Gene Literature Dashboard

IL-17 is a T cell-derived, proinflammatory cytokine that is suspected to be involved in the development of various inflammatory diseases. Although there are elevated levels of IL-17 in synovial fluid of patients with rheumatoid arthritis, the pathogenic role of IL-17 in the development of rheumatoid arthritis remains to be elucidated. In this report, the effects of IL-17 deficiency were examined in IL-1 receptor antagonist-deficient (IL-1Ra(-/-)) mice that spontaneously develop an inflammatory and destructive arthritis due to unopposed excess IL-1 signaling. IL-17 expression is greatly enhanced in IL-1Ra(-/-) mice, suggesting that IL-17 activity is involved in the pathogenesis of arthritis in these mice. Indeed, the spontaneous development of arthritis did not occur in IL-1Ra(-/-) mice also deficient in IL-17. The proliferative response of ovalbumin-specific T cells from DO11.10 mice against ovalbumin cocultured with antigen-presenting cells from either IL-1Ra(-/-) mice or wild-type mice was reduced by IL-17 deficiency, indicating insufficient T cell activation. Cross-linking OX40, a cosignaling molecule on CD4(+) T cells that plays an important role in T cell antigen-presenting cell interaction, with anti-OX40 Ab accelerated the production of IL-17 induced by CD3 stimulation. Because OX40 is induced by IL-1 signaling, IL-17 induction is likely to be downstream of IL-1 through activation of OX40. These observations suggest that IL-17 plays a crucial role in T cell activation, downstream of IL-1, causing the development of autoimmune arthritis.

Protein kinases with a conserved catalytic domain make up one of the largest 'superfamilies' of eukaryotic proteins and play many key roles in biology and disease. Efforts to identify and classify all the members of the eukaryotic protein kinase superfamily have recently culminated in the mining of essentially complete human genome data.

Posttranslational modifications and remodeling of nucleosomes are critical factors in the regulation of transcription. Higher-order folding of chromatin also is likely to contribute to the control of gene expression, but the absence of a detailed description of the structure of the chromatin fiber has impaired progress in this area. Mammalian somatic cells contain a set of H1 linker-histone subtypes, H1 (0) and H1a to H1e, that bind to nucleosome core particles and to the linker DNA between nucleosomes. To determine whether the H1 histone subtypes play differential roles in the regulation of gene expression, we combined mice lacking specific H1 histone subtypes with mice carrying transgenes subject to position effects. Because position effects result from the unique chromatin structure created by the juxtaposition of regulatory elements in the transgene and at the site of integration, transgenes can serve as exquisitely sensitive indicators of chromatin structure. We report that some, but not all, linker histones can attenuate or accentuate position effects. The results suggest that the linker-histone subtypes play differential roles in the control of gene expression and that the sequential arrangement of the linker histones on the chromatin fiber might regulate higher-order chromatin structure and fine-tune expression levels.

<h4>Objective</h4>The pharmacological profile of escitalopram, the S-(+)-enantiomer of citalopram, was studied and compared with citalopram and the R-(-)-enantiomer, R-citalopram.<h4>Methods</h4>Inhibition of the serotonin transporter (5-HTT) was studied in COS-1 cells expressing the human 5-HTT (h-5-HTT) and in rat brain synaptosomes. In vitro selectivity was studied relative to noradrenaline transporter (NAT) and dopamine transporter (DAT) function in rat brain synaptosomes, and affinities for other binding sites were determined. In vivo 5-HT activity was measured as inhibition of neuronal firing rate in rat dorsal raphe nucleus (DRN) and enhancement of 5-hydroxytryptophan (5-HTP)-induced behaviour (mouse and rat). Furthermore, studies were conducted in models of antidepressant (mouse forced-swim test), anxiolytic [foot-shock-induced ultrasonic vocalization (USV) in adult rats and mouse black and white box] and anti-aggressive activity (socially isolated mice).<h4>Results</h4>Escitalopram inhibited 5-HTT functions approximately 2 times more potently than citalopram and at least 40 times more potently than R-citalopram. Escitalopram showed insignificant activity at other monoamine transporters and 144 other binding sites. Escitalopram inhibited 5-HT neuronal firing in DRN and potentiated 5-HTP-induced behaviours more potently than citalopram; R-citalopram was inactive. Escitalopram and citalopram, but not R-citalopram, reduced forced-swimming-induced immobility and facilitated exploratory behaviour in the black and white box. Escitalopram and citalopram inhibited USV potently; R-citalopram was several times less potent. Escitalopram, citalopram and R-citalopram inhibited aggressive behaviour weakly. Escitalopram and citalopram had very potent anti-aggressive effects when co-administered with l-5-HTP.<h4>Conclusion</h4>Escitalopram is a very selective 5-HT reuptake inhibitor. It is more potent than its racemate citalopram and is effective in animal models predictive of antidepressant and anxiolytic activities.

Hemochromatosis is a genetic disease related to human leukocyte antigen (HLA) A3, B7, and B14 histocompatability antigens resulting in increased iron absorption from the gastrointestinal tract and deposition of iron in tissues. Arthropathy is not uncommon in the late stage of disease. Characteristic radiologic findings are commonly observed in the wrists and metacarpophalangeal joints as well as the hips, knees, and ankle joints. Presented here is a 34-year-old male with hemochromatosis and bilateral shoulder, knee, and ankle pain. Radiologic examination revealed osteoarthritic findings in both ankle joints and chondrocalcinosis in the knee joints. All the major criteria of hypermobility syndrome were observed on physical examination. The early-onset arthropathy seen with this hemochromatosis is thought to result from hypermobility syndrome.

Rho-GTPase has been implicated in axon outgrowth. However, not all of the critical steps controlled by Rho have been well characterized. Using cultured cerebellar granule neurons, we show here that stromal cell-derived factor (SDF)-1alpha, a neural chemokine, is a physiological ligand that can turn on two distinct Rho-dependent pathways with opposite consequences. A low concentration of the ligand stimulated a Rho-dependent pathway that mediated facilitation of axon elongation. In contrast, Rho/ROCK activation achieved by a higher concentration of SDF-1alpha caused repression of axon formation and induced no more increase in axon length. However, even at this higher concentration a Rho-dependent axon elongating activity could be recovered upon removal of ROCK activity using Y-27632. SDF-1alpha-induced axon elongating activity under ROCK inhibition was replicated by the dominant-active form of the mammalian homologue of the Drosophila gene Diaphanous (mDia)1 and counteracted by its dominant-negative form. Furthermore, RNAi knockdown of mDia1 abolished SDF-1alpha-induced axon elongation. Together, our results support a critical role for an SDF-1alpha/Rho/mDia1 pathway in mediating axon elongation.

PELP1 (proline-, glutamic acid-, and leucine-rich protein-1 (also referred to as MNAR, or modulator of nongenomic activity of estrogen receptor)), a recently identified novel coactivator of estrogen receptors, is widely expressed in a variety of 17 beta-estradiol (E2)-responsive reproductive tissues and is developmentally regulated in mammary glands. pRb (retinoblastoma protein), a cell cycle switch protein, plays a fundamental role in the proliferation, development, and differentiation of eukaryotic cells. To study the putative function of PELP1, we established stable MCF-7 breast cancer cell lines overexpressing PELP1. PELP1 overexpression hypersensitized breast cancer cells to E2 signaling, enhanced progression of breast cancer cells to S phase, and led to persistent hyperphosphorylation of pRb in an E2-dependent manner. Using phosphorylation site-specific pRb antibodies, we identified Ser-807/Ser-811 of pRb as a potential target site of PELP1. Interestingly, PELP1 was discovered to be physiologically associated with pRb and interacted via its C-terminal pocket domain, and PELP1/pRb interaction could be modulated by antiestrogen agents. Using mutant pRb cells, we demonstrated an essential role for PELP1/pRb interactions in the maximal coactivation functions of PELP1 using cyclin D1 as one of the targets. Taken together, these findings suggest that PELP1, a steroid coactivator, plays a permissive role in E2-mediated cell cycle progression, presumably via its regulatory interaction with the pRb pathway.

