Gene Literature Dashboard

The number of individuals with age-related cognitive impairment is rising dramatically in the UK and globally. There is considerable interest in the general hypothesis that improving the diet of older people may slow the progression of cognitive decline. To date, there has been little attention given to the possible protective role of n-3 long-chain polyunsaturated fatty acids (n-3 LCPs) most commonly found in oily fish, in age-related loss of cognitive function. The main research hypothesis of this study is that an increased dietary intake of n-3 LCPs will have a positive effect on cognitive performance in older people in the UK. To test this hypothesis, a double-blind randomised placebo-controlled trial will be carried out among adults aged 70-79 years in which the intervention arm will receive daily capsules containing n-3 LCP (0.5 g/day docosahexaenoic acid and 0.2 g/day eicosapentaenoic acid) while the placebo arm will receive daily capsules containing olive oil. The main outcome variable assessed at 24 months will be cognitive performance and a second major outcome variable will be retinal function. Retinal function tests are included as the retina is a specifically differentiated neural tissue and therefore represents an accessible window into the functioning of the brain. The overall purpose of this public-health research is to help define a simple and effective dietary intervention aimed at maintaining cognitive and retinal function in later life. This will be the first trial of its kind aiming to slow the decline of cognitive and retinal function in older people by increasing daily dietary intake of n-3 LCPs. The link between cognitive ability, visual function and quality of life among older people suggests that this novel line of research may have considerable public health importance.

<h4>Background</h4>Many cases of frontotemporal dementia (FTD) are familial, often with an autosomal dominant pattern of inheritance. Some are due to a mutation in the tau- encoding gene, on chromosome 17, and show an accumulation of abnormal tau in brain tissue (FTDP-17T). Most of the remaining familial cases do not exhibit tau pathology, but display neuropathology similar to patients with dementia and motor neuron disease, characterized by the presence of ubiquitin-immunoreactive (ub-ir), dystrophic neurites and neuronal cytoplasmic inclusions in the neocortex and hippocampus (FTLD-U). Recently, we described a subset of patients with familial FTD with autopsy-proven FTLD-U pathology and with the additional finding of ub-ir neuronal intranuclear inclusions (NII). NII are a characteristic feature of several other neurodegenerative conditions for which the genetic basis is abnormal expansion of a polyglutamine-encoding trinucleotide repeat region. The genetic basis of familial FTLD-U is currently not known, however the presence of NII suggests that a subset of cases may represent a polyglutamine expansion disease.<h4>Methods</h4>We studied DNA and post mortem brain tissue from 5 affected members of 4 different families with NII and one affected individual with familial FTLD-U without NII. Patient DNA was screened for CAA/CAG trinucleotide expansion in a set of candidate genes identified using a genome-wide computational approach. Genes containing CAA/CAG trinucleotide repeats encoding at least five glutamines were examined (n = 63), including the nine genes currently known to be associated with human disease. CAA/CAG tract sizes were compared with published normal values (where available) and with those of healthy controls (n = 94). High-resolution agarose gel electrophoresis was used to measure allele size (number of CAA/CAG repeats). For any alleles estimated to be equal to or larger than the maximum measured in the control population, the CAA/CAG tract length was confirmed by capillary electrophoresis. In addition, immunohistochemistry using a monoclonal antibody that recognizes proteins containing expanded polyglutamines (1C2) was performed on sections of post mortem brain tissue from subjects with NII.<h4>Results</h4>No significant polyglutamine-encoding repeat expansions were identified in the DNA from any of our FTLD-U patients. NII in the FTLD-U cases showed no 1C2 immunoreactivity.<h4>Conclusion</h4>We find no evidence to suggest that autosomal dominant FTLD-U with NII is a polyglutamine expansion disease.

<h4>Background</h4>Antidepressant-induced mania (AIM) has been described in bipolar disorder (BD) and has been associated with the short-allele of the serotonin transporter gene (5-HTT). We wished to investigate the frequency of and risk factors for AIM in pediatric patients with or at high risk for BD.<h4>Methods</h4>Fifty-two children and adolescents (30 with BD and 22 with subthreshold manic symptoms, 15.1 +/- 3.4 years old), all with a parent with BD, were interviewed with their parents for manic/depressive symptoms occurring before and after past antidepressant treatment. The 47 subjects with serotonin reuptake inhibitor (SSRI) exposure were genotyped for the 5-HTT polymorphism.<h4>Results</h4>Fifty percent of subjects were AIM+ and 25.5% had new onset of suicidal ideation. The AIM+ and AIM- groups did not differ significantly in relation to allele (p = .36) or genotype (p = .53) frequencies of the 5-HTT polymorphism. The AIM+ subjects were more likely to have more comorbidities (3.2 vs. 2.4; p = .02) and be BD type I (p = .04) than AIM- subjects.<h4>Conclusions</h4>Youth with or at high risk for BD may be particularly vulnerable to SSRI AIM and thus should be monitored if given SSRIs. In this preliminary study, we did not find that the 5-HTT polymorphism significantly influenced vulnerability to AIM.

<h4>Rationale</h4>3, 4-Methylenedioxymethamphetamine (MDMA or "ecstasy") is a popular drug of abuse known to result in depletions of the serotonin (5-HT) system. A number of studies have reported that ecstasy users differ from controls on a variety of measures of cognitive function. However, the literature is not consistent and many negative findings were also reported. One reason for such inconsistency might be interindividual variance in vulnerability to the deleterious effects of ecstasy due to a number of factors, both genetic and environmental.<h4>Objectives</h4>To investigate the hypothesis that carriers of the s allele at the 5-HT transporter gene-linked polymorphic region (5-HTTLPR), which was associated with reduced serotonergic neurotransmission relative to the l allele, would be most vulnerable to the effects of ecstasy on cognitive function.<h4>Methods</h4>We assessed memory, decision-making, and executive function in ecstasy users and controls, stratifying by genotype at the 5-HTTLPR.<h4>Results</h4>We observed that the 5-HTTLPR genotype groups differed on a number of measures in both the ecstasy users and the controls. While performing a risky decision-making task, ss and ls controls attended to differences in the probability of winning chosen gambles to a greater extent than the ll controls. However, this difference was dramatically attenuated in the ss ecstasy users. Furthermore, independent of ecstasy use, volunteers of the ss genotype outperformed the ll genotype on a visual planning task.<h4>Conclusions</h4>The results are consistent with the hypothesis that cognitive impairment in ecstasy users may depend on genetic variation at the 5-HTTLPR.

Although alteration to peripheral systems at the skeletal muscle level can contribute to one's ability to sustain endurance capacity, neural circuits regulating fatigue may also play a critical role. Previous studies demonstrated that increasing brain serotonin (5-HT) release is sufficient to hasten the onset of exercise-induced fatigue, while manipulations that increase brain dopamine (DA) release can delay the onset of fatigue. These results suggest that individual differences in endurance capacity could be due to factors capable of influencing the activity of 5-HT and DA systems. We evaluated possible differences in central fatigue pathways between two contrasting rat groups selectively bred for high (HCR) or low (LCR) capacity running. Using quantitative in situ hybridization, we measured messenger RNA (mRNA) levels of tryptophan hydroxylase (TPH), 5-HT transporter (5-HTT), 5-HT1A and 5-HT1B autoreceptors, dopamine receptor-D2 (DR-D2) autoreceptors and postsynaptic receptors, and dopamine receptor-D1 (DR-D1) postsynaptic receptors, in discrete brain regions of HCR and LCR. HCR expressed higher levels of 5-HT1B autoreceptor mRNA in the raphe nuclei relative to LCR, but similar levels of TPH, 5-HTT, and 5-HT1A mRNA in these areas. Surprisingly, HCR expressed higher levels of DR-D2 autoreceptor mRNA in the midbrain, while simultaneously expressing greater DR-D2 postsynaptic mRNA in the striatum compared to LCR. There were no differences in DR-D1 mRNA levels in the striatum or cortex between groups. These data suggest that central serotonergic and dopaminergic systems may be involved in the mechanisms by which HCR have delayed onset of exercise-induced fatigue compared to LCR.

Iron mediated oxidative stress is known to contribute to the neurodegenerative process in Parkinson's disease (PD). Although there are hints that genes involved in brain iron metabolism might be involved in the pathogenesis of PD in some instances, it is still not known whether the increase in brain iron content constitutes a primary or secondary event in the disease cascade. Recent studies on the role of hemochromatosis gene (HFE) mutations in PD vary from a protective effect of C282Y heterozygosity, no effect of the C282Y or H63D mutation to an increased risk for PD in C282Y mutation carriers. In this study, analyzing the whole coding region of the HFE gene by dHPLC in 278 PD patients, priorly characterized by transcranial sonography for increased iron content of the substantia nigra (SN), we did not find an association of the common HFE mutations and PD. However, we identified two novel variants (K92N and I217T) each in a single PD patient. These variations were not found in any of the controls. Future studies are necessary to reveal a possible functional relevance of these mutations for PD. Our results indicate that mutations in the HFE gene are not a common cause for PD with increased iron levels of the SN.

The native conformation of antithrombin III (ATIII) is a poor inhibitor of its coagulation pathway target enzymes because of the partial insertion of its reactive center loop (RCL) in its central A beta-sheet. This study focused on tyrosine 131, which is located at the helix D-sheet A interface, adjacent to the ATIII pentasaccharide and heparin cofactor-binding sites and some 17A away from the RCL insertion. Crystallographic structures show that the Tyr(131) ring is buried in native ATIII and then becomes exposed when pentasaccharide binds to the inhibitor and activates it. This change suggested that Tyr(131) might serve as a switch for ATIII conformational activation. The hypothesis is supported by results from this study, which progressively removed atoms from the Tyr(131) side chain. Rates of heparin-independent Y131L and Y131A factor Xa inhibition were 25 and 29 times faster than for the control and Y131F, suggesting that Tyr(131) ring interactions with neighboring helix D and strand 2A residues shift the uncatalyzed native-to-activated conformational equilibrium toward the RCL-inserted state. Thermal denaturation experiments showed Y131A and Y131L were less stable than the control and Y131F, implying an increased tendency toward A-sheet mobility in these genetically activated molecules. Thus, the tight Tyr(131)-Asn(127)-Leu(130)-Leu(140)-Ser(142) cluster at the helix D-strand 2A interface of native antithrombin contributes significantly to the stability of the ground state conformation, and tyrosine 131 serves as a heparin-responsive molecular switch during the allosteric activation of ATIII anticoagulant activity.

DEAD-box proteins are characterized by nine conserved motifs. According to these criteria, several hundreds of these proteins can be identified in databases. Many different DEAD-box proteins can be found in eukaryotes, whereas prokaryotes have small numbers of different DEAD-box proteins. DEAD-box proteins play important roles in RNA metabolism, and they are very specific and cannot mutually be replaced. In vitro, many DEAD-box proteins have been shown to have RNA-dependent ATPase and ATP-dependent RNA helicase activities. From the genetic and biochemical data obtained mainly in yeast, it has become clear that these proteins play important roles in remodeling RNP complexes in a temporally controlled fashion. Here, I shall give a general overview of the DEAD-box protein family.

The sea urchin embryo is a classical model system for studying the role of the cytoskeleton in such events as fertilization, mitosis, cleavage, cell migration and gastrulation. We have conducted an analysis of gene models derived from the Strongylocentrotus purpuratus genome assembly and have gathered strong evidence for the existence of multiple gene families encoding cytoskeletal proteins and their regulators in sea urchin. While many cytoskeletal genes have been cloned from sea urchin with sequences already existing in public databases, genome analysis reveals a significantly higher degree of diversity within certain gene families. Furthermore, genes are described corresponding to homologs of cytoskeletal proteins not previously documented in sea urchins. To illustrate the varying degree of sequence diversity that exists within cytoskeletal gene families, we conducted an analysis of genes encoding actins, specific actin-binding proteins, myosins, tubulins, kinesins, dyneins, specific microtubule-associated proteins, and intermediate filaments. We conducted ontological analysis of select genes to better understand the relatedness of urchin cytoskeletal genes to those of other deuterostomes. We analyzed developmental expression (EST) data to confirm the existence of select gene models and to understand their differential expression during various stages of early development.

