins as beta-amyloid or 3-nitropropionic acid, the currently available data suggest that calpain and cdk5 play a key role in neuronal cell death. It seems clear that the inappropriate activation of cysteine proteases occurs not only during neuronal cell death, but may also contribute to brain pathology in ischemia and traumatic brain disorders. Pharmacological modulation of calpain activation may, therefore, be useful in the treatment of neurodegenerative disorders. It is possible, although difficult, to develop synthetic inhibitors of cysteine proteases, specifically calpains. The inhibition of calpain activation has recently emerged as a potential therapeutic target for the treatment of neurodegenerative diseases.

A growing body of behavioral and molecular genetics literature has indicated that the development of attention-deficit/hyperactivity disorder (ADHD) may be attributed to both genetic and environmental factors. Family, twin, and adoption studies provide compelling evidence that genes play a strong role in mediating susceptibility to ADHD. Molecular genetic studies suggest that the genetic architecture of ADHD is complex, while the handful of genome-wide scans conducted thus far is not conclusive. In contrast, the many candidate gene studies of ADHD have produced substantial evidence implicating several genes in the etiology of the disorder. For the 8 genes for which the same variant has been studied in 3 or more case-control or family-based studies, 7 show statistically significant evidence of association with ADHD based on pooled odds ratios across studies: the dopamine D4 receptor gene (DRD4), the dopamine D5 receptor gene (DRD5), the dopamine transporter gene (DAT), the dopamine beta-hydroxylase gene (DBH), the serotonin transporter gene (5-HTT), the serotonin receptor 1B gene (HTR1B), and the synaptosomal-associated protein 25 gene (SNAP25). Recent pharmacogenetic studies have correlated treatment nonresponse with particular gene markers, while preclinical studies have increased our understanding of gene expression paradigms and potential analogs for human trials. This literature review discusses the relevance and implications of genetic associations with ADHD for clinical practice and future research.

<h4>Background</h4>Transcription factor binding sites (TFBS) impart specificity to cellular transcriptional responses and have largely been defined by consensus motifs derived from a handful of validated sites. The low specificity of the computational predictions of TFBSs has been attributed to ubiquity of the motifs and the relaxed sequence requirements for binding. We posited that the inadequacy is due to limited input of empirically verified sites, and demonstrated a multiplatform approach to constructing a robust model.<h4>Results</h4>Using the TFBS for the estrogen receptor (ER)alpha (estrogen response element [ERE]) as a model system, we extracted EREs from multiple molecular and genomic platforms whose binding to ERalpha has been experimentally confirmed or rejected. In silico analyses revealed significant sequence information flanking the standard binding consensus, discriminating ERE-like sequences that bind ERalpha from those that are nonbinders. We extended the ERE consensus by three bases, bearing a terminal G at the third position 3' and an initiator C at the third position 5', which were further validated using surface plasmon resonance spectroscopy. Our functional human ERE prediction algorithm (h-ERE) outperformed existing predictive algorithms and produced fewer than 5% false negatives upon experimental validation.<h4>Conclusion</h4>Building upon a larger experimentally validated ERE set, the h-ERE algorithm is able to demarcate better the universe of ERE-like sequences that are potential ER binders. Only 14% of the predicted optimal binding sites were utilized under the experimental conditions employed, pointing to other selective criteria not related to EREs. Other factors, in addition to primary nucleotide sequence, will ultimately determine binding site selection.

No abstract available.

Understanding the molecular and cellular mechanisms underlying membrane traffic pathways is crucial to the treatment and cure of human disease. Various human diseases caused by changes in cellular homeostasis arise through a single gene mutation(s) resulting in compromised membrane trafficking. Many pathogenic agents such as viruses, bacteria, or parasites have evolved mechanisms to subvert the host cell response to infection, or have hijacked cellular mechanisms to proliferate and ensure pathogen survival. Understanding the consequence of genetic mutations or pathogenic infection on membrane traffic has also enabled greater understanding of the interactions between organisms and the surrounding environment. This review focuses on human genetic defects and molecular mechanisms that underlie eukaryote exocytosis and endocytosis and current and future prospects for alleviation of a variety of human diseases.

