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Transferrin receptor 1 (TfR) plays a critical role in cellular iron import for most higher organisms. Cell surface TfR binds to circulating iron-loaded transferrin (Fe-Tf) and transports it to acidic endosomes, where low pH promotes iron to dissociate from transferrin (Tf) in a TfR-assisted process. The iron-free form of Tf (apo-Tf) remains bound to TfR and is recycled to the cell surface, where the complex dissociates upon exposure to the slightly basic pH of the blood. Fe-Tf competes for binding to TfR with HFE, the protein mutated in the iron-overload disease hereditary hemochromatosis. We used a quantitative surface plasmon resonance assay to determine the binding affinities of an extensive set of site-directed TfR mutants to HFE and Fe-Tf at pH 7.4 and to apo-Tf at pH 6.3. These results confirm the previous finding that Fe-Tf and HFE compete for the receptor by binding to an overlapping site on the TfR helical domain. Spatially distant mutations in the TfR protease-like domain affect binding of Fe-Tf, but not iron-loaded Tf C-lobe, apo-Tf, or HFE, and mutations at the edge of the TfR helical domain affect binding of apo-Tf, but not Fe-Tf or HFE. The binding data presented here reveal the binding footprints on TfR for Fe-Tf and apo-Tf. These data support a model in which the Tf C-lobe contacts the TfR helical domain and the Tf N-lobe contacts the base of the TfR protease-like domain. The differential effects of some TfR mutations on binding to Fe-Tf and apo-Tf suggest differences in the contact points between TfR and the two forms of Tf that could be caused by pH-dependent conformational changes in Tf, TfR, or both. From these data, we propose a structure-based model for the mechanism of TfR-assisted iron release from Fe-Tf.

No abstract available.

Colorectal cancer is a multi-step process characterized by a sequence of genetic alterations in cell growth regulatory genes, such as the adenomatous polyposis coli, KRAS, p53 and DCC genes. In the present study mutation analysis was performed with SSCA/direct sequencing of the hot-spot regions in exons 11 and 15 for the BRAF gene and exons 1-2 for the KRAS gene in 130 primary colorectal cancer tumors and correlated with clinico-pathological and mutational data. We also performed mutation analysis of the corresponding conserved regions in the ARAF and RAF-1 genes. Mutations in the BRAF and KRAS genes were found in 11.5 and 40% of the tumors, respectively. One germline exonic and nine germline intronic genetic variants were found in the ARAF and RAF-1 genes. All of the BRAF mutations were located in the kinase domain of the conserved region 3 in exon 15 of the BRAF gene. One novel somatic mutation was also identified in the BRAF gene. The majority of the BRAF mutations were found in colon compared with rectal tumors (P = 0.014). In agreement with others, a statistically significant correlation between BRAF mutations and microsatellite instability could be found. A negative correlation was also evident between mutations in the BRAF and KRAS genes, which supports earlier studies where somatic mutations in these genes are mutually exclusive. Collectively, our results provide support for the idea that activation of the MAP kinase pathway, especially via BRAF and KRAS mutations, is of critical importance for the development of colorectal cancer.

<h4>Introduction</h4>In order to gain a better understanding of the molecular processes that underlie apoptosis and tissue regression in mammary gland, we undertook a large-scale analysis of transcriptional changes during the mouse mammary pregnancy cycle, with emphasis on the transition from lactation to involution.<h4>Method</h4>Affymetrix microarrays, representing 8618 genes, were used to compare mammary tissue from 12 time points (one virgin, three gestation, three lactation and five involution stages). Six animals were used for each time point. Common patterns of gene expression across all time points were identified and related to biological function.<h4>Results</h4>The majority of significantly induced genes in involution were also differentially regulated at earlier stages in the pregnancy cycle. This included a marked increase in inflammatory mediators during involution and at parturition, which correlated with leukaemia inhibitory factor-Stat3 (signal transducer and activator of signalling-3) signalling. Before involution, expected increases in cell proliferation, biosynthesis and metabolism-related genes were observed. During involution, the first 24 hours after weaning was characterized by a transient increase in expression of components of the death receptor pathways of apoptosis, inflammatory cytokines and acute phase response genes. After 24 hours, regulators of intrinsic apoptosis were induced in conjunction with markers of phagocyte activity, matrix proteases, suppressors of neutrophils and soluble components of specific and innate immunity.<h4>Conclusion</h4>We provide a resource of mouse mammary gene expression data for download or online analysis. Here we highlight the sequential induction of distinct apoptosis pathways in involution and the stimulation of immunomodulatory signals, which probably suppress the potentially damaging effects of a cellular inflammatory response while maintaining an appropriate antimicrobial and phagocytic environment.

Common genetic polymorphisms may explain a portion of the heritable risk for common diseases. Within candidate genes, the number of common polymorphisms is finite, but direct assay of all existing common polymorphism is inefficient, because genotypes at many of these sites are strongly correlated. Thus, it is not necessary to assay all common variants if the patterns of allelic association between common variants can be described. We have developed an algorithm to select the maximally informative set of common single-nucleotide polymorphisms (tagSNPs) to assay in candidate-gene association studies, such that all known common polymorphisms either are directly assayed or exceed a threshold level of association with a tagSNP. The algorithm is based on the r(2) linkage disequilibrium (LD) statistic, because r(2) is directly related to statistical power to detect disease associations with unassayed sites. We show that, at a relatively stringent r(2) threshold (r2>0.8), the LD-selected tagSNPs resolve >80% of all haplotypes across a set of 100 candidate genes, regardless of recombination, and tag specific haplotypes and clades of related haplotypes in nonrecombinant regions. Thus, if the patterns of common variation are described for a candidate gene, analysis of the tagSNP set can comprehensively interrogate for main effects from common functional variation. We demonstrate that, although common variation tends to be shared between populations, tagSNPs should be selected separately for populations with different ancestries.

Colorectal cancer is the third most common cause of cancer-related death in both men and women in the western hemisphere. According to the American Cancer Society, an estimated 105,500 new cases of colon cancer with 57,100 deaths will occur in the U.S. in 2003, accounting for about 10% of cancer deaths. Among the colon cancer patients, hereditary risk contributes approximately 20%. The main inherited colorectal cancers are the familial adenomatous polyposis (FAP) and the hereditary nonpolyposis colorectal cancers (HNPCC). The FAP and HNPCC are caused due to mutations in the adenomatous polyposis coli (APC) and DNA mismatch repair (MMR) genes. The focus of this review is to summarize the functions of APC and MMR gene products in the development of colorectal cancers.

Molecular dynamics studies have been performed for 3.5 ns on the ETS domain of Ets-1 transcription factor bound to the 14-bp DNA, d(AGTGCCGGAAATGT), comprising the core sequence of high-affinity (GGAA), ETS-GGAA. In like manner, molecular dynamics simulations have been carried out for 3.9 ns on the mutant low-affinity core sequence, GGAG (ETS-GGAG). Analyses of the DNA backbone of ETS-GGAG show conformational interconversions from BI to BII substates. Also, crank shaft motions are noticed at the mutated nucleotide base pair step after 1500 ps of dynamics. The corresponding nucleotide of ETS-GGAA is characteristic of a BI conformation and no crank shaft motions are observed. The single mutation of ETS-GGAA to ETS-GGAG also results in variations of helical parameters and solvent-accessible surface area around the major and minor grooves of the DNA. The presence of water contacts during the entire simulation proximal to the fourth base pair step of core DNA sequence is a characteristic feature of ETS-GGAA. Such waters are more mobile in ETS-GGAG at 100 ps and distant after 1500 ps. Anticorrelated motions between certain amino acids of Ets-1 protein are predominant in ETS-GGAA but less so or absent in the mutant. These motions are reflected in the flexibility of amino acid residues of the protein backbone. We consider that these conformational features and water contacts are involved in stabilizing the hydrogen bond interactions between helix-3 residues of Ets-1 and DNA during the transcription process.

Hereditary hemochromatosis (HH) is an autosomal recessive disease characterized by iron accumulation in several organs, followed by organ damage and failure. The C282Y mutation in the HFE gene explains 80-90% of all diagnosed cases of HH in populations of northwestern European ancestry. Targeted disruption of the mouse Hfe gene (or introduction of the murine mutation analogous to the C282Y human mutation) produces a murine model of HH. Another mutation in the HFE gene, H63D, is more prevalent than C282Y. However, the physiological consequences of the H63D mutation (as well as C282Y/H63D compound heterozygosity) on iron homeostasis are less well established. To evaluate the phenotypic consequences of the C282Y/H63D and H63D/H63D genotypes, we produced H67D (corresponding to H63D in humans) and C294Y (corresponding to C282Y in humans) knock-in mice. H67D homozygous mice, C294Y homozygous mice, and H67D/C294Y compound heterozygous mice each demonstrated hepatic iron loading. Even on a standard diet, by 10 weeks of age, hepatic iron levels in mice of these three genotypes were significantly higher than those of wild-type littermates. The relative severity of hepatic iron loading was C294Y/C294Y > C294Y/H67D > H67D/H67D. We conclude that the H67D allele, when homozygous or combined with a more consequential mutation like C294Y, leads to hepatic iron loading. These observations indicate that the H67D mutation leads to partial loss of Hfe function and can contribute to murine HH.

Hereditary hemochromatosis is a genetically heterogeneous disease of iron metabolism. The most common form of the disorder is an adult-onset form that has mainly been associated with the HFE pC282Y/pC282Y genotype. The phenotypic expression of this genotype is very heterogeneous and could be modulated by both environmental factors and modifier genes. The non-HFE hereditary hemochromatosis forms include a juvenile onset form associated with mutations in HAMP. From a cohort of 392 C282Y homozygous patients, we found 5 carriers of an additional HAMP mutation at the heterozygous state (pR59G, pG71D, or pR56X). We found that iron indices of these 5 patients were among the most elevated of the cohort. Moreover, we specified that the HAMP mutations were not detected in 300 control subjects. These results revealed that mutations in HAMP might increase the phenotypic expression of the pC282Y/pC282Y genotype. From a cohort of 31 patients with at least one chromosome lacking an HFE mutation, we further identified 4 males carrying a heterozygous HAMP mutation (pR59G or pG71D). Based on a digenic model of inheritance, these data suggest that the association of heterozygous mutations in the HFE and HAMP genes could lead, at least in some cases, to an adult-onset form of primary iron overload.