The newly discovered proteins hemojuvelin (Hjv) and transferrin receptor type 2 (TfR2) are involved in iron metabolism. Mutations in the Hjv and TfR2 gene cause hemochromatosis. We investigated the expression and cellular localization of Hjv and TfR2 in rat and human liver. The expression of Hjv and TfR2 was shown on mRNA and protein level by RT-PCR and immunoblot experiments. Their cellular localization was studied by immunofluorescence with antibodies raised against Hjv and TfR2. Hjv and TfR2 are present in human and rat liver and in primary human hepatocytes. Antisera raised against Hjv identified immunoreactive proteins with an apparent size of 44 and 46 kDa in immunoblot experiments of rat and human liver extracts, which are in accordance with the putative membrane-bound and cleaved soluble forms of this protein, respectively. TfR2 was detected as a 105 kDa protein corresponding to the predicted size of glycosylated TfR2 monomers. In immunofluorescence experiments, Hjv and TfR2 were found in rat liver only in hepatocytes. At the subcellular level, both proteins were predominantly localized to the basolateral membrane domain of hepatocytes. The localization of Hjv and TfR2 at the same membrane domain renders a functional interaction of these two proteins in iron homeostasis possible.

The clinical response of sepsis to a systemic inflammatory infection may be complicated by disseminated intravascular coagulation or DIC. In order to experimentally study the syndrome of DIC, we aimed for a severe sepsis model complicated by disseminated coagulation. Most-simplified-experimental models describing coagulation abnormalities as a consequence of sepsis are based on single dose endotoxemia. The so called-Shwartzman reaction contrarily, is elicited by a low dose endotoxin priming followed by an LPS challenge and is characterized by pathological manifestations that represent the syndrome of DIC. In order to investigate whether the Shwartzman reaction is superior to a single endotoxin challenge as a model for sepsis-induced DIC and to determine what the pathological effect is of an encounter of low endotoxin prior to an LPS challenge, we undertook the present study. In this study we demonstrate that low-dose endotoxin priming prior to an LPS challenge in the Shwartzman reaction is accountable for micro-vascular thrombosis in lung and liver and subsequent (multi-) organ failure, not observed after a single-dose endotoxin challenge, which indicates that the Shwartzman reaction is well suited-model to study sepsis-induced DIC adversities. Remarkably, only minor differences in the innate immune response were established between the single-dose endotoxin challenge and the Shwartzman reaction.

UPF1 is an essential eukaryotic RNA helicase that plays a key role in various mRNA degradation pathways, notably nonsense-mediated mRNA decay (NMD). In combination with UPF2 and UPF3, it forms part of the surveillance complex that detects mRNAs containing premature stop codons and triggers their degradation in all organisms studied from yeast to human. We describe the 3 A resolution crystal structure of the highly conserved cysteine-histidine-rich domain of human UPF1 and show that it is a unique combination of three zinc-binding motifs arranged into two tandem modules related to the RING-box and U-box domains of ubiquitin ligases. This UPF1 domain interacts with UPF2, and we identified by mutational analysis residues in two distinct conserved surface regions of UPF1 that mediate this interaction. UPF1 residues we identify as important for the interaction with UPF2 are not conserved in UPF1 homologs from certain unicellular parasites that also appear to lack UPF2 in their genomes.

<h4>Background</h4>Although the short allele of the serotonin transporter promoter polymorphism (5-HTT) has been linked to increased risk of major depression in early adult life, its relationships with late-life depression and to changes in subcortical nuclei remain unclear.<h4>Methods</h4>5-HTT genotypes (SS, SL, LL) were determined for 45 older persons with major depression (mean age=52.0, sd=12.8) and 16 healthy controls (mean age=55.8, sd=10.3). MRI-derived volumes of the amygdala, hippocampus, caudate and putamen were determined by reliable tracing techniques.<h4>Results</h4>In those with depression, the short allele of 5-HTT was associated with smaller caudate nucleus volumes. Although hippocampal and amygdala volumes were smaller in those with depression as compared with control subjects, 5-HTT gene status did not predict this reduction in size.<h4>Limitations</h4>The findings are limited by the number of clinical and control participants.<h4>Conclusions</h4>Reduced caudate nucleus volume in older patients with major depression was associated with the short allele of the 5-HTT gene. This regional brain change may be a consequence of early developmental expression as well as later vascular or degenerative effects of this genotype.

In mice, dystrophic cardiovascular calcification (DCC) is controlled by a major locus on proximal mouse chromosome 7 named Dyscalc1. Here we present a strategy that combines in silico analysis, expression analysis, and extensive sequencing for ultrafine mapping of the Dyscalc1 locus. We subjected 15 laboratory mouse strains to freeze-thaw injury of the heart, and association with respective genotypes allowed condensation of the Dyscalc1 locus to 1 Mb. Within this region, 51 known and predicted genes were studied in DCC-susceptible C3H/He and DCC-resistant C57BL/6 mice with respect to mRNA expression in response to injury. Five genes displayed differential expression. Genotyping of seven novel single nucleotide polymorphisms (SNPs) within these genes revealed an 80-Kb region in NZB mice that were found positive for calcification though carrying otherwise alleles from DCC-resistant mice. This microheterogeneity in NZB mice was evolutionary conserved in all DCC-susceptible mouse strains and contains the genes EMP-3, BC013491, and Abcc6 (partially). The flanking SNPs are rs3703247 and NT_039420.5_2757991. mRNA levels of EMP-3 were found to be upregulated in response to injury in both C57BL/6 and C3H/He mice. Sequencing of EMP-3 revealed an SNP leading to an amino acid substitution (p.T153I) that was found in all mouse strains susceptible for DCC but not in resistant strains such as C57BL/6 mice. Thus, the p.T153I changes might affect the biological function of EMP-3 gene product after injury. Using this combined approach, we ultrafine-mapped the Dyscalc1 locus to an 80-Kb region and identified EMP-3 as a new candidate gene for DCC.

IL-10-producing CD4(+) type 1 regulatory T (Tr1) cells play a critical role in the maintenance of peripheral tolerance. Although immunosuppressive drugs, cytokines, costimulatory molecules, and immature dendritic cells are implicated in the induction of Tr1 cells, the signals that negatively regulate the generation and function of Tr1 cells have been elusive. We report that OX40 ligand (OX40L) completely inhibited the generation of IL-10-producing Tr1 cells from naïve and memory CD4(+) T cells induced by the immunosuppressive drugs dexamethasone and vitamin D3. This unique function of OX40L was not shared by two costimulatory TNF family members, GITR ligand and 4-1BB ligand. OX40L strongly inhibited the generation of IL-10-producing Tr1 cells induced by two physiologic stimuli, the inducible costimulatory ligand and immature dendritic cells. In addition, OX40L strongly inhibited IL-10 production and suppressive function of differentiated IL-10-producing Tr1 cells. These two novel functions of OX40L shed light on the mechanism by which OX40/OX40L regulates immunity and tolerance.

Huntington's disease (HD) is a hereditary and fatal disorder caused by an expanded CAG triplet repeat in the HD gene, resulting in a mutant form of the protein huntingtin. Wild-type and mutant huntingtin are expressed in most tissues of the body but the normal function of huntingtin is not fully known. In HD, the neuropathology is characterized by intranuclear and cytoplasmic inclusions of huntingtin aggregates, and cell death primarily in striatum and cerebral cortex. However, hypothalamic atrophy occurs at early stages of HD with loss of orexin- and somatostatin-containing cell populations. Several symptoms of HD such as sleep disturbances, alterations in circadian rhythm, and weight loss may be due to hypothalamic dysfunction. Endocrine changes including increased cortisol levels, reduced testosterone levels and increased prevalence of diabetes are found in HD patients. In HD mice, alterations in the hypothalamic-pituitary-adrenal axis occurs as well as pancreatic beta-cell and adipocyte dysfunction. Increasing evidence points towards important pathology of the hypothalamus and the endocrine system in HD. As many neuroendocrine factors are secreted into the cerebrospinal fluid, blood and urine, it is possible that their levels may reflect the disease state in the central nervous system. Investigating neuroendocrine changes in HD opens up the possibility of finding biomarkers to evaluate future therapies for HD, as well as of identifying novel targets for therapeutic interventions.

Chromosomal translocation is a common cause of leukaemia and the most common chromosome translocations found in leukaemia patients involve the mixed lineage leukaemia (MLL) gene. AF10 is one of more than 30 MLL fusion partners in leukaemia. We have recently demonstrated that the H3K79 methyltransferase hDOT1L contributes to MLL-AF10-mediated leukaemogenesis through its interaction with AF10 (ref. 5). In addition to MLL, AF10 has also been reported to fuse to CALM (clathrin-assembly protein-like lymphoid-myeloid) in patients with T-cell acute lymphoblastic leukaemia (T-ALL) and acute myeloid leukaemia (AML). Here, we analysed the molecular mechanism of leukaemogenesis by CALM-AF10. We demonstrate that CALM-AF10 fusion is both necessary and sufficient for leukaemic transformation. Additionally, we provide evidence that hDOT1L has an important role in the transformation process. hDOT1L contributes to CALM-AF10-mediated leukaemic transformation by preventing nuclear export of CALM-AF10 and by upregulating the Hoxa5 gene through H3K79 methylation. Thus, our study establishes CALM-AF10 fusion as a cause of leukaemia and reveals that mistargeting of hDOT1L and upregulation of Hoxa5 through H3K79 methylation is the underlying mechanism behind leukaemia caused by CALM-AF10 fusion.

<h4>Background</h4>In this paper we present a method for the statistical assessment of cancer predictors which make use of gene expression profiles. The methodology is applied to a new data set of microarray gene expression data collected in Casa Sollievo della Sofferenza Hospital, Foggia--Italy. The data set is made up of normal (22) and tumor (25) specimens extracted from 25 patients affected by colon cancer. We propose to give answers to some questions which are relevant for the automatic diagnosis of cancer such as: Is the size of the available data set sufficient to build accurate classifiers? What is the statistical significance of the associated error rates? In what ways can accuracy be considered dependant on the adopted classification scheme? How many genes are correlated with the pathology and how many are sufficient for an accurate colon cancer classification? The method we propose answers these questions whilst avoiding the potential pitfalls hidden in the analysis and interpretation of microarray data.<h4>Results</h4>We estimate the generalization error, evaluated through the Leave-K-Out Cross Validation error, for three different classification schemes by varying the number of training examples and the number of the genes used. The statistical significance of the error rate is measured by using a permutation test. We provide a statistical analysis in terms of the frequencies of the genes involved in the classification. Using the whole set of genes, we found that the Weighted Voting Algorithm (WVA) classifier learns the distinction between normal and tumor specimens with 25 training examples, providing e = 21% (p = 0.045) as an error rate. This remains constant even when the number of examples increases. Moreover, Regularized Least Squares (RLS) and Support Vector Machines (SVM) classifiers can learn with only 15 training examples, with an error rate of e = 19% (p = 0.035) and e = 18% (p = 0.037) respectively. Moreover, the error rate decreases as the training set size increases, reaching its best performances with 35 training examples. In this case, RLS and SVM have error rates of e = 14% (p = 0.027) and e = 11% (p = 0.019). Concerning the number of genes, we found about 6000 genes (p < 0.05) correlated with the pathology, resulting from the signal-to-noise statistic. Moreover the performances of RLS and SVM classifiers do not change when 74% of genes is used. They progressively reduce up to e = 16% (p < 0.05) when only 2 genes are employed. The biological relevance of a set of genes determined by our statistical analysis and the major roles they play in colorectal tumorigenesis is discussed.<h4>Conclusions</h4>The method proposed provides statistically significant answers to precise questions relevant for the diagnosis and prognosis of cancer. We found that, with as few as 15 examples, it is possible to train statistically significant classifiers for colon cancer diagnosis. As for the definition of the number of genes sufficient for a reliable classification of colon cancer, our results suggest that it depends on the accuracy required.