<h4>Background</h4>Two distinct classes of regulators have been implicated in regulating neuronal gene expression and mediating neuronal identity: transcription factors such as REST/NRSF (RE1 silencing transcription factor) and CREB (cAMP response element-binding protein), and microRNAs (miRNAs). How these two classes of regulators act together to mediate neuronal gene expression is unclear.<h4>Results</h4>Using comparative sequence analysis, here we report the identification of 895 sites (NRSE) as the putative targets of REST. A set of the identified NRSE sites is present in the vicinity of the miRNA genes that are specifically expressed in brain-related tissues, suggesting the transcriptional regulation of these miRNAs by REST. We have further identified target genes of these miRNAs, and discovered that REST and its cofactor complex are targets of multiple brain-related miRNAs including miR-124a, miR-9 and miR-132. Given the role of both REST and miRNA as repressors, these findings point to a double-negative feedback loop between REST and the miRNAs in stabilizing and maintaining neuronal gene expression. Additionally, we find that the brain-related miRNA genes are highly enriched with evolutionarily conserved cAMP response elements (CRE) in their regulatory regions, implicating the role of CREB in the positive regulation of these miRNAs.<h4>Conclusion</h4>The expression of neuronal genes and neuronal identity are controlled by multiple factors, including transcriptional regulation through REST and post-transcriptional modification by several brain-related miRNAs. We demonstrate that these different levels of regulation are coordinated through extensive feedbacks, and propose a network among REST, CREB proteins and the brain-related miRNAs as a robust program for mediating neuronal gene expression.

In nine members of polyglutamine (polyQ) diseases, CAG repeat expansions of their responsible genes are observed. The disease is considered to be caused by the formation of polyQ aggregates that sequester proteins essential for cell viability. To understand the pathological process of polyQ diseases, a proteomic approach was used to identify aggregate interacting proteins (AIPs). Constructs were designed to express EGFP-fused, CAG-expanded (150 Q) huntingtin exon1 under the control of an ecdysone-inducible promoter and either lacking or containing a nuclear localization signal (NLS). After induction of a stably transfected Neuro 2A cell line with ecdysone, aggregates form in either the cytoplasm or the nucleus. The aggregates in these two different compartments were isolated with different methods. Cytoplasmic aggregate particles were purified using a fluorescence-activated cell sorter (FACS) by monitoring EGFP fluorescence, whereas nuclear aggregates were purified by using the detergent insoluble nature of aggregates. The resulting highly pure aggregates were subjected to SDS-PAGE followed by Coomassie blue staining. Bands containing AIP candidates were excised, and, after in-gel digestion with trypsin, were analyzed by mass spectrometry to identify the proteins. Novel candidates were confirmed as AIPs by immunocytological analysis to observe colocalization with polyQ aggregates. This chapter describes methods for the establishment of stable mutant cells, the purification of polyQ aggregates, and sample preparation for mass spectrometry analysis in detail.

<h4>Introduction</h4>The role of the cellular microenvironment in breast tumorigenesis has become an important research area. However, little is known about gene expression in histologically normal tissue adjacent to breast tumor, if this is influenced by the tumor, and how this compares with non-tumor-bearing breast tissue.<h4>Methods</h4>To address this, we have generated gene expression profiles of morphologically normal epithelial and stromal tissue, isolated using laser capture microdissection, from patients with breast cancer or undergoing breast reduction mammoplasty (n = 44).<h4>Results</h4>Based on this data, we determined that morphologically normal epithelium and stroma exhibited distinct expression profiles, but molecular signatures that distinguished breast reduction tissue from tumor-adjacent normal tissue were absent. Stroma isolated from morphologically normal ducts adjacent to tumor tissue contained two distinct expression profiles that correlated with stromal cellularity, and shared similarities with soft tissue tumors with favorable outcome. Adjacent normal epithelium and stroma from breast cancer patients showed no significant association between expression profiles and standard clinical characteristics, but did cluster ER/PR/HER2-negative breast cancers with basal-like subtype expression profiles with poor prognosis.<h4>Conclusion</h4>Our data reveal that morphologically normal tissue adjacent to breast carcinomas has not undergone significant gene expression changes when compared to breast reduction tissue, and provide an important gene expression dataset for comparative studies of tumor expression profiles.

