Gene Literature Dashboard

Development of both dendrites and axons is important for the formation of neuronal circuits, because dendrites receive information and the axon is responsible for sending signals. In the past decade, extensive studies have revealed many molecules underlying axonal outgrowth and pathfinding. In contrast, much less is known about the molecular mechanisms that control dendrite development. Here we report the identification of an evolutionarily conserved Ig superfamily member, dendrite arborization and synapse maturation 1 (Dasm1), which plays a critical role in dendrite development. Dasm1 contains five Ig domains and two fibronectin III domains in the extracellular N terminus, a single transmembrane domain, and an intracellular C-terminal tail with a type I PDZ domain binding motif at the end. It is highly expressed in the brain and localized at the dendrites. Suppression of Dasm1 expression in hippocampal neurons via RNA interference or expression of Dasm1 without its cytoplasmic tail specifically impairs dendrite, but not axon, outgrowth. Together with its orthologues in other species, Dasm1 defines a family of molecules likely involved specifically in dendrite arborization.

Extensive investigation into the molecular basis of iron overload disorders has provided new insights into the complexity of iron metabolism and related cellular pathways. The possible involvement of genes affecting iron homeostasis, including HFE, SLC40A1, HAMP and CYBRD1, was investigated in individuals who were referred for confirmation or exclusion of a diagnosis of haemochromatosis, but who tested negative or were heterozygous for the causative HFE mutation, C282Y. Denaturing high performance liquid chromatography analysis of these genes revealed a unique spectrum of mutations in the South African study population, including 67 unrelated patients and 70 population-matched controls. Two novel CYBRD1 gene mutations, R226H and IVS1-4C-->G, were identified in 11% of South African Caucasian patient referrals. We identified a novel D270V mutation in the SLC40A1 gene in a Black South African female with iron overload. These mutations were absent in the control population. In Africans with iron overload not related to the HFE gene, the possible involvement of the SLC40A1 and CYBRD1 genes was demonstrated for the first time. This study confirms the genetic heterogeneity of haemochromatosis and highlights the significance of CYBRD1 mutations in relation to iron overload.

Gramicidin A was studied by continuous wave electron spin resonance (CW-ESR) and by double-quantum coherence electron spin resonance (DQC-ESR) in several lipid membranes (using samples that were macroscopically aligned by isopotential spin-dry ultracentrifugation) and vesicles. As a reporter group, the nitroxide spin-label was attached at the C-terminus yielding the spin-labeled product (GAsl). ESR spectra of aligned membranes containing GAsl show strong orientation dependence. In DPPC and DSPC membranes at room temperature the spectral shape is consistent with high ordering, which, in conjunction with the observed high polarity of the environment of the nitroxide, is interpreted in terms of the nitroxide moiety being close to the membrane surface. In contrast, spectra of GAsl in DMPC membranes indicate deeper embedding and tilt of the NO group. The GAsl spectrum in the DPPC membrane at 35 degrees C (the gel to Pbeta phase transition) exhibits sharp changes, and above this temperature becomes similar to that of DMPC. The dipolar spectrum from DQC-ESR clearly indicates the presence of pairs in DMPC membranes. This is not the case for DPPC, rapidly frozen from the gel phase; however, there are hints of aggregation. The interspin distance in the pairs is 30.9 A, in good agreement with estimates for the head-to-head GAsl dimer (the channel-forming conformation), which matches the hydrophobic thickness of the DMPC bilayer. Both DPPC and DSPC, apparently as a result of hydrophobic mismatch between the dimer length and bilayer thickness, do not favor the channel formation in the gel phase. In the Pbeta and Lalpha phases of DPPC (above 35 degrees C) the channel dimer forms, as evidenced by the DQC-ESR dipolar spectrum after rapid freezing. It is associated with a lateral expansion of lipid molecules and a concomitant decrease in bilayer thickness, which reduces the hydrophobic mismatch. A comparison with studies of dimer formation by other physical techniques indicates the desirability of using low concentrations of GA (approximately 0.4-1 mol %) accessible to the ESR methods employed in the study, since this yields non-interacting dimer channels.

Chromosomal rearrangements resulting in gene fusions are frequently involved in carcinogenesis. Here, we describe a semiautomatic procedure for identifying fusion gene transcripts by using publicly available mRNA and EST databases. With this procedure, we have identified 96 transcript sequences that are derived from 60 known fusion genes. Also, 47 or more additional sequences appear to be derived from 20 or more previously unknown putative fusion genes. We have experimentally verified the presence of a previously unknown IRA1/RGS17 fusion in the breast cancer cell line MCF7. The fusion gene encodes the full-length RGS17 protein, a regulator of G protein-coupled signaling, under the control of the IRA1 gene promoter. This study demonstrates that databases of ESTs can be used to discover fusion genes resulting from structural rearrangement of chromosomes.

Transferrin receptor 2 (TfR2) plays a critical role in iron homeostasis because patients carrying disabling mutations in the TFR2 gene suffer from hemochromatosis. In this study, iron-responsive regulation of TfR2 at the protein level was examined in vitro and in vivo. HepG2 cell TfR2 protein levels were up-regulated after exposure to holotransferrin (holoTf) in a time- and dose-responsive manner. ApoTf or high-iron treatment with non-Tf-bound iron failed to elicit similar effects, suggesting that TfR2 regulation reflects interactions of the iron-bound ligand. Hepatic TfR2 protein levels also reflected an adaptive response to changing iron status in vivo. Liver TfR2 protein levels were down- and up-regulated in rats fed an iron-deficient and a high-iron diet, respectively. TfR2 was also up-regulated in Hfe(-/-) mice, an animal model that displays liver iron loading. In contrast, TfR2 levels were reduced in hypotransferrinemic mice despite liver iron overload, supporting the idea that regulation of the receptor is dependent on Tf. This idea is confirmed by up-regulation of TfR2 in beta-thalassemic mice, which, like hypotransferrinemic mice, are anemic and incur iron loading, but have functional Tf. Based on these combined results, we hypothesize that TfR2 acts as a sensor of iron status such that receptor levels reflect Tf saturation.

The physiological role of transferrin (Tf) receptor 2 (TfR2), a homolog of the well-characterized TfR1, is unclear. Mutations in TfR2 result in hemochromatosis, indicating that this receptor has a unique role in iron metabolism. We report that HepG2 cells, which endogenously express TfR2, display a biphasic pattern of Tf uptake when presented with ligand concentrations up to 2 muM. The apparently nonsaturating pathway of Tf endocytosis resembles TfR1-independent Tf uptake, a process previously characterized in some liver cell types. Exogenous expression of TfR2 but not TfR1 induces a similar biphasic pattern of Tf uptake in HeLa cells, supporting a role for TfR2 in this process. Immunoelectron microscopy reveals that while Tf, TfR1, and TfR2 are localized in the plasma membrane and tubulovesicular endosomes, TfR2 expression is associated with the additional appearance of Tf in multivesicular bodies. These combined results imply that unlike TfR1, which recycles apo-Tf back to the cell surface after the release of iron, TfR2 promotes the intracellular deposition of ligand. Tf delivered by TfR2 does not appear to be degraded, which suggests that its delivery to this organelle may be functionally relevant to the storage of iron in overloaded states.

