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We report the initial sequencing and comparative analysis of the Macaca mulatta transcriptome. Cloned sequences from 11 tissues, nine animals, and three species (M. mulatta, M. fascicularis, and M. nemestrina) were sampled, resulting in the generation of 48,642 sequence reads. These data represent an initial sampling of the putative rhesus orthologs for 6,216 human genes. Mean nucleotide diversity within M. mulatta and sequence divergence among M. fascicularis, M. nemestrina, and M. mulatta are also reported.

Three HFE gene mutations (HFE 845 G-->A, 187 C-->G and 193 A-->T) are the most common mutations related to hereditary haemochromatosis (HH). The genotype for these mutations was analysed in 359 Spanish individuals with altered iron metabolism and iron overload. Various biochemical parameters were measured in serum samples from 96 of these individuals, and the effect of the genotype on these parameters was studied. Allele frequencies were 12.95% for the HFE C282Y variant, 28.97% for the HFE H63D variant and 0.69% for the HFE S65C variant, calculated in a total of 718 chromosomes. Multiple comparisons analysis showed very significant differences (p=0.001) in transferrin saturation index (TSI) between the HFE C282Y variant homozygous and control (ten healthy volunteers) groups. Highly significant (p=0.0001) and significant (p=0.005) differences in serum ferritin values were found between the HFE C282Y variant homozygous and control groups and between compound (HFE C282Y/H63D variant) heterozygous and control groups, respectively. Very significant differences (p=0.001) in serum iron values were observed between the HFE C282Y variant homozygous and control groups. TSI and serum ferritin values detected most HFE C282Y variant homozygotes and are recommended to facilitate the clinical diagnosis of HH.

Evidence of the existence of major prostate cancer (PC)-susceptibility genes has been provided by multiple segregation analyses. Although genomewide screens have been performed in over a dozen independent studies, few chromosomal regions have been consistently identified as regions of interest. One of the major difficulties is genetic heterogeneity, possibly due to multiple, incompletely penetrant PC-susceptibility genes. In this study, we explored two approaches to overcome this difficulty, in an analysis of a large number of families with PC in the International Consortium for Prostate Cancer Genetics (ICPCG). One approach was to combine linkage data from a total of 1,233 families to increase the statistical power for detecting linkage. Using parametric (dominant and recessive) and nonparametric analyses, we identified five regions with "suggestive" linkage (LOD score >1.86): 5q12, 8p21, 15q11, 17q21, and 22q12. The second approach was to focus on subsets of families that are more likely to segregate highly penetrant mutations, including families with large numbers of affected individuals or early age at diagnosis. Stronger evidence of linkage in several regions was identified, including a "significant" linkage at 22q12, with a LOD score of 3.57, and five suggestive linkages (1q25, 8q13, 13q14, 16p13, and 17q21) in 269 families with at least five affected members. In addition, four additional suggestive linkages (3p24, 5q35, 11q22, and Xq12) were found in 606 families with mean age at diagnosis of < or = 65 years. Although it is difficult to determine the true statistical significance of these findings, a conservative interpretation of these results would be that if major PC-susceptibility genes do exist, they are most likely located in the regions generating suggestive or significant linkage signals in this large study.

<h4>Background</h4>It has been suggested that iron metabolism may be involved in the pathogenesis of atherothrombotic cerebral infarction (ACI). The C282Y and H63D mutations in the hemochromatosis (HFE) gene are associated with increased serum iron levels and net iron accumulation. The aim of this study was to test the hypothesis that the C282Y and H63D mutations in the HFE gene are risk factors for ACI in a Slovene population.<h4>Material/methods</h4>The C282Y and H63D HFE gene mutations were tested in 96 Caucasian patients who had suffered an acute cerebral infarction, later confirmed as ACI, and 115 control subjects. Genotypes were determined by electrophoresis of the DNA digestion products from RsaI (C282Y) and MboI (H63D).<h4>Results</h4>We failed to demonstrate that the C282Y and H63D mutations were risk factors for ACI in Caucasians. The percentage of C282Y and H63D genotypes (dominant model) in ACI-cases (C282Y: 7.3%, n=7; H63D: 28.1%, n=27) did not differ significantly (P=0.9 and P=0.7 respectively) from that of the controls (C282Y: 7.0%, n=8; H63D: 26.1%, n=30). Logistic regression analysis revealed that arterial hypertension, diabetes, and cigarette smoking are independent risk factors for ACI, whereas the C282Y and H63D HFE gene mutations are not.<h4>Conclusions</h4>We provided evidence that the C282Y and H63D HFE gene mutations were not risk factors for ACI in this Slovene population.

<h4>Background</h4>Extrahepatic manifestations associated with Hepatitis C virus (HCV) such as arthritis, vasculitis, cryoglobulinemia, are well known. However, HCV related arthritis in the absence of clinical, biochemical and histological evidence of liver disease is not common. This article deals with such a case and its response to interferon therapy.<h4>Case report</h4>We present a case of a 32 year old Filipino male who presented with bilateral symmetrical painful swelling of multiple joints including, hands, elbows, shoulders, and knees. Serum rheumatoid factor, antinuclear antibodies and a comprehensive work-up for rheumatologic disorders were all negative. Both initially and subsequently, serological tests for hepatitis A, B, and autoimmune liver diseases, Wilson's disease, hemochromatosis, syphilis, human immunodeficiency virus (HIV) and cryoglobulinemia were negative, initially and subsequently. However, the hepatitis C antibody test was positive and hepatitis C viral RNA was detected in high titers. The joint symptoms did not improve despite therapy with nonsteroidal anti-inflammatory drugs and a short course of prednisone prescribed earlier by his primary care physician. The patient then requested and was subsequently treated with interferon alpha 2b.<h4>Results</h4>The patient responded rapidly to the interferon therapy with significant and sustained improvement in joint symptoms and disappearance of hepatitis C viral RNA from his serum.<h4>Conclusions</h4>HCV arthritis should be considered in the differential diagnosis of seronegative arthritis of undetermined etiology even in the setting of normal liver chemistries.