<h4>Objective</h4>To assess geographic differences in the frequencies of HFE C282Y and H63D genotypes in six racial/ethnic groups recruited in the Hemochromatosis and Iron Overload Screening (HEIRS) Study.<h4>Design</h4>HFE C282Y and H63D genotypes of 97,551 participants, ages > or = 25 years, who reported that they belonged to one of six racial/ethnic groups, were analyzed. HFE genotype frequencies were compared among the racial/ethnic groups and among the HEIRS Study field centers within each racial/ethnic group.<h4>Results</h4>The distribution of HFE C282Y and H63D genotypes differed among racial/ethnic groups (P<.0001) and among field centers in Hispanics, Asians, Whites, and Blacks (each P<.05). Genotype frequencies were similar among field centers in Native Americans and Pacific Islanders. Frequencies of C282Y and H63D genotypes were greatest in Whites. The lowest frequencies of C282Y genotypes were observed in Asians; Blacks had the lowest H63D genotype frequencies and the highest frequency of the wild-type genotype. Among racial/ethnic groups, Hispanics had the greatest variation in HFE genotypes across geographic regions.<h4>Conclusion</h4>HFE C282Y and H63D genotype frequencies vary significantly between racial/ethnic groups and within some racial/ethnic groups across geographic regions.

There are more cases of tuberculosis in the world today than at any other time in history. The global epidemic has generated intense interest into the immunological mechanisms that control infection. Although CD4+ T cells play a critical role in host immunity to Mycobacterium tuberculosis, there is considerable interest in understanding the role of other T cell subsets in preventing disease development following infection. CD8+ T cells are required for optimum host defense following M. tuberculosis infection, which has led to investigation into how this protective effect is mediated. A critical review of recent literature regarding the role of CD8+ T cells in protective immunity to M. tuberculosis infection is now required to address the strengths and weaknesses of these studies. In this article, we evaluate the evidence that CD8+ T cells are critical in immunity to M. tuberculosis infection. We discuss the specific mycobacterial proteins that are recognized by CD8+ T cells elicited during infection. Finally, we examine the effector mechanisms of CD8+ T cells generated during infection and synthesize recent studies to consider the protective roles that these T cells serve in vivo.

<h4>Background</h4>The neuronal synapse is a fundamental functional unit in the central nervous system of animals. Because synaptic function is evolutionarily conserved, we reasoned that functional sequences of genes and related genomic elements known to play important roles in neurotransmitter release would also be conserved.<h4>Results</h4>Evolutionary rate analysis revealed that presynaptic proteins evolve slowly, although some members of large gene families exhibit accelerated evolutionary rates relative to other family members. Comparative sequence analysis of 46 megabases spanning 150 presynaptic genes identified more than 26,000 elements that are highly conserved in eight vertebrate species, as well as a small subset of sequences (6%) that are shared among unrelated presynaptic genes. Analysis of large gene families revealed that upstream and intronic regions of closely related family members are extremely divergent. We also identified 504 exceptionally long conserved elements (> or =360 base pairs, > or =80% pair-wise identity between human and other mammals) in intergenic and intronic regions of presynaptic genes. Many of these elements form a highly stable stem-loop RNA structure and consequently are candidates for novel regulatory elements, whereas some conserved noncoding elements are shown to correlate with specific gene expression profiles. The SynapseDB online database integrates these findings and other functional genomic resources for synaptic genes.<h4>Conclusion</h4>Highly conserved elements in nonprotein coding regions of 150 presynaptic genes represent sequences that may be involved in the transcriptional or post-transcriptional regulation of these genes. Furthermore, comparative sequence analysis will facilitate selection of genes and noncoding sequences for future functional studies and analysis of variation studies in neurodevelopmental and psychiatric disorders.