<h4>Rationale</h4>Serotonergic mechanisms are thought to play an important role in the regulation of mood, motor activity and sleep patterns. Serotonin reuptake is controlled by the serotonin transporter (5-HTT) and by a common functional insertion/deletion polymorphism in the corresponding gene's promoter region (5-HTTLPR). Homozygosity for the long variant may confer a favourable response to treatment with serotonin reuptake inhibitors (SSRIs), and to sleep deprivation.<h4>Objectives</h4>The study assessed the role of the 5-HTTLPR genotype in determining motor side effects of antidepressant medication.<h4>Methods</h4>Motor activity patterns of 62 patients with major depression who were being treated with either SSRIs or tricyclic antidepressants (TCAs) were monitored over a 24-h period using a wrist-actograph. Additionally, motor activity was rated in a semi-structured interview using the motor agitation and retardation scale (MARS).<h4>Results</h4>Night-time motor activity was significantly increased in homozygous carriers of the long 5-HTTLPR allele (LL-genotype) who were being treated with SSRIs in comparison to short allele carriers (LS-genotype and SS-genotype), regardless of the type of antidepressant treatment (P<0.001). It was also significantly increased in comparison to patients with the LL-genotype who were being treated with TCAs (P<0.01). Differences in actographic motor activity were most prominent between 11 p.m. and 4 a.m. Clinical ratings of motor activity also showed a trend toward higher agitation scores in patients with the LL-genotype who received SSRI treatment.<h4>Conclusions</h4>Homozygosity for the long variant of the 5-HTTLPR may cause a predisposition to increased night-time motor activity in conjunction with SSRI treatment.

A panel of monoclonal antibodies specific for CD27 ligand (CD70), CD30 ligand (CD153), CD134 ligand (OX40L), and CD137 ligand (4-1BBL) were screened in vivo for their ability to affect the control of Mycobacterium avium infection in C57Bl/6 mice. Only the blocking of CD153 led to increased mycobacterial burdens. We then used CD30-deficient mice and found an increase in the proliferation of two strains of M. avium in these mice as compared with control animals. The increased mycobacterial growth was associated with decreased T cell expansion and reduced interferon-gamma (IFN-gamma) responses as a result of reduced polarization of the antigen-specific, IFN-gamma-producing T cells. At late times but not early in infection, the lymphoid cuff surrounding granulomas was depleted in the CD30-deficient animals. This report expands our knowledge about tumor necrosis factor superfamily members involved in the immune responses to mycobacterial infection by identifying CD30-CD153 interactions as required for optimal immune control of M. avium infection.

Alzheimer's disease-associated beta-amyloid peptides (Abeta) are generated by the sequential proteolytic processing of amyloid precursor protein (APP) by beta- and gamma-secretases. There is growing evidence that cholesterol- and sphingolipid-rich membrane microdomains are involved in regulating trafficking and processing of APP. BACE1, the major beta-secretase in neurons is a palmitoylated transmembrane protein that resides in lipid rafts. A subset of APP is subject to amyloidogenic processing by BACE1 in lipid rafts, and this process depends on the integrity of lipid rafts. Here we describe the association of all four components of the gamma-secretase complex, namely presenilin 1 (PS1)-derived fragments, mature nicastrin, APH-1, and PEN-2, with cholesterol-rich detergent insoluble membrane (DIM) domains of non-neuronal cells and neurons that fulfill the criteria of lipid rafts. In PS1(-/-)/PS2(-/-) and NCT(-/-) fibroblasts, gamma-secretase components that still remain fail to become detergent-resistant, suggesting that raft association requires gamma-secretase complex assembly. Biochemical evidence shows that subunits of the gamma-secretase complex and three TGN/endosome-resident SNAREs cofractionate in sucrose density gradients, and show similar solubility or insolubility characteristics in distinct non-ionic and zwitterionic detergents, indicative of their co-residence in membrane microdomains with similar protein-lipid composition. This notion is confirmed using magnetic immunoisolation of PS1- or syntaxin 6-positive membrane patches from a mixture of membranes with similar buoyant densities following Lubrol WX extraction or sonication, and gradient centrifugation. These findings are consistent with the localization of gamma-secretase in lipid raft microdomains of post-Golgi and endosomes, organelles previously implicated in amyloidogenic processing of APP.

<h4>Background</h4>We sought to evaluate the hypothesis that the high incidence of cutaneous melanoma in white persons in central Alabama is associated with a predominance of Irish and Scots descent.<h4>Methods</h4>Frequencies of country of ancestry reports were tabulated. The reports were also converted to scores that reflect proportional countries of ancestry in individuals. Using the scores, we computed aggregate country of ancestry indices as estimates of group ancestry composition. HLA-DRB1*04 allele frequencies and relationships to countries of ancestry were compared in probands and controls. Results were compared to those of European populations with HLA-DRB1*04 frequencies.<h4>Results</h4>Ninety evaluable adult white cutaneous melanoma probands and 324 adult white controls reported countries of ancestry of their grandparents. The respective frequencies of Ireland, and Scotland and "British Isles" reported countries of ancestry were significantly greater in probands than in controls. The respective frequencies of Wales, France, Italy and Poland were significantly greater in controls. 16.7% of melanoma probands and 23.8% of controls reported "Native American" ancestry; the corresponding "Native American" country of ancestry index was not significantly different in probands and controls. The frequency of HLA-DRB1*04 was significantly greater in probands, but was not significantly associated with individual or aggregate countries of ancestry. The frequency of DRB1*04 observed in Alabama was compared to DRB1*04 frequencies reported from England, Wales, Ireland, Orkney Island, France, Germany, and Australia.<h4>Conclusion</h4>White adults with cutaneous melanoma in central Alabama have a predominance of Irish, Scots, and "British Isles" ancestry and HLA-DRB1*04 that likely contributes to their high incidence of cutaneous melanoma.

<h4>Background</h4>Increased hepatic iron is assumed to potentiate progression towards liver fibrosis in chronic hepatitis C virus (HCV) infection. In this study we have evaluated the potentiating effect of marked hepatic iron overload and chronic HCV infection on hepatic fibrosis in thalassemic patients.<h4>Methods</h4>Liver biopsies of one group of patients with beta thalassemia major and chronic HCV infection (group 1) was compared with two groups of patients (groups 2&3) with either chronic HCV infection or thalassemia major, respectively (20 patients in each group). Necroinflammation, fibrosis, and iron overload were graded and compared.<h4>Results</h4>Stage of fibrosis in group 1 patients was significantly higher than the other two groups (p < 0.05). Necroinflammatory grade was significantly lower, but iron score was significantly higher in thalassemic patients (group 3) in comparison to groups 1 and 2 (p < 0.05).<h4>Conclusion</h4>Our results indicate that marked liver iron overload and HCV infection in thalassemic patients have potentiating effect on hepatic fibrogenesis.

The pathogenesis of diabetes associated with hemochromatosis is not known. We therefore examined glucose homeostasis and beta-cell function in mouse models of hemochromatosis. Mice with targeted deletion of the hemochromatosis gene (Hfe(-/-)) on the 129/Sv genetic background exhibited a 72% increase in iron content in the islets of Langerhans compared with wild-type controls. Insulin content was decreased in Hfe(-/-) mice by 35%/pancreas and 25%/islet. Comparable decreases were seen in the mRNA levels of beta-cell-specific markers, ins1, ins2, and glucose transporter 2. By 6-8 months, islets from Hfe(-/-) mice were 45% smaller, associated with increased staining for activated caspase 3 and terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end labeling. Islets from Hfe(-/-) mice were also desensitized to glucose, with half-maximal stimulation of insulin secretion seen at 16.7 +/- 0.9 mm glucose in perifused islets from Hfe(-/-) mice compared with 13.1 +/- 0.6 mm glucose in wild-type animals. Carbonyl protein modification, a marker for oxidative stress, was increased by 58% in Hfe(-/-) islets. Despite decreased islet size, Hfe(-/-) mice exhibited enhanced glucose tolerance. Fasting serum insulin levels were comparable between Hfe(-/-) and Hfe(+/+) mice, but were 48% lower in the Hfe(-/-) mice 30 min after challenge. Similar results were seen in mice carrying an Hfe mutation analogous to the common human mutation (C282Y) and in mice fed excess dietary iron. Hfe(-/-)mice on the C57BL6 background exhibited decreased glucose tolerance at 10-12 months due to an inability to increase insulin levels as they aged. We conclude that iron excess results in beta-cell oxidant stress and decreased insulin secretory capacity secondary to beta-cell apoptosis and desensitization of glucose-induced insulin secretion. This abnormality alone, however, is insufficient to cause diabetes.