Striatal degeneration in Huntington's disease (HD) is associated with increases in perikaryal calbindin immunolabeling in yet-surviving striatal projection neurons. Since similar increases have also been observed in surviving striatal projection neurons after intrastriatal injection of the excitotoxin quinolinic acid, the increased calbindin in HD striatum has been interpreted to suggest an excitotoxic process in HD. We used immunolabeling to assess if calbindin is elevated in striatal projection neurons of R6/2 HD transgenic mice. These mice bear exon 1 of the human huntingtin gene with 144 CAG repeats and show some of the neuropathological signs (e.g., neuronal intranuclear inclusions) and clinical traits (e.g., wasting prior to early death) of HD. We found an increased frequency of calbindin-immunoreactive neuronal perikarya in the striatum of 6- and 12-week-old R6/2 mice compared to wild-type controls. This increase was most notable in the normally calbindin-poor dorsolateral striatum. We found no significant changes in the total area of striatum occupied by the calbindin-negative striosomes and no consistent changes in striatal calbindin mRNA. The increase in calbindin in R6/2 striatal neurons was thus limited to the matrix compartment, and it may be triggered by increased Ca2+ entry due to the demonstrated heightened NMDA sensitivity of these neurons. The data further support the similarity of R6/2 mice to HD, and are consistent with the occurrence of an excitotoxic process in striatum in both.

There is significant evidence that energy production impairment and mitochondrial dysfunction play a role in the pathogenesis of Huntington disease. Nonetheless, the specific mitochondrial defects due to the presence of mutant huntingtin have not been fully elucidated. To determine the effects of mutant huntingtin on mitochondrial energy production, a thorough analysis of respiration, ATP production, and functioning of the respiratory complexes was carried out in clonal striatal cells established from Hdh(Q7) (wild-type) and Hdh(Q111) (mutant huntingtin knock-in) mouse embryos. Mitochondrial respiration and ATP production were significantly reduced in the mutant striatal cells compared with the wild-type cells when either glutamate/malate or succinate was used as the substrate. However, mitochondrial respiration was similar in the two cell lines when the artificial electron donor TMPD/ascorbate, which feeds into complex IV, was used as the substrate. The attenuation of mitochondrial respiration and ATP production when either glutamate/malate or succinate was used as the substrate was not due to impairment of the respiratory complexes, because their activities were equivalent in both cell lines. Intriguingly, in the striatum of presymptomatic and pathological grade 1 Huntington disease cases there is also no impairment of mitochondrial complexes I-IV (Guidetti, P., Charles, V., Chen, E. Y., Reddy, P. H., Kordower, J. H., Whetsell, W. O., Jr., Schwarcz, R., and Tagle, D. A. (2001) Exp. Neurol. 169, 340-350). To our knowledge, this is the first comprehensive analysis of the effects of physiological levels of mutant huntingtin on mitochondrial respiratory function within an appropriate cellular context. These findings demonstrate that the presence of mutant huntingtin impairs mitochondrial ATP production through one or more mechanisms that do not directly affect the function of the respiration complexes.

There are numerous studies on the immune response against malignant human tumors. This study was aimed to address the complexity and specificity of humoral immune response against a benign human tumor. We assembled a panel of 62 meningioma-expressed antigens that show reactivity with serum antibodies of meningioma patients, including 41 previously uncharacterized antigens by screening of a fetal brain expression library. We tested the panel for reactivity with 48 sera, including sera of patients with common-type, atypical, and anaplastic meningioma, respectively. Meningioma sera detected an average of 14.6 antigens per serum and normal sera an average of 7.8 antigens per serum (P = 0.0001). We found a decline of seroreactivity with malignancy with a statistical significant difference between common-type and anaplastic meningioma (P < 0.05). We detected 17 antigens exclusively with patient sera, including 12 sera that were reactive against KIAA1344, 9 against natural killer tumor recognition (NKTR), and 7 against SRY (sex determining region Y)-box2 (SOX2). More than 80% of meningioma patients had antibodies against at least one of the antigens KIAA1344, SC65, SOX2, and C6orf153. Our results show a highly complex but specific humoral immune response against a benign tumor with a distinct serum reactivity pattern and a decline of complexity with malignancy. The frequent antibody response against specific antigens offers new diagnostic and therapeutic targets for meningioma. We developed a statistical learning method to differentiate sera of meningioma patients from sera of healthy donors.

We report the identification and characterization of a potent regulator of genomic integrity, mouse and human FRAG1 gene, a conserved homolog of replication factor C large subunit that is homologous to the alternative replication factor C subunits Elg1, Ctf18/Chl12, and Rad24 of budding yeast. FRAG1 was identified in a search for key caretaker genes involved in the regulation of genomic stability under conditions of replicative stress. In response to stress, Atr participates in the down-regulation of FRAG1 expression, leading to the induction of apoptosis through the release of Rad9 from damaged chromatin during the S phase of the cell cycle, allowing Rad9-Bcl2 association and induction of proapoptotic Bax protein. We propose that the Frag1 signal pathway, by linking replication stress surveillance with apoptosis induction, plays a central role in determining whether DNA damage is compatible with cell survival or whether it requires cell elimination by apoptosis.

Mutations that affect the dynein motor machinery are sufficient to cause motor neuron disease. It is not known why there are aggregates or inclusions in affected tissues in mice with such mutations and in most forms of human motor neuron disease. Here we identify a new mechanism of inclusion formation by showing that decreased dynein function impairs autophagic clearance of aggregate-prone proteins. We show that mutations of the dynein machinery enhanced the toxicity of the mutation that causes Huntington disease in fly and mouse models. Furthermore, loss of dynein function resulted in premature aggregate formation by mutant huntingtin and increased levels of the autophagosome marker LC3-II in both cell culture and mouse models, compatible with impaired autophagosome-lysosome fusion.

In mammals, flies, and worms, sex is determined by distinctive regulatory mechanisms that cause males (XO or XY) and females (XX) to differ in their dose of X chromosomes. In each species, an essential X chromosome-wide process called dosage compensation ensures that somatic cells of either sex express equal levels of X-linked gene products. The strategies used to achieve dosage compensation are diverse, but in all cases, specialized complexes are targeted specifically to the X chromosome(s) of only one sex to regulate transcript levels. In C. elegans, this sex-specific targeting of the dosage compensation complex (DCC) is controlled by the same developmental signal that establishes sex, the ratio of X chromosomes to sets of autosomes (X:A signal). Molecular components of this chromosome counting process have been defined. Following a common step of regulation, sex determination and dosage compensation are controlled by distinct genetic pathways. C. elegans dosage compensation is implemented by a protein complex that binds both X chromosomes of hermaphrodites to reduce transcript levels by one-half. The dosage compensation complex resembles the conserved 13S condensin complex required for both mitotic and meiotic chromosome resolution and condensation, implying the recruitment of ancient proteins to the new task of regulating gene expression. Within each C. elegans somatic cell, one of the DCC components also participates in the separate mitotic/meiotic condensin complex. Other DCC components play pivotal roles in regulating the number and distribution of crossovers during meiosis. The strategy by which C. elegans X chromosomes attract the condensin-like DCC is known. Small, well-dispersed X-recognition elements act as entry sites to recruit the dosage compensation complex and to nucleate spreading of the complex to X regions that lack recruitment sites. In this manner, a repressed chromatin state is spread in cis over short or long distances, thus establishing the global, epigenetic regulation of X chromosomes that is maintained throughout the lifetime of hermaphrodites.