Cis-acting genetic variations can affect the amount and structure of mRNA/protein. Genomic surveys indicate that polymorphisms affecting transcription and mRNA processing, including splicing and turnover, may account for the main share of genetic factors in human phenotypic variability; however, most of these polymorphisms remain yet to be discovered. We use allelic expression imbalance (AEI) as a quantitative phenotype in the search for functional cis-acting polymorphisms in many genes, including ABCB1 (multidrug resistance 1 gene, MDR1, Pgp). Previous studies have shown that ABCB1 activity correlates with a synonymous polymorphism, C3435T; however, the functional polymorphism and molecular mechanism underlying this clinical association remained unknown. Analysis of allele-specific expression in liver autopsy samples and in vitro expression experiments showed that C3435T represents a main functional polymorphism, accounting for 1.5- to 2-fold changes in mRNA levels. The mechanism appears to involve increased mRNA turnover, probably as a result of different folding structures calculated for mRNA with the Mfold program. Other examples of the successful application of AEI analysis for studying functional polymorphism include 5-HTT (serotonin transporter, SLC6A4) and OPRM1 (mu opioid receptor). AEI is therefore a powerful approach for detecting cis-acting polymorphisms affecting gene expression and mRNA processing.

<h4>Background & aims</h4>Iron overload may cause or contribute to hepatic injury and fibrosis. Mutations in the HFE gene may influence development or progression of chronic liver disease by increasing iron stores or modulating immune responses. The aim of this work was to assess the influence of HFE mutations and serum and hepatic measures of iron status on baseline features and response to lead-in therapy in subjects with advanced chronic hepatitis C enrolled in the Hepatitis C Anti-viral Long-term Treatment to prevent Cirrhosis (HALT-C) Trial.<h4>Methods</h4>Entry criteria included an Ishak fibrosis score >2 and lack of iron overload (Scheuer iron grade <3+) according to local study pathologists. All baseline biopsy specimens were rescored by consensus of study pathologists, and detailed assessment of stainable iron was performed. Hepatic iron concentrations were measured on portions of 144 liver biopsy specimens. A total of 1051 out of 1145 subjects agreed to HFE mutational testing (C282Y, H63D, S65C).<h4>Results</h4>Thirty-five percent carried at least one HFE gene mutation. There were no significant differences in the prevalence of HFE gene mutations among subjects with fibrosis (35.5%) versus cirrhosis (32.9%). Thirty-three percent of subjects had end-of-treatment and 16% sustained virologic responses. Presence of HFE mutations, in particular the H63D variation, was associated with increased end-of-treatment (40% vs 29%, P = .0078) and sustained virologic responses (20% with HFE mutation vs 14% sustained virologic response without HFE mutation; P = .009).<h4>Conclusions</h4>Although HFE mutations (especially the most frequent H63D mutation) are associated with increased iron loading, they are also associated with increased sustained virologic responses in US patients with advanced chronic hepatitis C.

<h4>Background</h4>Mast cells are well established effectors of IgE-triggered allergic reactions and immune responses to parasitic infections. Recent studies indicate that mast cells may play roles in adaptive and innate immunity, suggesting an innovative view of the regulation of immune responses. Here, we profiled the transcriptome of human mast cells sensitized with IgE alone, or stimulated by FcepsilonRI aggregation.<h4>Results</h4>Our data show that among 8,793 genes examined, 559 genes are differentially regulated in stimulated mast cells when compared with resting/unstimulated mast cells. The major functional categories of upregulated genes include cytokines, chemokines, and other genes involved in innate and adaptive immune-responses. We observed the increased expression of over 63 gene-transcripts following IgE-sensitization alone. Our data was validated using Real-Time-PCR; ELISA and western blot. We confirmed that IgE alone does not trigger mast cell-immediate responses, such as calcium signals, degranulation or protein-phosphorylation.<h4>Conclusion</h4>This report represents a substantial advance in our understanding of the genome wide effects triggered by "passive sensitization" or active stimulation of human mast cells, supporting mast cells' potential involvement in a wide range of inflammatory responses.

Cancer classification is the critical basis for patient-tailored therapy, while pathway analysis is a promising method to discover the underlying molecular mechanisms related to cancer development by using microarray data. However, linking the molecular classification and pathway analysis with gene network approach has not been discussed yet. In this study, we developed a novel framework based on cancer class-specific gene networks for classification and pathway analysis. This framework involves a novel gene network construction, named ordering network, which exhibits the power-law node-degree distribution as seen in correlation networks. The results obtained from five public cancer datasets showed that the gene networks with ordering relationship are better than those with correlation relationship in terms of accuracy and stability of the classification performance. Furthermore, we integrated the ordering networks, classification information and pathway database to develop the topology-based pathway analysis for identifying cancer class-specific pathways, which might be essential in the biological significance of cancer. Our results suggest that the topology-based classification technology can precisely distinguish cancer subclasses and the topology-based pathway analysis can characterize the correspondent biochemical pathways even if there are subtle, but consistent, changes in gene expression, which may provide new insights into the underlying molecular mechanisms of tumorigenesis.

We describe a 50-year-old woman who developed chronic inflammatory demyelinating polyneuropathy (CIDP) one year after onset of hemochromatosis. Electrodiagnostic studies showed evidence of multifocal demyelination. Marked hypergammaglobulinemia with positive anti-nuclear and anti-DNA antibodies was found. Corticosteroid treatment resulted in a significant lessening of neurological symptoms. This is the first case of CIDP with hemochromatosis. The association may be coincidental, but the altered immune system by hemochromatosis was possibly related to the development of CIDP in this patient.

Patients with hereditary hemochromatosis (HH) have been reported to develop diastolic functional abnormalities detectable by echocardiography, but it is unknown whether these occur in asymptomatic subjects. Thus, this study tested whether echocardiographic left ventricular (LV) relaxation abnormalities are detectable in subjects with asymptomatic HH. Forty-three asymptomatic subjects with HH (C282Y homozygosity in the HFE gene) and 21 age- and gender-matched control subjects without known HFE mutations underwent echocardiography with comprehensive diastolic functional evaluations. Subjects with HH were in New York Heart Association functional class I and consisted of 22 newly diagnosed patients (group A) and 21 chronically phlebotomized subjects with stable iron levels (group B). Group A subjects showed significant iron overload compared with group B subjects and controls (group C) (ferritin 1,164 +/- 886 [p <0.05 vs groups B and C], 128 +/- 262, and 98 +/- 76 microg/L and transferrin saturation 79 +/- 19% [p <0.05 vs groups B and C], 42 +/- 21%, and 26 +/- 10% for groups A, B, and C, respectively). Echocardiographic evaluation revealed (1) no statistically significant abnormalities of Doppler LV relaxation in HH groups; (2) significant augmentation of atrial contractile function in subjects with HH compared with controls, which was not correlated with iron levels and treatment status; and (3) the preservation of overall LV systolic function in HH groups. In conclusion, the results of this study suggest that the augmentation of atrial contraction appears to be an early detectable echocardiographic cardiac manifestation of abnormal diastolic function in asymptomatic subjects with HH, which may reflect undetectable subclinical LV relaxation abnormalities.

Many studies have demonstrated a role for netrin-1-deleted in colorectal cancer (DCC) interactions in both axon guidance and neuronal migration. Neogenin, a member of the DCC receptor family, has recently been shown to be a chemorepulsive axon guidance receptor for the repulsive guidance molecule (RGM) family of guidance cues [Rajagopalan S, Deitinghoff L, Davis D, Conrad S, Skutella T, Chedotal A, Mueller B, Strittmatter S (2004) Neogenin mediates the action of repulsive guidance molecule. Nat Cell Biol 6:755-762]. Here we show that neogenin is present on neural progenitors, including neurogenic radial glia, in the embryonic mouse forebrain suggesting that neogenin expression is a hallmark of neural progenitor populations. Neogenin-positive progenitors were isolated from embryonic day 14.5 forebrain using flow cytometry and cultured as neurospheres. Neogenin-positive progenitors gave rise to neurospheres displaying a high proliferative and neurogenic potential. In contrast, neogenin-negative forebrain cells did not produce long-term neurosphere cultures and did not possess a significant neurogenic potential. These observations argue strongly for a role for neogenin in neural progenitor biology. In addition, we also observed neogenin on parvalbumin- and calbindin-positive interneuron neuroblasts that were migrating through the medial and lateral ganglionic eminences, suggesting a role for neogenin in tangential migration. Therefore, neogenin may be a multi-functional receptor regulating both progenitor activity and neuroblast migration in the embryonic forebrain.

<h4>Background</h4>In this study we present a single population test (Ewens-Waterson) applied in a genomic context to investigate the presence of recent positive selection in the Irish population. The Irish population is an interesting focus for the investigation of recent selection since several lines of evidence suggest that it may have a relatively undisturbed genetic heritage.<h4>Results</h4>We first identified outlier single nucleotide polymorphisms (SNPs), from previously published genome-wide data, with high FST branch specification in a European-American population. Eight of these were chosen for further analysis. Evidence for selective history was assessed using the Ewens-Watterson's statistic calculated using Irish genotypes of microsatellites flanking the eight outlier SNPs. Evidence suggestive of selection was detected in three of these by comparison with a population-specific genome-wide empirical distribution of the Ewens-Watterson's statistic.<h4>Conclusion</h4>The cystic fibrosis gene, a disease that has a world maximum frequency in Ireland, was among the genes showing evidence of selection. In addition to the demonstrated utility in detecting a signature of natural selection, this approach has the particular advantage of speed. It also illustrates concordance between results drawn from alternative methods implemented in different populations.

Predictive genetic testing for Huntington's disease (HD) has revealed early cognitive deficits in asymptomatic gene carriers, such as altered working memory, executive function and impaired recognition memory. The perirhinal cortex processes aspects of recognition memory and the underlying mechanism is believed to be long-term depression (LTD) of excitatory neurotransmission, the converse of long-term potentiation (LTP). We have used the R6/1 mouse model of HD to assess synaptic plasticity in the perirhinal cortex. We report here a progressive derailment of both LTD and short-term plasticity at perirhinal synapses. Layer II/III neurones gradually lose their ability to support LTD, show early nuclear localization of mutant huntingtin and display a progressive loss of membrane integrity (depolarization and loss of cell capacitance) accompanied by a reduction in the expression of D1 and D2 dopamine receptors visualized in layer I of the perirhinal cortex. Importantly, abnormalities in both short-term and long-term plasticity can be reversed by the introduction of a D2 dopamine receptor agonist (Quinpirole), suggesting that alterations in dopaminergic signalling may underlie early cognitive dysfunction in HD.

The neurotransmitter serotonin (5-HT) plays morphogenetic roles during development, and their alteration could contribute to autism pathogenesis in humans. To further characterize 5-HT's contributions to neocortical development, we assessed the thickness and neuronal cell density of various cerebral cortical areas in serotonin transporter (5-HTT) knockout (ko) mice, characterized by elevated extracellular 5-HT levels. The thickness of layer IV is decreased in 5-HTT ko mice compared with wild-type (wt) mice. The overall effect on cortical thickness, however, depends on the genetic background of the mice. Overall cortical thickness is decreased in many cortical areas of 5-HTT ko mice with a mixed c129-CD1-C57BL/6J background. Instead, 5-HTT ko mice backcrossed into the C57BL/6J background display increases in supragranular and infragranular layers, which compensate entirely for decreased layer IV thickness, resulting in unchanged or even enhanced cortical thickness. Moreover, significant increases in neuronal cell density are found in 5-HTT ko mice with a C57BL/6J background (wt:hz:ko ratio = 1.00:1.04:1.17) but not in the mixed c129-CD1-C57BL/6J 5-HTT ko animals. These results provide evidence of 5-HTT gene effects on neocortical morphology in epistatic interaction with genetic variants at other loci and may model the effect of functional 5-HTT gene variants on neocortical development in autism.