For more than three decades, the molecular composition of the interpodocyte slit diaphragm of the glomerular filtration barrier has remained elusive. The first electron microscopic studies described the slit diaphragm as a porous, 'zipper-like' structure, but it was not until 1998 that the first transmembrane molecule of the slit diaphragm was identified: nephrin is a cell surface receptor of the immunoglobulin superfamily participating in cell-cell adhesion and signaling functions. Mutations in nephrin lead to the congenital nephrotic syndrome of the Finnish type, suggesting that nephrin is of pivotal importance for maintaining the filtration barrier. In recent years, the mapping of the genetic background of other inherited and acquired nephropathies and generation of transgenic animal models have led to a beginning of a new era in nephrology, possibly promising new targeted therapies and advanced diagnostics. This review article will briefly summarize the main findings that explain the molecular architecture of the glomerular filter itself and causes of some glomerular diseases that lead to proteinuria and, eventually, to renal failure.

The extracellular matrix (ECM) plays a significant role in the mechanical behaviour of the lung parenchyma. The ECM is composed of a three-dimensional fibre mesh that is filled with various macromolecules, among which are the glycosaminoglycans (GAGs). GAGs are long, linear and highly charged heterogeneous polysaccharides that are composed of a variable number of repeating disaccharide units. There are two main types of GAGs: nonsulphated GAG (hyaluronic acid) and sulphated GAGs (heparan sulphate and heparin, chondroitin sulphate, dermatan sulphate, and keratan sulphate). With the exception of hyaluronic acid, GAGs are usually covalently attached to a protein core, forming an overall structure that is referred to as proteoglycan. In the lungs, GAGs are distributed in the interstitium, in the sub-epithelial tissue and bronchial walls, and in airway secretions. GAGs have important functions in lung ECM: they regulate hydration and water homeostasis; they maintain structure and function; they modulate the inflammatory response; and they influence tissue repair and remodelling. Given the great diversity of GAG structures and the evidence that GAGs may have a protective effect against injury in various respiratory diseases, an understanding of changes in GAG expression that occur in disease may lead to opportunities to develop innovative and selective therapies in the future.

Osteoblasts and chondrocytes, which derive from a common mesenchymal precursor (osteochondroprogenitor), are involved in bone formation and remodeling in vivo. Determination of osteochondroprogenitor fate is under the control of complex hormonal and local factors converging onto a series of temporospatial dependent transcription regulators. Sox9, together with L-Sox5 and Sox6, of the Sox family is required for chondrogenic differentiation commitment, while Runx2/Cbfa 1, a member of runt family and Osterix/Osx, a novel zinc finger-containing transcription factor play a pivotal role in osteoblast differentiation decision and hypertrophic chondrocyte maturation. Recent in vitro and in vivo evidence suggests beta-catenin, a transcriptional activator in the canonical Wnt pathway, can act as a determinant factor for controlling chondrocyte and osteoblast differentiation. Here we focus on several intensively studied transcription factors and Wnt/beta-catenin signal molecules to illustrate the regulatory mechanism in directing commitment between osteoblast and chondrocyte, which will eventually allow us to properly manipulate the mesenchymal progenitor cell differentiation on bone and regeneration of cartilage tissue engineering.

Maintenance of stable extracellular iron concentrations requires the coordinate regulation of iron transport into plasma from dietary sources in the duodenum, from recycled senescent red cells in macrophages and from storage in hepatocytes. Moreover, during fetal development, the iron requirements of the fetus must be matched by the transport of maternal iron across the placenta. Hepcidin is a 25-amino acid disulfide-rich peptide synthesized in the liver that acts as a systemic iron-regulatory hormone by regulating iron transport from iron-exporting tissues into plasma. Hepcidin inhibits the cellular efflux of iron by binding to, and inducing the degradation of, ferroportin, the sole iron exporter in iron-transporting cells. In turn, hepcidin synthesis is increased by iron loading and decreased by anemia and hypoxia. Additionally, hepcidin synthesis is greatly increased during inflammation, trapping iron in macrophages, decreasing plasma iron concentrations and causing iron-restricted erythropoiesis characteristic of anemia of inflammation (anemia of chronic disease). Recent studies indicate that hepcidin deficiency underlies most known forms of hereditary hemochromatosis. This implies that, with the exception of very rare mutations that affect the hepcidin gene itself or modify ferroportin to make it less responsive to hepcidin, hemochromatosis genes encode molecules that regulate hepcidin synthesis. The central involvement of hepcidin in iron regulation and its pathologies should make the eventual hepcidin assay useful for the diagnosis of iron disorders and the monitoring of their treatments. The development of hepcidin agonists and antagonists may provide useful therapeutics for the treatment of iron disorders.