The murine tumor cell DnaJ-like protein 1 or MTJ1/ERdj1 is a membrane J-domain protein enriched in microsomal and nuclear fractions. We previously showed that its lumenal J-domain stimulates the ATPase activity of the molecular chaperone BiP/GRP78 (Chevalier, M., Rhee, H., Elguindi, E. C., and Blond, S. Y. (2000) J. Biol. Chem. 275, 19620-19627). MTJ1/ERdj1 also contains a large carboxyl-terminal cytosolic extension composed of two tryptophan-mediated repeats or SANT domains for which the function(s) is unknown. Here we describe the cloning of the human homologue HTJ1 and its interaction with alpha(1)-antichymotrypsin (ACT), a member of the serine proteinase inhibitor (serpin) family. The interaction was initially identified in a two-hybrid screening and further confirmed in vitro by dot blots, native electrophoresis, and fluorescence studies. The second SANT domain of HTJ1 (SANT2) was found to be sufficient for binding to ACT, both in yeast and in vitro. Single tryptophan-alanine substitutions at two strictly conserved residues significantly (Trp-497) or totally (Trp-520) abolished the interaction with ACT. SANT2 binds to human ACT with an intrinsic affinity equal to 0.5 nm. Preincubation of ACT with nearly stoichiometric concentrations of SANT2 wild-type but not SANT2: W520A results in an apparent loss of ACT inhibitory activity toward chymotrypsin. Kinetic analysis indicates that the formation of the covalent inhibitory complex ACT-chymotrypsin is significantly delayed in the presence of SANT2 with no change on the catalytic efficiency of the enzyme. This work demonstrates for the first time that the SANT2 domain of MTJ1/HTJ1/ERdj1 mediates stable and high affinity protein-protein interactions.

No abstract available.

Symptoms of Huntington's disease may be caused by a toxic insult triggered by the mutant human huntingtin (Htt) protein itself, by a maladaptive protective mechanism initiated in response to an insult, or by a combination of these. We observed a protection from N-methyl-d-aspartate (NMDA) receptor-induced excitotoxicity in striata of symptomatic N171-82Q mice, a new transgenic model of Huntington's disease. The goal of this study was to determine if NMDA receptor-mediated signalling pathways are altered in these mice. Multiple proteins of NMDA receptor and dopamine D1 receptor pathways are being regulated in ways predictive of the protection we observe. Although examining NMDA receptor subunit proteins showed no change in NR1, NR2A, or NR2B in the striata of the symptomatic mice, we observed a decrease in phosphorylation of NR1 at Ser897, previously reported to decrease NMDA receptor current. The dopamine D1 receptor, responsible for protein kinase A activation and subsequent phosphorylation of Ser897 of NR1, also showed an age-related decrease. Other proteins regulated in this disease were associated with PSD-95-like scaffolding proteins of the NMDA receptor. Specifically, we observed a decrease in membrane-associated neuronal nitric oxide synthase (nNOS), a decrease in PSD-95-like proteins, which link nNOS to the NMDA receptor complex, and a decrease in citron, a protein associated with dendritic spine formation. From these data, we conclude that the N171-82Q mice seem to be regulating, in a protective direction, many of the known effector pathways of NMDA receptor-induced excitotoxicity. These regulations, although seemingly effective in decreasing neuronal death, may in fact be causing some of the symptoms associated with the disease.

Hereditary hemochromatosis (HH) type I is a disorder of iron metabolism caused by a mutation in the HFE gene. Whereas the prevalence of the mutation is very high, its penetrance seems very low. The goal of our study was to determine whether hepcidin, a recently identified iron-regulatory peptide, could be a genetic modifier contributing to the HH phenotype. In mice, deficiency of either HFE (Hfe(-/-)) or hepcidin (Usf2(-/-)) is associated with the same pattern of iron overload observed in patients with HH. We intercrossed Hfe(-/-) and Usf2(+/-) mice and asked whether hepcidin deficiency increased the iron burden in Hfe(-/-) mice. Our results showed that, indeed, liver iron accumulation was greater in the Hfe(-/-)Usf2(+/-) mice than in mice lacking Hfe alone. This result, in agreement with recent findings in humans, provides a genetic explanation for some variability of the HH phenotype.

Genetic causes of hereditary hemochromatosis (HH) include mutations in the HFE gene, coding for a beta2-microglobulin (beta2m)-associated major histocompatibility complex class I-like protein. However, iron accumulation in patients with HH can be highly variable. Previously, analysis of beta2mRag1(-/-) double-deficient mice, lacking all beta2m-dependent molecules and lymphocytes, demonstrated increased iron accumulation in the pancreas and heart compared with beta2m single knock-out mice. To evaluate whether the observed phenotype in beta2mRag1(-/-) mice was due solely to the absence of Hfe or to other beta2m-dependent molecules, we generated HfeRag1(-/-) double-deficient mice. Our studies revealed that introduction of Rag1 deficiency in Hfe knock-out mice leads to heightened iron overload, mainly in the liver, whereas the heart and pancreas are relatively spared compared with beta2mRag1(-/-) mice. These results suggest that other beta2m-interacting protein(s) may be involved in iron regulation and that in the absence of functional Hfe molecules lymphocyte numbers may influence iron overload severity.

The serotonin transporter (5-HTT) regulates serotonergic neurotransmission via clearance of extracellular serotonin. Abnormalities in 5-HTT expression or function are found in mood and anxiety disorders, and the 5-HTT is a major target for antidepressants and anxiolytics. The 5-HTT is further implicated in the pathophysiology of these disorders by evidence that genetic variation in the promoter region of the HTT (SLC6A4) is associated with individual differences in anxiety and neural responses to fear. To further evaluate the role of the 5-HTT in anxiety, we employed a mouse model in which the 5-HTT gene (htt) was constitutively inactivated. 5-HTT -/- mice were characterized for anxiety-related behaviors using a battery of tests (elevated plus maze, light<-->dark exploration test, emergence test, and open field test). Male and female 5-HTT -/- mice showed robust phenotypic abnormalities as compared to +/+ littermates, suggestive of increased anxiety-like behavior and inhibited exploratory locomotion. The selective 5-HT(1A) receptor antagonist, WAY 100635 (0.05-0.3 mg/kg), produced a significant anxiolytic-like effect in the elevated plus maze in 5-HTT -/- mice, but not +/+ controls. The present findings demonstrate abnormal behavioral phenotypes in 5-HTT null mutant mice in tests for anxiety-like and exploratory behavior, and suggest a role for the 5-HT(1A) receptor in mediating these abnormalities. 5-HTT null mutant mice provide a model to investigate the role of the 5-HTT in mood and anxiety disorders.

Deficiency of C1 Inhibitor leads to unopposed activation of complement, with localized, unpredictable, and sometimes life-threatening attacks of angioedema. Treatment with plasma-derived C1 Inhibitor rapidly aborts attacks, and may be lifesaving, but is expensive, requires use of a pooled blood product, may need to be repeated and may not be effective in autoantibody mediated angioedema. The antifibrinolytic agents aprotinin, tranexamic acid, and epsilon-aminocaproic acid are useful for prophylaxis and treatment of angioedema, likely by inhibiting plasmin. Specific drugs to replace the deficient C1 Inh have not been reported. The kallikrein inhibitor DX-88 (Dyax) has received orphan drug status in Europe and is undergoing clinical trial in Europe and the USA.

Assembly of compact mitotic chromosomes and resolution of sister chromatids are two essential processes for the correct segregation of the genome during mitosis. Condensin, a five-subunit protein complex, is thought to be required for chromosome condensation. However, recent genetic analysis suggests that condensin is only essential to resolve sister chromatids. To study further the function of condensin we have depleted DmSMC4, a subunit of the complex, from Drosophila S2 cells by dsRNA-mediated interference. Cells lacking DmSMC4 assemble short mitotic chromosomes with unresolved sister chromatids where Barren, a non-SMC subunit of the complex is unable to localise. Topoisomerase II, however, binds mitotic chromatin after depletion of DmSMC4 but it is no longer confined to a central axial structure and becomes diffusely distributed all over the chromatin. Furthermore, cell extracts from DmSMC4 dsRNA-treated cells show significantly reduced topoisomerase II-dependent DNA decatenation activity in vitro. Nevertheless, DmSMC4-depleted chromosomes have centromeres and kinetochores that are able to segregate, although sister chromatid arms form extensive chromatin bridges during anaphase. These chromatin bridges do not result from inappropriate maintenance of sister chromatid cohesion by DRAD21, a subunit of the cohesin complex. Moreover, depletion of DmSMC4 prevents premature sister chromatid separation, caused by removal of DRAD21, allowing cells to exit mitosis with chromatin bridges. Our results suggest that condensin is required so that an axial chromatid structure can be organised where topoisomerase II can effectively promote sister chromatid resolution.

We have previously shown that Nkx3.2, a transcriptional repressor that is expressed in the sclerotome and developing cartilage, can activate the chondrocyte differentiation program in somitic mesoderm in a bone morphogenetic protein (BMP)-dependent manner. In this work, we elucidate how BMP signaling modulates the transcriptional repressor activity of Nkx3.2. We have found that Nkx3.2 forms a complex, in vivo, with histone deacetylase 1 (HDAC1) and Smad1 and -4 in a BMP-dependent manner. The homeodomain and NK domain of Nkx3.2 support the interaction of this transcription factor with HDAC1 and Smad1, respectively, and both of these domains are required for the transcriptional repressor activity of Nkx3.2. Furthermore, the recruitment of an HDAC/Sin3A complex to Nkx3.2 requires that Nkx3.2 interact with Smad1 and -4. Indeed, Nkx3.2 both fails to associate with the HDAC/Sin3A complex and represses target gene transcription in a cell line lacking Smad4, but it performs these functions if exogenous Smad4 is added to these cells. While prior work has indicated that BMP-dependent Smads can support transcriptional activation, our findings indicate that BMP-dependent Smads can also potentiate transcriptional repression, depending upon the identity of the Smad-interacting transcription factor.