Diverse members of the G protein-coupled receptor (GPCR) superfamily participate in a variety of physiological functions and are major targets of pharmaceutical drugs. Here we report that the repertoire of GPCRs for endogenous ligands consists of 367 receptors in humans and 392 in mice. Included here are 26 human and 83 mouse GPCRs not previously identified. A direct comparison of GPCRs in the two species reveals an unexpected level of orthology. The evolutionary preservation of these molecules argues against functional redundancy among highly related receptors. Phylogenetic analyses cluster 60% of GPCRs according to ligand preference, allowing prediction of ligand types for dozens of orphan receptors. Expression profiling of 100 GPCRs demonstrates that most are expressed in multiple tissues and that individual tissues express multiple GPCRs. Over 90% of GPCRs are expressed in the brain. Strikingly, however, the profiles of most GPCRs are unique, yielding thousands of tissue- and cell-specific receptor combinations for the modulation of physiological processes.

Chondrogenesis leads to the formation of mature cartilage and generates initial skeletal elements that serve as templates for endochondral bone formation. Bone morphogenetic proteins (BMPs) are involved in several developmental and organogenetic processes and have been identified as key regulators in chondrogenesis. In the present study we sought to determine the transcriptional mechanisms contributing to the induction of chondrogenic markers by BMP-2. Time-course studies with BMP-2-stimulated C3H10T1/2 cells showed a dose-dependent appearance of Alcian-blue-positive material and up-regulated expression of type-II collagen mRNA. This last effect required new protein synthesis because addition of cycloheximide completely blocked the induction of type-II collagen mRNA. A region encompassing the chondrocyte-specific enhancer, localized in intron I of type-II collagen alpha1 chain (Col2a1) gene, is sufficient to confer BMP-2-dependent transcriptional induction of type-II collagen gene expression. Analysis of the expression levels of chondrogenic Sry-type high-mobility group (HMG) box proteins (SOX) transcription factors demonstrated a time-dependent induction of Sox6 expression by BMP-2 that correlated with the appearance of BMP-2- induced protein complexes bound to the chondrocyte-specific enhancer. Preincubation of nuclear extracts with SOX6 and SOX9 antibodies markedly reduced the intensity of these bands. Forced expression of SOX6 mimicked the BMP-2 effect, whereas coexpression of SOX9 promoted a synergistic interaction between both factors in transcription from the chondrocyte-specific enhancer. Moreover, overexpression of a SOX6 mutated form, devoid of its high-mobility group domain, was sufficient to prevent transcriptional induction of the chondrocyte-specific enhancer by BMP-2. Taken together, these results indicate that SOX6 is an important downstream mediator of BMP-2 signaling in chondrogenesis.

Behçet's disease is characterized by recurrent aphthous stomatitis, uveitis, genital ulcers, and skin lesions. The role of the HLA-B*51 gene has been confirmed in recent years, although its contribution to the overall genetic susceptibility to Behçet's disease was estimated to be only 19%. The production of a variety of cytokines by T cells activated with multiple antigens has been shown to play a pivotal role in the activation of neutrophils. As regards the treatment, anti-tumor necrosis factor alpha therapy has been shown to be effective for mucocutaneous symptoms as well as for sight-threatening panuveitis, although a randomized, controlled trial is required.

Chromosomal region 18q21 is frequently deleted in colorectal cancer (CRC) and is associated with poor prognosis. Potential tumor suppressor mechanisms altered by 18q21 deletion include mediation of TGFbeta signaling by SMADs. Following the definition of SMAD4 deletion as a negative predictive marker for chemotherapy benefit in patients with CRC, we aimed to evaluate the clinical relevance of the deletion of other SMAD genes clustered in this region: SMAD2 and SMAD7 in 264 CRC biopsies from a previous clinical study. In contrast to SMAD2 deletion, for which no clinical relevance was observed, hazard ratios (HR) in a multivariate analysis associated with SMAD7 deletion [overall survival (OS): HR = 0.43, p = 0.0012; disease-free survival (DFS): HR = 0.50, p = 0.0033] indicated a favorable outcome for these patients. In addition, SMAD7 duplication had a hazardous effect on survival [OS: HR = 2.10, p = 0.020; DFS: HR = 2.06, p = 0.015]. Moreover, the HRs associated with one additional copy of SMAD7 were 1.76, p = 0.00024 [OS] and 1.64, p = 0.00048 [DFS] respectively, showing a graded effect of SMAD7 on patient outcome depending on gene copy number that suggests a dose-and-effect basis. Since SMAD7 blocks TGFbeta signaling, these data are consistent with the loss of SMAD7 rendering carcinoma cells more sensitive to cell growth arrest/apoptotic effect of TGFbeta, whereas gain of SMAD7 function might result in TGFbeta resistance, thereby emphasizing the role of TGFbeta in tumor suppression.

<h4>Background</h4>Genetic haemochromatosis is a common hereditary iron loading disorder in humans. The disease is associated with loss of function mutations in the HFE gene. This is thought to change iron stores via increased iron absorption.<h4>Aims</h4>In this study we investigated how adaptation of mucosal reductase activity is engaged in this process and how the changes compare with adaptation seen when an iron deficient diet is fed.<h4>Methods</h4>Duodenal mucosal surface reductase was measured with nitroblue tetrazolium in age matched groups of male Hfe knockout mice (Hfe) and wild- type mice fed a purified diet containing normal (iron adequate), high (iron rich), or low (iron deficient) iron concentrations.<h4>Results</h4>Reductase activity increased when mice were fed an iron deficient diet and decreased when they were fed an iron rich diet. Total villus activity, as measured by the average area under the activity curve along the crypt-villus axis, was increased 2.8-2.9-fold by iron deficiency in both genotypes. Approximately half of this difference was attributable to the significantly increased length of the villi in mice on an iron deficient diet (p<0.05). Hfe knockout did not affect villus length but increased mucosal reductase activity near the villus tips. Similar increases (1.3-1.6-fold) were seen on all diets but the increase was significant for iron deficient and iron loaded diets only (p<0.05).<h4>Conclusion</h4>Hfe gene product and dietary iron downregulate villus reductase activity in mice.

Neonatal hemochromatosis is an enigmatic disease. Little is known about iron metabolism in this disease, including the tissue concentration of ferritin or its H and L subunit ratio. The authors report the tissue iron, ferritin, and ferritin subunit content of a child who died at 5 weeks of neonatal hemochromatosis. The child was born at 29 weeks gestation to a mother with lupus, sickle cell trait, and gestational diabetes. The child's severe liver dysfunction led to the clinical diagnosis of neonatal hemochromatosis at 1 week of age. Despite aggressive support, including red cell transfusions and chelation, the child died of an intracranial hemorrhage. Autopsy showed liver fibrosis and iron staining characteristic of neonatal hemochromatosis. Autopsy liver tissue was compared to age-matched control tissue. Soluble protein was analyzed by the Bradford method. Soluble iron (over 90% of total iron) was analyzed by the o-phenanthroline complex. Tissue ferritin and human ferritin controls (Calzyme) were analyzed by Western blotting after SDS-PAGE, identified with sheep anti-human ferritin antibodies (The BindingSite) secondary antibody-fluorescence for detection, and quantified using the Molecular Dynamics Storm 840 phosphorimager and ImageQuant software. Protein, iron, and total ferritin were similar in the normal and neonatal hemochromatosis liver tissues. Ferritin subunits, however, showed an increased H/L-subunit ratio compared to an age-matched control. This first report of a marked increase in the ferritin H/L-subunit ratio may point to an underlying mechanism of disease in this enigmatic disorder.

According to the hypothesis of initial conditions, drug response may be determined by different initial states of neurotransmitter protein recognition systems. Platelet serotonin (5-HT) transport kinetics were studied as initial-conditions predictors of antidepressant response in 24 depressed patients before and after 3 weeks of treatment with nortriptyline (75 mg). The initial affinity of the 5-HT transporter (5-HTT) correctly predicted 71% of the outcome. The pretreatment affinity constant ( Km) correlated (r = 0.61; p < 0.002) with that measured after 3 weeks of treatment (Kapp). Responding patients had a significantly higher initial Km before treatment and a significantly higher Kapp after treatment. Nonresponders had an initial Km significantly lower than that of 24 controls. Nortriptyline plasma levels were not statistically different between response groups. These results are consistent with two previously published observations, which indicate that the initial affinity of the 5-HTT predicted response to fluvoxamine or fluoxetine in the same way. Insofar as all three drugs increase the apparent affinity of the 5-HTT, it appears that a better response is related to those cases where the initial affinity is already higher before treatment.