The term hemochromatosis encompasses at least four types of genetic iron overload conditions, most of them recently distinguished from one another as a result of the identification of a series of genes related to iron metabolism. At least three of these entities (HFE hemochromatosis, juvenile hemochromatosis and transferrin receptor 2 hemochromatosis) involve systemic hepcidin deficiency as a key pathogenetic factor. Major advances in the management of hemochromatosis influence the diagnostic approach to the disease, with the development of an overall non invasive strategy, mainly based on clinical, biological (iron parameters and genetic testing), and imaging (especially magnetic resonance imaging) data. Therapeutic management remains, on the curative side, dominated by phlebotomy (venesection), practical aspects of which have been recently revisited by the Guidelines Department of the French "Haute Autorité de Santé." However, innovative treatment approaches, based on the improved pathophysiological understanding of these diseases and the progress in iron chelation therapy, are emerging. Preventive therapy, focused on family screening, remains a key part of the management of hemochromatosis.

In patients with porphyria cutanea tarda (PCT), hepatic iron accumulation associated to hereditary hemochromatosis (HH) could play a role in the etiology and in the clinical expression of the disease. The H63D and C282Y mutations of the HFE gene frequency were studied in a PCT group of patients and compared with the frequency observed in a group of volunteer blood donors. PCT patients were cataloged as hereditary or acquired PCT carriers, whether or not they presented uroporphyrinogen decarboxilase gene mutations. Fifty percent of PCT patients were carriers of the disease's genetic type. Such percentage is significantly higher than what other authors have previously informed. H63D and C282Y mutations were present in 23% and 2.4% of the volunteer blood donors, respectively. Similar frequencies were informed by others authors in Chilean white ethnic populations, and also in Spaniard and Argentinean populations, but significantly higher than that observed in Chile's Araucanean aboriginal population. Probably the frequency of H63D and C283Y mutations are related to the Spaniard ascendancy dominance of Chile's white ethnic population. The frequency of HFE gene mutations in PCT patients was not different than what was observed in volunteer blood donors. Similarly, there was no statistical difference in the frequency of these mutations among patients with acquired or genetic PCT disease. With the obtained results, it is not possible postulate an association between PCT and the hereditary hemochromatosis of HFE gene mutations carrier conditions.

In a group of 10,156 patients with neurological diseases, the IgM level was assessed (using laser nephelometry) both in cerebrospinal fluid (CSF) and serum; concentration of other 17 protein fractions was also simultaneously determined: albumin, immunoglobulins, acute phase reactants, complement components, apolipoproteins, proteinase inhibitors and alpha1-microglobulin. Total protein, element counts and glucose level were also evaluated. In patients with normal CSF findings, only limited statistically significant correlations were demonstrated between IgM and other CSF protein fractions while, in the group of patients with pathological CSF findings, significant correlations were found between CSF(IgM) and other immunoglobulins, complement fractions and the rate of intrathecal synthesis of immunoglobulins. Correlations were also found between CSF(IgM) and CSF antithrombin-III and alpha1-microglobulin. Correlations between CSF(IgM) and CSF apolipoproteins support the theory of CNS tissue destruction whenever the concentration of CSF apolipoproteins is elevated. Our data substantially contribute to establishing the diagnosis in patients with neurological diseases; simultaneous measurement of a high number of CSF proteins is becoming inevitable for a reasonable assessment of the CSF Protein Status.