<h4>Background</h4>The tear film is a thin layer of fluid that covers the ocular surface and is involved in lubrication and protection of the eye. Little is known about the protein composition of tear fluid but its deregulation is associated with disease states, such as diabetic dry eyes. This makes this body fluid an interesting candidate for in-depth proteomic analysis.<h4>Results</h4>In this study, we employ state-of-the-art mass spectrometric identification, using both a hybrid linear ion trap-Fourier transform (LTQ-FT) and a linear ion trap-Orbitrap (LTQ-Orbitrap) mass spectrometer, and high confidence identification by two consecutive stages of peptide fragmentation (MS/MS/MS or MS3), to characterize the protein content of the tear fluid. Low microliter amounts of tear fluid samples were either pre-fractionated with one-dimensional SDS-PAGE and digested in situ with trypsin, or digested in solution. Five times more proteins were detected after gel electrophoresis compared to in solution digestion (320 versus 63 proteins). Ontology classification revealed that 64 of the identified proteins are proteases or protease inhibitors. Of these, only 24 have previously been described as components of the tear fluid. We also identified 18 anti-oxidant enzymes, which protect the eye from harmful consequences of its exposure to oxygen. Only two proteins with this activity have been previously described in the literature.<h4>Conclusion</h4>Interplay between proteases and protease inhibitors, and between oxidative reactions, is an important feature of the ocular environment. Identification of a large set of proteins participating in these reactions may allow discovery of molecular markers of disease conditions of the eye.

Small organic molecules termed osmolytes are harnessed by a variety of cell types in a wide range of organisms to counter unfavorable physiological conditions that challenge protein stability and function. Using a well characterized reporter system that we developed to allow in vivo observations, we have explored how the osmolyte proline influences the stability and aggregation of a model aggregation-prone protein, P39A cellular retinoic acid-binding protein. Strikingly, we find that the natural osmolyte proline abrogates aggregation both in vitro and in vivo (in an Escherichia coli expression system). Importantly, proline also prevented aggregation of constructs containing exon 1 of huntingtin with extended polyglutamine tracts. Although compatible osmolytes are known to stabilize the native state, our results point to a destabilizing effect of proline on partially folded states and early aggregates and a solubilizing effect on the native state. Because proline is believed to act through a combination of solvophobic backbone interactions and favorable side-chain interactions that are not specific to a particular sequence or structure, the observed effect is likely to be general. Thus, the osmolyte proline may be protective against biomedically important protein aggregates that are hallmarks of several late-onset neurodegenerative diseases including Huntington's, Alzheimer's, and Parkinson's. In addition, these results should be of practical importance because they may enable protein expression at higher efficiency under conditions where aggregation competes with proper folding.

HFE and transferrin receptor 2 (TFR2) are membrane proteins integral to mammalian iron homeostasis and associated with human hereditary hemochromatosis. Here we demonstrate that HFE and TFR2 interact in cells, that this interaction is not abrogated by disease-associated mutations of HFE and TFR2, and that TFR2 competes with TFR1 for binding to HFE. We propose a new model for the mechanism of iron status sensing that results in the regulation of iron homeostasis.

Huntington's disease (HD) is a progressive neurodegenerative disorder for which only symptomatic treatments of limited effectiveness are available. Preventing early misfolding steps and thereby aggregation of the polyglutamine (polyQ)-containing protein huntingtin (htt) in neurons of patients may represent an attractive therapeutic strategy to postpone the onset and progression of HD. Here, we demonstrate that the green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) potently inhibits the aggregation of mutant htt exon 1 protein in a dose-dependent manner. Dot-blot assays and atomic force microscopy studies revealed that EGCG modulates misfolding and oligomerization of mutant htt exon 1 protein in vitro, indicating that it interferes with very early events in the aggregation process. Also, EGCG significantly reduced polyQ-mediated htt protein aggregation and cytotoxicity in an yeast model of HD. When EGCG was fed to transgenic HD flies overexpressing a pathogenic htt exon 1 protein, photoreceptor degeneration and motor function improved. These results indicate that modulators of htt exon 1 misfolding and oligomerization like EGCG are likely to reduce polyQ-mediated toxicity in vivo. Our studies may provide the basis for the development of a novel pharmacotherapy for HD and related polyQ disorders.

Although the development of sea urchin embryos has been studied extensively and clearly involves both cell adhesion and cell migration, rather little is known about the adhesion receptors and extracellular matrix molecules involved. The completion of the genome of Strongylocentrotus purpuratus allows a comprehensive survey of the complement of cell-cell and cell-matrix adhesion molecules in this organism. Furthermore, the phylogenetic position of echinoderms offers the opportunity to compare the complement of adhesion proteins between protostome and deuterostome invertebrates and between invertebrate and vertebrate deuterostomes. Many aspects of development and cell interactions differ among these different taxa and it is likely that analysis of the spectrum of adhesion receptors and extracellular matrix proteins can open up new insights into which molecules have evolved to suit particular developmental processes. In this paper, we report the results of an initial analysis along these lines. The echinoderm adhesome (complement of adhesion-related genes/proteins) is similar overall to that of other invertebrates although there are significant deuterostome-specific innovations and some interesting features previously thought to be chordate or vertebrate specific.

Rubinstein-Taybi syndrome (RTS) is a rare human genetic disorder characterized by mental retardation and physical abnormalities. Many RTS patients have a genetic mutation which has been mapped to chromosome 16p13.3, a genomic region encoding cyclic AMP (cAMP) response element binding protein (CREB) binding protein (CBP). CBP is a transcriptional co-activator that binds to CREB when the latter is phosphorylated and promotes gene transcription. CREB-dependent gene transcription has been shown to underlie long-term memory formation. In this review we will focus on recent findings regarding the biology of CBP and its role in memory formation and cognitive dysfunction in RTS. We will also review the role of CBP in other neurological disorders, including Alzheimer's disease, Huntington's disease and amyotrophic lateral sclerosis. Finally, we will discuss novel therapeutic approaches targeted to CBP/CREB function for treating the cognitive dysfunction of RTS and other neurological disorders.

Contact-dependent interactions between EphB receptors and ephrin-B ligands mediate a variety of cell-cell communication events in the developing and mature central nervous system (CNS). These predominantly repulsive interactions occur at the interface between what are considered to be mutually exclusive EphB and ephrin-B expression domains. We previously used receptor and ligand affinity probes to show that ephrin-B ligands are expressed in the floor plate and within a dorsal region of the embryonic mouse spinal cord, while EphB receptors are present on decussated segments of commissural axons that navigate between these ephrin-B domains. Here we present the generation and characterization of two new monoclonal antibodies, mAb EfB1-3, which recognizes EphB1, EphB2, and EphB3, and mAb efrnB1, which is specific for ephrin-B1. We use these reagents and polyclonal antibodies specific for EphB1, EphB2, EphB3, or ephrin-B1 to describe the spatiotemporal expression patterns of EphB receptors and ephrin-B1 in the vertebrate spinal cord. Consistent with affinity probe binding, we show that EphB1, EphB2, and EphB3 are each preferentially expressed on decussated segments of commissural axons in vivo and in vitro, and that ephrin-B1 is expressed in a dorsal domain of the spinal cord that includes the roof plate. In contrast to affinity probe binding profiles, we show here that EphB1, EphB2, and EphB3 are present on the ventral commissure, and that EphB1 and EphB3 are expressed on axons that compose the dorsal funiculus. In addition, we unexpectedly find that mesenchymal cells, which surround the spinal cord and dorsal root ganglion, express ephrin-B1.

By RT-PCR, we isolated a partial cDNA clone for the chick Semaphorin7A (Sema7A) gene. We further analyzed its expression patterns and compared them with those of the Sema3D gene, in chick embryonic development. Sema3D and Sema7A appeared to be expressed in distinct cell populations. In mesoderm-derived structures, Sema7A expression was detected in the newly formed somites, whereas Sema3D expression was found in the notochord. In ectoderm-derived tissues, Sema3D is expressed broadly in the surface ectoderm, lens and nasal placodes. Sema3D is also expressed in the developing nervous system including diencephalon, dorsal neural tube, optical and otic vesicles. In the limb bud, Sema3D expression was found throughout the ectoderm excluding the apical ectoderm ridge (AER), where Sema7A is concentrated. Although both genes appeared to be expressed in the migrating neural crest cells, Sema3D expression is limited to neural crest cells migrating out of the midbrain/hindbrain regions, while Sema7A expression is widespread in both cranial and trunk neural crest cells.

Liver cancer is one of the leading causes of cancer death worldwide. To identify novel target genes that are related to liver carcinogenesis, we examined new genes that are differentially expressed in human hepatocellular carcinoma (HCC) cell lines and tissues based on the expressed sequence tag (EST) frequency. Eleven libraries were constructed from seven HCC cell lines and three normal liver tissue samples obtained from Korean patients. An analysis of gene expression profiles for HCC was performed using the frequency of ESTs obtained from these cDNA libraries. Genes were identified (n=120) as being either up- or down-regulated in human liver cancer cells. Among these, 14 genes (FTL, K-ALPHA1, LDHA, RPL4, ENO1, ANXA2, RPL9, RPL10, RPL13A, GNB2L1, AMBP, GC, A1BG, and SERPINC1), in addition to previously well-known liver cancer related genes, were confirmed to be differentially expressed in seven liver cancer cell lines and 17 HCC tissues by semi-quantitative RT-PCR. In addition, 73 genes, in which there was a significant difference (P>0.99) between HBV- and HCV-associated HCC cells, were selected. Of these, expression patterns of 14 (RPLP0, AKR1C, KRT8, GPX4, RPS15, ID1, RPS21, VIM, EEF1G, EIF4A1, HLA-C, FN1, CD44, and RPS10) were confirmed by semi-quantitative RT-PCR in four of HBV- and three of HCV-associated HCC cell lines. Among those genes, an immunohistochemical analysis for ANXA2 showed that it is expressed at high levels in HCC. Using an analysis of EST frequency, the newly identified genes, especially ANXA2, represent potential biomarkers for HCC and useful targets for elucidating the molecular mechanisms associated with HCC involving virological etiology.

Huntington's disease is a neurological disorder caused by the expansion of a polyglutamine tract in the protein huntingtin. Several other neurological diseases also result from the expansion of polyglutamine regions in different proteins. Despite intense efforts, no definitive biochemical or physiological role for huntingtin has been described, nor has a function been assigned to the polyglutamine region in unaffected individuals. This article presents the hypothesis that polyglutamine expansions within huntingtin and other polyglutamine proteins provide a function in and of themselves. Incorporating multiple glutamine residues into a protein during synthesis, and releasing them during protein turnover, may represent a means of minimizing interruptions in brain levels of glutamine and glutamate during periods of malnutrition. The number and variety of different proteins containing polyglutamine expansions can be interpreted as a series of evolutionary "experiments" toward a nontoxic form for glutamine storage.

Mutations in the HFE gene result in hereditary hemochromatosis, a disorder of iron metabolism characterized by increased intestinal iron absorption. Based on the observation that ectopic expression of HFE strongly inhibits apical iron uptake (Arredondo et al., 2001, FASEB J 15, 1276-1278), a negative regulation of HFE on the apical membrane transporter DMT1 was proposed as a mechanism by which HFE regulates iron absorption. To test this hypothesis, we investigated: (i) the effect of HFE antisense oligonucleotides on apical iron uptake by polarized Caco-2 cells; (ii) the apical/basolateral membrane distribution of HFE, beta-2 microglobulin and DMT1; (iii) the putative molecular association between HFE and DMT1. We found that HFE antisense treatment reduced HFE expression and increased apical iron uptake, whereas transfection with wild-type HFE inhibited iron uptake. Thus, an inverse relationship was established between HFE levels and apical iron uptake activity. Selective apical or basolateral biotinylation indicated preferential localization of DMT1 to the apical membrane and of HFE and beta-2 microglobulin (beta2m) to the basolateral membrane. Ectopic expression of HFE resulted in increased distribution of HFE-beta2m to the apical membrane. The amount of HFE-beta2m in the apical membrane inversely correlated with apical iron uptake rates. Immunoprecipitations of HFE or beta2m with specific antibodies resulted in the co-precipitation of DMT1. These results sustain a model by which direct interaction between DMT1 and HFE-beta2m in the apical membrane of Caco-2 cells result in down-regulation of apical iron uptake activity.