Huntington's disease (HD) is caused by an expansion of a polyglutamine (polyQ) tract within huntingtin (htt) protein. To examine the cytotoxic effects of polyQ-expanded htt, we overexpressed an enhanced green fluorescent protein (EGFP)-tagged N-terminal fragment of htt with 150 glutamine residues (Nhtt150Q-EGFP) in Aplysia neurons. A combined confocal and electron microscopic study showed that Aplysia neurons expressing Nhtt150Q-EGFP displayed numerous abnormal aggregates (diameter 0.5-5 microm) of filamentous structures, which were formed rapidly (approximately 2 h) but which were sustained for at least 18 days in the cytoplasm. Furthermore, the overexpression of Nhtt150Q-EGFP in sensory cells impaired 5-hydroxytryptamine (5-HT)-induced long-term synaptic facilitation in sensori-motor synapses without affecting basal synaptic strength or short-term facilitation. This study demonstrates the stability of polyQ-based aggregates and their specific effects on long-term synaptic plasticity.

Iron overload in the liver may occur in the clinical conditions hemochromatosis and transfusion-dependent thalassemia or by long-term consumption of large amounts of dietary iron. As iron concentrations increase in the liver, cirrhosis develops, and subsequently the normal architecture of the liver deteriorates. The underlying mechanisms whereby iron loading of hepatocytes leads to the pathology of the liver are not understood. Similarly, a direct relationship between the expression levels of paracellular junction genes and altered hepatocellular physiology has been reported; however, no relationship has been identified between iron loading and the expression of paracellular junction genes. Here, we report that the expression of numerous paracellular junction genes was decreased in iron-loaded hepatocytes, leading to increased cellular permeability, increased baculovirus-mediated gene transfer, and decreased gap junction communication. Iron loading of hepatocytes resulted in decreased E-cadherin promoter activity and subsequently decreased E-cadherin mRNA and protein expression. The data presented in this study describe a clear relationship between iron overload and decreased expression of paracellular junction genes in hepatic cells of rat and human origin.

<h4>Objectives</h4>While body iron status may influence platelets, little information is available about platelet expression of proteins regulating iron homeostasis. HFE, the protein defective in hereditary hemochromatosis, and transferrin receptor 2 (TfR2) are two novel protein candidates that could be involved in mechanisms of iron transport across the platelet plasma membrane.<h4>Methods</h4>The expression and localization of HFE, TfR1 and TfR2 proteins in human platelets were examined using Western blotting and immunocytochemistry.<h4>Results</h4>Human platelets expressed HFE and TfR2, whereas no signal for TfR1 was found. The positive reactions for HFE and TfR2 were mainly confined to the platelet plasma membrane.<h4>Conclusions</h4>Expression of HFE and TfR2 proteins in human platelets may indicate that the mutations in the corresponding genes could influence platelet count, size and/or activation. The presence of TfR2 and absence of TfR1 suggests that HFE may serve a different function in platelets compared with the other HFE-positive cell types, e.g. enterocytes, macrophages and syncytiotrophoblasts.

Galectin-3, a beta-galactoside-binding lectin, is overexpressed in many neoplasms and may be useful when differentiating between benign and malignant thyroid neoplasms. Recently, interest has focused on the classification and biologic behavior of hyalinizing trabecular tumors (HTTs). In this study we compared galectin-3 expression in a number of different thyroid neoplasms to gain insight into the biologic behavior of HTT. Formalin-fixed, paraffin-embedded tissues from 153 thyroid neoplasms were stained with a monoclonal antibody to galectin-3. These tumors included 58 HTTs, 60 papillary carcinomas, 21 follicular carcinomas, and 14 follicular adenomas. Reactivity was graded as negative, weak, or strong by three observers. The average patient age was similar in the patients with HTTs, papillary carcinomas, and follicular adenomas. The patients with follicular carcinomas were approximately a decade older than all other groups of patients. All groups of thyroid neoplasms occurred more frequently in female patients. Follow-up revealed metastatic disease in patients with papillary (36.6%) and follicular carcinomas (19%) but not in patients with follicular adenomas or HTTs. Galectin-3 immunostaining showed that 60% of the HTTs were negative or had weak (H) (1+) staining and 40% had strong (2-3+) staining. In the majority of the reactive cases, staining was diffuse and predominantly cytoplasmic. Fifty of the 60 (83%) papillary carcinomas and 11 of the 21 (52%) follicular carcinomas showed strong immunostaining. The immunostaining was also diffuse in the majority of papillary and follicular carcinomas. The strong immunoreactivity seen in most of the carcinomas was in contrast to the relatively weak or negative immunostaining in the majority of follicular adenomas (93%). The immunophenotype of HTT, as characterized by galectin-3 expression, is intermediate between that of benign and malignant thyroid tumors, suggesting that some tumors with strong staining may behave like carcinomas, although this was not noted in our cases. Our study suggests that the variable pattern of galectin-3 expression may reflect a difference in biologic behavior between HTT and papillary thyroid carcinoma.

The dopamine transporter (DAT) and the serotonin transporter (5-HTT) play important roles in methamphetamine (METH) dependence because they are the target of METH action. For this study, the association between the DAT and 5-HTT polymorphisms and METH dependence were investigated for a Chinese-male sample population. The investigated polymorphisms included those of the DAT 3'-variable number tandem repeat, the 5-HTT gene promoter and a 5-HTT variable number tandem repeat polymorphisms. No significant difference was demonstrated for genotype or allele frequency, when comparing METH dependent and control cases for the DAT and the 5-HTT polymorphisms. The findings of this study suggest that these polymorphisms do not play major roles in METH dependence in the Chinese-male population.

2-Hydroxy-6-oxo-6-(2(')-aminophenyl)-hexa-2,4-dienoate hydrolases (CarC enzymes) from two carbazole-degrading bacteria were purified using recombinant Escherichia coli strains with the histidine (His)-tagged purification system. The His-tagged CarC (ht-CarC) enzymes from Pseudomonas resinovorans strain CA10 (ht-CarC(CA10)) and Janthinobacterium sp. strain J3 (ht-CarC(J3)) exhibited hydrolase activity toward 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate as the purified native CarC(CA10) did. ht-CarC(J3) was crystallized in the space group I422 with cell dimensions of a=b=130.3A, c=84.5A in the hexagonal setting, and the crystal structure of ht-CarC(J3) was determined at 1.86A resolution. The final refined model of ht-CarC(J3) yields an R-factor of 21.6%, although the electron-density corresponding to Ile146 to Asn155 was ambiguous in the final model. We compared the known structures of BphD from Rhodococcus sp. strain RHA1 and CumD from Pseudomonas fluorescens strain IP01. The backbone conformation of ht-CarC(J3) was better superimposed with CumD than with BphD(RHA1). The side-chain directions of Arg185 and Trp262 residues in the substrate binding pockets of these enzymes were different among these proteins, suggesting that these residues may take a conformational change during the catalytic cycles.