The serotonin transporter (5-HTT) gene is a firm candidate to explain eating disorders. In this association study, two different polymorphisms were analysed: a variable number of tandem repeat (VNTR) polymorphism in intron 2 and a deletion/insertion polymorphism (5-HTTLPR) in the promoter region. The hypothesis that these gene polymorphisms may be a susceptibility factor in bulimia nervosa (BN) was explored in a female population of 102 purgative bulimics. BN patients who have suffered preceding anorexia nervosa (AN) episodes formed the so-called previous AN bulimic patient group. In our sample of normal-eater controls and purging type bulimics, regardless of whether or not the BN patients had suffered prior AN episodes, no differences were found considering the frequencies of genotypes, alleles or haplotypes of both polymorphic regions of the 5-HTT gene.

<h4>Context</h4>Bioelectronic sensors, which combine microchip and biological components, are an emerging technology in clinical diagnostic testing. An electronic detection platform using DNA biochip technology (eSensor) is under development for molecular diagnostic applications. Owing to the novelty of these devices, demonstrations of their successful use in practical diagnostic applications are limited.<h4>Objective</h4>To assess the performance of the eSensor bioelectronic method in the validation of 6 Epstein-Barr virus-transformed blood lymphocyte cell lines with clinically important mutations for use as sources of genetic material for positive controls in clinical molecular genetic testing. Two cell lines carry mutations in the CFTR gene (cystic fibrosis), and 4 carry mutations in the HFE gene (hereditary hemochromatosis).<h4>Design</h4>Samples from each cell line were sent for genotype determination to 6 different molecular genetic testing facilities, including the laboratory developing the DNA biochips. In addition to the bioelectronic method, at least 3 different molecular diagnostic methods were used in the analysis of each cell line. Detailed data were collected from the DNA biochip output, and the genetic results were compared with those obtained using the more established methods.<h4>Results</h4>We report the successful use of 2 applications of the bioelectronic platform, one for detection of CFTR mutations and the other for detection of HFE mutations. In all cases, the results obtained with the DNA biochip were in concordance with those reported for the other methods. Electronic signal output from the DNA biochips clearly differentiated between mutated and wild-type alleles. This is the first report of the use of the cystic fibrosis detection platform.<h4>Conclusions</h4>Bioelectronic sensors for the detection of disease-causing mutations performed well when used in a "real-life" situation, in this case, a validation study of positive control blood lymphocyte cell lines with mutations of public health importance. This study illustrates the practical potential of emerging bioelectronic DNA detection technologies for use in current molecular diagnostic applications.

Teratomas of the testis in post-pubertal patients are histologically diverse tumors that often coexist with other types of germ cell tumors. Using laser capture microdissection and loss of heterozygosity analysis, we investigated the clonality of mature teratoma and its relationship to other components of malignant mixed germ cell tumors to gain potential insight into the histogenetic relationship of teratoma with other germ cell tumor components. All 16 patients had mature teratoma as one component of their mixed germ cell tumors. The other histological subtypes included immature teratoma, seminoma, embryonal carcinoma, yolk sac tumor, and choriocarcinoma. Laser-assisted microdissection was performed on the formalin-fixed, paraffin-embedded tissue. Polymerase chain reaction was used to amplify genomic DNA at specific loci on chromosome 1p36.2 (D1S508), 2q22-32 (D2S156), 9p21-22 (D9S162), 11p13 (D11S903), 12q22-23 (D12S1051), and 18q21 (D18S46). Fourteen of 16 (88%) cases showed allelic loss in one or more components of the mixed germ cell tumors. Fourteen of 16 mature teratomas showed allelic loss in at least one of six microsatellite polymorphic markers analyzed. The frequency of allelic loss in mature teratoma was 50% (7 of 14) with D1S508, 33% (5 of 15) with D2S156, 58% (7 of 12) with D9S162, 43% (6 of 14) with D11S903, 20% (3 of 15) with D12S1051, and 33% (5 of 15) with D18S46. Completely concordant allelic loss patterns between mature teratoma and all of the other germ cell tumor components were seen in 10 of 14 tumors in which mature teratoma showed loss of heterozygosity. Our data support the common clonal origin of mature teratoma with other components of malignant mixed germ cell tumors of the testis.

<h4>Background</h4>Hereditary hemochromatosis is a recessive disorder characterized by iron accumulation in parenchymal cells, followed by organ damage and failure. The disorder is mainly attributable to the C282Y and H63D mutations in the HFE gene, but additional mutations in the HFE, transferrin receptor 2 (TfR2), and hepcidin genes have been reported. The copresence of mutations in different genes may explain the phenotypic heterogeneity of the disorder and its variable penetrance.<h4>Methods</h4>We used denaturing HPLC (DHPLC) for rapid DNA scanning of the HFE (exons 2, 3, and 4), hepcidin, and TfR2 (exons 2, 4 and 6) genes in a cohort of 657 individuals with altered indicators of iron status.<h4>Results</h4>DHPLC identification of C282Y and H63D HFE alleles was in perfect agreement with the restriction endonuclease assay. Fourteen DNA samples were heterozygous for the HFE S65C mutation. In addition, we found novel mutations: two in HFE (R66C in exon 2 and R224G in exon 4), one in the hepcidin gene (G71D), and one in TfR2 (V22I), plus several intronic or silent substitutions. Six of the seven individuals with hepcidin or TfR2 coding mutations carried also HFE C282Y or S65C mutations.<h4>Conclusion</h4>DHPLC is an efficient method for mutational screening for the genes involved in hereditary hemochromatosis and for the study of their copresence.

<h4>Background</h4>Survival at the intermediate stage of colorectal cancer (CRC) is less predictable than in the early and advanced stages. Several genetic markers possibly involved in growth and progression of CRC can be used for prognosis.<h4>Aims</h4>This study investigated the proportion of allelic loss (loss of heterozygosity (LOH)) at the BRCA1 locus in sporadic CRC and its value in patient prognosis.<h4>Patients and methods</h4>A total of 314 patients were investigated for LOH at the BRCA1 locus using polymerase chain reaction by means of three intragenic polymorphic microsatellite markers. Allelic losses were compared with clinicopathological characteristics of patients, recurrence rate, disease free survival (DFS), and overall survival.<h4>Results</h4>Twenty six patients were excluded because of microsatellite instability. Of the remaining 288 cases, 244 (84.7%) were informative, with 97 (39.8%) patients bearing BRCA1 LOH. Recurrence rate was higher in patients with LOH (p=0.0003), and DFS was 73.3% (SEM 5.7) at five years in patients without LOH, and 49.2% (7.1) in cases with positive allelic loss (p=0.0004). Retention of alleles at the BRCA1 locus was associated with a favourable DFS in stages I and II (p<0.05). The presence of LOH was also significantly associated with short overall survival (p=0.02). Multivariate analysis in the complete series showed that stage (p=0.006) and lymph node metastases (> or =4 nodes, p=0.0001; 1-3 nodes, p=0.038) were independent prognostic factors. However, multivariate study by stages revealed that BRCA1 LOH was an independent prognostic factor in stages I and II (p=0.001).<h4>Conclusions</h4>BRCA1 LOH is a molecular alteration present in CRC, with unfavourable repercussions for overall survival, that could be considered as an outstanding independent prognostic factor in stages I and II.

Interactions between APC and T lymphocytes have been implicated as a major factor contributing to inflammatory bowel disease. To test whether OX40/OX40L interaction plays a role in chronic intestinal inflammation, we induced chronic colitis using dextran sulfate sodium and treated the mice with a murine fusion protein (OX40-IgG). Treatment resulted in a dose-dependent and significant reduction of intestinal inflammation (46%) as measured by a histologic score. IL-10 and IL-5 production from mesenteric lymph node cells increased 20-fold and 18-fold, respectively. In colonic tissue, IL-10 mRNA levels increased and the expression of T-bet was decreased to 30%. IL-10 neutralization partly inhibited the beneficial effects of OX40-IgG treatment. Surprisingly, despite the reduction of inflammation we found the number and size of colonic lymphoid follicles increased, with an accumulation of CD4(+) cells in the mantle area. In contrast, the number of CD4(+) cells infiltrating the mucosa was significantly reduced, as was their CXCR5 expression (24-fold). We conclude that OX40/OX40L interaction contributes to the perpetuation of chronic colitis partly by suppressing IL-10 production. Furthermore, our data suggest that the OX40/OX40L-induced CXCR5 expression on CD4(+) cells may be important for the inflammatory process by allowing migration to the germinal center for further differentiation of CD4(+) cells before they infiltrate the chronically inflamed mucosa.

Three polymorphic gene mutations in the human hemochromatosis (HFE) gene (C282Y, H63D, S65C) are associated with non-transfusion-related iron overload in Caucasians. More recently, these mutations have also been identified in African-Americans. However, the prevalence of HFE gene mutations in African-Americans with sickle cell disease (SCD) has not been described. The presence of these mutations in this population is particularly important, because patients with SCD may be placed on chronic red cell transfusion therapy and are thus at further risk for iron overload. Thus, we attempted to establish the gene mutation prevalence in African-Americans with SCD, to compare this frequency with published gene frequencies in African-Americans, and to evaluate their significance with regard to transfusion-related iron overload. Eighty-nine African-American patients with SCD, all of whom were receiving chronic red cell transfusion therapy, were screened by DNA analysis for the three HFE gene mutations. Two patients were heterozygous for the C282Y HFE mutation (2.3%), six were heterozygous for the H63D mutation (6.8%), none carried the S65C mutation (0.0%), and no homozygous or compound heterozygous subjects were identified. The prevalence of C282Y and H63D in the SCD population was similar to that observed in the general African-American population. In addition, there was no increased mutation prevalence when comparing those SCD patients on chronic transfusion therapy who had ferritin levels greater than 2,500 ng/mL and those less than 2,500 ng/mL. This study represents the first identification of the known HFE gene mutations by DNA analysis in the SCD population. We conclude that the presence of recognized HFE coding region mutations do not seem to have an impact on the degree of iron overload in patients with SCD receiving chronic transfusion therapy.

No abstract available.