<h4>Background</h4>Estrogens and their receptors are important in human development, physiology and disease. In this study, we utilized an integrated genome-wide molecular and computational approach to characterize the interaction between the activated estrogen receptor (ER) and the regulatory elements of candidate target genes.<h4>Results</h4>Of around 19,000 genes surveyed in this study, we observed 137 ER-regulated genes in T-47D cells, of which only 89 were direct target genes. Meta-analysis of heterogeneous in vitro and in vivo datasets showed that the expression profiles in T-47D and MCF-7 cells are remarkably similar and overlap with genes differentially expressed between ER-positive and ER-negative tumors. Computational analysis revealed a significant enrichment of putative estrogen response elements (EREs) in the cis-regulatory regions of direct target genes. Chromatin immunoprecipitation confirmed ligand-dependent ER binding at the computationally predicted EREs in our highest ranked ER direct target genes, NRIP1, GREB1 and ABCA3. Wider examination of the cis-regulatory regions flanking the transcriptional start sites showed species conservation in mouse-human comparisons in only 6% of predicted EREs.<h4>Conclusions</h4>Only a small core set of human genes, validated across experimental systems and closely associated with ER status in breast tumors, appear to be sufficient to induce ER effects in breast cancer cells. That cis-regulatory regions of these core ER target genes are poorly conserved suggests that different evolutionary mechanisms are operative at transcriptional control elements than at coding regions. These results predict that certain biological effects of estrogen signaling will differ between mouse and human to a larger extent than previously thought.

Members of the Sox family of transcription factors are involved in a number of crucial developmental processes, including sex determination, neurogenesis and skeletal development. LSox5 is a member of the group D Sox factors that, in conjunction with Sox6 and Sox9, promotes chondrogenesis by activating the expression of cartilage-specific extracellular matrix molecules. We have cloned the chicken homologue of LSox5 and found that it is initially expressed in the premigratory and migratory neural crest after Slug and FoxD3. Subsequently, the expression of LSox5 is maintained in cephalic crest derivatives, and it appears to be required for the development of the glial lineage, the Schwann cells and satellite glia in cranial ganglia. Misexpression of LSox5 in the cephalic neural tube activated RhoB expression throughout the dorsoventral axis. Furthermore, the prolonged forced expression of LSox5 enlarged the dorsal territory in which the neural crest is generated, extended the 'temporal window' of neural crest segregation, and led to an overproduction of neural crest cells in cephalic regions. In addition to HNK-1, the additional neural crest cells expressed putative upstream markers (Slug, FoxD3) indicating that a regulatory feedback mechanism may operate during neural crest generation. Thus, our data show that in addition to the SoxE genes (Sox9 and Sox10) a SoxD gene (Sox5) also participates in neural crest development and that a cooperative interaction may operate during neural crest generation, as seen during the formation of cartilage.

Phenotype-based functional genomic methods are useful for the identification of genes that are related to a particular biological function or disease. Essential to this approach is the ability to regulate the expression of selected genes. Artificial transcription factors (ATFs) are key molecular tools that selectively regulate gene expression in vivo. Here, we use an ATF library to identify genes that participate in rendering a cell resistant to the drug Taxol, a potent anti-cancer drug that binds to tubulin and inhibits cell division. The library, which encodes ATFs that activate (rather than inhibit) transcription, was introduced into a HeLa cell line, and Taxol-resistant cells were selected. After eight rounds of selection, we identified two ATFs that significantly increased the level of Taxol resistance (TR) in HeLa cells. Gene expression microarray experiments using these ATFs identified 37 co-regulated genes, including genes already known to participate in TR. This study demonstrates that ATF libraries can be used to induce phenotypic alterations in eukaryotic cells and then identify specific genes that are associated with the phenotype of choice.

Spinal muscular atrophy (SMA) is the leading genetic cause of infant mortality. SMA is caused by the homozygous loss of the survival motor neuron 1 (SMN1) gene. A nearly identical copy gene exists known as SMN2, however, due to an aberrant splicing event, the SMN2 gene fails to produce sufficient full-length protein to protect against disease development in the absence of SMN1. While a number of compounds have recently been identified that can stimulate full-length survival motor neuron (SMN) expression from the nearly identical copy SMN2, one of the difficulties has been the lack of a highly reproducible and quantitative means to measure the levels of SMN protein. To develop a technique that allows the rapid and highly sensitive measurement of SMN protein, a Surface Plasmon Resonance (SPR) application has been developed. The ability to quantify unassociated SMN protein and monitor the binding of SMN with other proteins in solution using a SPR sensor in less than 15 min and at low ng mL(-1) levels in HEPES Buffer Saline (HBS) has been achieved. The detection limit for the specific binding of SMN in HBS pH 7.4 solution is 0.99 ng mL(-1) with non-specific binding accounting for approximately 30% of the signal. Quantification of SMN is based on an immunoassay performed on the gold surface of the SPR sensor. 16-mercaptohexadecanoic acid (MHA) was reacted with dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) to form a pre-activated thiol (MHA-NHS). Antibodies for SMN were then coupled to the sensor with the pre-activated thiol. Sensor specificity was examined with mixtures of myoglobin (MG) and SMN. SMN sensor response decreases by more than 60% when MG was added to SMN. The decrease in sensor response can be attributed to non-specific binding of SMN to MG, verified with a sensor for MG.

The phosphatidylethanolamine (PE)-binding proteins (PEBPs) are an evolutionarily conserved family of proteins with pivotal biological functions. Here we describe the cloning and functional characterization of a novel family member, human phosphatidylethanolamine-binding protein 4 (hPEBP4). hPEBP4 is expressed in most human tissues and highly expressed in tumor cells. Its expression in tumor cells is further enhanced upon tumor necrosis factor (TNF) alpha treatment, whereas hPEBP4 normally co-localizes with lysosomes, TNFalpha stimulation triggers its transfer to the cell membrane, where it binds to Raf-1 and MEK1. L929 cells overexpressing hPEBP4 are resistant to both TNFalpha-induced ERK1/2, MEK1, and JNK activation and TNFalpha-mediated apoptosis. Co-precipitation and in vitro protein binding assay demonstrated that hPEBP4 interacts with Raf-1 and MEK1. A truncated form of hPEBP4, lacking the PE-binding domain, maintains lysosomal co-localization but has no effect on cellular responses to TNFalpha. Given that MCF-7 breast cancer cells expressed hPEBP4 at a high level, small interfering RNA was used to silence the expression of hPEBP4. We demonstrated that down-regulation of hPEBP4 expression sensitizes MCF-7 breast cancer cells to TNFalpha-induced apoptosis. hPEBP4 appears to promote cellular resistance to TNF-induced apoptosis by inhibiting activation of the Raf-1/MEK/ERK pathway, JNK, and PE externalization, and the conserved region of PE-binding domain appears to play a vital role in this biological activity of hPEBP4.

A polymorphism in the human serotonin transporter gene promoter (5-HTTLPR) is associated with anxiety and increased risk for developing depression in the face of adversity. Here, we report that among infant rhesus macaques, an orthologous polymorphism (rh5-HTTLPR) interacts with adversity in the form of peer rearing to influence adrenocorticotropic hormone (ACTH) response to stress and, further, that this interaction is sexually dichotomous. ACTH responses to separation are higher in l/s than in l/l males. In females, however, it is only among those with a history of adversity that the s allele is associated with increased ACTH responses to stress. Of interest, peer-reared animals, in particular females carrying the s allele, also exhibit lower cortisol responses to stress, a pattern that has been recognized in association with certain stress-related neuropsychiatric disorders. By extension, our findings suggest the intriguing possibility that human females carrying the 5-HTTLPR s allele could be more vulnerable to the effects of early adversity. This interactive effect may underlie the increased incidence of certain stress-related disorders in women.