Protein kinase C (PKC)-dependent regulation of voltage-gated Ca (Ca(v); with alpha(1)beta1Balpha2/delta subunits) channel 2.3 was investigated using phorbol 12-myristate 13-acetate (PMA), or by M(1) muscarinic receptor activation in Xenopus oocytes. The inward Ca(2+)-current with Ba(2+) (I(Ba)) as the charge carrier was potentiated by PMA or acetyl-beta-methylcholine (MCh). The inactivating [I(inact)] and non-inactivating [I(noninact)] components of I(Ba) and the time constant of inactivation tau(inact) were all increased by MCh or PMA. This may be a PKC-dependent action since the effect of MCh and PMA was blocked by Ro-31-8425 or beta-pseudosubstrate. MCh effect was blocked by atropine, guanosine-5'-O-(2-thiodiphosphate) trilithium (GDPbetaS) or U-73122. The effect of MCh but not PMA was blocked by the inhibition of inositol-1,4,5-trisphosphate (IP3) receptors, intracellular Ca(2+) ([Ca(2+)](i)) or the translocation of conventional PKC (cPKC) with heparin, BAPTA and betaC2.4, respectively. While a lower concentration (25 nM) of Ro-31-8425 blocked MCh, a higher concentration (500 nM) of Ro-31-8425 was required to block PMA action. This differential susceptibility of MCh and PMA to heparin, BAPTA, betaC2.4 or Ro-31-8425 is suggestive of the involvement of Ca(2+)-dependent cPKC in MCh action, whereas cPKC and Ca(2+)-independent novel PKC (nPKC) in PMA action. PMA led to additional increase in I(Ba) that was already potentiated by preadministered MCh (1 or 10 microM), leading to the suggestion that differential phosphorylation sites for cPKC and nPKC may be present in the alpha(1)2.3 subunit of Ca(v) 2.3 channels.

Human cytomegalovirus (HCMV) glycoprotein US2 increases the proteasome-mediated degradation of major histocompatibility complex (MHC) class I heavy chain (HC), class II DR-alpha and DM-alpha proteins, and HFE, a nonclassical MHC protein. US2-initiated degradation of MHC proteins apparently involves the recruitment of cellular proteins that participate in a process known as endoplasmic reticulum (ER)-associated degradation. ER-associated degradation is a normal process by which misfolded proteins are recognized and translocated into the cytoplasm for degradation by proteasomes. It has been demonstrated that truncated forms of US2, especially those lacking the cytoplasmic domain (CT), can bind MHC proteins but do not cause their degradation. To further assess how the US2 CT domain interacts with the cellular components of the ER-associated degradation pathway, we constructed chimeric proteins in which the US2 CT domain or the CT and transmembrane (TM) domains replaced those of the HCMV glycoprotein US3. US3 also binds both class I and II proteins but does not cause their degradation. Remarkably, chimeras containing the US2 CT domain caused the degradation of both MHC class I and II proteins although this degradation was less than that by wild-type US2. Therefore, the US2 CT and TM domains can confer on US3 the capacity to degrade MHC proteins. We also analyzed complexes containing MHC proteins and US2, US3, US11, or US3/US2 chimeras for the presence of cdc48/p97 ATPase, a protein that binds polyubiquitinated proteins and likely functions in the extraction of substrates from the ER membrane before the substrates meet proteasomes. p97 ATPase was present in immunoprecipitates containing US2, US11, and two chimeras that included the US2 CT domain, but not in US3 complexes. Therefore, it appears that the CT domain of US2 participates in recruiting p97 ATPase into ER-associated degradation complexes.

Huntington's disease (HD) is one of a number of familial polyglutamine (polyQ) repeat diseases. These neurodegenerative disorders are caused by expression of otherwise unrelated proteins that contain an expansion of a polyQ tract, rendering them toxic to specific subsets of vulnerable neurons. These expanded repeats have an inherent propensity to aggregate; insoluble neuronal nuclear and cytoplasmic polyQ aggregates or inclusions are hallmarks of the disorders [1,2]. In HD, inclusions in diseased brains often precede onset of symptoms, and have been proposed to be involved in pathogenicity [3-5]. Various strategies to block the process of aggregation have been developed in an effort to create drugs that decrease neurotoxicity. A discussion of the effect of antibodies, caspase inhibitors, chemical inhibitors, heat-shock proteins, suppressor peptides and transglutaminase inhibitors upon aggregation and disease is presented.

Hereditary hyperferritinaemia cataract syndrome is an autosomal dominant disorder caused by heterogeneous mutations of the iron regulatory element (IRE) in the ferritin l-chain mRNA. The mutations are rare and fast DNA scanning would facilitate diagnosis. The aim of the study was to compare the analytical performances of two fast DNA scanning techniques: denaturing high-performance liquid chromatography (DHPLC) and double-gradient denaturing gradient gel electrophoresis (DG-DGGE). We analysed the sequence encoding the 5' untranslated flanking region of ferritin l-chain mRNA, which includes an IRE stem loop structure. The two systems unambiguously identified all the 12 accessible mutations in a single run, including the difficult C-G transversions. DHPLC and DG-DGGE identified seven abnormal patterns in DNA samples from 47 subjects with unexplained hyperferritinaemia; all had mutations in the IRE sequence, including two not reported before: C36G and A37G. The scanning of 250 DNA samples from subjects genotyped for HFE led to the identification of four new mutations, all outside the IRE structure: C10T, C16T, C90T and del-T156. We conclude that DHPLC, similar to DG-DGGE, detects all the mutations in the l-ferritin 5'UTR sequence in a single run, and that various mutations occur outside the IRE structure.

We have determined the complete sequence of 951,695 bp from the class I region of H2, the mouse major histocompatibility complex (Mhc) from strain 129/Sv (haplotype bc). The sequence contains 26 genes. The sequence spans from the last 50 kb of the H2-T region, including 2 class I genes and 3 class I pseudogenes, and includes the H2-M region up to Gabbr1. A 500-kb stretch of the H2-M region contains 9 class I genes and 4 pseudogenes, which fall into two subfamilies, M1 and M10, distinct from other mouse class I genes. This M1/M10 class I gene-cluster is separated from the centromeric H2-T and the telomeric H2-M4, -5 and -6 class I genes by "nonclass I genes". Comparison with the corresponding 853-kb region of the human Mhc, which includes the HLA-A region, shows a mosaic of conserved regions of orthologous nonclass I genes separated by regions of species-specific expansion of paralogous Mhc class I genes. The analysis of this mosaic structure illuminates the dynamic evolution of the Mhc class I region among mammals and provides evidence for the framework hypothesis.

Endothelial cell production of anticoagulant heparan sulfate (HS(act)) is controlled by the Hs3st1 gene, which encodes the rate-limiting enzyme heparan sulfate 3-O-sulfotransferase-1 (3-OST-1). In vitro, HS(act) dramatically enhances the neutralization of coagulation proteases by antithrombin. The in vivo role of HS(act) was evaluated by generating Hs3st1(-/-) knockout mice. Hs3st1(-/-) animals were devoid of 3-OST-1 enzyme activity in plasma and tissue extracts. Nulls showed dramatic reductions in tissue levels of HS(act) but maintained wild-type levels of tissue fibrin accumulation under both normoxic and hypoxic conditions. Given that vascular HS(act) predominantly occurs in the subendothelial matrix, mice were subjected to a carotid artery injury assay in which ferric chloride administration induces de-endothelialization and occlusive thrombosis. Hs3st1(-/-) and Hs3st1(+/+) mice yielded indistinguishable occlusion times and comparable levels of thrombin.antithrombin complexes. Thus, Hs3st1(-/-) mice did not show an obvious procoagulant phenotype. Instead, Hs3st1(-/-) mice exhibited genetic background-specific lethality and intrauterine growth retardation, without evidence of a gross coagulopathy. Our results demonstrate that the 3-OST-1 enzyme produces the majority of tissue HS(act). Surprisingly, this bulk of HS(act) is not essential for normal hemostasis in mice. Instead, 3-OST-1-deficient mice exhibited unanticipated phenotypes suggesting that HS(act) or additional 3-OST-1-derived structures may serve alternate biologic roles.

Bronchial asthma is characterized by massive infiltration of eosinophils and airway hyperreactivity (AHR), which are caused by overproduction of Th2 cytokines (IL-4, IL-5, and IL-13) by allergen-specific T cells. We recently demonstrated a critical contribution of OX40 ligand (OX40L) to the development of Th2-mediated experimental leishmaniasis. In this study, we have examined the role of OX40L in the development of Th2-mediated pulmonary inflammation by utilizing OX40L-deficient mice and a neutralizing anti-OX40L mAb in a murine model of asthma. Sensitization and airway challenge with ovalbumin in wild-type BALB/c mice induced a typical allergic asthma characterized by AHR, accumulation of eosinophils, increased mucus production, and high levels of Th2 cytokines in the lung. All these asthmatic responses were not induced in OX40L-deficient BALB/c mice. Administration of neutralizing anti-OX40L mAb in wild-type BALB/c mice during the sensitization period also abolished the induction of asthmatic responses. In contrast, administration of anti-OX40L mAb during the challenge period did not inhibit the asthmatic responses. These results indicate a critical role for OX40L in the induction phase, which leads to the development of pathogenic Th2 cells, but not in the effector phase, which includes migration and activation of pathogenic Th2 cells in the lung.