EID1 (E1A-like inhibitor of differentiation 1) functions as an inhibitor of nuclear receptor-dependent gene transcription by directly binding to co-regulators. Alternative targets include the co-repressor small heterodimer partner (SHP, NR0B2) and the co-activators CBP/p300, indicating that EID1 utilizes different inhibitory strategies. Recently, EID2 was characterized as an inhibitor of muscle differentiation and as an antagonist of both CBP/p300 and HDACs. Here, we describe a third family member designated EID3 that is highly expressed in testis and shows homology to a region of EID1 implicated in binding to CBP/p300. We demonstrate that EID3 acts as a potent inhibitor of nuclear receptor transcriptional activity by a mechanism that is independent of direct interactions with nuclear receptors, including SHP. Furthermore, EID3 directly binds to and blocks the SRC-1 interacting domain of CBP, which has been implicated to act as the interaction surface for nuclear receptor co-activators. Consistent with this idea, EID3 prevents recruitment of CBP to a natural nuclear receptor-regulated promoter. Our study suggests that EID-family members EID3 and EID1 act as inhibitors of CBP/p300-dependent transcription in a tissue-specific manner.

Huntington's disease (HD) is a hereditary neurodegenerative disorder characterized by progressive psychiatric, cognitive, and motor disturbances. We studied the expression of synaptic vesicle proteins in the R6/1 transgenic mouse model of HD. We observed that the levels of rabphilin 3A, a protein involved in exocytosis, is substantially decreased in synapses of most brain regions in R6/1 mice. The appearance of the reduction coincides with the onset of motor deficits and behavioral disturbances. Double immunohistochemistry did not show colocalization between rabphilin 3A and huntingtin aggregates in the HD mice. Using in situ hybridization, we demonstrated that rabphilin 3A mRNA expression was substantially reduced in the R6/1 mouse cortex compared to wild-type mice. Our results indicate that a decrease in mRNA levels underlie the depletion of protein levels of rabphilin 3A, and we suggest that this reduction may be involved in causing impaired synaptic transmission in R6/1 mice.

In Caucasians, 4-35% of hemochromatosis patients carry at least one chromosome without a common HFE mutation (i.e. C282Y, H63D and S65C). Several studies have now shown that iron overload phenotypes in such patients can be associated with uncommon HFE mutations. We previously supported implication of the C282Y/Q283P compound heterozygous genotype in hemochromatosis phenotypes and, based on molecular dynamics simulations, proposed that the Q283P substitution prevents normal folding of the HFE alpha3-domain. In the current work, we have used HeLa cells carrying wild-type or Q283P-mutant HFE cDNA under the control of a tetracycline-sensitive promoter to functionally characterise the Q283P mutation. Experiments using cells over-expressing wild-type HFE confirm the existence of beta2microglobulin(beta2m)/HFE and HFE/transferrin receptor 1 (TfR1) interactions, as well as the capacity of HFE to reduce transferrin-mediated iron uptake. In contrast, neither beta2m/HFE nor HFE/TfR1 complex formation was detected in cells over-expressing the mutated form of HFE. Moreover, the 283P HFE protein was found to have a very limited effect on the major cellular iron uptake pathway. Combined, our results indicate that the Q283P mutation leads to structural and functional consequences similar to those described for the main hereditary hemochromatosis mutation. As a consequence, our study has implications for the screening of hemochromatosis patients that have one or two copies of HFE which lack the main mutations. It also highlights that protein structure prediction methods could be more generally used to better interpret relationships between rare genotypes and molecular diagnosis of a human inherited disorder.

Mammalian nonheme iron absorption requires reduction of dietary iron for uptake by the divalent metal ion transport system in the intestine. This was thought to be mediated by duodenal cytochrome b (Cybrd1), a ferric reductase enzyme resident on the luminal surface of intestinal absorptive cells. To test its importance in vivo, we inactivated the murine Cybrd1 gene and assessed tissue iron stores in Cybrd1-null mice. We found that loss of Cybrd1 had little or no impact on body iron stores, even in the setting of iron deficiency. We conclude that other mechanisms must be available for the reduction of dietary iron.

Nuclear aggregates of polyglutamine (polyQ)-expanded proteins are associated with a number of neurodegenerative diseases including Huntington's disease (HD) and spinocerebellar ataxias (SCAs). The nuclear deposition of polyQ proteins correlates with rearrangements of nuclear matrix, transcriptional dysregulation, and cell death. To explore the requirement for polyQ tracks in educing such cellular responses, we examined whether a non-polyQ protein can deposit as nuclear aggregates and elicit similar responses. We report that a protein chimera (GFP170*) composed of the green fluorescent protein (GFP) fused to an internal fragment of the Golgi Complex Protein (GCP-170) forms nuclear aggregates analogous to those formed by polyQ proteins. Like the polyQ nuclear aggregates, GFP170* inclusions recruit molecular chaperones and proteasomal components, alter nuclear structures containing the promyelocytic leukemia protein (PML), recruit transcriptional factors such as CREB-binding protein (CBP) and p53, repress p53 transcriptional activity, and induce cell death. Our results indicate that nuclear aggregation and transcriptional effects are not unique to polyQ-containing proteins and may represent a general response to misfolded proteins in the nucleus.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent developmental toxicant in most vertebrates. However, frogs are relatively insensitive to TCDD toxicity, especially during early life stages. Toxicity of TCDD and related halogenated aromatic hydrocarbons is mediated by the aryl hydrocarbon receptor (AhR), and specific differences in properties of the AhR signaling pathway can underlie in TCDD toxicity in different species. This study investigated the role of AhR in frog TCDD insensitivity, using Xenopus laevis as a model system. X. laevis, a pseudotetraploid species, expresses two distinct AhR1 genes, AhR1alpha and AhR1beta. Sharing 86% amino acid identity, these likely represent distinct genes, both orthologous to mammalian AhR and paralogous to the AhR2 gene(s) in most fish. Both AhR1alpha and AhR1beta exhibit TCDD-dependent binding of cognate DNA sequences, but they bind TCDD with at least 20-fold lower affinity than the mouse AhR(b-1) protein, and they are similarly less responsive in TCDD-induced reporter gene induction in conjunction with the mouse CYP1A1 promoter. Furthermore, CYP1A6 and CYP1A7 induction by TCDD in cultured X. laevis A6 cells appears much less responsive than CYP1A induction in cell lines derived from more sensitive animals. Taken together, these data suggest that low affinity binding by X. laevis AhRs plays an important mechanistic role in the insensitivity of frogs to TCDD. An understanding of these molecular mechanisms should aid amphibian ecotoxicology and refine the use of frog embryos as a model [e.g. in FETAX (Frog Embryo Teratogenesis Assay-Xenopus)] for determining developmental toxicity of samples containing dioxin-like compounds.