Human TFR2 (transferrin receptor 2) is a membrane-bound protein homologous with TFR1. High levels of TFR2 mRNA were found mainly in the liver and, to a lesser extent, in erythroid precursors. However, although the presence of the TFR2 protein in hepatic cells has been confirmed in several studies, evidence is lacking about the presence of the TFR2 protein in normal erythroid cells. Using two anti-TFR2 monoclonal antibodies, G/14C2 and G/14E8, we have provided evidence that TFR2 protein is not expressed in normal erythroid cells at any stage of differentiation, from undifferentiated CD34+ cells to mature orthochromatic erythroblasts. In contrast, erythroleukaemic cells (K562 cells) exhibited a high level of expression of TFR2 at both the mRNA and the protein level. We can therefore conclude that an elevated expression of TFR2 protein is observed in leukaemic cells, but not in normal erythroblasts. The implications of this observation for the understanding of the phenotypic features of haemochromatosis due to mutation of the TFR2 gene are discussed.

A 37-year-old Japanese man with systemic hemochromatosis due to multiple transfusions was referred to us for the treatment of severe aplastic anemia (SAA), from which he had been suffering for 24 years. The patient had diabetes arising from the hemochromatosis, chronic anal fissures, and a kidney abscess due to neutropenia. He was treated with a nonmyeloablative preconditioning regimen followed by non-T-cell-depleted (non-TCD) allogeneic peripheral blood stem cell transplantation (PBSCT) from his human leukocyte antigen (HLA)-haploidentical 2-loci-mismatched sibling. Prompt engraftment of granulocytes and platelets was observed, and graft-versus-host disease was easy to control. Noninherited maternal antigens in the donor were confirmed prior to PBSCT, and they were also detected in small quantities in the recipient. This report describes the first successful nonmyeloablative hematopoietic stem cell transplant in a heavily transfused SAA patient from an HLA-haploidentical 2-loci-mismatched sibling donor. The result suggests that a long-term fetomaternal microchimerism-positive sibling can be a second-line donor if an alternative HLA-identical donor is not available.

<h4>Background and aims</h4>While upregulation of divalent metal transporter 1 (DMT1) and iron regulated gene 1 (IREG1) within duodenal enterocytes is reported in patients with hereditary haemochromatosis (HH), these findings are controversial. Furthermore, the effect of HFE, the gene mutated in HH, on expression of these molecules is unclear. This study examines duodenal expression of these three molecules in HH patients (prior to and following phlebotomy), in patients with iron deficiency (ID), and in controls.<h4>Methods</h4>DMT1, IREG1, and HFE mRNA were measured in duodenal tissue of C282Y homozygous HH patients, in ID patients negative for the C282Y mutation with a serum ferritin concentration less than 20 microg/l, and in controls negative for C282Y and H63D mutations with normal iron indices, using real time polymerase chain reaction.<h4>Results</h4>DMT1 and IREG1 mRNA levels were not significantly different in non-phlebotomised (untreated) HH patients compared with controls. DMT1 expression was significantly increased in HH patients who had undergone phlebotomy therapy (treated) and in patients with ID compared with controls. IREG1 was significantly increased in ID patients relative to controls, and while IREG1 expression was 1.8-fold greater in treated HH patients, this was not statistically significant. HFE mRNA expression was not significantly different in any of the groups investigated relative to controls.<h4>Conclusions</h4>These findings demonstrate that untreated HH patients do not have increased duodenal DMT1 and IREG mRNA, but rather phlebotomy increases expression of these molecules, reflecting the effect of phlebotomy induced erythropoiesis. Finally, HFE appears to play a minor role in the regulation of iron absorption by the duodenal enterocyte.

No abstract available.

The transcriptional activity of estrogen receptor alpha (ER-alpha) is modified by regulatory action and interactions of coactivators and corepressors. Recent studies have shown that the metastasis-associated protein 1 (MTA1) represses estrogen receptor element (ERE)-driven transcription in breast cancer cells. With a yeast two-hybrid screen to clone MTA1-interacting proteins, we identified a known nuclear receptor coregulator (NRIF3) as an MTA1-binding protein. NRIF3 interacted with MTA1 both in vitro and in vivo. NRIF3 bound to the C-terminal region of MTA1, while MTA1 bound to the N-terminal region of NRIF3, containing one nuclear receptor interaction LXXLL motif. We showed that NRIF3 is an ER coactivator, hyperstimulated ER transactivation functions, and associated with the endogenous ER and its target gene promoter. MTA1 repressed NRIF3-mediated stimulation of ERE-driven transcription and interfered with NRIF3's association with the ER target gene chromatin. In addition, NRIF3 deregulation enhanced the responsiveness of breast cancer cells to estrogen-induced stimulation of growth and anchorage independence. Furthermore, we found that NRIF3 is an estrogen-inducible gene and activated ER associated with the ER response element in the NRIF3 gene promoter. These findings suggest that NRIF3, an MTA1-interacting protein, is an estrogen-inducible gene and that regulatory interactions between MTA1 and NRIF3 might be important in modulating the sensitivity of breast cancer cells to estrogen.

ARHGAP family genes, such as FNBP2, SRGAP1/ARHGAP13, SRGAP2/ARHGAP14, ARHGAP4 and AHRGAP20/KIAA1391, encode GTPase activating proteins for Rho family proteins (RhoGAPs). Here, we identified and characterized the ARHGAP23 gene by using bioinformatics. KIAA1501 (AB040934.1) was a 5'-truncated partial cDNA derived from the ARHGAP23 gene. Complete coding sequence of human ARHGAP23 cDNA was determined by assembling BM806021 EST, BQ718622 EST, KIAA1501 partial cDNA, and AC115090.8 genome sequence corresponding to exons 7 and 25. ARHGAP23 gene encoded 1491-aa isoform 1 (without exon 23) and 1144-aa isoform 2 (with exon 23) due to alternative splicing. Isoform 2 was C-terminally truncated due to frame-shift within 23-bp exon 23. ARHGAP23 mRNA was expressed in placenta, prostate, hippocampus, brain medulla as well as in brain tumor, salivary gland tumor, head and neck tumor. Mouse 4933428G20 (NM_021493.1) was a 5'-truncated partial cDNA derived from Arhgap23 gene at mouse chromosome 11D. Human ARHGAP23, ARHGAP21 and Xenopus rGAP shared the common domain structure consisting of PDZ, Pleckstrin homology (PH), and RhoGAP domains. ARHGAP23-KIAA1684-MLLT6-RNF110-PIP5K2B-LASP1-PLXDC1-CACNB1 locus at human chromosome 17q12 and CACNB2-PLXDC2-LASP2-MLLT10-BMI1-PIP5K2A-KIAA1217-ARHGAP21 locus at human chromosome 10p12 were paralogous regions (paralogons) with internal inversion. MLLT6, MLLT10 and LASP1 genes are fusion partners of MLL gene in hematological malignancies, while RNF110, PIP5K2B, LASP1 and BMI1 genes are amplified in human tumors. Evolutionary recombination hotspots and oncogenomic recombination hotspots were co-localized around the ARHGAP23-CACNB1 locus and the ARHGAP21-CACNB2 locus.

Linker histones have long been known to facilitate chromatin condensation in vitro. For many years, this activity led to speculation that these proteins function as global repressors of transcription. However, work from numerous systems has demonstrated that this is not the case, and that linker histones appear to affect only the transcription of a small subset of genes, and, in fact, act to activate as well as repress transcription. Furthermore, there is evidence that suggests that linker histones are not required for mitotic condensation. In this review, we focus on studies of biological functions of linker histones primarily performed in unicellular eukaryotes. We highlight recent developments pointing towards linker histone involvement in DNA repair and ageing, which raise the possibility that the biological functions of linker histones are more varied than previously imagined.

The effect of HFE inactivation on iron homeostasis during an acute phase response was investigated in mice. HFE knockout, beta2-microglobulin knockout and C57BL/6J mice were injected with Freund's Complete Adjuvant to induce an acute phase response and hepatic hepcidin expression and serum transferrin saturation was determined 16 h later. Hepcidin mRNA increased in all strains in response to an acute phase stimulus when compared with untreated control animals. Hypoferraemia also occurred in all strains, indicating that both the upregulation of hepcidin and the decrease in transferrin saturation associated with an acute phase response is not dependent on HFE function.