<h4>Aim</h4>To appraise the correlation of mutation and methylation of hMSH1 with microsatellite instability (MSI) in gastric cancers.<h4>Methods</h4>Mutation of hMLH1 was detected by Two-dimensional electrophoresis (Two-D) and DNA sequencing; Methylation of hMLH1 promoter was measured with methylation-specific PCR; MSI was analyzed by PCR-based methods.<h4>Results</h4>Sixty-eight cases of sporadic gastric carcinoma were studied for mutation and methylation of hMLH1 promoter and MSI. Three mutations were found, two of them were caused by a single bp substitution and one was caused by a 2 bp substitution, which displayed similar Two-D band pattern. Methylation of hMLH1 promoter was detected in 11(16.2 %) gastric cancer. By using five MSI markers, MSI in at least one locus was detected in 17/68(25 %) of the tumors analyzed. Three hMLH1 mutations were all detected in MSI-H (>=2 loci, n=8), but no mutation was found in MSI-L (only one locus, n=9) or MSS (tumor lacking MSI or stable, n=51). Methylation frequency of hMLH1 in MSI-H (87.5 %, 7/8) was significantly higher than that in MSI-L (11.1 %, 1/9) or MSS (5.9 %, 3/51) (P<0.01-0.001), but no difference was found between MSI-L and MSS (P>0.05).<h4>Conclusion</h4>Both mutation and methylation of hMLH1 are involved in the MSI pathway but not related to the LOH pathway in gastric carcinogenesis.

<h4>Aims</h4>To assess the level of undertreatment of hypercholesterolaemia in the general population, taking intra-person variability in serum cholesterol concentrations into account, and to identify determinants of undertreatment of hypercholesterolaemia.<h4>Methods</h4>In this cross-sectional study, data from two population-based surveys on cardiovascular disease risk factors conducted between 1987 and 1997 in the Netherlands were used. For all 64 757 respondents aged 20-59 years, treatment eligibility for lipid-lowering drug use was established according to the Dutch Cholesterol Consensus. Multivariate logistic models were used to identify determinants of undertreatment.<h4>Results</h4>During the study period, 56.8% of the study population had undesirable cholesterol concentrations (serum total cholesterol> 5 mmol l(-1)) and 5.5% of those were eligible for pharmacological treatment based on their absolute risk of coronary heart disease. Of those eligible for pharmacological treatment, 16.3% were treated, and 19.6% of those treated had their serum total cholesterol concentration controlled. Only 3.2% of those eligible for pharmacological treatment were both treated and controlled. We identified several determinants for undertreatment, e.g. male gender and younger age for primary prevention and female gender and older age for secondary prevention. Treatment has improved slightly in more recent years.<h4>Conclusions</h4>Over 95% of the population eligible for the pharmacological treatment of hypercholesterolaemia was either untreated or was uncontrolled. To decrease undertreatment, identification of high-risk patients should be increased. Those who are treated with lipid-lowering medication could further benefit from more aggressive treatment, especially with statins.

<h4>Background and objectives</h4>Hereditary hemochromatosis is a recessive condition characterized by iron accumulation in several organs, followed by organ damage and failure. The disorder is prevalently due to C282Y and H63D mutations in the HFE gene, but additional HFE and TFR2 mutations have been reported. Early iron overload may be assessed by biochemical parameters such as increased transferrin saturation and serum ferritin.<h4>Design and methods</h4>Taking advantage of the collection of 178 DNA samples selected for increased transferrin saturation (>50% in males and >45% in females) from a previous large scale screening of Italian blood donors, we simultaneously assessed the presence of 14 hemochromatosis-associated molecular defects (11 of HFE and 3 of TFR2) by a reverse hybridization-based strip assay.<h4>Results</h4>In the series studied the overall C282Y allele frequency was 9% and that of the H63D and S65C was 22.2% and 1.4%, respectively. One rare HFE allele (E168Q), but no TFR2 mutation was detected. When checked at a second examination, transferrin saturation was significantly higher in C282Y homozygotes, H63D/ C282Y compound heterozygotes and H63D homozygotes as compared to wild-type subjects (p<0.05).<h4>Interpretation and conclusions</h4>Our results confirm previous findings on C282Y and H63D mutations in Italy, show that the C282Y allele frequency is enriched in samples selected for altered iron parameters, and that a few rare genotypes are present in Northern Italy. None of the known TFR2 mutations was identified in this series confirming the preliminary indication of their rare occurrence. Subjects with hemochromatosis-associated genotypes show a persistently higher mean transferrin saturation than do those with wild type genotypes.

The nasopharyngeal Haemophilus influenzae flora of healthy children under the age of 3 years attending day care centers in three distinct French geographic areas was analyzed by sampling during two periods, spring 1999 (May and June) and fall 1999 (November and December). The average carrier rate among 1,683 children was 40.9%. The prevalence of capsulated H. influenzae carriers was 0.4% for type f and 0.6% for type e. No type b strains were found among these children, of whom 98.5% had received one or more doses of anti-Haemophilus b vaccine. Among the strains, 44.5% were TEM-type beta-lactamase producers and nine (1.3%) were beta-lactamase-negative ampicillin-resistant strains. Pulsed-field gel electrophoresis restriction patterns showed a large diversity with 366 SmaI patterns from 663 strains. Among the strains isolated during a given period, 33% were isolated simultaneously in more than one area. In each area, depending on the sampling period, 68 to 72% of the strains had new pulsotypes and persistence of 28 to 32% of the strains was noted. For the 297 beta-lactamase-producing strains, 194 patterns were found. The genomic diversity of these strains was comparable to that of the whole set of strains and does not suggest a clonal diffusion. Among the beta-lactamase-producing strains isolated in November and December, depending on the area, 66 to 73% had new pulsotypes with persistence of only 27 to 33% of the strains. In any given geographic area, colonization by H. influenzae appears to be a dynamic process involving a high degree of genomic heterogeneity among the noncapsulated colonizing strains.

We show that Neurospora crassa has a single histone H1 gene, hH1, which encodes a typical linker histone with highly basic N- and C-terminal tails and a central globular domain. A green fluorescent protein-tagged histone H1 chimeric protein was localized exclusively to nuclei. Mutation of hH1 by repeat-induced point mutation (RIP) did not result in detectable defects in morphology, DNA methylation, mutagen sensitivity, DNA repair, fertility, RIP, chromosome pairing, or chromosome segregation. Nevertheless, hH1 mutants had mycelial elongation rates that were lower than normal on all tested carbon sources. This slow linear growth phenotype, however, was less evident on medium containing ethanol. The pyruvate decarboxylase gene, cfp, was abnormally derepressed in hH1 mutants on ethanol-containing medium. This derepression was also found when an ectopically integrated fusion of the cfp gene promoter to the reporter gene hph was analyzed. Thus, Neurospora histone H1 is required for the proper regulation of cfp, a gene with a key role in the respiratory-fermentative pathway.

The TUC (TOAD-64/Ulip/CRMP) proteins are homologs of UNC-33, a protein that is required for axon extension and guidance in Caenorhabditis elegans. The TUC proteins are expressed in newly born neurons in the developing nervous system and have been implicated in semaphorin signaling and neuronal polarity. Here, we identify several new variants of the TUC family, each of which is expressed during distinct periods of neural development. We cloned and characterized TUC-4b, a variant of TUC-4a that includes a unique N-terminal extension. The functional relevance of this N-terminal domain is demonstrated by the finding that overexpression of TUC-4b, but not TUC-4a, results in increased neurite length and branching. Furthermore, whereas TUC-4a is expressed throughout life, TUC-4b is expressed exclusively during embryonic development. TUC-4b is localized to SV2 (synaptic vesicle protein 2)-positive vesicles in the central domain of the growth cone, suggesting a potential role in growth cone vesicle transport. Furthermore, TUC-4b interacts with the SH3A (Src homology 3A) domain of intersectin, an endocytic-exocytic adaptor protein. Together, these data suggest that TUC-4b can regulate neurite extension and branching through a mechanism that may involve membrane transport in the growth cone.