The discovery of the HFE, HJV, HAMP, TfR2, and SLC40A1 genes and preliminary understanding of their roles in iron homeostasis have contributed tremendously to our understanding of the pathogenesis of genetic hemochromatosis. Although several new models of iron metabolism have been proposed, some key "sensor" steps of iron absorption in the enterocytes and of iron storage in hepatocytes and other cells remain unclear. A diagnosis of non-HFE genetic hemochromatosis should be considered in patients with unexplained iron overload who do not have the common mutations in the HFE genes. Phenotypic evaluation such as liver biopsy and measurement of hepatic iron concentration remain important in non-HFE hemochromatosis because mutations in other genes are rare and there are no other available noninvasive tests to confirm the diagnosis. Phlebotomy remains the mainstay of therapy also for non-HFE hemochromatosis. However, phlebotomy may not be well tolerated in certain forms of non-HFE hemochromatosis such as "ferroportin disease."

No abstract available.

Each biomedical system has its own way of naming the pieces of information it contains, i.e., of defining its data elements (DEs). Integrating DEs facilitates the integration of biomedical resources. However, the mapping of DEs to the UMLS is ambiguous in many cases, when any correspondence is found at all. We propose to evaluate the potential contribution of a more general terminology: WordNet. Our method is based on synonyms, definitions, and structural properties of the terminologies. We applied it to a set of 474 DEs extracted from eleven biomedical sources. We show that WordNet can improve the direct mapping of DEs to UMLS when used to validate and disambiguate UMLS direct mappings. WordNet can also help identify indirect mappings of DEs to the UMLS.

Patients with porphyria cutanea tarda (PCT) reveal a susceptibility to reversible inactivation of hepatic uroporphyrinogen decarboxylase, which might be triggered by alcohol, hepatitis C virus infection, and iron overload. Inherited factors that may predispose to clinically overt PCT also include sequence deviations in the HFE gene that is mutated in classical hemochromatosis. Here, we studied the prevalence of both common and rare hemochromatosis gene variations in 51 PCT patients and 54 healthy controls of German origin. The frequency of the common HFE gene mutation C282Y was 15.7 % in PCT patients and 2.8 % in healthy control individuals (P < 0.001). By contrast, the frequencies of the common H63D mutation did not differ, and the allele frequencies of the less frequently observed sequence deviations as substitution S65C in the HFE gene and mutation Y250X in the TFR2 gene underlying hemochromatosis type 3 (HFE3) were < 0.02 both in PCT patients and controls. Our results comprise the first molecular studies of both common and rare hemochromatosis gene variants in German PCT patients, indicating a significant role of the C282Y mutation in the pathogenesis of PCT.

The present in vitro study created nanometer crystalline hydroxyapatite (HA) and amorphous calcium phosphate for novel orthopedic applications. Specifically, nano-crystalline HA and amorphous calcium phosphate nanoparticles were synthesized by a wet chemical process followed by hydrothermal treatment for 2 hours at 200 degrees C and 70 degrees C, respectively. Resulting particles were then pressed into compacts. For the preparation of control conventional HA particles (or those currently used in orthopedics with micron diameters), the aforementioned calcium phosphate particles were pressed into compacts and sintered at 1100 degrees C for 2 hours. All calcium phosphate-based particles were fully characterized. Results showed that although there was an initial weight gain for all the compacts studied in this experiment, higher eventual degradation rates up to 3 weeks were observed for nano-amorphous calcium phosphate compared with nano-crystalline HA which was higher than conventional HA. Peptide functionalization (with the cell adhesive peptide lysine-arginine-serine-arginine [KRSR] and the non-cell-adhesive peptide lysine-serine-arginine-arginine [KSRR]) was accomplished by means of a three-step reaction procedure: silanization with 3-aminopropyltriethoxysilane (APTES), cross-linking with N-succinimidyl-3-maleimido propionate (SMP), and finally peptide immobilization. The peptide functionalization was fully characterized. Results demonstrated increased osteoblast (bone-forming cell) adhesion on non-functionalized and functionalized nano-crystalline HA compacts compared with nano amorphous calcium phosphate compacts; both increased osteoblast adhesion compared with conventional HA. To further exemplify the novel properties of nano crystalline HA, results also showed similar osteoblast adhesion between non-functionalized nano crystalline HA and KRSR functionalized conventional HA. Thus, results provided evidence that nanocrystalline HA should be further studied for orthopedic applications.