Intracellular transport by microtubule-dependent motors is crucial for neuronal survival and function. Recent advances reveal novel strategies for the regulation of transport and the attachment of motors to cargoes. Current findings also illustrate the importance of directed transport in neuronal biology, including microtubule-motor-dependent transduction of neurotrophic signals and axonal damage signal complexes. Furthermore, recent data implicating the dysfunction of microtubule-dependent transport in the cause and development of several neurodegenerative diseases provides evidence for the vital role of transport in neuronal and organismal function.

Sphingomonas paucimobilis SYK-6 is able to grow on various lignin-derived biaryls as the sole source of carbon and energy. These compounds are degraded to vanillate and syringate by the unique and specific enzymes in this strain. Vanillate and syringate are converted to protocatechuate (PCA) and 3-O-methylgallate (3MGA), respectively, by the tetrahydrofolate-dependent O-demethylases. Previous studies have suggested that these compounds are further degraded via the PCA 4,5-cleavage pathway. However, our subsequent analysis of the ligB insertion mutant, which encodes the beta subunit of PCA 4,5-dioxygenase, suggested that at least one alternative route is involved in 3MGA degradation. In the present study, we isolated the desZ gene, which confers 3MGA degradation activity on Escherichia coli. The deduced amino acid sequence of desZ showed ca. 20 to 43% identity with the type II extradiol dioxygenases. Gas chromatography-mass spectrometry analysis suggested that DesZ catalyzes the 3,4-cleavage of 3MGA. Disruption of both desZ and ligB in SYK-6 resulted in loss of the dioxygen-dependent 3MGA transformation activity, but the resulting mutant retained the ability to grow on syringate. We found that the cell extract of the desZ ligB double mutant was able to convert 3MGA to gallate when tetrahydrofolate was added to the reaction mixture, and the cell extract of this mutant degraded gallate to the same degree as the wild type did. All these results suggest that syringate is degraded through multiple 3MGA degradation pathways in which ligAB, desZ, 3MGA O-demethylase, and gallate dioxygenase are participants.

Serum bilirubin is an important antioxidant that is found at increased levels in hereditary hemochromatosis patients. We hypothesized that increased levels of serum bilirubin may play a protective role against oxidative stress induced by iron overload in carriers of mutations in the hereditary hemochromatosis gene (HFE). We studied the relation between serum total bilirubin, serum iron levels, the HFE C282Y and H63D mutations, and mortality. The study was conducted in 2,332 randomly selected subjects from the Rotterdam Study, a population-based follow-up study of people aged 55 years or over. Serum bilirubin levels were significantly correlated with serum iron (Pearson's correlation coefficient (r) = 0.4, P < 0.001), transferrin saturation (r = 0.4, P < 0.001), and serum ferritin (r = 0.2, P < 0.05). Carriers of the HFE mutations had higher levels of bilirubin compared to wild-type homozygotes. The relation was the strongest in H63D heterozygotes or homozygotes and C282Y heterozygotes. High levels of serum bilirubin were associated with a 2.8 (95% CI 0.9-8.8) fold reduction in mortality in H63D homozygotes and a 2.2 (1.0-4.7) fold reduction in mortality in C282Y heterozygotes. Taken together, our data suggest that the high levels of the antioxidant bilirubin may counteract the adverse effect of oxidative stress induced by iron overload. This may explain in part the reduced penetrance of the HFE mutations.

The metastatic cascade is a complicated process that involves many steps from gain of the metastatic phenotype in the primary tumor cells through establishment of macroscopic tumor at the distant target organ. A group of genes, termed metastasis suppressor genes (MSG), encode for proteins that inhibit various steps of the metastatic cascade. Accordingly, loss of MSG promotes the metastatic phenotype. Although several MSG have been identified, the mechanisms through which they enhance metastasis are not clearly defined. Gene array analysis of a low metastatic LNCaP prostate cancer cell line compared to its highly metastatic derivative C4-2B prostate cancer cell line revealed decreased expression of raf kinase inhibitor protein (RKIP) in the C4-2B cell line. RKIP blocks the activation of several signaling pathways including MEK, G-proteins and NFkappaB. Immunohistochemical analysis of prostate cancer primary tumors and metastases revealed that RKIP protein expression was decreased in metastases. Restoration of RKIP expression in the C4-2B cell line diminished metastasis in a murine model. These results demonstrate that RKIP is a MSG. Loss of RKIP enhanced both angiogenesis and vascular invasion, and protected against apoptosis. These findings suggest that targeting the RKIP pathway may diminish the metastatic cascade. However, challenges exist as to the best method to target RKIP expression. Restoration of RKIP expression in all cancer cells in vivo is challenging. A plausible strategy is to use small molecules that target proteins in signaling pathways that are dysregulated due to loss of RKIP.

Alzheimer's disease (AD) has become linked to inflammation and metal biology. Metals (copper, zinc and iron) and inflammatory cytokines are significant factors that increase the onset of sporadic late onset forms of the dementia. The genetic discovery that alleles in the hemochromatosis gene accelerate the onset of disease by five years has certainly validated interest in the metallobiology of AD as originally described by biochemical criteria. Also the presence of an Iron-Responsive Element (IRE) in the 5'UTR of the Amyloid Precursor Protein transcript (APP 5'UTR) provided the first molecular biological support for the current model that APP of AD is a metaloprotein. At the biochemical level, copper, zinc and iron were shown to accelerate the aggregation of the Abeta peptide and enhance metal catalyzed oxidative stress associated with amyloid plaque formation. These amyloid associated events remain the central pathological hallmark of AD in the brain cortex region of AD patients. The involvement of metals in the plaque of AD patients and the demonstration of metal dependent translation of APP mRNA have encouraged the development of chelators as a major new therapeutic strategy for the treatment of AD, running parallel to the development of a vaccine. The other notable pathological feature of AD discussed here is inflammation. The presence of neuro-inflammatory events during AD was supported by clinical trials wherein use of non steroidal anti-inflammatory drugs (NSAIDs) was shown to reduce the risk of developing AD. Drug targets that address inflammation include the use of small molecules that prevent Abeta peptide from activating microglia, the use of cytokine suppressive anti-inflammatory drugs (CSAIDS), and the continued search for a vaccine directed to Abeta sub-fragments (even though the full-length Abeta immunogen generated brain-inflammation and encephalitis in some patients). Our laboratory currently uses a transfection-based assay to screen for small molecule drugs that selectively suppress the capacity of the APP 5'UTR to confer expression to a downstream reporter gene. Based on the presence of both an Interleukin-1 (IL-1) responsive acute box domain and an IRE in the APP 5'UTR, we predict that our APP 5'UTR directed drug screens will identify both novel metal chelators and novel NSAIDS. These lead drugs are readily testable to measure APP holoprotein expression in a cell based secondary assay, and by use of an APP transgenic mouse model to test potential beneficial effects of lead drug treatments on amyloid burden.

Morphological analysis of a conditional yeast mutant in acetyl-CoA carboxylase acc1ts/mtr7, the rate-limiting enzyme of fatty acid synthesis, suggested that the synthesis of C26 VLCFAs (very-long-chain fatty acids) is important for maintaining the structure and function of the nuclear membrane. To characterize this C26-dependent pathway in more detail, we have now examined cells that are blocked in pathways that require C26. In yeast, ceramide synthesis and remodelling of GPI (glycosylphosphatidylinositol)-anchors are two pathways that incorporate C26 into lipids. Conditional mutants blocked in either ceramide synthesis or the synthesis of GPI anchors do not display the characteristic alterations of the nuclear envelope observed in acc1ts, indicating that the synthesis of another C26-containing lipid may be affected in acc1ts mutant cells. Lipid analysis of isolated nuclear membranes revealed the presence of a novel C26-substituted PI (phosphatidylinositol). This C26-PI accounts for approx. 1% of all the PI species, and is present in both the nuclear and the plasma membrane. Remarkably, this C26-PI is the only C26-containing glycerophospholipid that is detectable in wild-type yeast, and the C26-substitution is highly specific for the sn-1 position of the glycerol backbone. To characterize the biophysical properties of this lipid, it was chemically synthesized. In contrast to PIs with normal long-chain fatty acids (C16 or C18), the C26-PI greatly reduced the bilayer to hexagonal phase transition of liposomes composed of 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE). The biophysical properties of this lipid are thus consistent with a possible role in stabilizing highly curved membrane domains.