No abstract available.

<h4>Objective</h4>AMD3100 is a new CXCR4 antagonist that induces a rapid release of hematopoietic progenitors from the bone marrow to the peripheral blood. We conducted a clinical study where patients with multiple myeloma and non-Hodgkin's lymphoma were treated with AMD3100 (A) to increase the number of peripheral blood progenitor cells (PBPCs) when given a mobilization regimen of granulocyte colony-stimulating factor (G-CSF, G). Because experimental data suggest that A+G-mobilized PBPCs are functionally different from G-mobilized PBPCs, we were interested in an intraindividual comparison of the gene expression profile of CD34+ cells in the two different settings.<h4>Methods</h4>To this end peripheral blood CD34+ cells of three patients (three G, three A+G samples) were isolated by immunomagnetic followed by flow cytometric sorting to a purity of >99%. Total RNA was purified. Differentially expressed genes were analyzed by using the Affymetrix GeneChip Human Genome U133 Plus2.0 and the software package Micro Array Solutions 1.3 (SAS Institute Inc.).<h4>Results</h4>We found a pattern of unanimously higher (81 genes, log2 ratio > 0.5; p < 0.0001) or lower (29 genes, log2 ratio < -0.4; p < 0.0001) expressed genes in the A+G-mobilized vs G-mobilized CD34+ PBPCs. Significant changes of four selected genes noted in the microarray analysis were validated by quantitative real-time polymerase chain reaction. Genes were grouped according to gene function. Only increased expression was found in the categories antiapoptosis (e.g., MPO, HSPA1B), cell cycle (e.g., MS4A3, RRM2), replication/DNA repair (e.g., MPO, HSPA1B), cell motility (e.g., TNFSF4, HMMR), and oxygen transport. Decreased expression occurred in the proapoptosis gene group (e.g., MDA5, BCL10). CXCR4 receptor gene expression itself was significantly 1.5-fold higher in the A+G vs G group.<h4>Conclusion</h4>We conclude that A+G-mobilized CD34+ PBPCs express significantly higher amounts of genes that potentially promote superior engraftment after myeloablative therapy than G-mobilized CD34+ PBPCs.

The current study was undertaken to assess whether fibrosis could regress under venesection therapy in patients with C282Y homozygous genetic hemochromatosis. The 36 patients studied were recruited from a subfile of our database consisting of 125 C282Y homozygotes with either severe fibrosis or cirrhosis (F3 or F4 fibrosis stage, respectively, according to the METAVIR grading system). The second liver biopsy was performed for management of liver cancer, extrahepatic surgery, or assessment of liver fibrosis. All paired biopsies were reviewed by two pathologists without knowledge of clinical data. Among the 13 patients who had F3 fibrosis on their initial liver biopsy, 3 had F0, 6 had F1, and 2 had F2 on their second liver biopsy. Among the 23 patients with cirrhosis on their initial liver biopsy, 1 had F0, 4 had F1, 3 had F2, and 2 had F3 on their second liver biopsy. When defining regression of fibrosis as a decrease of at least 2 METAVIR units, fibrosis regressed in 9 of 13 (69%) F3 and in 8 of 23 (35%) F4. When the ratio of gammaglobulins (g/L) to (platelets [n/mm(3)] x prothrombin activity [%]) was greater than 7.5, fibrosis never regressed. In conclusion, these data extend the concept of regression of fibrosis to patients with treated genetic hemochromatosis and suggest that some simple biochemical tests would be predictive of further regression of fibrosis as a result of venesection therapy. If confirmed on larger series, this could modify the ultrasound screening policy of hepatocellular carcinoma in genetic hemochromatosis.

Hepatic iron overload in hemochomatosis patients can be highly variable but in general it develops in older patients. The purpose of this study was to compare development of iron load in of beta2m-/- and Hfe-/- mice paying special attention to liver pathology in older age groups. Liver iron content of beta2m-/-, Hfe-/- and control B6 mice of different ages (varying from 3 weeks to 18 months) was examined. Additional parameters (haematology indices, histopathology, lipid content and ferritin expression) were also studied in 18-month-old mice. The beta2m-/- strain presents higher hepatic iron content, hepatocyte nuclear iron inclusions, mitochondria abnormalities. In addition, hepatic steatosis was a common observation in this strain. In the liver of Hfe-/- mice, large mononuclear infiltrates positive for ferritin staining were commonly observed. The steatosis commonly observed the beta2m-/- mice may be a reflection of its higher hepatic iron content. The large hepatic mononuclear cell infiltrates seen in Hfe-/- stained for ferritin, may point to the iron sequestration capacity of lymphocytes and contribute to the clarification of the differences found in the progression of hepatic iron overload and steatosis in older animals from the two strains.

<h4>Background</h4>Evidence from neuroimaging post-mortem, and genetic studies suggests that bipolar disorder (BD) is associated with abnormalities of the serotonin-transporter (5-HTT) system. Because of various limitations of these studies, however, it has remained unclear whether 5-HTT binding is abnormal in unmedicated BD-subjects. This study used PET and [(11)C]DASB, a radioligand that afforded higher sensitivity and specificity for the 5-HTT than previously available radioligands, to compare 5-HTT binding between BD and control subjects.<h4>Methods</h4>The 5-HTT binding-potential (BP) was assessed in 18 currently-depressed, unmedicated BD-subjects and 37 healthy controls using PET and [(11)C]DASB.<h4>Results</h4>In BD, the mean 5-HTT BP was increased in thalamus, dorsal cingulate cortex (DCC), medial prefrontal cortex and insula and decreased in the brainstem at the level of the pontine raphe-nuclei. Anxiety ratings correlated positively with 5-HTT BP in insula and DCC, and BP in these regions was higher in subjects manifesting pathological obsessions and compulsions relative to BD-subjects lacking such symptoms. Subjects with a history of suicide attempts showed reduced 5-HTT binding in the midbrain and increased binding in anterior cingulate cortex versus controls and to BD-subjects without attempts.<h4>Conclusions</h4>This is the first study to report abnormalities in 5-HTT binding in unmedicated BD-subjects. The direction of abnormality in the brainstem was opposite to that found in the cortex, thalamus, and striatum. Elevated 5-HTT binding in the cortex may be related to anxiety symptoms and syndromes associated with BD.

This study shows that solitary, dormant human cancer cells, retrieved from metastasis-free organs of animals carrying spontaneously metastatic primary tumors, can reactivate their tumorigenic and metastatic potency. The tumors were produced by MDA-MB-435 CL16 breast cancer cells permanently labeled with green fluorescent protein and the neomycin resistance gene. This enabled unequivocal identification of tumor cells emerging from organ explants cultured in neomycin to eliminate nonneoplastic host cells. Rescued cells resumed proliferation and generated lines that were tumorigenic and metastatic in fresh animals. All resulting primary and secondary tumors were uniformly labeled. Cells recovered from bone marrows and spleens, where there were no metastases, were as tumorigenic and metastatic as cells recovered from lungs and lymph nodes, which are the preferred sites of colonization for this tumor line. This evidence that malignant growth of disseminated cancer cells is suspended indefinitely by microenvironmental conditions in metastasis-free organs, although it is still active in others of the same host, shows that neoplastic progression can be arrested and has far-reaching biological and clinical implications. Specifically, it predicts the existence of natural, nonimmune host mechanisms that stimulate or inactivate tumor growth in different anatomical sites, which may be exploitable for therapeutic benefit.

Systemic lupus erythematosus (SLE) is characterized by a deviation of the immune system that involves T cell-dependent autoantibody production. The aim of this study was to investigate the role of co-stimulatory markers on T cells in this disease. Twenty-eight patients with SLE as defined by the American College of Rheumatology (ACR) criteria and 11 healthy controls were included into the study. Eleven patients had biopsy-proven lupus nephritis while 17 patients had no clinical evidence of lupus nephritis. Clinical disease activity was assessed according to the systemic lupus erythematosus disease index (SLEDAI). CD4+ T cell populations in the peripheral blood were analysed for the expression of co-stimulatory markers CD45RO, CD70, CD80, CD86, CD137, CD137L, CD134, CD152, CD154 and ICOS. SLE patients showed an increased frequency of peripheral CD4+ T cells expressing high levels of CD80, CD86 and CD134 compared to healthy controls (7.1 +/- 1.5% versus 1.7 +/- 0.9%; P < 0.005; 2.3 +/- 0.4% versus 1.0 +/- 0.2%; P = 0.008, 20.2 +/- 2.0% versus 10.6 +/- 1.9%; P < 0.005, respectively). Significantly higher levels of CD80 on CD4+ T cells were detected in SLE patients with lupus nephritis compared to patients without nephritis (11.9 +/- 3.3% versus 4.0 +/- 0.7%; P < 0.005). There was an increased presence of CD134+ CD4+ cells in SLE patients with lupus nephritis (27.5 +/- 4.0% versus 15.5 +/- 1.3%; P < 0.005). CD80 and CD134 expression was significantly correlated with SLEDAI (r = 0.42, P = 0.03; r = 0.56, P < 0.005). Co-stimulatory molecules on CD4+ T cells are associated with renal disease and disease activity in patients with systemic lupus erythematosus.

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase, initially discovered as part of the NPM-ALK fusion protein, resulting from the t(2;5) translocation that is frequently associated with anaplastic large-cell lymphomas. The native ALK protein is normally expressed in the developing and, at a weaker level, adult nervous system. We recently demonstrated that the oncogenic, constitutively kinase-activated NPM-ALK protein was antiapoptotic when expressed in Jurkat lymphoblastic cells treated with cytotoxic drugs. In contrast, we now show that Jurkat cells overexpressing the wild-type ALK receptor are more sensitive to doxorubicin-induced apoptosis than parental cells. Moreover, the ALK protein is cleaved during apoptosis in a caspase-dependent manner. Mutation of aspartic residues to asparagine allowed us to map the caspase cleavage site in the juxtamembrane region of ALK. In order to assess the role of ALK in neural cell-derived tissue, we transiently expressed ALK in the 13.S.1.24 rat neuroblast immortalized cell line. ALK expression led to apoptotic cell death of the neuroblasts. ALK ligation by specific activating antibodies decreased ALK-facilitated apoptosis in both lymphoid and neuronal cell lines. Moreover, ALK transfection reduced the survival of primary cultures of cortical neurons. Thus, ALK has a proapoptotic activity in the absence of ligand, whereas it is antiapoptotic in the presence of its ligand and when the kinase is intrinsically activated. These properties place ALK in the growing family of dependence receptors.

<h4>Objective</h4>To investigate the hepatic expression of immunological markers relevant to a cytotoxic response in relation to viral genotype.<h4>Methods</h4>The frozen liver biopsies were obtained from 28 HF genotyped patients and made the sections stained. The morphometry was used to analyze the major histocompatibility complex class I (MHC-I), CD8, beta(2)-microglobulin (beta(2) -mG), HFE and CD68 in the stained sections. Biopsy data of response to therapy with interferon were available in 18 cases.<h4>Results</h4>CD8+ was usually clustered together and localized in portal tracts and sinusoids, and seen to interact with MHC I positive lining cells. MHC-I and beta(2) -mG were expressed mainly in endothelial and Kupffer cells. HFE was expressed in most round and dendritic CD68+ cells. Patients with virus genotype 3a had higher hepatic MHC-I and HFE expression, and a better sustained response to interferon (IFN) therapy than patients without.<h4>Conclusion</h4>The MHC-I expression in the liver of patient with chronic hepatitis C virus infection seems to relate to viral-genotype. The hepatic MHC-I and HFE expression are higher in patients with virus genotype 3a than that in patients with non-3a genotype.