Topoisomerase IIalpha (topoIIalpha) and 13S condensin are both required for mitotic chromosome assembly. Here we show that they constitute the two main components of the chromosomal scaffold on histone-depleted chromosomes. The structural stability and chromosomal shape of the scaffolding toward harsh extraction procedures are shown to be mediated by ATP or its nonhydrolyzable analogs, but not ADP. TopoIIalpha and 13S condensin components immunolocalize to a radially restricted, longitudinal scaffolding in native-like chromosomes. Double staining for topoIIalpha and condensin generates a barber pole appearance of the scaffolding, where topoIIalpha- and condensin-enriched "beads" alternate; this structure appears to be generated by two juxtaposed, or coiled, chains. Cell cycle studies establish that 13S condensin appears not to be involved in the assembly of prophase chromatids; they lack this complex but contain a topoIIalpha-defined (-mediated?) scaffolding. Condensin associates only during the pro- to metaphase transition. This two-step assembly process is proposed to generate the barber pole appearance of the native-like scaffolding.

<h4>Background</h4>DNA-based HFE gene testing can confirm hereditary hemochromatosis in most people of Northern European descent. However, liver biopsy is important to detect cirrhosis.<h4>Objective</h4>To develop noninvasive criteria to predict the presence or absence of advanced hepatic fibrosis or cirrhosis in Americans with hemochromatosis.<h4>Design</h4>Cross-sectional study.<h4>Setting</h4>Six tertiary care referral clinics.<h4>Patients</h4>182 patients with phenotypically defined hemochromatosis.<h4>Measurements</h4>Liver histopathology and serum ferritin, aspartate aminotransferase, and alanine aminotransferase levels. Multivariate logistic regression analysis was used to examine factors associated with cirrhosis (defined as bridging fibrosis or unequivocal cirrhosis on biopsy).<h4>Results</h4>Cirrhosis was present in 40 of 182 (22%) patients in the overall group and in 35 of 147 (24%) of C282Y homozygotes. Only 1 of 93 patients with a serum ferritin level less than 1000 microg/L had cirrhosis compared with 39 of 89 patients with serum ferritin levels greater than 1000 microg/L (P < 0.001). No C282Y homozygotes or C282Y/H63D compound heterozygotes with serum ferritin levels less than 1000 microg/L had cirrhosis. Elevated serum aminotransferase levels (P = 0.001) and serum ferritin levels greater than 1000 microg/L (P = 0.001), but not age older than 40 years (P = 0.2), were independently associated with cirrhosis. In a multivariate model, the probability of cirrhosis was 7.4% among patients with serum ferritin levels less than 1000 microg/L compared with 72% among patients with serum ferritin levels greater than 1000 microg/L after adjustment for age and elevated serum liver enzyme levels.<h4>Conclusions</h4>Patients with hemochromatosis and serum ferritin levels less than 1000 microg/L are unlikely to have cirrhosis. Liver biopsy to screen for cirrhosis may be unnecessary in such patients, regardless of age or serum liver enzyme levels.

<h4>Context</h4>Serotonin transporters (5-HTT) are regarded as one of the major therapeutic targets of antidepressants. However, there have only been a few studies about 5-HTT occupancy, and in particular, data concerning classical antidepressants are still limited.<h4>Objective</h4>To investigate the relationship between 5-HTT occupancy and a wide range of antidepressant dosing protocols.<h4>Design, setting, and participants</h4>Antidepressant occupancies of 5-HTT were measured using positron emission tomography (PET) with [11C](+)McN5652. Twenty-seven healthy volunteers were measured with and without pretreatment with single low doses of antidepressants, and long-term doses were evaluated in 10 patients. Scan data were collected between December 12, 1995, and August 7, 2002, and data were analyzed during the 2001-2002 period at the National Institute of Radiological Sciences (Chiba, Japan). Intervention Four different doses of clomipramine hydrochloride (5-50 mg) and 3 different doses of fluvoxamine maleate (12.5-50 mg) were used for single administration. Long-term doses were 20 to 250 mg per day for clomipramine hydrochloride, and 25 to 200 mg per day for fluvoxamine maleate. Main Outcome Measure Occupancies in the thalamus were calculated using the individual baseline of [11C](+)McN5652 for single-dose studies and 2 long-term-dose studies, and the mean value of healthy volunteers as the baseline for 8 long-term-dose studies. The average data from inactive enantiomers [11C](-)McN5652 were used for the estimation of nonspecific binding.<h4>Results</h4>Occupancy of 5-HTT increased in a curvilinear manner. Even 10 mg of clomipramine hydrochloride showed approximately 80% occupancy, which was comparable with that of 50 mg of fluvoxamine maleate. Estimated median effective dose (ED50) of clomipramine hydrochloride was 2.67 mg for oral dose and 1.42 ng/mL for plasma concentration; those of fluvoxamine maleate were 18.6 mg and 4.19 ng/mL, respectively.<h4>Conclusions</h4>Clinical doses of clomipramine and fluvoxamine occupied approximately 80% of 5-HTT, and dose escalation would have minimal effect on 5-HTT blockade. Ten milligrams of clomipramine hydrochloride was enough to occupy 80% of 5-HTT in vivo.

Zona pellucida (ZP)-induced acrosome reaction in sperm is a required step for mammalian fertilization. However, the precise mechanism of the acrosome reaction remains unclear. We previously reported that PLCdelta4 is involved in the ZP-induced acrosome reaction in mouse sperm. Here we have monitored Ca2+ responses in single sperm, and we report that the [Ca2+]i increase in response to ZP, which is essential for driving the acrosome reaction in vivo, is absent in PLCdelta4-/- sperm. Progesterone, another physiological inducer of the acrosome reaction, failed to induce sustained [Ca2+]i increases in PLCdelta4-/- sperm, and consequently the acrosome reaction was partially inhibited. In addition, we observed oscillatory [Ca2+]i increases in wild-type sperm in response to these acrosome inducers. Calcium imaging studies revealed that the [Ca2+]i increases induced by exposure to ZP and progesterone started at different sites within the sperm head, indicating that these agonists induce the acrosome reaction via different Ca2+ mechanisms. Furthermore, store-operated channel (SOC) activity was severely impaired in PLCdelta4-/- sperm. These results indicate that PLCdelta4 is an important enzyme for intracellular [Ca2+]i mobilization in the ZP-induced acrosome reaction and for sustained [Ca2+]i increases through SOC induced by ZP and progesterone in sperm.

The Pulmonary adenoma resistance 2 (Par2) locus of the BALB/cByJ mouse, located within 0.5 cM of chromosome 18, is responsible for reducing the mean multiplicity of urethane-induced lung tumors relative to those in C57BL/6J, A/J and C3H/HeJ mice. Thus, BALB/B6-Par2 congenic strain genetically identical to BALB/cByJ except carrying C57BL/6J Par2 alleles develops seven times more tumors than BALB/cByJ. To gain clues for identification of Par2 candidate genes, we analysed lung tumorigenesis in BALB/cByJ<-->BALB.B6-Par2 chimeric animals. Of 100 tumors induced by urethane in 16 chimeras, 82 originated from BALB.B6-Par2 cells, indicating the Par2 phenotype to be cell-autonomous. In addition, the BALB.B6-Par2- and BALB/cByJ-derived tumors were similar in mean size, implying that the phenotype is primarily expressed during initiation rather than in the promotion stage of carcinogenesis. Given these results, we surveyed a comprehensive mouse genome database and physically mapped Par2 within a 2.3 Mbp segment containing three known genes, Poli, Mbd2 and Dcc. Among those, the Poli seemed to be the most reasonable Par2 candidate, since it encodes an extremely error-prone DNA polymerase preferentially incorporating G or T opposite template T in vitro, reminiscent of the Kras2 activation because of an A to G or T point mutation within codon 61 with which most urethane-induced lung tumors are initiated. Indeed, our sequencing of Poli cDNAs from BALB/cByJ, C57BL/6J, A/J and C3H/HeJ lungs revealed 21 BALB/cByJ-specific single-nucleotide polymorphisms in the coding region accompanied by seven amino-acid substitutions and an elevated frequency of alternative splicing, while no polymorphisms associated with tumor susceptibility were found for either Mbd2 or Dcc. Notably, we obtained evidence that BALB/cByJ Par2 alleles may selectively decrease the frequency of Kras2-mutated tumors compared with C57BL/6J alleles. Consequently, the Poli is an intriguing Par2 candidate clearly deserving further evaluation.