<h4>Background</h4>Hereditary haemochromatosis, a disease that affects iron metabolism, progresses with a greater or lesser tendency to induce iron overload, possibly leading to severe organ dysfunction. Most elite endurance athletes take iron supplements during their active sporting life, which could aggravate this condition.<h4>Objective</h4>To determine the prevalence and discuss potential clinical implications of mutations of HFE (the gene responsible for hereditary haemochromatosis) in endurance athletes.<h4>Methods</h4>Basal concentrations of iron, ferritin, and transferrin and transferrin saturation were determined in the period before competition in 65 highly trained athletes. Possible mutations in the HFE gene were evaluated in each subject by extracting genomic DNA from peripheral blood. The restriction enzymes SnaBI and BclI were used to detect the mutations 845G-->A (C282Y) and 187C-->G (H63D).<h4>Results</h4>Our findings indicate a high prevalence of HFE gene mutations in this population (49.2%) compared with sedentary controls (33.5%). No association was detected in the athletes between mutations and blood iron markers.<h4>Conclusions</h4>The findings support the need to assess regularly iron stores in elite endurance athletes.

Changes in the function of the serotonergic system influence both panic phobic symptoms and carbon dioxide (CO2) reactivity in patients with panic disorder. Schmidt et al. [2000: J Abnorm Psychol 109(2):308-320] recently reported a predictive role of the genetic variants of the 5-HTTLPR on the fearful response to CO2 in healthy controls. We tested the hypothesis that the heterogeneity of CO2 reactivity in patients with panic disorder could be related to the allelic variation of the 5-HTT promoter. Ninety-five patients with panic disorder were challenged with 35% CO2. 5-HTTLPR allelic variation in each subject was determined using a PCR-based method. There were no differences for all the measures of CO2 reactivity among the genotype groups. CO2 reactivity of patients with panic disorder seems not to be influenced by the genetic variants of the 5-HTTLPR; this finding does not support a role for the serotonin transporter in the etiopathogenesis of CO2 reactivity in panic disorder.

Early diagnosis and immediate therapeutic interventions are crucial factors to reduce the damage extent and the risk of death. Currently, the diagnosis of stroke relies on neurological assessment of the patient and neuro-imaging techniques including computed tomography and/or magnetic resonance imaging scan. An early diagnostic marker of stroke, ideally capable to discriminate ischemic from hemorrhagic stroke would considerably improve patient acute management. Using surface-enhanced laser desorption/ionization (SELDI) technology, we aimed at finding new early diagnostic plasmatic markers of stroke. Strong anionic exchange (SAX) SELDI profiles of plasma samples from 21 stroke patients were compared to 21 samples from healthy controls. Seven peaks appeared to be differentially expressed with significant p values (p < 0.05). Proteins were stripped from the SAX chips, separated on a one-dimensional electrophoresis (1-DE) gel and stained using mass spectrometry (MS)-compatible silver staining. Following in-gel tryptic digestion, the peptides were analyzed by MS. Four candidate proteins were identified as apolipoprotein CI (ApoC-I), apolipoprotein CIII (ApoC-III), serum amyloid A (SAA), and antithrombin-III fragment (AT-III fragment). Assessment of ApoC-I and ApoC-III levels in plasma samples using a sandwich enzyme-linked immunosorbent assay (ELISA) allowed to distinguish between hemorrhagic (n = 15) and ischemic (n = 16) stroke (p < 0.001). To the best of our knowledge, ApoC-I and ApoC-III are the first reported plasmatic biomarkers capable to accurately distinguish between ischemic and hemorrhagic stroke in a small number of patients. It requires further investigation in a large cohort of patients.

In invertebrates, lectins play relevant roles in innate immunity; however, their regulatory mechanisms have not been identified yet. In this work, we purified, by gel filtration and affinity chromatography, lectin aggregates circulating in the hemolymph of the freshwater prawn Macrobrachium rosenbergii and compared their physicochemical properties with a previously described lectin (MrL). High-molecular weight MrL aggregates (MrL-I) lack hemagglutinating activity and showed bands of 62.1, 67.1 and 81.4 kDa, whereas MrL-III, which corresponds to MrL, showed hemagglutinating activity and is constituted by a single 9.6-kDa band as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. MrL-I and MrL-III showed similar amino acid composition but different carbohydrates concentration. Edman degradation indicated NH2-terminal sequence of five amino acids for the 9.6-kDa MrL-III (DVPLL/A) and eleven for the main 81.4-kDa band identified in MrL-I (DVPLL/AXKQQQD); analysis by MALDI-TOF indicated a different tryptic pattern for MrL-I and MrL-III. MrL-I was recognized by monoclonal antibodies against MrL-III. Circular dichroism indicated that the secondary structure in both proteins is similar and contains 23% of beta-sheet and 24% of alpha-helix. Our results suggest that differential posttranslational processes that favor aggregation are involved in regulating the activity of the lectin.

Patients with Crohn's disease have an increased risk of developing intestinal tumours. However, the carcinogenic mechanisms remain poorly understood. To address this question, this report describes an unusual case of Crohn's disease complicated by synchronous small intestinal and colonic adenocarcinomas. Genetic events in both the tumours and their adjacent mucosae were evaluated and the tumorigenesis of these cancers is discussed.

<h4>Background</h4>An increased frequency of hereditary hemochromatosis gene mutations occurs in patients with porphyria cutanea tarda. Polymerase chain reaction analysis of peripheral blood for hemochromatosis gene (HFE) mutations is available for clinical use. Early detection and treatment of hereditary hemochromatosis limit disease progression and improve life expectancy.<h4>Objective</h4>We present 8 patients with porphyria cutanea tarda subsequently found to have hereditary hemochromatosis or mutations in the HFE gene.<h4>Methods</h4>Retrospective review of patients in whom both porphyria cutanea tarda and hereditary hemochromatosis or HFE gene mutations were diagnosed between 1976 and 2000.<h4>Results</h4>Eight patients with porphyria cutanea tarda (6 males, 2 females; age range, 4-60 years; mean age at diagnosis of porphyria cutanea tarda, 42 years) were subsequently found to have hepatic iron overload or HFE gene mutations. Two patients had liver biopsy findings compatible with homozygous hereditary hemochromatosis. In the other 6 patients, HFE gene analysis revealed 3 homozygous C282Y, 1 compound heterozygous C282Y/H63D, and 2 heterozygous C282Y mutations. Seven patients (88%) had no specific signs or symptoms of hereditary hemochromatosis at diagnosis. In 5 patients (63%), the diagnosis of hereditary hemochromatosis or HFE gene mutation was initially suspected by the dermatologist.<h4>Conclusion</h4>Porphyria cutanea tarda can be an important cutaneous marker for patients with mutations of the HFE gene. HFE gene analysis should be done in patients who present with porphyria cutanea tarda. The dermatologist may play a key role in the early diagnosis of subclinical hereditary hemochromatosis in patients who present with porphyria cutanea tarda.