We investigated whether cell proliferation and neurogenesis are altered in R6/2 transgenic Huntington's disease mice. Using bromodeoxyuridine (BrdU), we found a progressive decrease in the number of proliferating cells in the dentate gyrus of R6/2 mice. This reduction was detected in pre-symptomatic mice, and by 11.5 weeks, R6/2 mice had 66% fewer newly born cells in the hippocampus. The results were confirmed by immunohistochemistry for the cell cycle markers Ki-67 and proliferating cell nuclear antigen (PCNA). We did not observe changes in cell proliferation in the R6/2 subventricular zone, indicating that the decrease in cell proliferation is specific for the hippocampus. This decrease corresponded to a reduction in actual hippocampal neurogenesis as assessed by double immunostaining for BrdU and the neuronal marker neuronal nuclei (NeuN) and by immunohistochemistry for the neuroblast marker doublecortin. Reduced hippocampal neurogenesis may be a novel neuropathological feature in R6/2 mice that could be assessed when evaluating potential therapies.

Mutations in the iron exporter ferroportin (Fpn) (IREG1, SLC40A1, and MTP1) result in hemochromatosis type IV, a disorder with a dominant genetic pattern of inheritance and heterogeneous clinical presentation. Most patients develop iron loading of Kupffer cells with relatively low saturation of plasma transferrin, but others present with high transferrin saturation and iron-loaded hepatocytes. We show that known human mutations introduced into mouse Fpn-GFP generate proteins that either are defective in cell surface localization or have a decreased ability to be internalized and degraded in response to hepcidin. Studies using co-immunoprecipitation of epitope-tagged Fpn and size-exclusion chromatography demonstrated that Fpn is multimeric. Both WT and mutant Fpn participate in the multimer, and mutant Fpn can affect the localization of WT Fpn, its stability, and its response to hepcidin. The behavior of mutant Fpn in cell culture and the ability of mutant Fpn to act as a dominant negative explain the dominant inheritance of the disease as well as the different patient phenotypes.

Minerals are essential for a wide variety of metabolic and physiologic processes in the human body. Some of the physiologic roles of minerals important to athletes are their involvement in: muscle contraction, normal hearth rhythm, nerve impulse conduction, oxygen transport, oxidative phosphorylation, enzyme activation, immune functions, antioxidant activity, bone health, and acid-base balance of the blood. The two major classes of minerals are the macrominerals and the trace elements. The scope of this article will focus on the ergogenic theory and the efficacy of such mineral supplementation.

Developmental abnormalities of the heart are the underlying cause of many congenital heart malformations. The embryological development of the integrated cardiovascular tissue is the result of multiple tissue and cell-to-cell interactions involving temporal and spatial events under genetic control. Recent technological advances, like microarray analysis of gene expression, are providing new tools to aid in deciphering the complex networks of gene expression that regulate cardiac development. Here, we review our current understanding of the genetics of congenital heart disorders with emphasis on gene expression studies and report preliminary data from infants with conotruncal defects. We report our microarray analysis showing over- and underexpression of individual genes and gene network interactions from dysplastic pulmonic tissue from two infants with tetralogy of Fallot compared with normal pulmonic tissue from an unaffected control infant.

Three isoforms of carbonic anhydrase-related protein (CA-RP) are evolutionally well conserved among the CA gene family but lack classical CA activity. Although the biological function of CA-RPs is unknown, overexpression of CA-RP VIII has been reported in certain tumor types. Based on the finding that CA-RPs are commonly expressed in the neuronal cells, we investigated expression of all three CA-RPs in gastrointestinal stromal tumor (GIST). In contrast to no detectable signal of any of the three CA-RPs in intestinal cells of Cajal, immunohistochemical analysis showed distinct cytoplasmic expressions of CA-RPs VIII and XI in 13 (59%) and 20 (91%) of 22 GIST tissue specimens, respectively. The positive signals for both CA-RPs VIII and XI were more intense in the periphery than in the central part of GISTs, whereas no significant signal for CA-RP X expression was observed in any of the GISTs. These expression patterns of CA-RPs were consistently observed by reverse transcription-polymerase chain reaction-Southern blot and immunocytochemistry in the cultured GIST cell line GIST-T1. Ectopic expression of CA-RP XI in GIST-T1 cells induced cell proliferation and invasion in vitro. These findings indicate that CA-RP XI plays a role in the development of GIST.

The embryonic genome is formed by fusion of a maternal and a paternal genome. To accommodate the resulting diploid genome in the fertilized oocyte dramatic global genome reorganizations must occur. The higher order structure of chromatin in vivo is critically dependent on architectural chromatin proteins, with the family of linker histone proteins among the most critical structural determinants. Although somatic cells contain numerous linker histone variants, only one, H1FOO, is present in mouse oocytes. Upon fertilization H1FOO rapidly populates the introduced paternal genome and replaces sperm-specific histone-like proteins. The same dynamic replacement occurs upon introduction of a nucleus during somatic cell nuclear transfer. To understand the molecular basis of this dynamic histone replacement process, we compared the localization and binding dynamics of somatic H1 and oocyte-specific H1FOO and identified the molecular determinants of binding to either oocyte or somatic chromatin in living cells. We find that although both histones associate readily with chromatin in nuclei of somatic cells, only H1FOO is capable of correct chromatin association in the germinal vesicle stage oocyte nuclei. This specificity is generated by the N-terminal and globular domains of H1FOO. Measurement of in vivo binding properties of the H1 variants suggest that H1FOO binds chromatin more tightly than somatic linker histones. We provide evidence that both the binding properties of linker histones as well as additional, active processes contribute to the replacement of somatic histones with H1FOO during nuclear transfer. These results provide the first mechanistic insights into the crucial step of linker histone replacement as it occurs during fertilization and somatic cell nuclear transfer.