Relatively little has been published on the pharmacology of R-type and T-type Ca(2+) channels. Here, whole-cell Ca(2+) channel currents were recorded from human embryonic kidney 293 cell-lines transfected with either alpha1E subunits (R-type currents) or alpha1G or alpha1I subunits (T-type currents). R-type currents were inhibited by sipatrigine and the related compound 202W92 (R-(-)-2,4-diamino-6-(fluromethyl)-5-(2,3,5-trichlorophenyl)pyrimidine) with IC(50) 10 and 56 microM, respectively. A therapeutic concentration of lamotrigine (10 microM) inhibited R-type currents (30%) but was without effect on alpha1I-mediated T-type currents. Lamotrigine was also a weak inhibitor of T-type currents mediated by alpha1G subunits (<10% inhibition by 100 microM).

Primary haemochromatosis is an autosomal recessive disorder with a high prevalence (1 in 200-400) among North-Europeans. Approximately 64-100% of patients with primary haemochromatosis are homozygous for a missense mutation that alters a major-histocompatibility-complex class I-like protein designated HFE (from 'haemochromatosis'). This predominant mutation is a substitution of cysteine to tyrosine at amino acid residue 282 (Cys282Tyr) in the alpha 3-domain of the HFE protein. This mutation in the HFE protein in the intestinal crypt cells is supposed to lead to up-regulation of iron transporters for dietary iron in the mucosal cells at the tip of the duodenal villi. As a consequence, an excess of absorbed iron accumulates in the major organs of the HFE-mutated patients, leading to multi-organ dysfunction. A second mutation, His63Asp, in the HFE-gene is enriched in primary haemochromatosis patients who are concomitantly heterozygous for the Cys282Tyr-mutation. However, its role in the pathophysiology of primary haemochromatosis is not yet clear. A small subgroup of patients with primary haemochromatosis do not have mutations in HFE; for some of them the genetic basis has been determined. Homozygosity for the Cys282Tyr-mutation plus biochemical and clinical evidence of iron overload renders the diagnosis of HFE-related primary haemochromatosis indisputable. In other cases, liver biopsy remains the gold standard for the diagnosis of primary haemochromatosis. First-degree relatives of index patients should be screened for the disease.

Mutations in the class I-like Major Histocompatibility Complex gene HFE are associated with hereditary hemochromatosis (HH), a disorder caused by excessive iron uptake. Three common mutations have been found: C282Y, H63D, and S65C. Moreover, several studies have suggested that HFE mutations may be involved in several age-related chronic diseases such as Alzheimer's disease (AD) and coronary heart disease, but apparently paradoxically also with longevity. In particular, in AD, patients carrying the H63D allele have been suggested to have a mean age at onset of 72 vs. 77 years for those who were homozygous for the wild-type allele. Thus, it seems that H63D mutations may anticipate sporadic AD clinical presentation in susceptible individuals. In the present study, we analysed the HFE genotype in 123 patients with sporadic AD and 152 age-matched controls from Northern Italy. Samples were typed for C282Y, H63D and S65C alleles using the polymerase chain reaction and sequence specific primers. No significant differences were observed in frequencies of the different alleles between controls and AD both for the whole group and when the data were analysed according to gender. In addition, we failed to observe any difference in the age at onset between patients carrying the mutant HFE-H63D allele and those homozygous for the wild-type allele, either in men or women. Also taking into account the presence or absence of the APOE-epsilon 4 allele, no significant differences were observed between carriers of the mutant HFE-H63D allele and those homozygous for the wild-type allele. Thus, our study does not support the suggestion that H63D mutations may anticipate sporadic AD clinical presentation in susceptible individuals.

Hereditary hemochromatosis is an HLA-linked inherited disease characterised by inappropriately high absorption of iron by the gastrointestinal mucosa. The cysteine-to-tyrosine substitution at codon 282 of the HFE encoding gene sequence is responsible for the disease, although other variants, as H63D and S65C, may modify the affinity of the protein for transferrin receptors. We have recently reported that C282Y mutation is significantly increased in very old (>90 years) Sicilian women, suggesting a role in attainment of longevity. In addition, an increase of H63D polymorphism was also observed in these women but the difference was not significant. To validate and extend these results we investigated the distribution of these three common HFE gene mutations in Sardinian centenarians and controls. DNA samples, obtained from 61 controls and 57 Centenarians, were typed for HFE polymorphisms using sequence specific primers. Among the controls, none was heterozygous for the C282Y mutation, 15 were heterozygous for H63D mutation and one for S65C. Among the centenarians, none was heterozygous for the C282Y mutation whereas 25 were heterozygous for H63D mutation and four for the S65C mutation. No significant differences were observed in frequencies of the different alleles between young and centenarians both on the whole and when the data were analysed according to gender. However, there was a trend for an increased frequency of H63D allele in centenarian women (24 vs. 17%, i.e. 19/80 vs. 13/78). It is noteworthy that the cumulative frequency of H63D mutations in Centenarian and very old women from Sardinia and Sicily is 22 vs. 11%, i.e. 30/136 vs. 23/210, P=0.008. These findings are consistent with the hypothesis that there may be a survival difference for centenarian women, among carriers and non-carriers of alleles involved in iron sparing.

The identities of ligands interacting with protein tyrosine phosphatase (PTP) receptors to regulate neurite outgrowth remain mainly unknown. Analysis of cDNA and genomic clones encoding the rat leukocyte common antigen-related (LAR) PTP receptor predicted a small, approximately 11 kDa ectodomain isoform, designated LARFN5C, containing a novel N terminal followed by a C-terminal segment of the LAR fifth fibronectin type III domain. RT-PCR and Northern blot analysis confirmed the presence of LARFN5C transcripts in brain. Transfection of COS cells with LARFN5C-Fc cDNA resulted in expression of the predicted protein, and Western blot analysis verified expression of approximately 11 kDa LARFN5C protein in vivo and its developmental regulation. Beads coated with rLARFN5C demonstrated aggregation consistent with homophilic binding, and pull-down and immunoprecipitation assays demonstrated that rLARFN5C associates with the LAR receptor. rLARFN5C binding to COS cells was dependent on LAR expression, and rLARFN5C binding to LAR +/+ hippocampal neurons was fivefold greater than that found by using LAR-deficient (-/-) neurons. Substratum-bound rLARFN5C had potent neurite-promoting effects on LAR +/+ neurons, with a fivefold loss in potency with the use of LAR -/- neurons. rLARFN5C in solution at low nanomolar concentrations inhibited neurite outgrowth induced by substratum-bound rLARFN5C, consistent with receptor-based function. These studies suggest that a small ectodomain isoform of a PTP receptor can function as a ligand for the same receptor to promote neurite outgrowth.