<h4>Background</h4>Chronic hepatitis C is frequently associated with increased hepatic iron stores. It remains controversial whether heterozygous mutations of hemochromatosis genes affect fibrosis progression. Therefore our aim was to assess associations between HFE mutations and hepatic inflammation and stage of fibrosis in German hepatitis C patients.<h4>Methods</h4>Liver biopsies from 166 patients were scored for inflammatory activity (A0-4) and hepatic fibrosis (F0-4). Gene mutations were determined by LightCycler, restriction fragment length polymorphism analysis, or direct sequencing.<h4>Results</h4>The frequencies of common HFE mutations C282Y and H63D are 4.2% and 21.3%, whereas the recently described S65C substitution and the Y250X mutation in the transferrin receptor 2 gene are very rare. In regression analysis, heterozygous carriers of C282Y or H63D mutations display significantly (P < 0.05) higher inflammatory activities and more advanced fibrosis than patients without mutations. For C282Y heterozygous patients, the odds ratios for marked inflammatory activity (A2-4) and advanced liver fibrosis or cirrhosis (F2-4) are 4.9 and 4.6, respectively, compared with patients carrying homozygous wild-type alleles. C282Y mutations are associated with significantly (P < 0.05) increased serum iron and aminotransferase levels, whereas H63D heterozygotes display higher transferrin saturation, serum iron, and ferritin concentrations compared to wild-type (P < 0.01).<h4>Conclusions</h4>Common heterozygous hemochromatosis mutations are associated with higher grades of inflammation and more severe hepatic fibrosis. Our findings support a role of HFE mutations as primary risk factors for fibrogenesis and disease progression in chronic hepatitis C.

<h4>Background/aims</h4>Host factors that may influence progression of hepatitis C infection to chronic hepatitis include T-cell responses and iron accumulation. We evaluated the hepatic expression of immunological markers relevant for a cytotoxic response in relation to viral and HFE genotype.<h4>Methods</h4>Frozen liver biopsies were obtained at diagnosis from 28 HFE genotyped patients. Sections stained for CD8, MHC-I, beta(2)m, HFE and CD68 were analyzed blind by morphometry. Response to therapy was available in 12 cases.<h4>Results</h4>A negative correlation was found between the number of CD8(+) cells and fibrosis. CD8(+) cells localized as clusters in portal tracts and sinusoids and were seen interacting with MHC-I positive lining cells. MHC-I and beta(2)m were expressed mainly in the endothelial and Kupffer cells. HFE was expressed in most, but not all, round and dendritic CD68(+) cells. Patients with virus genotype 3a had higher hepatic MHC-I and HFE expression, and a better-sustained response to IFN therapy than other patients.<h4>Conclusions</h4>In chronic hepatitis C virus infection MHC-I expression in the liver seems to relate to viral-genotype. In addition, the expression of MHC-I molecules by Kupffer cells places them as probable important players in the host response to HCV.

Mutations in the Raf signaling pathway are known to play a pivotal role in the progression of malignant melanoma. In this study, we provide evidence that the Raf-1 kinase inhibitory protein (RKIP) and its effects on Raf-1-mediated activation of mitogen-activated protein/extracellular signal-regulated kinase kinase are important for the metastatic potential of malignant melanoma. Screening nine melanoma cell lines at mRNA and protein levels, we detected significant down-regulation of RKIP expression in comparison with normal melanocytes. Loss of RKIP expression in transformed cells in vivo was confirmed in immunohistochemical analyses demonstrating reduction of RKIP expression already in primary melanoma and even stronger down-regulation or complete loss in melanoma metastases. Stable transfection of the melanoma cell line Mel Im with an RKIP expression plasmid blocked the Raf kinase pathway, resulting in down-regulation of extracellular signal-regulated kinase 1/2 and activator protein 1 activity. In very good agreement with the in vivo finding that down-regulation of RKIP expression is most obvious in melanoma metastasis, overexpression of RKIP in the highly invasive Mel Im cell line leads to a significant inhibition of invasiveness in vitro. Taken together, our results suggest that loss of RKIP in malignant melanoma contributes to enhanced invasiveness of transformed cells and therefore to progression of the disease.

The recently described family of dependence receptors is a new family of functionally related receptors. These proteins have little sequence similarity but display the common feature of inducing two completely opposite intracellular signals depending on ligand availability: in the presence of ligand, these receptors transduce a positive signal leading to survival, differentiation or migration, while in the absence of ligand, the receptors initiate or amplify a negative signal for apoptosis. Thus, cells that express these proteins manifest a state of dependence on their respective ligands. The mechanisms that trigger cell death induction in the absence of ligand are in large part unknown, but typically require cleavage by specific caspases. In this review we will present the proposed mechanisms for cell death induction by these receptors and their potential function in nervous system development and regulation of tumorigenesis.

Our data suggest a novel mechanism whereby pathological-length polyglutamine (polyQ) proteins promote the spermine synthetic pathway, increasing polyQ-aggregation and cell death. As detected in a cell-free turbidity assay, spermine promotes aggregation of thio-polyQ62 in a dose-dependent manner. Using a stable neuronal cell line expressing pathological-length [polyQ57-yellow fluorescent protein (YFP) (Q57)] or non-pathological-length [polyQ19-YFP (Q19)] polyglutamine protein, we show that multiple steps in the production of polyamines are affected in Q57 cells, suggesting dysfunctional spermine homeostasis. As the building block for spermine synthesis, arginine transport is significantly increased in neuronal cell lines stably expressing Q57. Q57 lines displayed upregulated basal and inducible arginase I activities that were not seen in polyQ19-YFP lines. Normal induction of spermidine/spermine N-acetyltransferase in Q19 lines regulating back-conversion of spermine, thereby reducing spermine levels, however, was not observed in Q57 lines. Pharmacological activation of ornithine decarboxylase (ODC), a key enzyme of the polyamine synthetic pathway, increased cellular aggregates and increased cell death in Q57 cells not observed in Q19 cells. Inhibition of ODC by difluoromethylornithine prevented basal and induced cell death in Q57 cells, demonstrating a central role for polyamines in this process.

Respiratory infections cause significant morbidity and mortality worldwide. Although an immune response is required to eliminate respiratory pathogens, if unchecked, it can damage surrounding tissues and block primary lung function. Based on our knowledge of immune T-cell activation, there are several pathways to which immune intervention could be applied. However, relatively few interventions target only those immune cells that are responding to antigens. OX40 and 4-1BB are members of the tumour necrosis factor receptor family and are expressed on the surface of T cells in several inflammatory conditions. Recently, the inhibition of OX40 has proved beneficial during influenza virus infection. This review highlights the recent advances in the manipulation of such molecules and how they have been applied to inflammatory conditions that are caused by viruses in the lung.

More than a decade ago, the DCC (deleted in colorectal cancer) gene was proposed as a putative tumor suppressor gene. Data supporting this proposal included observations that one DCC allele was deleted in roughly 70% of colorectal cancers, some cancers had somatic mutations of the DCC gene, and DCC expression was often reduced or absent in colorectal cancer tissues and cell lines. Despite subsequent studies which have supported DCC's potential role as a tumor suppressor gene, the rarity of point mutations identified in DCC coding sequences, the lack of a tumor predisposition phenotype in mice heterozygous for DCC inactivating mutations, and the presence of other known and candidate tumor suppressor genes on chromosome 18q have raised questions about DCC's candidacy. Following its initial characterization, the DCC protein was identified as a transmembrane receptor for netrins, key factors in axon guidance in the developing nervous system. At first glance, the established role of DCC and netrin-1 during organization of the spinal cord could be viewed as a further challenge to the position that DCC inactivation might play a significant role in tumorigenesis. However, recent observations on DCC's functions in intracellular signaling have renewed interest in the potential contribution of DCC inactivation to cancer. In particular, data indicate that, when engaged by netrin ligands, DCC may activate downstream signaling pathways. Moreover, in settings where netrin is absent or at low levels, DCC can promote apoptosis. Here, we review DCC's candidacy as a tumor suppressor gene, with an emphasis on how recent molecular analyses of DCC have offered support for the notion that DCC may function as a tumor suppressor gene.