<h4>Introduction</h4>Epidemiological evidence strongly links fish oil, which is rich in docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), with low incidences of several types of cancer. The inhibitory effects of omega-3 polyunsaturated fatty acids on cancer development and progression are supported by studies with cultured cells and animal models. Propofol (2,6-diisopropylphenol) is the most extensively used general anesthetic-sedative agent employed today and is nontoxic to humans at high levels (50 microg/ml). Clinically relevant concentrations of propofol (3 to 8 microg/ml; 20 to 50 microM) have also been reported to have anticancer activities. The present study describes the synthesis, purification, characterization and evaluation of two novel anticancer conjugates, propofol-docosahexaenoate (propofol-DHA) and propofol-eicosapentaenoate (propofol-EPA).<h4>Methods</h4>The conjugates linking an omega-3 fatty acid, either DHA or EPA, with propofol were synthesized and tested for their effects on migration, adhesion and apoptosis on MDA-MB-231 breast cancer cells.<h4>Results</h4>At low concentrations (25 microM), DHA, EPA or propofol alone or in combination had minimal effect on cell adhesion to vitronectin, cell migration against serum and the induction of apoptosis (only 5 to 15% of the cells became apoptotic). In contrast, the propofol-DHA or propofol-EPA conjugates significantly inhibited cell adhesion (15 to 30%) and migration (about 50%) and induced apoptosis (about 40%) in breast cancer cells.<h4>Conclusion</h4>These results suggest that the novel propofol-DHA and propofol-EPA conjugates reported here may be useful for the treatment of breast cancer.

<h4>Background</h4>Primary ciliary dyskinesia (PCD) is a severe inherited disorder characterised by chronic respiratory disease, male infertility, and, in approximately 50% of affected individuals, a left-right asymmetry defect called situs inversus. PCD is caused by defects in substructures of the ciliary and flagellar axoneme, most commonly loss of the outer dynein arms. Although PCD is believed to involve mutations in many genes, only three have been identified.<h4>Methods</h4>To facilitate discovery of new PCD genes, we have used database searching and analysis to systematically identify the human homologues of proteins associated with the Chlamydomonas reinhardtii outer dynein arm, the best characterised outer arm of any species.<h4>Results</h4>We find that 12 out of 14 known Chlamydomonas outer arm subunits have one or more likely orthologues in humans. The results predict a total of 24 human genes likely to encode outer dynein arm subunits and associated proteins possibly necessary for outer arm assembly, plus 12 additional closely related human genes likely to encode inner dynein arm subunits.<h4>Conclusion</h4>These genes, which have been located on the human chromosomes for easy comparison with known or suspected PCD loci, are excellent candidates for screening for disease-causing mutations in PCD patients with outer and/or inner dynein arm defects.

Hepcidin is the principal iron regulatory hormone and its overproduction contributes to anemia of inflammation (AI). In vitro, hepcidin binds to and induces the degradation of the exclusive iron exporter ferroportin. We explored the effects and distribution of synthetic hepcidin in the mouse. A single intraperitoneal injection of hepcidin caused a rapid fall of serum iron in a dose-dependent manner, with a 50-microg dose resulting in iron levels 80% lower than in control mice. The full effect was seen within only 1 hour, consistent with a blockade of iron export from tissue stores and from macrophages involved in iron recycling. Serum iron remained suppressed for more than 48 hours after injection. Using radiolabeled hepcidin, we demonstrated that the serum concentration of hepcidin at the 50-microg dose was 1.4 microM, consistent with the inhibitory concentration of 50% (IC50) of hepcidin measured in vitro. Radiolabeled hepcidin accumulated in the ferroportin-rich organs, liver, spleen, and proximal duodenum. Our study highlights the central role of the hepcidin-ferroportin interaction in iron homeostasis. The rapid and sustained action of a single dose of hepcidin makes it an appealing agent for the prevention of iron accumulation in hereditary hemochromatosis.

Netrins are guidance cues that play a fundamental role in organizing the developing brain. The netrin receptor, DCC (deleted in colorectal cancer), is highly expressed by dopaminergic (DA) neurons. DCC may therefore participate in the organization of DA circuitry during development and also influence DA function in the adult. Here we show that adult dcc heterozygous mice exhibit a blunted behavioral response to the indirect DA agonist amphetamine and do not develop sensitization to its effects when treated repeatedly. These behavioral alterations are associated with profound changes in DA function. In the medial prefrontal cortex, dcc heterozygotes exhibit increased tyrosine hydroxylase (TH) protein levels and dramatic increases in basal concentrations of DA and DA metabolites. In contrast, in the nucleus accumbens, dcc heterozygotes show no changes in either TH or DA levels, but exhibit decreased concentrations of DA metabolites, suggesting reduced DA activity. In addition, dcc heterozygous mice exhibit a small, but significant reduction in total number of TH-positive neurons in midbrain DA cell body regions. These results demonstrate for the first time that alterations in dcc expression lead to selective changes in DA function and, in turn, to differences in DA-related behaviors in adulthood. These findings raise the possibility that changes in dcc function early in life are implicated in the development of DA dysregulation observed in certain psychiatric disorders, such as schizophrenia, or following chronic use of drugs of abuse.

Mutations in the Hfe gene can be associated with the iron overload disorder known as hemochromatosis. A number of recent studies suggest that carrying an Hfe mutation is a risk factor or genetic modifier for Alzheimer's disease (AD). In AD, Hfe protein expression is induced on cells associated with neuritic plaques and on neurons in the periplaque area. In this study, the factors that may be responsible for induction of Hfe in AD brain were determined using BV-2 cells. Hfe expression was induced by serum deprivation, menadione and beta-amyloid. The labile iron pool was consistently decreased when Hfe expression increased. However, the changes in expression of Hfe appeared independent of the expression of transferrin receptor and ferritin. These data provide insight into the induction of Hfe in AD and indicate that Hfe expression may be a protective function to limit cellular iron exposure during cell stress. These results are the first in a series of studies to understand how mutations in Hfe can be a risk factor for AD.