N-Methyl-D-aspartate receptor (NMDAR)-mediated excitotoxicity has been proposed to play a role in Huntington disease (HD), caused by expansion of a polyglutamine tract in the protein huntingtin. HD is characterized by selective neurodegeneration most severely affecting striatal medium-sized spiny projection neurons (MSNs), where expression of the NMDAR subunit NR2B is increased relative to other NR2 subunits. Here, we review our data that NR2B-type NMDAR currents are selectively potentiated by mutant huntingtin in transfected non-neuronal cells and acutely dissociated striatal MSNs from the YAC72 transgenic mouse model of HD. As well, we report increased striatal MSN NMDAR-mediated synaptic currents in corticostriatal slice recordings from YAC72 compared with wild-type mice. This effect was associated with a larger NMDAR- to AMPAR-mediated current ratio, suggesting specific potentiation of postsynaptic NMDARs. Enhanced NMDAR current likely involves increased surface receptor numbers or activity, since we observed no differences between genotypes in striatal NR2B expression. Potentiation of NR2B-containing NMDAR current in striatal MSNs expressing mutant huntingtin may help explain the exquisite vulnerability of these neurons to degeneration in HD.

5-hydroxytryptamine (5-HT or serotonin) is a charged molecule and must be transported across biological membranes. Enzymes that catabolize 5-HT are all intracellular; therefore, 5-HT inactivation requires a high affinity transporter, known as serotonin transporter (SERT or 5-HTT). In the central and enteric nervous systems, SERT is located in serotonergic neurons; however, these neurons are not present in the gastrointestinal mucosa, where 5-HT initiates peristaltic and secretory reflexes. Instead, SERT is expressed by enterocytes. The severity of gastrointestinal effects caused by drugs that inhibit SERT, such as tricyclic antidepressants, selective serotonin reuptake inhibitors and cocaine, does not usually prevent their therapeutic or recreational use because backup transporters and alterations in receptor gene expression allow the gut to adapt, albeit imperfectly, to their toxicity.

The conformation of mononucleosome complexes reconstituted with recombinant core histones on a 614-basepair-long DNA fragment containing the Xenopus borealis 5S rRNA nucleosome positioning sequence was studied by scanning/atomic force microscopy in the absence or presence of linker histone H1. Imaging without prior fixation was conducted with air-dried samples and with mononucleosomes that were injected directly into the scanning force microscopy fluid cell and visualized in buffer. From a quantitative analysis of approximately 1,700 complexes, the following results were obtained: i), In the absence of H1, a preferred location of the nucleosome at the X. borealis 5S rRNA sequence in the center of the DNA was detected. From the distribution of nucleosome positions, an energy difference of binding to the 5S rRNA sequence of DeltaDeltaG approximately 3 kcal mol(-1) as compared to a random sequence was estimated. Upon addition of H1, a significantly reduced preference of nucleosome binding to this sequence was observed. ii), The measured entry-exit angles of the DNA at the nucleosome in the absence of H1 showed two maxima at 81 +/- 29 degrees and 136 +/- 18 degrees (air-dried samples), and 78 +/- 25 degrees and 137 +/- 25 degrees (samples imaged in buffer solution). In the presence of H1, the species with the smaller entry-exit angle was stabilized, yielding average values of 88 +/- 34 degrees for complexes in air and 85 +/- 10 degrees in buffer solution. iii), The apparent contour length of the nucleosome complexes was shortened by 34 +/- 13 nm as compared to the free DNA due to wrapping of the DNA around the histone octamer complex. Considering an 11 nm diameter of the nucleosome core complex, this corresponds to a total of 145 +/- 34 basepairs that are wound around the nucleosome.

<h4>Background</h4>Primary adenocarcinomas of the small intestine are rare, and the genetic mechanisms involved in their carcinogenesis remain unclear.<h4>Aim</h4>To examine the expression of candidate proteins in small intestinal adenocarcinomas by immunohistochemistry performed on tissue microarrays (TMAs).<h4>Methods</h4>Twenty seven primary sporadic small intestinal adenocarcinomas were analysed. The TMA technique was validated by comparing immunohistochemical labelling of hMLH1 and hMSH2 on TMAs and the tissue sections they derived from. The expression of Smad4, hMSH6, beta catenin, and p53 was investigated and results compared with those obtained in 14 malignant ampullary tumours.<h4>Results</h4>TMA technology with threefold redundancy adequately represented the immunohistochemical pattern of small intestinal adenocarcinomas. Loss of hMLH1 expression, but not hMSH2 or hMSH6, was seen in two of 27 small intestinal adenocarcinomas. All ampullary tumours showed nuclear staining for hMSH2 and hMSH6. One case showed lack of immunostaining for hMLH1. Smad4 expression was absent in five small intestinal adenocarcinomas and two ampullary tumours. Overexpression of p53 was detected in the nuclei of 14 of the 27 small intestinal adenocarcinomas, and five of the 14 ampullary tumours. Nuclear or cytoplasmic expression of beta catenin was present in all specimens.<h4>Conclusion</h4>Inactivation of the SMAD4/DPC4 gene seems to be involved in small intestinal adenocarcinoma tumorigenesis. Overexpression of p53 and abnormal expression of beta catenin are two common events, unlike the loss of expression of the DNA mismatch repair proteins (hMLH1, hMSH2, and hMSH6). The carcinogenetic process appears to be similar in small intestinal adenocarcinomas and malignant ampullary tumours.

Siah proteins function as E3 ubiquitin ligase enzymes to target the degradation of diverse protein substrates. To characterize the physiological roles of Siah2, we have generated and analyzed Siah2 mutant mice. In contrast to Siah1a knockout mice, which are growth retarded and exhibit defects in spermatogenesis, Siah2 mutant mice are fertile and largely phenotypically normal. While previous studies implicate Siah2 in the regulation of TRAF2, Vav1, OBF-1, and DCC, we find that a variety of responses mediated by these proteins are unaffected by loss of Siah2. However, we have identified an expansion of myeloid progenitor cells in the bone marrow of Siah2 mutant mice. Consistent with this, we show that Siah2 mutant bone marrow produces more osteoclasts in vitro than wild-type bone marrow. The observation that combined Siah2 and Siah1a mutation causes embryonic and neonatal lethality demonstrates that the highly homologous Siah proteins have partially overlapping functions in vivo.

Serotonin (5-hydroxytryptamine; 5-HT) is a potent modulator of the physiology and behavior involved in generating appropriate responses to environmental cues such as danger or threat. Furthermore, genetic variation in 5-HT subsystem genes can impact upon several dimensions of emotional behavior including neuroticism and psychopathology, but especially anxiety traits. Recently, functional neuroimaging has provided a dramatic illustration of how a promoter polymorphism in the human 5-HT transporter (5-HTT) gene, which has been weakly related to these behaviors, is strongly related to the engagement of neural systems, namely the amygdala, subserving emotional processes. In this commentary, we discuss how functional neuroimaging can be used to characterize the effects of polymorphisms in 5-HT subsystem genes on the response of neural circuits underlying the generation and regulation of mood and temperament as well as susceptibility to affective illness. We argue that in time, such knowledge will allow us to not only transcend phenomenological diagnosis and represent mechanisms of disease, but also identify at-risk individuals and biological pathways for the development of new treatments.

Current evidence indicates that virtually all neuropsychiatric disorders, like many other common medical disorders, are genetically complex, with combined influences from multiple interacting genes, as well as from the environment. However, additive or epistatic gene interactions have proved quite difficult to detect and evaluate in human studies. Mouse phenotypes, including behaviors and drug responses, can provide relevant models for human disorders. Studies of gene-gene interactions in mice could thus help efforts to understand the molecular genetic bases of complex human disorders. The serotonin transporter (SERT, 5-HTT, SLC6A4) provides a relevant model for studying such interactions for several reasons: human variants in SERT have been associated with several neuropsychiatric and other medical disorders and quantitative traits; SERT blockers are effective treatments for a number of neuropsychiatric disorders; there is a good initial understanding of the phenotypic features of heterozygous and homozygous SERT knockout mice; and there is an expanding understanding of the interactions between variations in SERT expression and variations in the expression of a number of other genes of interest for neuropsychiatry and neuropharmacology. This paper provides examples of experimentally-obtained interactions between quantitative variations in SERT gene expression and variations in the expression of five other mouse genes: DAT, NET, MAOA, 5-HT(1B) and BDNF. In humans, all six of these genes possess polymorphisms that have been independently investigated as candidates for neuropsychiatric and other disorders in a total of > 500 reports. In the experimental studies in mice reviewed here, gene-gene interactions resulted in either synergistic, antagonistic (including 'rescue' or 'complementation') or more complex, quantitative alterations. These were identified in comparisons of the behavioral, physiological and neurochemical phenotypes of wildtype mice vs. mice with single allele or single gene targeted disruptions and mice with partial or complete disruptions of multiple genes. Several of the descriptive phenotypes could be best understood on the basis of intermediate, quantitative alterations such as brain serotonin differences. We discuss the ways in which these interactions could provide models for studies of gene-gene interactions in complex human neuropsychiatric and other disorders to which SERT may contribute, including developmental disorders, obesity, polysubstance abuse and others.

Serotonin transporter (5-HTT) null mutant mice provide a model system to study the role genetic variation in the 5-HTT plays in the regulation of emotion. Anxiety-like behaviors were assessed in 5-HTT null mutants with the mutation placed on either a B6 congenic or a 129S6 congenic background. Replicating previous findings, B6 congenic 5-HTT null mutants exhibited increased anxiety-like behavior and reduced exploratory locomotion on the light <--> dark exploration and elevated plus-maze tests. In contrast, 129S6 congenic 5-HTT null mutant mice showed no phenotypic abnormalities on either test. 5-HTT null mutants on the 129S6 background showed reduced 5-HT(1A) receptor binding (as measured by quantitative autoradiography) and reduced 5-HT(1A) receptor function (as measured by 8-OH-DPAT-induced hypothermia). These data confirm that the 5-HTT null mutation produced alterations in brain 5-HT function in mice on the 129S6 background, thereby discounting the possibility that the absence of an abnormal anxiety-like phenotype in these mice was due to a suppression of the mutation by 129 modifier genes. Anxiety-like behaviors in the light <--> dark exploration and elevated plus-maze tests were significantly higher in 129S6 congenic +/+ mice as compared to B6 congenic +/+ mice. This suggests that high baseline anxiety-like behavior in the 129S6 strain might have precluded detection of the anxiety-like effects of the 5-HTT null mutation on this background. Present findings provide further evidence linking genetic variation in the 5-HTT to abnormalities in mood and anxiety. Furthermore, these data highlight the utility of conducting behavioral phenotyping of mutant mice on multiple genetic backgrounds.