Neurotransmitter regulation of bone metabolism has been a subject of increasing interest and investigation. We reported previously that osteoblastic cells express a functional serotonin (5-HT) signal transduction system, with mechanisms for responding to and regulating uptake of 5-HT. The clonal murine osteocytic cell line, MLO-Y4, demonstrates expression of the serotonin transporter (5-HTT), and the 5-HT1A, and 5-HT2A receptors by real-time RT-PCR and immunoblot analysis. Immunohistochemistry using antibodies for the 5-HTT, and the 5-HT1A and 5-HT2A receptors reveals expression of all three proteins in both osteoblasts and osteocytes in rat tibia. 5-HTT binding sites were demonstrated in the MLO-Y4 cells with nanomolar affinity for the stable cocaine analog [125I]RTI-55. Imipramine and fluoxetine, antagonists with specificity for 5-HTT, show the highest potency to antagonize [125I]RTI-55 binding in the MLO-Y4 cells. GBR-12935, a relatively selective dopamine transporter antagonist, had a much lower potency, as did desipramine, a selective norepinephrine transporter antagonist. The maximal [3H]5-HT uptake rate in MLO-Y4 cells was 2.85 pmol/15 min/well, with a Km value of 290 nM. Imipramine and fluoxetine inhibited specific [3H]5-HT uptake with IC50 values in the nanomolar range. 5-HT rapidly stimulated PGE2 release from MLO-Y4 cells; the EC50 for 5-HT was 0.1 microM, with a 3-fold increase seen at 60 min. The rate-limiting enzyme for serotonin synthesis, tryptophan hydroxylase, is expressed in MLO-Y4 cells as well as osteoblastic MC3T3-E1 cells. Thus, osteocytes, as well as osteoblasts, are capable of 5-HT synthesis, and express functional receptor and transporter components of the 5-HT signal transduction system.

Mutations in ferroportin (Fpn) result in iron overload. We correlate the behavior of three Fpn mutants in vitro with patients' phenotypes. Patients with Fpn mutations A77D or N174I showed macrophage iron loading. In cultured cells, FpnA77D did not reach the cell surface and cells did not export iron. Fpn mutant N174I showed plasma membrane and intracellular localization, and did not transport iron. Fpn mutation G80S was targeted to the cell surface and was transport competent, however patients showed macrophage iron. We suggest that FpnG80S represents a class of Fpn mutants whose behavior in vitro does not explain the patients' phenotype.

Hepatomegaly is commonly observed in hepatic iron overload due to human hemochromatosis and in animal models of iron loading, but the mechanisms underlying liver enlargement in these conditions have received scant attention. In this study, male rats were treated with iron dextran or dextran alone for 6 months. Chronic iron loading resulted in a > 50-fold increase in hepatic iron concentration. Both liver weights and liver/body weight ratios were increased approximately 2-fold in the iron-loaded rats (P < 0.001 for both). Hepatocyte nuclei expressing proliferating cell nuclear antigen (PCNA), a marker of S phase, were significantly increased in the iron-loaded livers, suggesting enhanced proliferation. To assess the mechanisms by which iron promotes proliferation, the expression of tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-6, hepatocyte growth factor (HGF), and transforming growth factor-alpha (TGF-alpha) were assessed by reverse transcription-polymerase chain reaction (RT-PCR). Of these growth-associated factors, only TNF-alpha messenger RNA (mRNA) was significantly increased by iron loading (about 3-fold; P = 0.005). Because cyclin D1 is required for entry of hepatocytes into the cell cycle after partial hepatectomy or treatment with direct mitogens, levels of immunoreactive cyclin D1 were examined and found to be significantly increased in the iron-loaded livers. The increase in cyclin D1 protein in the iron-loaded livers was paralleled by an increase in the abundance of its transcript as measured by real-time PCR. Taken together, these results suggest that iron is a direct mitogen in the liver and raise the possibility that chronic stimulation of hepatocyte proliferation may play a role in the pathophysiology of iron overload states.

Genetic alterations have been recognized as an important event in the carcinogenesis of gastric cancer (GC). We conducted high resolution bacterial artificial chromosome array-comparative genomic hybridization, to elucidate in more detail the genomic alterations, and to establish a pattern of DNA copy number changes with distinct clinical variables in GC. Our results showed some correlations between novel amplified or deleted regions and clinical status. Copy-number gains were frequently detected at 1p, 5p, 7q, 8q, 11p, 16p, 20p and 20q, and losses at 1p, 2q, 4q, 5q, 7q, 9p, 14q, and 18q. Losses at 4q23, 9p23, 14q31.1, or 18q21.1 as well as a gain at 20q12 were correlated with tumor-node-metastasis tumor stage. Losses at 9p23 or 14q31.1 were associated with lymph node status. Metastasis was determined to be related to losses at 4q23 or 4q28.2, as well as losses at 4q15.2, 4q21.21, 4q 28.2, or 14q31.1, with differentiation. One of the notable aspects of this study was that the losses at 4q or 14q could be employed in the evaluation of the metastatic status of GC. Our results should provide a potential resource for the molecular cytogenetic events in GC, and should also provide clues in the hunt for genes associated with GC.

The rate of electron transfer is measured to two ferrocene and one iron tetraphenylporphyrin redox species coupled through terminal acetylenes to azide-terminated thiol monolayers by the Cu(I)-catalyzed azide-alkyne cycloaddition (a Sharpless "click" reaction) to form the 1,2,3-triazole linkage. The high yield, chemoselectivity, convenience, and broad applicability of this triazole formation reaction make such a modular assembly strategy very attractive. Electron-transfer rate constants from greater than 60,000 to 1 s(-1) are obtained by varying the length and conjugation of the electron-transfer bridge and by varying the surrounding diluent thiols in the monolayer. Triazole and the triazole carbonyl linkages provide similar electronic coupling for electron transfer as esters. The ability to vary the rate of electron transfer to many different redox species over many orders of magnitude by using modular coupling chemistry provides a convenient way to study and control the delivery of electrons to multielectron redox catalysts and similar interfacial systems that require controlled delivery of electrons.

We report a patient in whom hepatosiderosis was diagnosed at the age of 55 years and who has since been treated by regular bleeding. The H63D mutation was found in the heterozygous state in the HFE gene. No mutation was recorded in the SLC11A3 gene (ferroportin). Hepatosiderosis did not seem primary, nevertheless its cause long remained elusive. Only 2 years ago did we find the responsible condition, a very mildly expressed form of dehydrated hereditary stomatocytosis (DHS). This genetic disease is a strongly iron-loading condition. Haemolysis was fully compensated. Kalaemia was slightly elevated, suggesting a pseudohyperkalaemia that may be associated with DHS. Osmotic gradient ektacytometry allowed to assess the diagnosis of DHS. The red cell monovalent Na+ and K+ concentrations were moderately elevated and reduced respectively. The temperature dependence of the ouabain + bumetanide-resistant K+ influx produced a shallow slope, above and parallel to the control curve. These features were consistent with the diagnosis of DHS. The pronounced hepatosiderosis contrasted with the mildly expressed DHS, and with the ferritinaemia that was slightly elevated, if at all, prior to bleeding. Bleeding caused ferritinaemia to decrease and hepatosiderosis to recede. The whole picture accounts for a misleading presentation of DHS, in which the primary condition long remained hidden behind one of its remotest complications, hepatosiderosis.

Src homology 2 (SH2)-domain-mediated interactions with phosphotyrosine (pY)-containing ligands are critical for the regulation of SHP-1 phosphatase activity. Peptides based on a binding site from receptor tyrosine kinase Ros (EGLN-pY2267-MVL, 1) have recently been shown to bind to the SHP-1 N-terminal SH2 domain (N-SH2) with considerably high affinity. In addition, two peptides cyclized between positions -1 and +2 relative to pY (EGLc[K(COCH(2)NH)pYMX]L-NH(2), 2: X=D, 3: X=E) bound to the N-SH2 domain, but did not activate the enzyme and even partially prevented stimulation of SHP-1 activity by the physiological ligand 1. These findings prompted us to further examine the determinants for optimal binding to the N-SH2 domain and for the stimulation and inhibition of SHP-1 activity. Herein we demonstrate that combining the preferred residues in both pY+1 (such as Phe or norleucine, Nle) and pY+3 (such as homophenylalanine, Hfe) leads to highly efficient activating ligands of SHP-1. Particularly in the context of the cyclic peptides 7 (EGLc[K(COCH(2)NH)pYFD]Hfe-NH(2)) and 8 (EGLc[K(COCH(2)NH)pYNleD]HfeL-NH(2)), the incorporation of these residues resulted in high-affinity ligands with a significantly increased ability to stimulate SHP-1 activity. We suggest that different binding modes (according to consensus sequences class I and II) are responsible for obtaining either activating (7 and 8) or nonactivating (2 and 3) ligands. Peptides such as 7 and 8 that bind in the extended fashion of the type II mode activate the phosphatase through complete filling of the cavity for pY+3. In contrast, peptides such as 2 and 3 that bind in the class I mode do not activate the enzyme because they allow more conformational space at pY+3. Therefore, their binding does not force the conformational transition necessary to trigger the dissociation of N-SH2 and the catalytic domain.

OX40 is a T cell costimulator activated by OX40L. Blockade of the OX40L-OX40 interaction has ameliorative effects in animal models of T cell pathologies. In order to better understand the interaction between OX40 and OX40L, we have determined the crystal structure of murine OX40L and of the human OX40-OX40L complex at 1.45 and 2.4 A, respectively. These structures show that OX40L is an unusually small member of the tumor necrosis factor superfamily (TNFSF). The arrangement of the OX40L protomers forming the functional trimer is atypical and differs from that of other members by a 15 degrees rotation of each protomer with respect to the trimer axis, resulting in an open assembly. Site-directed changes of the interfacial residues of OX40L suggest this interface lacks a single "hot spot" and that instead, binding energy is dispersed over at least two distinct areas. These structures demonstrate the structural plasticity of TNFSF members and their interactions with receptors.

Genomic instability is a major feature of neoplastic development in colorectal carcinoma and other cancers. Specific genomic instability events, such as deletions in chromosomes and other alterations in gene copy number, have potential utility as biologically relevant prognostic biomarkers. For example, genomic deletions on chromosome arm 18q are an indicator of colorectal carcinoma behavior and potentially useful as a prognostic indicator. Adapting a novel genomic technology called molecular inversion probes which can determine gene copy alterations, such as genomic deletions, we designed a set of probes to interrogate several hundred individual exons of >200 cancer genes with an overall distribution covering all chromosome arms. In addition, >100 probes were designed in close proximity of microsatellite markers on chromosome arm 18q. We analyzed a set of colorectal carcinoma cell lines and primary colorectal tumor samples for gene copy alterations and deletion mutations in exons. Based on clustering analysis, we distinguished the different categories of genomic instability among the colorectal cancer cell lines. Our analysis of primary tumors uncovered several distinct categories of colorectal carcinoma, each with specific patterns of 18q deletions and deletion mutations in specific genes. This finding has potential clinical ramifications given the application of 18q loss of heterozygosity events as a potential indicator for adjuvant treatment in stage II colorectal carcinoma.

The role of iron metabolism in Alzheimer's disease (AD) is well documented. Regulation of the proteins that maintain cellular iron metabolism is mediated by two cytoplasmic RNA-binding proteins, the Iron Regulatory Proteins (IRP1 and IRP2), that function through post-transcriptional interactions with RNA stem loop structures called iron-responsive elements. As the primary mediator of iron homeostasis in neuronal cells, IRP2 is a strong candidate for polymorphisms that could impact AD pathogenesis. Thus, we performed a pilot study to assess polymorphisms in the gene encoding IRP2 (IREB2) on clinically well-characterized, post-mortem samples (50 AD and 50 controls). DNA sequence analysis of the IREB2 gene region revealed 14 polymorphisms. Two (rs2656070 and rs13180) showed statistically significant skewing of allelic and genotypic distributions between AD patients and controls. In silico analyses revealed that rs2656070 lies within a probable promoter and disrupts the binding sites of at least two known transcription factors. Though silent and likely not functionally relevant, rs13180 is in complete LD with rs2656070 (D' > 0.999), creating an IREB2-haplotype that is significantly associated with AD. Confirmation of this association in a larger cohort of cases and controls would further support the role of iron regulation in the pathogenesis of this catastrophic and increasingly common neurodegenerative disorder.