As described in this volume, our knowledge of mammalian iron metabolism has advanced dramatically over the past decade. While basic physiological concepts were described through elegant experiments in the 1950s and 1960s, many molecular details remained unknown. Modern techniques in genetics, biochemistry and molecular biology have allowed for the identification and characterization of most, but probably not all, of the key molecules that move iron into, out of, and between cells. Insight into the complex regulation of these iron transport proteins has been accelerated through the identification of hemochromatosis disease genes. Although much of the information has been gleaned from experiments in animal models, it is likely that it can be directly extrapolated to humans.

Hepcidin is a recently discovered peptide made in the liver, distributed in plasma and excreted in urine. This peptide hormone is the homeostatic regulator of intestinal iron absorption, iron recycling by macrophages, and iron mobilization from hepatic stores. Hepcidin acts by inhibiting the efflux of iron through ferroportin, the sole known iron exporter of enterocytes, macrophages and hepatocytes. As befits an iron-regulatory hormone, hepcidin synthesis is increased by iron loading and decreased by anemia and hypoxia. Hepcidin is markedly induced during infections and inflammation, causing iron to be sequestered in macrophages, hepatocytes and enterocytes. The resulting decrease in plasma iron levels eventually contributes to the anemia associated with infection and inflammation. These alterations in iron metabolism probably have a role in host defense by limiting the availability of iron to invading microorganisms. At the opposite extreme, early studies indicate that hepcidin deficiency--due to the dysregulation of its synthesis or mutations in the hepcidin gene itself--is the immediate cause of most forms of hemochromatosis.

Since its original description as a rare disease of iron overload resulting in liver disease, diabetes mellitus, and bronzing of the skin ('bronze diabetes'), hereditary hemochromatosis has undergone several redefinitions leading to widely varying estimates of its prevalence. Over the last decade, the finding of a relatively high prevalence of the C282Y polymorphism of the HFE gene associated with hemochromatosis in Northern European populations suggested that the disease may be much more common than previously thought. However, several large population-based studies have now shown that the penetrance of the C282Y/C282Y genotype is very low, indicating that C282Y homozygosity is a necessary but not sufficient factor in causation of the disease. Studies are now focusing on other genetic and environmental factors, including alcohol, that may contribute to differential expression of C282Y homozygosity.

Non-HFE haemochromatosis is a negative definition applied to all those haemochromatosis disorders that are unrelated to HFE mutations. Four genes are responsible for the distinct types of non-HFE haemochromatosis: hepcidin and hemojuvelin are the genes involved in type 2 or juvenile haemochromatosis, transferrin receptor 2 is involved in type 3 haemochromatosis, and ferroportin 1 is mutated in type 4, the atypical dominant form of primary iron overload. Molecular genetic studies of these conditions have greatly contributed to our understanding of the regulation of iron absorption. A milestone was the discovery that hepcidin, the key iron regulator in mice, is the gene mutated in the most severe, juvenile form of haemochromatosis. This finding indicates a fundamental role of hepcidin in inhibiting both iron absorption from duodenal cells and iron release from macrophages, and has opened up a new view of haemochromatosis as a disorder of hepcidin.

A large body of evidence indicates that the level of serum ferritin parallels the concentration of storage iron within the body, regardless of the cell type in which it is stored. Elevated serum ferritin levels, in the absence of inflammation and liver disease, are currently taken to indicate increased iron stores and require further investigation to determine the site of iron overload. Until recently, the only genetic disorder with elevated serum ferritin levels known in Western countries was hereditary HLA-related HFE-related genetic haemochromatosis in Caucasians (HFE, OMIM 235200), and a high serum ferritin in apparently healthy persons was considered suggestive of this disease. In the last few years, at least two novel genetic disorders of iron metabolism presenting as unexplained hyperferritinaemia have been recognized. The first one is hereditary hyperferritinaemia/cataract syndrome (HHCS, OMIM 600886). HHCS arises from various point mutations or deletions within a protein binding sequence in the 5'-UTR of the L-ferritin mRNA that results in increased efficiency of L-ferritin translation. The second one is haemochromatosis type 4, or HFE4 (OMIM 606069), or ferroportin disease. In this latter condition, reticuloendothelial iron overload and hyperferritinaemia are caused by loss-of-function mutations in the SLC11A3 gene that mainly impair macrophage iron recycling. These genetic disorders should be taken into account in the differential diagnosis of unexplained hyperferritinaemia.

Members of the LAP protein family, LET-413 in Caenorhabditis elegans, Scribble in Drosophila melanogaster, and Erbin, Lano, Densin-180 and hScrib in mammals, have conserved structural features. LET-413 and Scribble are junctional proteins involved in establishing and maintaining epithelial cell polarity. scribble also behaves as a neoplastic tumor suppressor gene. We show here that, in epithelial cells, hScrib is recruited at cell-cell junctions in an E-cadherin-dependent manner as shown by calcium switch assays in MDCK cells, re-expression of E-cadherin in MDA-231 cells treated by 5-Aza-2'-deoxycytidine (5Aza), and siRNA experiments. hScrib is restricted at the basolateral membrane of epithelial cells by its LRR domain, and is enriched in Triton X-100-insoluble fractions. In breast cancers, most lobular tumors did not express hScrib and E-cadherin while ductal tumors had a less frequent downregulation of hScrib. Our data provide additional insights on the modalities of recruitment of hScrib at the cell-cell junctions, and establish a potential link between the E-cadherin and hScrib tumor suppressors.

Axon guidance represents a key stage in the formation of neuronal network. Axons are guided by a variety of guidance factors, such as semaphorins, ephrins and netrin. Plexins function as receptors for the repulsive axonal guidance molecules semaphorins. Intracellular domains of plexins are responsible for initiating cellular signal transduction inducing axon repulsion. Recent advances have revealed molecular mechanisms for plexin-mediated cytoskeletal reorganization, leading to repulsive responses, and small GTPases play important roles in this signaling. Plexin-B1 activates Rho through Rho-specific guanine nucleotide exchange factors, leading to neurite retraction. Plexin-B1 possesses an intrinsic GTPase-activating protein activity for R-Ras and induces growth cone collapse through R-Ras inactivation. In this review we survey current understanding of the signaling mechanisms of plexins.