There is evidence that HMGB proteins facilitate, while linker histones inhibit chromatin remodelling, respectively. We have examined the effects of HMG-D and histone H1/H5 on accessibility of nucleosomal DNA. Using the 601.2 nucleosome positioning sequence designed by Widom and colleagues we assembled nucleosomes in vitro and probed DNA accessibility with restriction enzymes in the presence or absence of HMG-D and histone H1/H5. For HMG-D our results show increased digestion at two spatially adjacent sites, the dyad and one terminus of nucleosomal DNA. Elsewhere varying degrees of protection from digestion were observed. The C-terminal acidic tail of HMG-D is essential for this pattern of accessibility. Neither the HMG domain by itself nor in combination with the adjacent basic region is sufficient. Histone H1/H5 binding produces two sites of increased digestion on opposite faces of the nucleosome and decreased digestion at all other sites. Our results provide the first evidence of local changes in the accessibility of nucleosomal DNA upon separate interaction with two linker binding proteins.

Hho1p is assumed to serve as a linker histone in Saccharomyces cerevisiae and, notably, it possesses two putative globular domains, designated HD1 (residues 41-118) and HD2 (residues 171-252), that are homologous to histone H5 from chicken erythrocytes. We have determined the three-dimensional structure of globular domain HD1 with high precision by heteronuclear magnetic resonance spectroscopy. The structure had a winged helix-turn-helix motif composed of an alphabetaalphaalphabetabeta fold and closely resembled the structure of the globular domain of histone H5. Interestingly, the second globular domain, HD2, in Hho1p was unstructured under physiological conditions. Gel mobility assay demonstrated that Hho1p preferentially binds to supercoiled DNA over linearized DNA. Furthermore, NMR analysis of the complex of a deletion mutant protein (residues 1-118) of Hho1p with a linear DNA duplex revealed that four regions within the globular domain HD1 are involved in the DNA binding. The above results suggested that Hho1p possesses properties similar to those of linker histones in higher eukaryotes in terms of the structure and binding preference towards supercoiled DNA.

Feedback inhibition is a fundamental principle in signal transduction allowing rapid adaptation to different stimuli. In mammalian cells, the major feedback inhibitor for G-protein-coupled receptors (GPCR) is G-protein-coupled receptor kinase 2 (GRK-2), which phosphorylates activated receptors, uncouples them from G proteins and initiates their internalization. The functions of GRK-2 are indispensable and need to be tightly controlled. Dysregulation promotes disorders such as hypertension or heart failure. In our search for a control mechanism for this vital kinase, here we show that the Raf kinase inhibitor protein (RKIP) is a physiological inhibitor of GRK-2. After stimulation of GPCR, RKIP dissociates from its known target, Raf-1 (refs 6-8), to associate with GRK-2 and block its activity. This switch is triggered by protein kinase C (PKC)-dependent phosphorylation of the RKIP on serine 153. The data delineate a new principle in signal transduction: by activating PKC, the incoming receptor signal is enhanced both by removing an inhibitor from Raf-1 and by blocking receptor internalization. A physiological role for this mechanism is shown in cardiomyocytes in which the downregulation of RKIP restrains beta-adrenergic signalling and contractile activity.

Low-copy repeats, or segmental duplications, are highly dynamic regions in the genome. The low-copy repeats on chromosome 22q11.2 (LCR22) are a complex mosaic of genes and pseudogenes formed by duplication processes; they mediate chromosome rearrangements associated with velo-cardio-facial syndrome/DiGeorge syndrome, der(22) syndrome, and cat-eye syndrome. The ability to trace the substrates and products of recombination events provides a unique opportunity to identify the mechanisms responsible for shaping LCR22s. We examined the genomic sequence of known LCR22 genes and their duplicated derivatives. We found Alu (SINE) elements at the breakpoints in the substrates and at the junctions in the truncated products of recombination for USP18, GGT, and GGTLA, consistent with Alu-mediated unequal crossing-over events. In addition, we were able to trace a likely interchromosomal Alu-mediated fusion between IGSF3 on 1p13.1 and GGT on 22q11.2. Breakpoints occurred inside Alu elements as well as in the 5' or 3' ends of them. A possible stimulus for the 5' or 3' terminal rearrangements may be the high sequence similarities between different Alu elements, combined with a potential recombinogenic role of retrotransposon target-site duplications flanking the Alu element, containing potentially kinkable DNA sites. Such sites may represent focal points for recombination. Thus, genome shuffling by Alu-mediated rearrangements has contributed to genome architecture during primate evolution.

By altering chromatin structure, histone acetyltransferases (HATs) act as transcriptional regulators. We observed in a model of primary neurons that histone acetylation levels decreased at the onset of apoptosis. The CREB-binding protein (CBP) is a HAT of particular interest because it also acts as a co-activator controlling, among others, CREB-dependent transcriptional activity. It has been demonstrated that CREB exerts neuroprotective functions, but the fate of CBP during neuronal apoptosis remained unclear till now. This work provided evidence that CBP is specifically targeted by caspases and calpains at the onset of neuronal apoptosis, and CBP was futher identified as a new caspase-6 substrate. This ultimately impinged on the CBP/p300 HAT activity that decreased with time during apoptosis entry, whereas total cellular HAT activity remained unchanged. Interestingly, CBP loss and histone deacetylation were observed in two different pathological contexts: amyloid precursor protein-dependent signaling and amyotrophic lateral sclerosis model mice, indicating that these modifications are likely to contribute to neurodegenerative diseases. In terms of function, we demonstrated that fine-tuning of CBP HAT activity is necessary to ensure neuroprotection.

Action potentials (APs) are the principal physiological stimuli for neurotransmitter secretion in neurons. Most studies on stimulus-secretion coupling have been performed under voltage clamp using artificial electrical stimuli. To investigate the modulatory effects of AP codes on neural secretion, we introduce a capacitance method to study AP-induced secretion in single cells. The action potential pattern was defined by a four-parameter "code function:" F(n, m, f, d). With this method, cell secretion evoked by stimulation with an AP code was quantified in real time by membrane capacitance (Cm) in adrenal chromaffin cells. We found, in addition to AP frequency (f), for a given number of APs, another parameter of the AP code, the number of AP bursts (m) in which the set of APs occurs, can effectively modulate cell secretion. Possible mechanisms of the m effect are depletion of the readily releasable pool and inactivation of Ca2+ channels during a burst of APs. The physiological m effect may play a key role in AP-mediated neural information transfer within a single neuron and among the elements of a neural network.

CatSper family proteins are putative ion channels expressed exclusively in membranes of the sperm flagellum and required for male fertility. Here, we show that mouse CatSper1 is essential for depolarization-evoked Ca2+ entry and for hyperactivated movement, a key flagellar function. CatSper1 is not needed for other developmental landmarks, including regional distributions of CaV1.2, CaV2.2, and CaV2.3 ion channel proteins, the cAMP-mediated activation of motility by HCO3-, and the protein phosphorylation cascade of sperm capacitation. We propose that CatSper1 functions as a voltage-gated Ca2+ channel that controls Ca2+ entry to mediate the hyperactivated motility needed late in the preparation of sperm for fertilization.

Mucin-type O-linked glycoproteins are involved in a variety of biological interactions in higher eukaryotes. The biosynthesis of these glycoproteins is initiated by a family of polypeptide N-acetyl-alpha-galactosaminyltransferases (ppGalNAcTs) that modify proteins in the secretory pathway. The lack of a defined consensus sequence for the ppGalNAcTs makes the prediction of mucin-type O-linked glycosylation difficult based on primary sequence alone. Herein we present a method for labeling mucin-type O-linked glycoproteins with a unique chemical tag, the azide, which permits their selective covalent modification from complex cell lysates. From a panel of synthetic derivatives, we identified an azido GalNAc analog (N-azidoacetylgalactosamine, GalNAz) that is metabolized by numerous cell types and installed on mucin-type O-linked glycoproteins by the ppGalNAcTs. The azide serves as a bioorthogonal chemical handle for selective modification with biochemical or biophysical probes using the Staudinger ligation. The approach was validated by labeling a recombinant glycoprotein that is known to possess O-linked glycans with GalNAz. In addition, GalNAz efficiently labeled mucin-type O-linked glycoproteins expressed at endogenous levels. The ability to label mucin-type O-linked glycoproteins with chemical tags should facilitate their identification by proteomic strategies.

Genome-wide screens were performed in yeast to identify genes that enhance the toxicity of a mutant huntingtin fragment or of alpha-synuclein. Of 4850 haploid mutants containing deletions of nonessential genes, 52 were identified that were sensitive to a mutant huntingtin fragment, 86 that were sensitive to alpha-synuclein, and only one mutant that was sensitive to both. Genes that enhanced toxicity of the mutant huntingtin fragment clustered in the functionally related cellular processes of response to stress, protein folding, and ubiquitin-dependent protein catabolism, whereas genes that modified alpha-synuclein toxicity clustered in the processes of lipid metabolism and vesicle-mediated transport. Genes with human orthologs were overrepresented in our screens, suggesting that we may have discovered conserved and nonoverlapping sets of cell-autonomous genes and pathways that are relevant to Huntington's disease and Parkinson's disease.

Alpha-synuclein is implicated in several neurodegenerative disorders, such as Parkinson's disease and multiple system atrophy, yet its functions remain obscure. When expressed in yeast, alpha-synuclein associated with the plasma membrane in a highly selective manner, before forming cytoplasmic inclusions through a concentration-dependent, nucleated process. Alpha-synuclein inhibited phospholipase D, induced lipid droplet accumulation, and affected vesicle trafficking. This readily manipulable system provides an opportunity to dissect the molecular pathways underlying normal alpha-synuclein biology and the pathogenic consequences of its misfolding.