Porous bone tissue engineering scaffolds were fabricated using both nano hydroxyapatite (nano HA) powder (20 nm average particle size) and micro HA powder (10 microm average particle size), resulting in sintered scaffolds of 59 vol% porosity and 8.6 +/- 1.9 microm average grain size and 72 vol% porosity and 588 +/- 55nm average grain size, respectively. Scanning electron microscopy was used to measure both the grain size and pore size. MC3T3-E1 osteoblast (OB) attachment and proliferation on both nano HA and micro HA porous scaffolds were quantified. As expected, OB cell number was greater on nano HA scaffolds compared with similarly processed micro HA scaffolds 5 days after seeding, while OB attachment did not appear greater on the nano HA scaffolds (p < 0.05).

Autophagy is an intracellular phenomenon in which a cell digests its own constituents. Autophagy is well conserved in nature from lower eukaryotes to mammals and has been attributed to disparate physiological events - including cell death, the mechanism of which is different from apoptosis. However, unlike in apoptosis, in which a family of cysteine proteases (caspases) and a number of other regulatory proteins have been identified and characterized, the mechanism of autophagic cell death remains unclear. In addition, the general mechanisms by which autophagy is initiated and modulated are just emerging, and several lines of evidence indicate that activated class I phosphatidylinositol 3-kinase and mammalian target of rapamycin (mTOR) inhibit autophagy, while class III phosphatidylinositol 3-kinase acts as a facilitator. Autophagy has been attributed to a number of cardiac disorders, such as ischemic cardiomyopathy, cardiac hypertrophy, hemochromatosis and myocardial aging. Induction of ventricular hypertrophy is associated with decreased autophagy, whereas it is enhanced during the regression of hypertrophy. Induction of acute cardiotoxicity by the anticancer drug anthracycline is also associated with massive cardiomyocyte loss due to autophagy (and apoptosis). Myocyte loss due to autophagy has also been reported during progression from compensated hypertrophy to heart failure in a pressure-overloaded model. Although the depth and dimension of the regulatory network that modulates autophagy in mammalian cells has yet to emerge, existing evidence suggests that it is an integral part of maintaining cellular metabolism, organelle homeostasis and redox equilibrium. Thus, it is a likely possibility that autophagy plays a crucial role in maintaining healthy myocytes in the myocardium.

Although there have been tremendous advances in the management of colorectal cancer (CRC), there is still a need for improved therapeutic approaches. On a molecular genetic level, CRC is one of the best-understood solid malignancies, and these insights can serve as a foundation for the design of novel targeted therapies. We present new genetic and epigenetic pathways that highlight the heterogeneous mechanisms in CRC pathogenesis, including the roles of the MYH DNA repair gene and of aberrant DNA hypermethylation and imprinting. We then describe some of the successful targeted therapies that inhibit COX2, EGFR, and VEGF as well as potential new targets that have been revealed by studies of molecular genetics.

No abstract available.

No abstract available.

Candidates for OLT must have irreversible acute or chronic end stage liver disease (table 41b.1). Virus or alcohol-induced liver cirrhosis constitute the most common disease indications in adults [1] (fig. 41b.1). In our department 28% of cirrhotic liver transplant recipients are transplanted for hepatitis C virus (HCV)-related liver disease and 26% undergo OLT for alcohol-related liver disease. Other indications include cholestatic liver disorders [primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), biliary atresia], hepatitis B virus (HBV) infection, autoimmune hepatitis, cystic fibrosis, inherited metabolic diseases (Wilson’s disease, hemochromatosis, alpha-1-antitrypsin deficiency), nonalcoholic steatohepatitis, nonmetastatic hepatocellular carcinoma, and acute virally-, toxin-, or drug-induced hepatic failure. The most common indications in children comprise biliary atresia and metabolic liver diseases

No abstract available.