<h4>Background</h4>The mechanism of the antithrombotic action of statins is unclear. We evaluated the effects of atorvastatin on the coagulation parameters and homocysteine levels of patients with primary hypercholesterolemia.<h4>Materials and methods</h4>Forty-four patients with primary hypercholesterolemia were treated with atorvastatin 10 mg/d for 24 weeks at Adnan Menderes University Medical Faculty, Division of Hematology, Aydin, Turkey. We evaluated the effects of atorvastatin on homocysteine; lipid parameters such as total cholesterol, low-density-lipoprotein (LDL) cholesterol, very-low-density-lipoprotein (VLDL) cholesterol, triglycerides, high-density-lipoprotein (HDL) cholesterol, lipoprotein (a), apolipoprotein Al and apolipoprotein B; and coagulation parameters such as fibrinogen, antithrombin-III, protein C, protein S, von Willebrand factor, D-dimer, partial thromboplastin time and prothrombin time; and hematological parameters such as hemoglobin, white blood cell and platelet counts, vitamin B12 and folic acid.<h4>Results</h4>Atorvastatin significantly decreased the levels of total cholesterol, LDL cholesterol (p < 0.001), VLDL cholesterol, triglycerides and apo B (p < 0.001). The level of HDL cholesterol significantly increased with atorvastatin treatment (p < 0.001). Atorvastatin significantly increased the levels of fibrinogen (p < 0.001), but it had no effect on other coagulation factors and homocysteine (p > 0.05). After treatment, while vitamin B12 levels significantly increased (p < 0.05), other hematological parameters were not changed with atorvastatin (p > 0.05).<h4>Conclusion</h4>Although there were beneficial effects of atorvastatin on lipid parameters, atorvastatin did not significantly change the level of homocysteine and hematological, and coagulation parameters, with the exception of fibrinogen and vitamin B12 levels.

Raf kinase inhibitory protein (RKIP or PEBP) is an inhibitor of the Raf/MEK/MAP kinase signaling cascade and a suppressor of cancer metastasis. We now show that RKIP associates with centrosomes and kinetochores and regulates the spindle checkpoint in mammalian cells. RKIP depletion causes decreases in the mitotic index, the number of metaphase cells, and traversal times from nuclear envelope breakdown to anaphase, and an override of mitotic checkpoints induced by spindle poisons. Raf-1 depletion or MEK inhibition reverses the reduction in the mitotic index, whereas hyperactivation of Raf mimics the RKIP-depletion phenotype. Finally, RKIP depletion or Raf hyperactivation reduces kinetochore localization and kinase activity of Aurora B, a regulator of the spindle checkpoint. These results indicate that RKIP regulates Aurora B kinase and the spindle checkpoint via the Raf-1/MEK/ERK cascade and demonstrate that small changes in the MAP kinase (MAPK) pathway can profoundly impact the fidelity of the cell cycle.

Corticotropin-releasing factor (CRF) and the related urocortin peptides mediate behavioral, cognitive, autonomic, neuroendocrine and immunologic responses to aversive stimuli by activating CRF(1) or CRF(2) receptors in the central nervous system and anterior pituitary. Markers of hyperactive central CRF systems, including CRF hypersecretion and abnormal hypothalamic-pituitary-adrenal axis functioning, have been identified in subpopulations of patients with anxiety, stress and depressive disorders. Because CRF receptors are rapidly desensitized in the presence of high agonist concentrations, CRF hypersecretion alone may be insufficient to account for the enhanced CRF neurotransmission observed in these patients. Concomitant dysregulation of mechanisms stringently controlling magnitude and duration of CRF receptor signaling also may contribute to this phenomenon. While it is well established that the CRF(1) receptor mediates many anxiety- and depression-like behaviors as well as HPA axis stress responses, CRF(2) receptor functions are not well understood at present. One hypothesis holds that CRF(1) receptor activation initiates fear and anxiety-like responses, while CRF(2) receptor activation re-establishes homeostasis by counteracting the aversive effects of CRF(1) receptor signaling. An alternative hypothesis posits that CRF(1) and CRF(2) receptors contribute to opposite defensive modes, with CRF(1) receptors mediating active defensive responses triggered by escapable stressors, and CRF(2) receptors mediating anxiety- and depression-like responses induced by inescapable, uncontrollable stressors. CRF(1) receptor antagonists are being developed as novel treatments for affective and stress disorders. If it is confirmed that the CRF(2) receptor contributes importantly to anxiety and depression, the development of small molecule CRF(2) receptor antagonists would be therapeutically useful.

The p.M172K TFR2 mutation was identified in two Italian siblings aged 32 and 40 years old with primary iron overload. The two patients showed a severe increase in serum iron indices. From the age of 25, the male sib also revealed abnormal levels of hepatic enzymes, presumably in relation to iron induced liver damage. Clinical findings seem to evidence that type 3 hemochromatosis can be more serious than classic hemochromatosis. This report adds two more type 3 hereditary hemochromatosis cases which suggest that TFR2 mutations could be more frequently involved in non-HFE hemochromatosis than has been actually thought.

Recent studies, using cytometric techniques based on fluorescence microscopy, have provided new information on how linker histones interact with chromatin in vivo or in situ. In particular, the use of green fluorescent proteins (GFPs) has enabled detailed studies of how individual H1 subtypes, and specific motifs in them, interact with chromatin in vivo. Furthermore, the development of cytochemical methods to study the interaction between linker histones and chromatin using DNA-binding fluorochromes as indirect probes for linker histone affinity in situ, in combination with highly sensitive and specific analytical methods, has provided additional information on the interactions between linker histones and chromatin in several cell systems. Such results verified that linker histones have a substantially higher affinity for chromatin in mature chicken erythrocytes than in frog erythrocytes, and they also indicated that the affinity decreased during differentiation of the frog erythrocytes. Furthermore, in cultured human fibroblasts, the linker histones showed a relatively high affinity for chromatin in interphase, whereas it showed a significantly lower affinity in highly condensed metaphase chromosomes. This method also enables the analysis of linker histone affinity for chromatin in H1-depleted fibroblasts reconstituted with purified linker histones. No consistent correlation between linker histone affinity and chromatin condensation has so far been detected.

Although they are one of the oldest family of proteins known (first described in 1884 by Kossel), histones continue to surprise researchers with their ever expanding roles in biology. In the past 25 years, the view of core histone octamers as a simple spool around which DNA in the nucleus is wound and linker histones as mere fasteners clipping it all together has transformed into the realization that histones play a vital role in transcriptional regulation. Through post-translational modifications, histones control the accessibility of transcription factors and a host of other proteins to multiple, conceivably thousands of, genes at once. While researchers have spent decades deciphering the role of histones in the overall structure of chromatin, it might surprise some to find that an entirely separate faction of scientists have focused on the role of histones beyond the confines of the nuclear envelope. In the past decade, there has been an accumulation of observations that suggest that histones can be found at the mitochondrion during the onset of apoptotic signaling and even at the cell surface, acting as a receptor for bacterial and viral proteins. More provocatively, immunologists are becoming convinced that they can also be found in the lumen of several tissues, acting as antimicrobial agents--critical components of an ancient innate immune system. Perhaps nowhere is this observation as dramatic as in the ability of neutrophils to entrap bacterial pathogens by casting out "nets" of DNA and histones that not only act as a physical barrier, but also display bactericidal activity. As our views regarding the role of histones inside and outside the cell evolve, some have begun to develop therapies that either utilize or target histones in the fight against cancer, microbial infection, and autoimmune disease. It is our goal here to begin the process of merging the dichotomous lives of histones both within and without the nuclear membrane.

<h4>Aim</h4>To evaluate possible risk factors for the spread of hepatitis C infection and to analyze the characteristics of the epidemiological and clinical patterns among the patients with hepatitis C infection.<h4>Methods</h4>During a five-year period a cross-sectional study was conducted among HCV positive individuals referred to the Ahwaz JundiShapour University Hospitals (AJSUH) and Hepatitis Clinic from 1 Sept 1999 to 1 Sept 2003. The control group consisted of first time blood donors referred to the Regional Blood Transfusion organization. Enzyme-linked immunosorbent assay and recombinant immunoblot assay anti-hepatitis C virus (HCV) tests were performed for two groups. Positive serum specimens were retested using polymerase chain reaction (PCR) for HCV RNA. Risk factors were evaluated using a questionnaire. Reported risk factors among infected subjects ("HCV-positive") were compared to those of subjects never exposed ("HCV-negative") to HCV.<h4>Results</h4>A total of 514 subjects were studied for HCV, of which 254 were HCV-positive and 260 HCV-negative donors comprised the control group. Mean age of the patients was 28.4 (Std 15.22) years. HCV-positive subjects were more likely to be of male gender (63% versus 37%). Transfusion 132 (52%), non-intravenous (n-i.v.) drug abuse and i.v. drug abuse 37 (14.5%), haemodialysis 25 (10%), receiving wounds at war and extramarital sexual activities (2.4%), tattooing (3.6%) were found to be independent risk factors of being HCV-positive. No apparent risk factors could be demonstrated in 29 (11.2%) of the positive cases.<h4>Conclusion</h4>Our data indicate that a history of transfusion and i.v. drug abuse and haemodialysis are important risk factors for HCV infection in our area and that more careful pretransfusion screening of blood for anti-HCV must be introduced in our blood banks. Improvements in certain lifestyle patterns, and customs in this area may be essential to prevent transmission of the infection.

<h4>Aim</h4>To evaluate the association among hepatic fibrosis, serum iron indices, and hepatic iron stores in patients with Chronic Hepatitis C (CHC).<h4>Methods</h4>Thirty-two CHC patients were included in our study. The histological degree of fibrosis and inflammation activity was assessed according to the Metavir system. The serum iron indices including ferritin, iron and transferrin saturation were measured. Hepatic iron deposition was graded by Perls' stain.<h4>Results</h4>The CHC patients with severe hepatic fibrosis (n = 16) were significantly older than CHC patients with mild fibrosis (n = 16) (P = 0.024). The serum iron indices, increased serum iron store and positive hepatic iron stain were not significantly different between the two groups. In multivariate logistic regression analysis, the age at biopsy was an independent predictor of severe hepatic fibrosis (Odds ratio = 1.312; P = 0.035). The positive hepatic iron stain was significantly associated with the values of alanine aminotransferase (ALT) (P = 0.017), ferritin (P = 0.008), serum iron (P = 0.019) and transferrin saturation (P = 0.003). The ferritin level showed significant correlation with the value of ALT (r = 0.531; P = 0.003), iron (r = 0.467; P = 0.011) and transferrin saturation (r = 0.556; P = 0.002).<h4>Conclusion</h4>Our findings suggest that the severity of hepatitis C virus (HCV)-related liver injury is associated with patient age at biopsy. Both serum iron indices and hepatic iron deposition show correlation with serum indices of chronic liver disease but are not related to grade and stage of liver histology.