Besides neuronal transmission, serotonin (5-HT) also acts as a trophic signal during the development of the central nervous and neural crest systems. In this study, we report that in addition to trophic effect, 5-HT increases the proliferation of fetal heart cells. We showed for the first time that the cultured heart cells, express serotonin transporter (5-HTT), which confirmed the previously observed accumulation of 5-HT in developing heart. The influence of 5-HT on developing heart cells is studied throughout the dosage. We found that 5-HT concentration at physiological level, 4 microM, permits an optimal proliferation of heart cells as indicated by the number of 5-bromo-deoxyuridine immunoreactive (BrdU-im) cells and myosin heavy chain immunoreactive cells (MF20-im); fluctuation towards either concentrations reduce the proliferation. We hypothesized that 5-HTT plays a role in the heart development. Our study indicated that the blockade of 5-HT uptake by paroxetine decreased the number of BrdU-im cells and MF20-im cells. These data indicate a role of 5-HT and 5-HTT on heart development. Abnormal 5-HT level or misuse of 5-HT uptake blocker may alter the heart development.

A girl with congenital agammaglobulinemia and minor facial anomalies lacked B cells in peripheral blood: karyotypic analysis of white blood cells showed balanced translocation, t(9;20)(q33.2;q12). In the current study, we isolated a novel gene, leucine-rich repeat-containing 8 (LRRC8), at the translocation site on chromosome 9. It has four transmembrane helices with one isolated and eight sequentially located leucine-rich repeats (LRRs) and constitutes a new protein family. It is expressed on T cells as well as on B-lineage cells. Translocation truncates the LRRC8 gene, resulting in deletion of the eighth, ninth, and half of the seventh LRR domains located close to the C-terminal. The truncated form of the LRRC8 gene is transcribed with sequences from the noncoding region adjacent to the truncated seventh LRR. Protein products derived from the truncated gene are coexpressed on white blood cells with the intact LRRC8 protein from the untranslocated allele. Transplantation experiments with murine bone marrow cells that were forced to express the truncated LRRC8 show that expression of the truncated protein inhibited B cell development. These results indicate that LRRC8 is responsible for the B cell deficiency in this patient and is required for B cell development.

The peroxiredoxin (PRDX) family is a recently identified family of peroxidases found in organisms ranging from bacteria to mammals. In mammals, six PRDX isoforms have been characterized in human (Homo sapiens), rat (Rattus norvegicus) and mouse (Mus musculus). PRDXs are cytosolic, secreted or targeted to organelles such as peroxisomes, mitochondria and the nucleus. Some PRDXs are synthesized as larger precursor proteins with a presequence that is cleaved to produce the mature form. To study the expression of the six PRDXs in bovine (Bos taurus), we first cloned cDNAs coding for PRDX1, PRDX2, PRDX4 and PRDX5. PRDX3 and PRDX6 had previously been cloned and characterized in bovine. The comparison of bovine PRDXs with their rat, mouse and primate orthologues reveals a minimum of 95% similarity of mature proteins. Even though mitochondrial or export signal presequences are normally less conserved, the unprocessed proteins still present a minimum of 84% similarity. Nevertheless, a major divergence lies at the N-terminus of bovine PRDX2, where a Cys-Val-Cys motif was identified. The expression of the six PRDXs in 22 bovine tissues has been studied by RT-PCR. Our results point out the ubiquity of the different PRDX transcripts in bovine tissues. The important conservation of the different PRDXs, the multiple processes they have been associated with, as well as the ubiquity of all the members of the family analyzed in this study for the first time altogether, suggest that they play a major role in the basal metabolism of mammalian cells.

Condensin is an evolutionarily conserved protein complex that helps mediate chromosome condensation and segregation in mitotic cells. Here, we show that condensin has two activities that contribute to meiotic chromosome condensation in Saccharomyces cerevisiae. One activity, common to mitosis, helps mediate axial length compaction. A second activity promotes chromosome individualization with the help of Red1 and Hop1, two meiotic specific components of axial elements. Like Red1 and Hop1, condensin is also required for efficient homologue pairing and proper processing of double strand breaks. Consistent with these functional links condensin is necessary for proper chromosomal localization of Red1 and Hop1 and the subsequent assembly of the synaptonemal complex. Finally, condensin has a Red1/Hop1-independent role in the resolution of recombination-dependent linkages between homologues in meiosis I. The existence of distinct meiotic activities of condensin (axial compaction, individualization, and resolution of recombination-dependent links) provides an important framework to understand condensin's role in both meiotic and mitotic chromosome structure and function.

Costimulatory signals received by diabetogenic T cells during priming by or upon secondary encounter with autoantigen are decisive in determining the outcome of autoimmune attack. The OX40-OX40 ligand (OX40L) costimulatory pathway is known to influence T cell responses, prompting us to examine its role in autoimmune diabetes. A null allele at OX40L completely prevented diabetes development in nonobese diabetic mice and strongly reduced its incidence in a TCR transgenic model (BDC2.5). However, somewhat paradoxically, the initial activation of T cells responsive to islet beta cell Ag was slightly faster and more efficient in the absence of OX40L, with an increased degree of cell proliferation and survival in the deficient hosts. Activated T cell migration into and retention within the islets was also slightly accelerated. When challenged in vitro, splenocytes from BDC2.5.OX40L(o/o) mice showed no altered reactivity to exogenously added peptide, no bias to the Th1 or Th2 phenotype, and no alteration in T cell survival. Thus, the OX40/OX40L axis has the paradoxical effect of dampening the early activation and migration of autoimmune T cells, but sustains the long-term progression to autoimmune destruction.

Recent advances in molecular biology have extended our understanding of the metabolic processes related to microbial transformation of petroleum hydrocarbons. The physiological responses of microorganisms to the presence of hydrocarbons, including cell surface alterations and adaptive mechanisms for uptake and efflux of these substrates, have been characterized. New molecular techniques have enhanced our ability to investigate the dynamics of microbial communities in petroleum-impacted ecosystems. By establishing conditions which maximize rates and extents of microbial growth, hydrocarbon access, and transformation, highly accelerated and bioreactor-based petroleum waste degradation processes have been implemented. Biofilters capable of removing and biodegrading volatile petroleum contaminants in air streams with short substrate-microbe contact times (<60 s) are being used effectively. Microbes are being injected into partially spent petroleum reservoirs to enhance oil recovery. However, these microbial processes have not exhibited consistent and effective performance, primarily because of our inability to control conditions in the subsurface environment. Microbes may be exploited to break stable oilfield emulsions to produce pipeline quality oil. There is interest in replacing physical oil desulfurization processes with biodesulfurization methods through promotion of selective sulfur removal without degradation of associated carbon moieties. However, since microbes require an environment containing some water, a two-phase oil-water system must be established to optimize contact between the microbes and the hydrocarbon, and such an emulsion is not easily created with viscous crude oil. This challenge may be circumvented by application of the technology to more refined gasoline and diesel substrates, where aqueous-hydrocarbon emulsions are more easily generated. Molecular approaches are being used to broaden the substrate specificity and increase the rates and extents of desulfurization. Bacterial processes are being commercialized for removal of H(2)S and sulfoxides from petrochemical waste streams. Microbes also have potential for use in removal of nitrogen from crude oil leading to reduced nitric oxide emissions provided that technical problems similar to those experienced in biodesulfurization can be solved. Enzymes are being exploited to produce added-value products from petroleum substrates, and bacterial biosensors are being used to analyze petroleum-contaminated environments.

We sought to establish the relationship of quantitative hepatic iron measurements and phlebotomy-mobilized iron in a large sample of HFE C282Y homozygotes with a hemochromatosis phenotype. Thus, we analyzed data from 79 unrelated C282Y homozygotes from treatment centers in Rochester, NY and Birmingham, AL who had undergone liver biopsy with measurement of hepatic iron content and who had achieved iron depletion (serum ferritin <25 ng/l) with quantitative phlebotomy. The sample consisted of 57 men and 22 women; their median age at diagnosis was 47 years (range 23-76 years). Sixty-three of 79 (79.7%) had hepatic iron index (HII; μmol/g dry weight of liver divided by age in years) ≥1.9, a conventional phenotypic definition of hemochromatosis. The mean quantity of phlebotomy-mobilized iron (± 1 sd) was 6.4 g (±4.0 g) in men (range 2.0-18.0 g) and 6.2 g (±5.8) in women (range 0.7-22.5 g). There was a significant positive correlation of liver iron levels with phlebotomy-mobilized iron in this patient sample (Pearson coefficient 0.75; R<PRE>2</PRE>=55.5%). This relationship was also demonstrable when data from males and females were analyzed separately. We calculated a phlebotomy-mobilized iron index (MII: phlebotomy-mobilized iron in mg divided by age in years) using the corresponding regression equations and evaluated its use as a surrogate for HII. Thus, a phlebotomy-mobilized iron of 3.5 g corresponds to liver iron levels of 80 μmol/g dry weight, and a MII of 80 corresponds to HII of 1.9. Forty-six of 79 subjects met all four phenotypic criteria for hemochromatosis (liver iron levels ≥80 μmol/g, HII≥1.9, phlebotomy-mobilized iron ≥3.5 g and MII≥80). Of the 20 subjects with MII<80, 9 had a HII≥1.9. Conversely, 5 of 16 subjects with HII<1.9 had MII≥80 and 8 had phlebotomy-mobilized iron ≥3.5 g. Most patients with a hemochromatosis phenotype and evidence of moderate or severe iron overload (>80%) are homozygous for the common HFE missense mutation C282Y. Thus, clinicians rely increasingly on HFE mutation analysis to diagnose hemochromatosis and on quantitative phlebotomy to estimate the severity of iron overload in many cases. Liver biopsy is now employed in selected patients to visualize fibrosis or cirrhosis and to identify coincidental hepatic disease. We conclude that the use of the MII permits a retrospective estimation of the age-adjusted severity of iron overload that has a diagnostic value similar to that of the HII in hemochromatosis patients with C282Y homozygosity.