During the last few years, the availability of endothelial cell cultures isolated from different vascular regions contributed to a significant increase in understanding the complex pro- and antithrombotic mechanisms in our circulatory system. The most important key enzyme is thrombin. Due to its haemostatic properties this serin protease catalyzes fibrin formation, activation of factors V, VIII and XIII as well as irreversible platelet aggregation. These processes may occur even within the circulatory system in case of endothelial stimulation (e. g. by inflammation mediators) for expression of binding sites for factors IX, IXa, X, Xa, von Willebrand protein and PAF. Thus not only catalytically activated coagulatory cascades, but also enhanced cooperation of platelets and granulocytes will occur. Paradoxically, in low intravascular concentration, thrombin, in combination with a healthy endothelial layer, may be the critical factor for the inhibition of thromboses. Respective antithrombogenic properties will mainly affect pre-venous microvascular circulation. In detail, they include thrombin-induced endothelial formation of antiaggregatory autacoids from platelet release products, anticoagulatory activation of protein C and absorption of active coagulation factors at endothelial heparan/ATIII complexes as well as release of profibrinolytic plasminogen activator of endothelial origin. The understanding of these complex regulatory functions enables not only a critical evaluation of actually discussed haemostasiologic risk factors (enhanced platelet reactivity, high concentrations of factor VII, VIII, fibrinogen, PAI, ATIII), but also the development of new pharmacologic strategies for prevention of thrombosis.

Synaptic feedback from rhythmically active neuronal circuits commonly causes their descending inputs to exhibit the rhythmic activity pattern generated by that circuit. In most cases, however, the function of this rhythmic feedback is unknown. In fact, generally these inputs can still activate the target circuit when driven in a tonic activity pattern. We are using the crab stomatogastric nervous system (STNS) to test the hypothesis that the neuronal circuit-mediated rhythmic activity pattern in projection neurons contributes to intercircuit regulation. The crab STNS contains an identified projection neuron, modulatory commissural neuron 1 (MCN1), whose tonic stimulation activates and modulates the gastric mill (chewing) and pyloric (filtering of chewed food) motor circuits in the stomatogastric ganglion (STG). During tonic stimulation of MCN1, the pyloric circuit regulates both gastric mill cycle frequency and gastropyloric coordination via a direct synapse onto a gastric mill neuron in the STG. However, when MCN1 is spontaneously active, it has a pyloric-timed activity pattern attributable to synaptic input from the pyloric circuit. This pyloric-timed activity in MCN1 provides the pyloric circuit with a second pathway for regulating the gastric mill rhythm. At these times, the direct STG synapse from the pyloric circuit to the gastric mill circuit is not necessary for pyloric regulation of the gastric mill rhythm. However, in the intact system, these two pathways play complementary roles in this intercircuit regulation. Thus, one role for rhythmicity in modulatory projection neurons is to enable them to mediate the interactions between distinct but related neuronal circuits.

<h4>Unlabelled</h4>Inactivation of tumor suppressor genes has been frequently observed in gastric carcinogenesis. Our purpose was to study the involvement of p53, APC, DCC, and Rb genes in gastric carcinoma.<h4>Method</h4>Loss of heterozygosity of the p53, APC, DCC and Rb genes was studied in 22 gastric cancer tissues using polymerase chain reaction; single-strand conformation polymorphism of the p53 gene exons 5-6 and exons 7-8 was studied using 35S-dATP, and p53 expression was detected using a histological immunoperoxidase method with an anti-p53 clone.<h4>Results and discussion</h4>No loss of heterozygosity was observed in any of these tumor suppressor genes; homozygous deletion was detected in the Rb gene in 23% (3/13) of the cases of intestinal-type gastric carcinoma. Eighteen (81.8%) cases showed band mobility shifts in exons 5-6 and/or 7-8 of the p53 gene. The presence of the p53 protein was positive in gastric cancer cells in 14 cases (63.6%). Normal gastric mucosa showed negative staining for p53; thus, the immunoreactivity was likely to represent mutant forms. The correlation of band mobility shift and the immunoreactivity to anti-p53 was not significant (P =.90). There was no correlation of gene alterations with the disease severity.<h4>Conclusions</h4>The inactivation of Rb and p53 genes is involved in gastric carcinogenesis in our environment. Loss of the Rb gene observed only in the intestinal-type gastric cancer should be further evaluated in association with Helicobacter pylori infection. The p53 gene was affected in both intestinal and diffuse histological types of gastric cancer.

Homologues of c-fos and c-jun from total DNA of Taenia crassiceps and Taenia solium were cloned and sequenced. The amino acid alignment analysis revealed that c-fos DNAs from T. crassiceps and T. solium were highly homologous (96%), and both have high homology compared to several mammalian c-fos proteins (93% to mouse, 96% to rat and 86% to human). The c-jun protein alignment showed higher homology (T. crassiceps and T. solium have 98%), when compared with mouse, rat and human, being 92%, 98% and 93% respectively. RT-PCR amplification of the parasite's total RNA, showed that T. crassiceps expressed both AP-1 complex genes, while T. solium only expressed c-fos. Southern blot hybridization analysis confirmed the true origin of each amplified gene. AP-1 transcription gene expression is regulated by oestradiol in the same fashion as their mammalian counterparts only in T. crassiceps. To study if AP-1 genes are involved in a physiological function of the cyst, reproduction was studied in vitro. Oestradiol treatment stimulated reproduction in T. crassiceps but not in T. solium cysticerci. This is the first report of the detection and functionality of AP-1 transcription factor genes in any species of helminth parasite.

Recent studies provide evidence that anxiety disorders may be linked to malfunction of serotonin neurotransmission or impaired activity of enzymes metabolising the catecholamines. Functional polymorphisms in the MAO-A uVNTR promoter gene, the COMT gene (Val158Met) exon 4, and the 5-HTT promoter gene (44 bp ins/del) were investigated in 101 patients with phobic disorders of the anxiety spectrum and 202 controls matched to the patients for sex, age and ethnicity. There were no significant differences between controls and patients in the allele and genotype frequencies of the 5-HTT and COMT gene polymorphisms. The frequency of >3 repeat alleles of the MAO-A gene polymorphism was significantly higher in female patients suffering from anxiety disorders, specifically panic attacks and generalized anxiety disorder. There was also a trend for the more frequent presence of >3 repeat alleles in female patients with agoraphobia and specific phobia, in contrast to female patients with social phobia, who did not differ from controls. The results support a possible role of the MAO-A gene in anxiety disorders.

Direct molecular genetic testing carried out in 59 Huntington's disease patients belonging to 46 families from Bashkortostan revealed the (CAG)n repeat expansion in exon 1 of the IT15 gene in 57 of them. By use of this analysis the disease status was not confirmed in two patients with atypical form of the disease and negative family history. The (CAG)n repeat expansion was identified in 27 out of 127 asymptomatic at-risk individuals. Analysis of the mutant (CAG)n allele inheritance demonstrated extremely high instability and high mutation rate predominantly leading to the appearance of the alleles with increasing number of (CAG)n repeats in subsequent generations. The instability was mostly observed in cases of paternal transmission. Almost complete linkage disequilibrium between the (CCG)7 mutant alleles and the del2642 deletion was demonstrated. Three major haplotypes revealed, (CCG)7/del-, (CCG)7/del+, and (CCG)10/del-, implied the existence of at least three sources of the origin of Huntington's disease in Bashkortostan. The identified haplotype frequency distribution patterns displayed similarities with those in European populations. The contribution of a number of genetic factors to the age of onset of Huntington's disease was analyzed.

The prevalence of the C282Y homozygous HFE genotype is high, approximately 1 in 200 in populations of Anglo-Celtic descent, and most authorities assumed this mutation would have a high clinical penetrance. Recent studies report the clinical penetrance of C282Y homozygous hereditary haemochromatosis is much lower than its prevalence, with possibly less than 5% developing clinical disease, although there is lack of consensus on a precise estimate. This review discusses reasons for this paradigm shift, including controversy on various definitions of clinical penetrance. It is inescapable that there are pronounced variations in clinical penetrance, and that certain C282Y homozygous individuals will not develop the clinical phenotype. This has prompted a search for modifier gene mutations amongst iron-metabolism genes, especially the known non- HFE haemochromatosis genes, and for possible environmental factors which might explain the observed variation in clinical penetrance.