Hepatic veno-occlusive disease (HVOD) is a serious complication of hematopoietic stem cell transplantation (HSCT). Since the liver is a major site of iron deposition in HFE-associated hemochromatosis, and iron has oxidative toxicity, we hypothesized that HFE genotype might influence the risk of HVOD after myeloablative HSCT. We determined HFE genotypes in 166 HSCT recipients who were evaluated prospectively for HVOD. We also tested whether a common variant of the rate-limiting urea cycle enzyme, carbamyl-phosphate synthetase (CPS), previously observed to protect against HVOD in this cohort, modified the effect of HFE genotype. Risk of HVOD was significantly higher in carriers of at least one C282Y allele (RR=3.7, 95% CI 1.2-12.1) and increased progressively with C282Y allelic dose (RR=1.7, 95% CI 0.4-6.8 in heterozygotes; RR=8.6, 95% CI 1.5-48.5 in homozygotes). The CPS A allele, which encodes a more efficient urea cycle enzyme, reduced the risk of HVOD associated with HFE C282Y. We conclude that HFE C282Y is a risk factor for HVOD and that CPS polymorphisms may counteract its adverse effects. Knowledge of these genotypes and monitoring of iron stores may facilitate risk-stratification and testing of strategies to prevent HVOD, such as iron chelation and pharmacologic support of the urea cycle.

Amino-terminal fragments of huntingtin (htt) appear to result from proteolytic processing of the full-length protein in Huntington's disease (HD), and fragments containing pathological expansions of polyglutamine elicit toxicity in model systems. Such fragments are sequestered into insoluble aggregates, which may initially serve a cellular protective mechanism, while soluble fragments and/or oligomers may be a more acute toxic species. Agents which enhance mutant htt clearance have shown therapeutic potential in animal models of HD. Here, we present the first evidence of an htt-specific single-chain Fv intrabody (C4) that selectively targets the soluble fraction of amino-terminal htt fragments. Our findings suggest that the C4 intrabody binds weakly, but does not alter the levels of endogenous, full-length htt. C4 appears to decrease the steady-state levels of amino-terminal htt fragments by binding to non-aggregated, but not aggregated, htt species. Intrabodies may be used as potential curative agents, and as drug discovery tools, for HD and other misfolded protein disorders.

Hematologic malignancies are characterized by fusion genes of biological/clinical importance. Immortalized cell lines with such aberrations are today widely used to model different aspects of leukemogenesis. Using cDNA microarrays, we determined the gene expression profiles of 40 cell lines as well as of primary leukemias harboring 11q23/MLL rearrangements, t(1;19)[TCF3/PBX1], t(12;21)[ETV6/RUNX1], t(8;21)[RUNX1/CBFA2T1], t(8;14)[IGH@/MYC], t(8;14)[TRA@/MYC], t(9;22)[BCR/ABL1], t(10;11)[PICALM/MLLT10], t(15;17)[PML/RARA], or inv(16)[CBFB/MYH11]. Unsupervised classification revealed that hematopoietic cell lines of diverse origin, but with the same primary genetic changes, segregated together, suggesting that pathogenetically important regulatory networks remain conserved despite numerous passages. Moreover, primary leukemias cosegregated with cell lines carrying identical genetic rearrangements, further supporting that critical regulatory pathways remain intact in hematopoietic cell lines. Transcriptional signatures correlating with clinical subtypes/primary genetic changes were identified and annotated based on their biological/molecular properties and chromosomal localization. Furthermore, the expression profile of tyrosine kinase-encoding genes was investigated, identifying several differentially expressed members, segregating with primary genetic changes, which may be targeted with tyrosine kinase inhibitors. The identified conserved signatures are likely to reflect regulatory networks of importance for the transforming abilities of the primary genetic changes and offer important pathogenetic insights as well as a number of targets for future rational drug design.

<h4>Background</h4>We determined the effect of regional citrate versus systemic heparin anticoagulation for continuous renal replacement therapy in critically ill subjects suffering from acute renal failure who were not at high risk for hemorrhagic complications.<h4>Methods</h4>Between April 1999 and June 2002, 30 critically ill subjects requiring continuous renal replacement therapy and using 79 hemofilters were randomly assigned to receive regional citrate or systemic heparin anticoagulation.<h4>Results</h4>The median hemofilter survival time was 124.5 hours (95% CI 95.3 to 157.4) in the citrate group, which was significantly longer than the 38.3 hours (95% CI 24.8 to 61.9) in the heparin group (P < 0.001). Increasing illness severity score, male gender, and decreasing antithrombin-III levels were independent predictors of an increased relative hazard of hemofilter failure. After adjustment for illness severity, antithrombin-III levels increased significantly more over the period of study in the citrate as compared to the heparin group (P= 0.038). Moreover, after adjustment for antithrombin-III levels and illness severity score, the relative risk of hemorrhage with citrate anticoagulation was significantly lower than that with heparin (relative risk of 0.14; 95% CI 0.02 to 0.96, P= 0.05).<h4>Conclusion</h4>Compared with systemic heparin anticoagulation, regional citrate anticoagulation significantly increases hemofilter survival time, and significantly decreases bleeding risk in critically ill patients suffering from acute renal failure and requiring continuous renal replacement therapy.

Peroxiredoxin 6 (Prdx6), a bifunctional 25-kDa protein with both GSH peroxidase and phospholipase A2 activities, is the only mammalian 1-Cys member of the peroxiredoxin superfamily and is expressed in all major organs, with a particularly high level in lung. Prdx6 uses GSH as an electron donor to reduce H2O2 and other hydroperoxides including phospholipid hydroperoxides at approximately 5 micromol/mg protein/min with K1 approximately 3 x 10(6) M(-1) s(-1). Oxidation of the Cys47 to a sulfenic acid during catalysis requires piGST-catalyzed glutathionylation and reduction with GSH to complete the enzymatic cycle. Prdx6 stably overexpressed in cells protected against oxidative stress, whereas antisense treatment resulted in oxidant stress and apoptosis. Adenoviral-mediated overexpression of Prdx6 in mouse lungs protected against the toxicity of hyperoxia, whereas Prdx6-null mice were more sensitive to the effects of hyperoxia or paraquat. We postulate that Prdx6 functions in antioxidant defense mainly by facilitating repair of damaged cell membranes via reduction of peroxidized phospholipids. The PLA2 activity of Prdx6 is Ca2+ independent and maximal at acidic pH. Inhibition of PLA2 activity results in alterations of lung surfactant phospholipid synthesis and turnover. Thus, Prdx6, a unique mammalian peroxiredoxin, is an important antioxidant enzyme and has a major role in lung phospholipid metabolism.