Since the first detection of aberrant crypt foci (ACF) in carcinogen-treated mice, there have been numerous studies focusing on these microscopically visible lesions both in rodents and in humans. ACF have been generally accepted as precancerous lesions in regard to histopathological characteristics, biochemical and immunohistochemical alterations, and genetic and epigenetic alterations. ACF show variable histological features, ranging from hyperplasia to dysplasia. ACF in human colon are more frequently located in the distal parts than in the proximal parts, which is in accordance with those in colorectal cancer (CRC). The immunohistochemical expressions of carcinoembryonic antigen (CEA), beta-catenin, placental cadherin (P-cadherin), epithelial cadherin (E-cadherin), inducible nitric oxide synthase (iNOS), cyclooxygenase (COX-2), and P16INK4a are found to be altered. Genetic mutations of K-ras, APC and p53, and the epigenetic alterations of CpG island methylation of ACF have also been demonstrated. Genomic instabilities due to the defect of mismatch repair (MMR) system are detectable in ACF. Two hypotheses have been proposed. One is the "dysplasia ACF-adenoma-carcinoma sequence", the other is "heteroplastic ACF-adenoma-carcinoma sequence". The malignant potential of ACF, especially dysplastic ACF, makes it necessary to reveal the nature of these lesions, and to prevent CRC from the earliest possible stage. The technique of magnifying chromoscope makes it possible to detect "in vivo" ACF, which is beneficial to colon cancer research, identifying high-risk populations for CRC, and developing preventive procedures.

Netrin-1 is a bifunctional guidance cue that directs migrating neurons and axons based on specific receptors expressed on the cell surface. Attraction occurs through the receptor Deleted in Colorectal Cancer (DCC) and repulsion occurs through a receptor complex of DCC and UNC5H, the vertebrate homolog to Caenorhabditis elegans UNC-5, but how the specific surface expression of these receptors is achieved remains unknown. Here, we demonstrate that surface expression of UNC5H1 is regulated in neurons by protein interacting with C kinase-1 (PICK1) and protein kinase C (PKC), and show that one mechanism by which cells control their response to netrin-1 is by changing the surface availability of receptors. We identified PICK1 as a binding partner for UNC5H1 using the yeast two-hybrid system and found that the extreme three C-terminal amino acids of UNC5H1 interact with the PSD-95/Dlg/ZO-1 (PDZ) domain of PICK1. Coexpression of UNC5H1 and PICK1 in heterologous cells results in the recruitment of PICK1 to UNC5H1 clusters. Endogenous UNC5H1 and PICK1 coimmunoprecipitate from extracts of cultured hippocampal neurons and P4 cortices, and immunohistochemistry shows that UNC5H1, PICK1, and PKC are all present in growth cones. PKC activation induces the formation of UNC5H1/PICK1/PKC complexes and leads to the specific removal of UNC5H1, but not DCC, from the surface of neurons and growth cones via a PICK1/PKC-dependent mechanism. Lastly, we demonstrate that activating PKC, which decreases surface expression of UNC5H1, inhibits netrin-1-dependent collapse of hippocampal growth cones. Together, our results suggest that by regulating the surface expression of UNC5Hs, an axon can modulate its repellent response to netrin-1.

<h4>Purpose</h4>Pancreatic endocrine neoplasms (PENs) are rare, mostly well-differentiated endocrine neoplasms, whose biology has been poorly characterized. Global expression microarrays can document abnormal pathways that impact on tumorigenesis and disease progression.<h4>Experimental design</h4>RNA was extracted from eight well-differentiated PENs and three highly enriched pancreatic islet cell samples (80-90% purity), and examined using the Affymetrix U133A oligonucleotide microarray. Microarray data were normalized using dCHIP for identification of differentially expressed genes. PEN tissue microarrays were constructed from 53 archival PENs for immunohistochemical validation of microarray data.<h4>Results</h4>Sixty-six transcripts were overexpressed > or =3-fold in PENs compared with normal islet cells, including putative oncogenes (MLLT10/AF10), growth factors [insulin-like growth factor-binding protein 3 (IGFBP3)], cell adhesion and migration molecules (fibronectin), and endothelial elements (MUC18/MelCAM and CD31). A total of 119 transcripts were underexpressed < or =3-fold in PENs compared with normal islet cells, including cell cycle checkpoint proteins (p21/Cip1), the MIC2 (CD99) cell surface glycoprotein, putative metastasis suppressor genes (NME3), and junD, a MEN1-regulated transcription factor. Using PEN tissue microarrays, we confirmed the differential up-regulation of IGFBP3 (70%) and fibronectin (22%) and differential down-regulation of p21 (46%) and MIC2 (CD99; 91%) in PENs versus normal pancreatic islets. IGFBP3 overexpression was significantly more common in metastatic (93%) versus primary PEN lesions (60%), P=0.022. Fibronectin overexpression demonstrated a trend toward significance in lymphatic PEN metastases (55%) compared with primary PEN lesions (24%; P=0.14).<h4>Conclusions</h4>Global expression analysis provides insight into tumorigenic pathways in PENs and may identify potential prognostic and therapeutic markers for these uncommon neoplasms.

We analyzed NMDA receptor subunit mRNAs, proteins, and anchoring proteins in mice transgenic for exon 1 of the HD gene. R6/2 mice had decreased levels of mRNAs encoding epsilon1 and epsilon2 NMDA receptor subunits (mouse orthologs of rat NR2A and NR2B subunits), but not the zeta1 subunit (mouse ortholog of NR1), as assessed by gene expression profiling and Northern blotting. In situ hybridization resolved mRNA decreases spatially to the CA1 field of hippocampus. Western blotting revealed decreases in plasma membrane-associated epsilon1 and epsilon2 subunits in hippocampus, and decreases in plasma membrane-associated zeta1 subunit in cortex and hippocampus. In addition, PSD-95 and alpha-actinin-2, proteins essential for anchoring NMDA receptors, were decreased. Finally, we found a decreased level of tyrosine-phosphorylated epsilon1 subunit, another determinant of NMDA receptor trafficking, in R6/2 hippocampus. Taken together, these data demonstrate multiple levels of NMDA receptor dysregulation, including abnormalities in mRNA expression levels, receptor stoichiometry, protein phosphorylation, and receptor trafficking.

The isoforms distribution of the glycoprotein antithrombin III (ATIII) derived from human plasma was investigated by means of isoelectric focusing (IEF) in polyacrylamide gels with immobilized pH gradients (IPG) and two-dimensional gel electrophoresis (2-DE) as well as capillary electrophoretic methods. It turned out that the presence of high concentrations of chaotropics (urea, thiourea) and zwitterionic detergents (3-[(3-cholamidepropyl)dimethylammonio]-1-propanesulfonate (CHAPS)) was decisive for attaining good resolution of the protein isoforms. Resolution by IPG-IEF was obtained with excellent reproducibility and pI differences down to 0.01 pH units could be distinguished. ATIII-alpha and ATIII-beta-fractions preseparated by heparin affinity chromatography showed an analogous but shifted spot pattern consisting each of one major and three minor isoforms. The main isoforms of ATIII-alpha and ATIII-beta exhibit pI values of 5.18 and 5.32, respectively, both values determined in the presence of high concentrations of urea. The pI difference of 0.14 pH units correspond to the effect of two sialic acids absent in ATIII-beta. The formation and occurrence of ATIII dimers and trimers turned out to be dependent on the sample preparation. The results obtained by 2-DE were compared with those of capillary zone electrophoresis (CZE) and capillary IEF (CIEF). Quantitative analysis regarding the CZE separated isoforms of plasma derived ATIII yielded a content of about 70% ATIII-alpha main isoform and about 6.6% of ATIII-beta. The pI values of ATIII determined by CIEF with internal calibration were in fair agreement with the pI values of the main isoforms achieved with 2-DE.

Huntington's disease (HD) inclusions are stained with anti-ubiquitin and anti-proteasome antibodies. This, together with proteasome activity studies on transfected cells, suggest that an impairment of the ubiquitin-proteasome system (UPS) may be key in HD pathogenesis. To test whether proteasome activity is impaired in vivo, we performed enzymatic assays for the three peptidase activities of the proteasome in brain extracts from the HD94 conditional mouse model of HD. We found no inhibition of any of the activities, suggesting that if UPS impairment happens in vivo, it is not at the level of the proteasome catalytic core. Intriguingly, the chymotrypsin- and trypsin-like activities increased selectively in the affected and aggregate-containing regions: cortex and striatum. Western blot analysis revealed no difference in total proteasome content whereas an increase in the interferon-inducible subunits of the immunoproteasome, LMP2 and LMP7, was observed. These subunits confer to the proteasome catalytic properties that are optimal for MHC-I peptide presentation. Immunohistochemistry in control mouse brain revealed LMP2 and LMP7 mainly in neurons. Accordingly, their increase in HD94 mice predominantly took place in neurons, and 5% of the ubiquitin-positive cortical aggregates were also LMP2-positive. Ultrastructural analysis of neurons with high level of immunoproteasome subunits revealed signs of neurodegeneration like nuclear indentation or fragmentation and dark cell appearance. The neuronal induction of LMP2 and LMP7 and the associated signs of neurodegeneration were also found in HD postmortem brains. Our results indicate that LMP2 and LMP7 participate in normal neuronal physiology and suggest a role in HD neurodegeneration.

Hepatitis C virus infection is often associated with an elevation of iron parameters. Free liver iron causes liver damage and liver fibrosis preferentially through induction of reactive oxygen species. With an allele frequency of 5-10% for the C282Y mutation and 6-30% for the H63D mutation, there is a frequent coincidence of hemochromatosis (HFE) mutations and chronic hepatitis C. There is increasing evidence that HFE homozygosity and even HFE heterozygosity are associated with an increased liver iron concentration and liver fibrosis progression in chronic hepatitis C. In addition, present data suggest an impact of iron on the outcome of interferon therapy. Thus, HFE mutations and liver iron stores seem to be important comorbid factors in chronic hepatitis C. Screening for iron parameters and HFE mutations should be considered in patients with hepatitis C.