Many epidemiological studies demonstrate that treatment with non-steroidal anti-inflammatory drugs (NSAIDs) reduce the incidence and mortality of certain malignancies, especially gastrointestinal cancer. The cyclooxygenase (COX) enzymes are well-known targets of NSAIDs. However, conventional NSAIDs non-selectively inhibit both the constitutive form COX-1, and the inducible form COX-2. Recent evidence indicates that COX-2 is an important molecular target for anticancer therapies. Its expression is undetectable in most normal tissues, and is highly induced by pro-inflammatory cytokines, mitogens, tumor promoters and growth factors. It is now well-established that COX-2 is chronically overexpressed in many premalignant, malignant, and metastastic cancers, including hepatocellular carcinoma (HCC). Overexpression of COX-2 in patients with HCC is generally higher in well-differentiated HCCs compared with less-differentiated HCCs or histologically normal liver, suggesting that COX-2 may be involved in the early stages of hepatocarcinogenesis, and increased expression of COX-2 in noncancerous liver tissue has been significantly associated with shorter disease-free survival in patients with HCC. In tumors, overexpression of COX-2 leads to an increase in prostaglandin (PG) levels, which affect many mechanisms involved in carcinogenesis, such as angiogenesis, inhibition of apoptosis, stimulation of cell growth as well as the invasiveness and metastatic potential of tumor cells. The availability of novel agents that selectively inhibit COX-2 (COXIB), has contributed to shedding light on the role of this molecule. Experimental studies on animal models of liver cancer have shown that NSAIDs, including both selective and non-selective COX-2 inhibitors, exert chemopreventive as well as therapeutic effects. However, the key mechanism by which COX-2 inhibitors affect HCC cell growth is as yet not fully understood. Increasing evidence suggests the involvement of molecular targets other than COX-2 in the anti-proliferative effects of COX-2 selective inhibitors. Therefore, COX-inhibitors may use both COX-2-dependent and COX-2-independent mechanisms to mediate their antitumor properties, although their relative contributions toward the in vivo effects remain less clear. Here we review the features of COX enzymes, the role of the expression of COX isoforms in hepatocarcinogenesis and the mechanisms by which they may contribute to HCC growth, the pharmacological properties of COX-2 selective inhibitors, the antitumor effects of COX inhibitors, and the rationale and feasibility of COX-2 inhibitors for the treatment of HCC.

<h4>Aim</h4>To analyze the relationship between smoking and nonalcoholic fatty liver disease (NAFLD).<h4>Methods</h4>This is a cross-sectional study of a healthy population, carried out in a check-up unit of a university hospital in Mexico City. We enrolled 933 subjects, 368 current smokers (cases) and 565 persons who had never smoked (controls). Demographic, metabolic and biochemical variables were measured in the two groups. NAFLD was determined by ultrasound and metabolic syndrome according to ATPIII.<h4>Results</h4>A total of 548 men (205 cases and 343 controls) and 337 women (114 cases and 223 controls) were included in the analysis. Statistical differences between cases and controls were observed only in high blood pressure prevalence (6.6% vs 11.3%, P < 0.05; cases and controls respectively), high-density lipoproteins (1.00 +/- 0.26 vs 1.06 +/- 0.28 mmol/L, P < 0.005), triglycerides (2.18 +/- 1.49 vs 1.84 +/- 1.1 mmol/L, P < 0.001), and erythrocyte sedimentation rate (11.3 +/- 9.3 vs 13.5 +/- 11.9 mm/h, P < 0.001). No differences were observed in the prevalence of NAFLD (22.27% vs 29.68%, P = NS) and metabolic syndrome (41.69% vs 36.74%, P = NS). Univariate analysis showed that smoking was not a risk factor for NAFLD (OR = 0.89, 95% CI 0.65-1.21).<h4>Conclusion</h4>No differences in NAFLD prevalence were observed between current smokers and nonsmokers, and furthermore, no differences were observed in heavy smokers (more than 20 packs/year), indicating that there is no relationship between smoking and NAFLD.

A commonly occurring polymorphic variant of the human 5-hydroxytryptamine (5-HT) transporter (5-HTT) gene that increases 5-HTT expression has been associated with reduced anxiety levels in human volunteer and patient populations. However, it is not known whether this linkage between genotype and anxiety relates to variation in 5-HTT expression and consequent changes in 5-HT transmission. Here we test this hypothesis by measuring the neurochemical and behavioral characteristics of a mouse genetically engineered to overexpress the 5-HTT. Transgenic mice overexpressing the human 5-HTT (h5-HTT) were produced from a 500 kb yeast artificial chromosome construct. These transgenic mice showed the presence of h5-HTT mRNA in the midbrain raphe nuclei, as well as a twofold to threefold increase in 5-HTT binding sites in the raphe nuclei and a range of forebrain regions. The transgenic mice had reduced regional brain whole-tissue levels of 5-HT and, in microdialysis experiments, decreased brain extracellular 5-HT, which reversed on administration of the 5-HTT inhibitor paroxetine. Compared with wild-type mice, the transgenic mice exhibited a low-anxiety phenotype in plus maze and hyponeophagia tests. Furthermore, in the plus maze test, the low-anxiety phenotype of the transgenic mice was reversed by acute administration of paroxetine, suggesting a direct link between the behavior, 5-HTT overexpression, and low extracellular 5-HT. In toto, these findings demonstrate that associations between increased 5-HTT expression and anxiety can be modeled in mice and may be specifically mediated by decreases in 5-HT transmission.

<h4>Aim</h4>The aim of this paper was to establish the physiologic changes in the coagulation and fibrinolytic systems during normal pregnancy.<h4>Methods</h4>One-hundred and twenty normal pregnant women were investigated in a longitudinal study involving 3 measurements: blood samples were collected at 12, 24 and 36 weeks of gestation and were assayed for prothrombin time, antithrombin III (ATIII) activity, protein C activity, protein S (PS) activity, prothrombin fragments 1+2, type 1 plasminogen activator inhibitor activity, tissue plasminogen activator antigen, plasminogen, activated protein C resistance, factors VII and VIII levels and D dimer. Student t-test, one way analysis of variance (ANOVA) and Fisher test were used for statistical analysis.<h4>Results</h4>Factor VII and factor VIII were always increased with respect to controls. Variance analysis showed a statistically significant reduction for anticoagulants (PS) and a rise for F1+2 and D dimer. With regard to fibrinolysis, there was an increase both for t-PA and PA1-1 during pregnancy. Moreover, the increased activity of factors of haemostasis was accompanied by an increase of activity and concentration of ATIII and acquired activated protein C resistance.<h4>Conclusions</h4>These findings suggest that normal pregnancy is associated with an hypercoagulable state, resulting into a moderate risk for thrombosis during the different trimesters of pregnancy. Also broad spectrum assays which measure a range of trombin/fibrin formation in serum have become an established mean to identify activation of blood coagulation and/or fibrinolysis. There is a considerable interest in the application of these assays to the diagnosis of other acquired hypercoagulable states; such as thrombophilia during pregnancy. From the viewpoint of coagulation/fibrinolysis changes, the follow-up of thrombophilia markers could be recommended when levels of coagulation parameters exceed the normal values during pregnancy.

<h4>Background</h4>Disulone (dapsone + iron oxalate) is a sulfone used in the treatment of numerous skin diseases. We report two cases of hepatosiderosis secondary to long-term administration of Disulone.<h4>Patients and methods</h4>Case n degrees 1. A 51-year-old man was treated with Disulone for a neutrophilic skin disease. After 17 years of treatment, elevated serum ferritin and free iron with hemolysis were found. Liver biopsy confirmed hepatosiderosis. A diagnosis of genetic hemochromatosis was ruled out by the absence of C282Y mutation of the HFE gene. Case n degrees 2. A 52-year-old man receiving Disulone for dermatitis herpetiformis for 25 years presented elevated serum ferritin and free iron with hemolysis. Hepatic iron overload was confirmed by liver biopsy. The absence of C282Y mutation (HFE gene) ruled out a diagnosis of genetic hemochromatosis.<h4>Discussion</h4>In our two cases, hepatosiderosis was noted after long-term administration of Disulone. This complication has been reported only rarely. In murine models, a relationship was found between prolonged administration of dapsone and hepatic iron overload as revealed by hemolysis. Although it is difficult to extrapolate this relationship to humans with any certainty, our patients had also chronic hemolysis and iron overload secondary to administration of Disulone. Moreover in France, dapsone is marketed in combination with iron oxalate, with the attendant risk of iron overload. These cases raise the question of the need for serum ferritin analysis during Disulone therapy.

Exposure to high doses of methamphetamine (METH), a major drug of abuse, may cause neuronal damage. Previous studies have implicated the role of peroxynitrite, produced by nitric oxide (NO) and reactive oxygen species, in dopaminergic neurotoxicity produced by METH in mice. The present article was undertaken to investigate if a neurotoxic regimen of METH is associated with changes in tissue levels of nitrate and nitrite, which are the stable products of NO. Administration of METH (5 mg/kg x 3) to Swiss Webster mice resulted in marked depletion of dopamine (DA) and DA transporter (DAT) binding sites but no change in 5-hydroxytryptamine (5-HT) and 5-HT transporter (5-HTT) binding sites in the striatum, amygdala, frontal cortex, and hippocampus, suggesting that METH causes selective neurotoxicity to DA nerve terminals. The concentration of nitrate in the striatum was increased by about two-fold after METH administration; however, no changes in nitrate concentration were detected in other brain regions that endured dopaminergic neurotoxicity. These findings suggest that (a) a neurotoxic regimen of METH produces selective increase in NO in the striatum, which may generate toxic species such as peroxynitrite, and (b) toxins other than NO-related derivatives may mediate dopaminergic neurotoxicity in the amygdala and frontal cortex.

Fine needle aspiration cytology is a widely practiced technique in the diagnosis of breast carcinoma and it is the only, diagnostic procedure performed before definitive treatment, at most institutions. While the histological grading of Duct Cell Carcinoma (DCC), NOS is done routinely in many centers worldwide the cytopathological grading of DCC, NOS is not commonly done. Grading of breast carcinoma, while the tumour is still in vivo, would be the most ideal and desirable situation, as it would be helpful in the selection of appropriate therapy for the patient. This study was undertaken with the aim to determine the grade of DCC, NOS by simple cytological scoring system as a method of predicting histological grade. We selected 80 cases of DCC, NOS which were initially diagnosed by Fine Needle Aspiration Cytology (FNAC) with subsequent histological confirmation. For cytological grading Papanicolou's stained smears and for histological grading Haematoxylin and Eosin stained sections were examined under light microscope. For histological grading Scraff Bloom Richardson's grading and for cytological grading Robinson's grading were followed. In both system grading were grade I, grade II and grade III of DCC, NOS. On cytological grading 39 (48.8%) cases were grade-I, 38 (47.5%) cases were grade-II and 3 (3.8%) cases were grade-III. On histological grading 33 (41.3%) cases were grade-I, 39 (48.8%) cases were grade-II and 8 (10%) cases were grade-III. In this study cytological grade correlated with histological grade in 69 (86.3%) cases. For grade-I tumour, sensitivity was 96.96%, efficiency was 94%. For grade-II tumour the sensitivity was 87.18% and efficiency was 77%. For grade-III tumour the sensitivity was 37.50% and efficiency was 23%. The overall accuracy was 96.3%. Significant association between histological and cytological grading of breast carcinoma, NOS was noted. Our observations in this study revealed that grading of duct cell carcinoma, NOS on FNAC material can be a useful in preoperative assessment of DCC, NOS prognosis and can be useful as an alternative to histological grading when neoadjuvant therapy is planned.

Changes in mitochondrial DNA have been reported in cancer cells. Since little information exists regarding mt DNA mutations in head and neck, the present study focused on ten head and neck cancer cell lines in the attempt to detect alterations in the ND4 gene sequence. DNA was extracted from 10 head and neck squamous cell carcinoma lines from 9 patients. MtDNA sequences were compared in normal and tumour cell line DNA. In ten head and neck squamous cell carcinoma cell lines, 8 somatic mutations and 5 polymorphisms of the mitochondrial gene for ND4 were found. All 5 polymorphisms were silent. Of the 8 somatic mutations, 3 altered the amino acid sequence suggesting a possible effect on enzyme function. The mitochondrial mutations and polymorphisms found demonstrated that these can serve as clonal markers for individual cell lines and demonstrate that the mitochondrial genome remains stable in the cell lines during in vitro culture.