<h4>Objective</h4>We investigated the relation between hepatocellular carcinoma (HCC) etiology and biological and clinical parameters indicative of severity of liver disease and/or tumor characteristics and patient survival.<h4>Methods</h4>We prospectively recruited 384 patients (82.3% male) with first diagnosis of HCC from 1995 to 1998 in Brescia, Italy. Etiology was assessed by interviewing patients regarding their history of alcohol intake and by testing sera for hepatitis B surface antigen and anti-hepatitis C virus (HCV) antibodies and HCV RNA.<h4>Results</h4>Heavy alcohol intake (>60 g of ethanol per day for at least 1 decade) was found in 33.1% of cases, hepatitis B virus (HBV) infection in 9.4%, HCV in 19.8%, hemochromatosis in 1.3%, alcohol and HBV in 12.0%, alcohol and HCV in 16.1%, HBV and HCV in 3.1%, and no factor in 5.2%. Patients with HBV infection with or without heavy alcohol intake were significantly younger than the others (61.7 vs 64.7 yr, p < 0.001). The proportion of males was significantly higher in patients with heavy alcohol intake alone than in the other patient groups (93.7% vs 77.3%, p < 0.001). Among patients with HCV infection with or without heavy alcohol intake, fewer patients had maximum tumor diameter > 5 cm than the others (12% vs 29.1%, p < 0.001). Eighty patients (20.8%) were alive at the end of follow-up (median survival, 17.7 months), and no differences were observed in survival rates by HCC risk factor.<h4>Conclusions</h4>Although some differences were observed in severity of liver disease or tumor characteristics according to etiology, patient survival was not influenced by HCC etiology.

To elucidate adsorption mechanism of alizarin red S (ARS), which is often used for staining bones in histology, adsorption of ARS on hydroxyapatite, Ca10(PO4)6(OH)2 (HAP), was investigated by a batch method, compared with alizarin, phenols, and benzenesulfonates. We found that ionized 1-, 2-OH groups (1-, 2-O(-)) of ARS can be electrostatically bound to Ca2+ on HAP, but that the 3-SO3(-) group of ARS hardly participates in adsorption on HAP. ARS-adsorbed HAP (ARS-HAP) in dark reddish violet was also prepared and analyzed by FT-IR microspectroscopy to gain structural information on bonding between ARS and HAP. The obtained spectrum, which was converted to difference spectra, indicated a single band of nu(C=O) at 1627 cm(-1) and two types of symmetric C=O stretching bands of nu(s)(C=O) + nu(C=C) at 1345 cm(-1) and nu(s)(C=O) + delta(O-C=C) at 1272 cm(-1). These bands imply the existence of a salt form in ARS-HAP via 1-, 2-OH groups of ARS. As a result of the existence of a chelate form in ARS-HAP via 1-OH and 9-C=O groups of ARS, two bands of nu(C=C) + nu(C=O) at 1572 cm(-1) and nu(C=O) + nu(C=C) at 1537 cm(-1) were also observed. In addition, ARS was almost desorbed from colored ARS-HAP at 50 degrees C by using neutral phosphate buffer to recover slightly pale pinkish HAP, or De-ARS-HAP. The desorbed ARS belongs to ARS previously adsorbed on HAP by salt formation, while the remaining color on De-ARS-HAP indicates ARS still adsorbed on HAP by chelate formation. Consequently, we elucidated two adsorption mechanisms of ARS on HAP: The major adsorption is salt formation made up with 1-, 2-O(-) of ARS and Ca2+ on HAP, and the minor adsorption is chelate formation made up with 1-O(-) and 9-C=O of ARS and Ca2+ on HAP.

HFE was discovered as the hereditary hemochromatosis (HH) gene. It is located on chromosome 6 (6p21.3), 4Mb telomeric to the HLA-A locus, and its product has a structure similar to MHC class I molecules. HFE encodes two frequent mutations: C282Y and H63D. One of these (C282Y) is present in a large proportion of Caucasian HH patients. HFE has a tissue distribution compatible with a role in iron absorption (intestine), recycling (macrophages) and transport to the fetus (placenta).

Although HLA-DQ genes are the major celiac disease (CD) susceptibility genes, results from Finnish families suggest that not all DQ2-encoding haplotypes confer equal susceptibility to CD, implying the effect of other gene(s) in the HLA region. The aim of the present work was to extend and confirm the aforementioned results in a southern European population ( Italian) and to better localize the additional risk factor/s. The association of nine loci spanning the HLA region from DR to HFE, 4.5-Mb telomeric of HLA-A, was tested. The analysis was performed by comparing marker frequencies in DR3-DQ2 haplotypes transmitted and non-transmitted to the affected offspring in 156 Italian CD families selected for having at least one DR3-positive parent. The same analysis was performed independently in 101 Finnish CD families selected with the same criteria. Three alleles, MICA-A5.1, MICB-CA24 and MIB-350, all characteristic of the B8-DR3 extended haplotype, showed a significantly increased frequency in DR3 transmitted haplotypes in the Italian families. DR3 haplotypes carrying the combination of these alleles conferred an approximate fourfold increased CD risk. B8-DR3 transmitted haplotypes were significantly more conserved telomerically down to the MIC-Class I region. Similar results were seen in the Finnish families. The major conclusion that holds true in both populations is that, while DQ2 is an absolute requirement for the development of CD, the presence of an additional genetic factor within the MIC-Class I region confers an approximate 4-fold increased risk of the disease.

Multiple genetic changes including activation of proto-oncogenes and inactivation of tumor suppressor gene are involved in the development of human ovarian cancer. We describe such genetic alterations with specific reference to histological subtypes. K-ras activation is specific for mucinous tumors including adenomas. Borderline tumors and carcinomas, suggesting that K-ras activation may be associated with the mucinous differentiation rather than malignant transformation. Inactivation of p53 is detected in 30-40% of ovarian carcinoma. Mutations are more frequently observed in serous carcinomas, but not found in adenomas or rarely found in borderline tumors, suggesting that p53 mutations may be directly involved in malignant transformation. TGFbeta-2 mutations are found in 50% of endometrioid carcinoma, but rarely in other type. Loss of DCC mRNA expression is found in 50% of serous carcinomas but less frequently in other type. Loss of DCC expression is rare in borderline tumors and adenomas, suggesting that inactivation of DCC may be directly involved in malignant transformation. Microsatellite instability (MI) is found in 17% of ovarian carcinomas, and is frequently found in endometrioid carcinomas. Although inactivation of p16 by point mutation or deletion is rare, p16 inactivation by loss of expression is relatively common.

<h4>Objective</h4>The purpose of this study was to explore the relationship between the loss of the gene deleted in colorectal carcinoma (DCC) gene expression in ovarian carcinoma and the transformation, progression of the tumor and its clinicopathological factors.<h4>Methods</h4>DCC gene mRNA expression were examined by reverse transcription polymerase chain reaction (RT-PCR) in 34 malignant, 10 benign and 10 normal ovarian samples. To clarify the expression of DCC gene by the DNA cloning and the DNA sequencing analysis in normal ovarian sample.<h4>Results</h4>The expression of DCC gene was lost in no normal ovarian tissues, in 2 (2/10) benign lesions, while the loss of DCC gene expression was found in 19 (19/34, 56%) carcinomas (P < 0.05). Similar findings were also noted when subdivision was into serous (14/18) and mucinous (4/11) categories (P < 0.05). The percentage of the loss of DCC gene expression in stage III - IV was 68%, in stage I - II was 22%, as well as 42% in grade I - II and 90% in grade III. 61% of the carcinoma with metastasis presented the loss of DCC gene expression, then there was 33% in which without metastasis.<h4>Conclusions</h4>In ovarian carcinomas, loss of DCC gene expression was significantly related to the serous phenotype, a high histological grade and a more advanced stage. The results indicated that loss of DCC gene expression may play an important role in ovarian carcinomas and its progression.

A case of a 51-year-old male with previously unsuspected genetic hemochromatosis (GH) associated with hepatocellular carcinoma (HCC), for which MR imaging was useful in diagnosis, is presented. Contrast-enhanced ultrasonography depicted a mass in segment 8 of the liver and a liver tumor was suspected. The mass could not be revealed on unenhanced or enhanced CT examination. MR imaging with unenhanced T1-weighted FLASH and T2-weighted gradient echo sequences clearly depicted the tumor; the tumor appeared hyperintense against a background of low hepatic signal intensity caused by iron overload. The iron-overloaded liver provided a natural source of paramagnetic contrast for detection of HCC. Studies are required to further assess the accuracy of these sequences in detection of HCC in patients with GH.