<h4>Aim</h4>To assess the frequency of the C282Y and H63D mutations on the HFE gene in Danish patients with clinical hereditary haemochromatosis initially diagnosed by phenotypic methods.<h4>Methods</h4>In the period 1950-1985, an epidemiological survey in Denmark identified 179 patients with clinical idiopathic haemochromatosis diagnosed by phenotypic methods (serum transferrin saturation, serum ferritin, liver biopsy and mobilisable body iron stores). In 32 unrelated patients, frozen blood samples were available for genetic analysis. In a subsequent series of 26 unrelated Danish patients, a phenotypic diagnosis of clinical idiopathic haemochromatosis was made before blood samples were taken for HFE genotyping. The total series consisted of 58 patients (40 men and 18 women) with a median age of 60 yrs (range 18-74). HFE genotyping was performed by the polymerase chain reaction (PCR) technique.<h4>Results</h4>Among the patients, 55 of 58 (94.8%) were C282Y/C282Y homozygous. One 63-year-old woman (1.7%) was compound C282Y/H63D heterozygous. Two women (3.4%), aged 42 and 43 yrs were negative for both the C282Y and the H63D mutation.<h4>Conclusion</h4>In the Danish population, homozygosity for the C282Y mutation appears to be the prevailing cause of clinically overt genetic haemochromatosis. This finding has implications both for the evaluation of patients with iron overload disorders and for the strategy in future population screening surveys.

<h4>Objectives</h4>Beta-thalassaemia minor (BTM) alone does not lead to iron overload, however, some gene modifiers and acquired causes are reported. When it is inherited together with a mutation in the HFE (HLA-H) gene associated with hereditary haemochromatosis, iron overload may ensue. To analyse the relationship between iron status and HFE mutations in Iranian BTM patients, we compared the frequency of the C282Y and H63D HFE mutations and ferritin level in a group of BTM patients from the National Thalassaemia Transfusion and Care Centre with that of healthy individuals.<h4>Patients and methods</h4>Ninety-three (56 females) documented BTM cases and 104 (54 females) controls were enrolled in the study. Serum ferritin level was measured in all subjects by immuno-radiometric assay and HFE genotypes were determined using restriction fragment length polymorphism analysis of PCR-amplified HFE gene fragment.<h4>Results</h4>Eighteen (19.4%) BTM patients vs. 12 (11.5%) controls were H63D heterozygotes, while there were three (3.2%) cases and three (2.9%) controls with H63D homozygosity. All three C282Y mutations were found in BMT patients with one of them being a compound heterozygote. A significant difference was observed in the total number of HFE mutations in favour of BTM patients over the controls (P < 0.05, OR = 2.064). The H63D and C282Y allele frequencies were 12.9 and 1.61 in patients and 8.65 and 0 in controls, respectively. The mean ferritin level in cases with HFE mutations showed no significant difference from that of the patients without mutations (P > 0.05).<h4>Conclusions</h4>Our results suggest that HFE mutations C282Y and H63D are more frequent in Iranian BTM patients than in the normal population, causing no significant changes in serum ferritin level.

While the role of the mutated Huntington's disease (HD) protein in the pathogenesis of HD has been the focus of intensive investigation, the normal protein has received less attention. Nonetheless, the wild-type HD protein appears to be essential for embryogenesis, since deletion of the HD gene in mice results in early embryonic lethality. This early lethality is due to a critical role the HD protein, called huntingtin (Htt), plays in extraembryonic membrane function, presumably in vesicular transport of nutrients. Studies of mutant mice expressing low levels of Htt and of chimeric mice generated by blastocyst injection of Hdh-/- embryonic stem cells show that wildtype Htt plays an important role later in development as well, specifically in forebrain formation. Moreover, various lines of study suggest that normal Htt is also critical for survival of neurons in the adult forebrain. The observation that Htt plays its key developmental and survival roles in those brain areas most affected in HD raises the possibility that a subtle loss of function on the part of the mutant protein or a sequestering of wild-type Htt by mutant Htt may contribute to HD pathogenesis. Regardless of whether this is so, the prosurvival role of Htt suggests that HD therapies that block production of both wild-type and mutant Htt may themselves be harmful.

The purpose of the present study was to elucidate the expression of human organic anion transporter 1 (hOAT1) and hOAT3 in the choroid plexus of the human brain and their interactions with neurotransmitter metabolites using stable cell lines. Immunohistochemical analysis revealed that hOAT1 and hOAT3 are expressed in the cytoplasmic membrane and cytoplasm of human choroid plexus. Neurotransmitter metabolites, namely, 5-methoxyindole-3-acetic acid (5-MI-3-AA), homovanillic acid (HVA), vanilmandelic acid (VMA), 3,4-dihydroxyphenylacetic acid (DOPAC), 5-hydroxyindole-3-acetic acid (5-HI-3-AA), N-acetyl-5-hydroxytryptamine (NA-5-HTT), melatonin, 5-methoxytryptamine (5-MTT), 3,4-dihidroxymandelic acid (DHMA), 5-hydroxytryptophol, and 5-methoxytryptophol (5-MTP), but not methanephrine (MN), normethanephrine (NMN), and 3-methyltyramine (3-MT), at 2 mM, inhibited para-aminohippuric acid uptake mediated by hOAT1. On the other hand, melatonin, 5-MI-3-AA, NA-5-HTT, 5-MTT, 5-MTP, HVA, 5-HI-3-AA, VMA, DOPAC, 5-hydroxytryptophol, and MN, but not 3-MT, DHMA, and NMN, at 2 mM, inhibited estrone sulfate uptake mediated by hOAT3. Differences in the IC(50) values between hOAT1 and hOAT3 were observed for DHMA, DOPAC, HVA, 5-HI-3-AA, melatonin, 5-MI-3-AA, 5-MTP, 5-MTT, and VMA. HOAT1 and hOAT3 mediated the transport of VMA but not HVA and melatonin. These results suggest that hOAT1 and hOAT3 are involved in the efflux of various neurotransmitter metabolites from the cerebrospinal fluid to the blood across the choroid plexus.

Serotoninergic neurons, astrocytes and nitrergic system play an important role in central nervous system (CNS) development. These systems are altered in prenatal ethanol exposure (PEE) but ethanol (EtOH) effects may be very diverse under different conditions. In this study, we analyzed morphologically two serotoninergic mesencephalic nuclei and three prosencephalic areas of serotoninergic innervation in a model of pre- and postnatal low-ethanol exposure. Female Wistar rats were orally exposed to EtOH 6.6% (v/v), ad libitum, for 6 weeks before mating and during gestation and lactation while control group received water ad libitum. Twenty-day-old offspring (P21) brains were processed and immunoreactivity (IR) using antibodies against tryptophan hydroxylase (TPH), 5-HT, 5-HT transporter (5HTT), glial fibrillary acidic protein (GFAP), S-100B protein, 200-kDa neurofilaments (Nf-200) and neuronal nitric oxide synthase (nNOS) was evaluated. Dorsal and median raphe nucleus (DRN and MRN), hippocampus (Hipp), striatum (Strt) and frontal cortex (FCx) were studied by computer-assisted image analysis. Relative optical density (ROD) of TPH-IR, 5-HT-IR and nNOS-IR neurons; cell area of GFAP-IR astrocytes; relative area of 5HTT-IR fibers and Nf-200-IR were evaluated. TPH-IR was increased in DRN and MRN and 5-HT-IR was increased only in MRN. 5-HTT-IR fibers and ROD of S-100B-IR astrocytes were increased in the three prosencephalic areas while GFAP-IR astrocytes were hypertrophied only in Hipp and FCx. Nf-200 expression was increased in Hipp and Strt and morphologically altered in the FCx. ROD of nNOS-IR neurons was increased in Strt and FCx but was not detected in Hipp. We have also detected morphological changes resembling accelerated development and maturation, and early aging. Considering the evidences of a close 5-HT-astroglial-NO relationship during CNS development the differential response of the studied regions is an interesting result that could be due to different gradients of development in the studied areas and/or different responses of those areas to the effects of a low pre- and postnatal ethanol exposure.

The presence of neuroendocrine cells is putative in thyroid hyalinizing trabecular tumor (HTT), although this entity requires a differential diagnosis from paraganglioma or medullary carcinoma of the thyroid, due to similarity of the growth pattern of the cells. Here, we present a case of HTT with a mixture of endocrine cells stained positive for somatostatin, chromogranin A and Grimelius' silver impregnation. The histology and results of other immunostainings were consistent with the features typical for HTT. In the world literature to date, only two cases of HTT with endocrine cells, including the current case, have been reported. Nevertheless, the cases may indicate the diversified differentiation of cells in HTT and may, in part, account for the resemblance of HTT to paraganglioma and medullary carcinoma.

<h4>Objective</h4>To study whether any relationship exists between the C282Y and H63D mutations of the HFE gene, iron liver content, and the severity of histological damage in patients with hepatitis C virus (HCV)-induced chronic hepatitis.<h4>Material and methods</h4>In 72 patients diagnosed with HCV-chronic infection, naïve for antiviral therapy, and undergoing liver biopsy, the Knodell index was established, a morphometric evaluation of hepatic hemosiderin deposits was performed by using a semiautomatic method of image analysis, and mutations of the HFE gene were identified through a polymerase chain reaction on leukocyte genomic DNA by using specific restriction enzymes. The control group for the distribution of HFE genetic variants was composed of 181 healthy individuals with the same ethnic and geographical (white Spaniards) origin.<h4>Results</h4>(Cases/controls): 1. Genotype distribution: a) mutation C282Y: no homozygotes, 6/23 heterozygotes, 66/158 without the mutation (not significant, n.s.); b) mutation H63D: 2/5 homozygotes, 26/52 heterozygotes, 44/124 without the mutation (n.s.). compound heterozygotes 2/6. 2. Allele frequencies: a) mutation C282Y: 0.042/0.064 (n.s.); b) mutation H63D: 0.208/0.171 (n.s.). Four C282Y heterozygous patients had stainable liver iron (p=0.015 vs patients without mutations). Sixty-six patients were not carriers of the C282Y mutation; among them, 26.9% of 26 carriers and 15% of 40 non-carriers of the H63D mutation had liver stainable iron (n.s.). Knodell index score, gender, age at diagnosis, mode of transmission, and serum and liver iron values were not related to the HFE genotype.<h4>Conclusions</h4>our results suggest that the C282Y mutation, but not the H63D mutation, of the HFE gene is frequently associated with stainable iron in the liver in HCV-related chronic hepatitis. The HFE genotype is not related to the histological severity of the disease.

No abstract available.