In this report, we have examined the expression of the T cell survival signals, OX40 ligand (OX40L) and CD30 ligand (CD30L) on CD4(+)CD3(-)CD11c(-)B220(-)IL-7Ralpha(+) inducer cells from birth to adulthood in mice. We found that adult but not neonatal inducer cells expressed high levels of OX40L and CD30L, whereas their expression of TNF-related activation-induced cytokine (TRANCE) and receptor activator of NF-kappaB (RANK) was comparable. The failure of neonatal inducer cells to express the ligands that rescue T cells helps to explain why exposure to Ag in neonatal life induces tolerance rather than immunity. The expression of OX40L and CD30L on inducer cells increased gradually in the first few weeks of life achieving essentially normal levels around the time mice were weaned. We found that IL-7 signaling through the common cytokine receptor gamma-chain was critical for the optimal expression of both TNF-related activation-induced cytokine and CD30L but not OX40L. Furthermore, glucocorticoids, which potently suppress T effector function, did not influence the expression of OX40L and CD30L in the presence of IL-7.

According to some authors the obsessive-compulsive (OC) spectrum includes on one extreme, the Obsessive-Compulsive Disorder (OCD) and on the other extreme the most impulsive behaviors. This is a controversial idea and other authors define the OC spectrum in different ways. The serotonin transporter (5-HTT) gene is one of the main genes that control serotonergic function. A polymorphism in the promoter area of this gene classifies subjects with low expression as S individuals (s/s or s/l) and subjects with high expression as L individuals (l/l). This polymorphism was studied in female OCD patients (n = 24), non-impulsive controls (n = 112) and impulsive suicidal patients (n = 118) to support the OC spectrum hypothesis from a genetic perspective. A linear association exists among the serotonin transporter promoter functional genotypes (S versus L individuals) (chi2 linear by linear association = 8.9; df = 1; p = 0.003). The frequency of S individuals (s/l or s/s) was lowest in OCD (54%, 13/24); intermediate in non-impulsive controls (71%, 80/112) and highest in impulsive suicide attempters (82%, 96/117). More importantly, future studies need to consider that genetics may be related to behavioral dimensions (compulsivity to impulsivity) instead of to specific psychiatric disorders defined in clinical terms.

Epstein-Barr virus (EBV) genomes persist indefinitely in latently infected human cells, in part due to their ability to stably segregate during cell division. This process is mediated by the viral EBNA1 protein, which tethers the viral episomes to the cellular mitotic chromosomes. We have previously identified a mitotic chromosomal protein, human EBNA1 binding protein 2 (hEBP2), which binds to EBNA1 and enables EBNA1 to partition EBV-based plasmids in Saccharomyces cerevisiae. Using an RNA silencing approach, we show that hEBP2 is essential for the proliferation of human cells and that repression of hEBP2 severely decreases the ability of EBNA1 and EBV-based plasmids to bind mitotic chromosomes. When expressed in yeast, hEBP2 undergoes the same cell cycle-regulated association with the mitotic chromatin as in human cells, and using yeast temperature-sensitive mutant strains, we found that the attachment of hEBP2 to mitotic chromosomes was dependent on the Ipl1 kinase. Both RNA silencing of the Ipl1 orthologue in human cells (Aurora B) and specific inhibition of the Aurora B kinase activity with a small molecule confirmed a role for this kinase in enabling hEBP2 binding to human mitotic chromosomes, suggesting that this kinase can regulate EBV segregation.

HFE hemochromatosis is the most frequent genetic iron overload disease. It is linked to the C282Y mutation of the HFE protein, protein encoded by the HFE gene, which is located on chromosome 6. The mechanisms accounting for iron excess are not only digestive hyperabsorption of iron but also excessive recycling of macrophagic iron coming from erythrophagocytosis and secreted into the blood. Both mechanisms are linked to an HFE-related hepatic failure in producing hepcidin, a key hormone of body iron regulation. The marked phenotypic variability of C282Y homozygosity expression is likely related to both genetic and environmental factors. The HFE gene discovery has rendered non invasive the positive diagnostic of HFE hemochromatosis, which is now based first on an increased level of plasma transferrin saturation leading to the request of the HFE mutation. Then, hepatic MRI is a reliable method to quantify iron overload. The HFE gene discovery has also paved the road of an enlarged field of differential diagnoses corresponding to novel entities of non-HFE related genetic iron overload syndromes.

Most of the iron required for erythropoiesis is provided by heme iron recycling following degradation of senescent erythrocytes by tissue macrophages. Accumulation of biochemical modifications at the red blood cell membrane during ageing (externalisation of phosphatidyl-serine, peroxydation of membrane-bound lipoproteins, loss of sialic acid residues and formation of senescence neoantigens) constitute a series of signals that will allow the macrophage to identify the red blood cells to be eliminated, through interaction with specific receptors. After this initial recognition step, the red blood cell is internalised by phagocytosis, and phagosome maturation, which can comprise recruitment of the endoplasmic reticulum, will favour degradation of red blood cell constituents. Heme is catabolised by heme oxygenase 1, anchored in the endoplasmic reticulum membrane. A fraction of the released iron will be recycled back to the plasma through ferroportin, a membrane-bound Fe (II) export molecule, and a fraction will retained within the ferritin molecules, to be released at later stages. Multiple evidence coming from human diseases (type 4 hemochromatosis) and animal models indicate that ferroportin is essential for heme iron recycling by macrophages. Furthermore, ferroportin seems to be the molecular target of hepcidin, this circulating peptide synthesized by the liver and acting as a negative regulator of intestinal iron absorption and iron recycling by macrophages. Perturbations in erythrophagocytosis play a physiopathological role in several diseases, including hemochromatosis, anemia of chronic disorders and thalassemia.

Genetic hemochromatosis (GH) is an iron overload disorder mainly due to the C282Y mutation of the HFE gene. The possibility of bone involvement was only recently recognized. The aims of this study were to assess bone mineral density (BMD) and bone remodeling in men with GH, and to examine the influence of iron overload. Thirty-eight men (mean age 47.2+/-9.4 years) with well-defined HFE-related GH were studied. They had an important iron overload with liver iron concentration to age ratio >2.5, no previous venesection therapy and were C282Y homozygotes (n=37) or compound C282Y/H63D heterozygote (n=1). BMD measured by DXA was 0.925+/-0.15 g/cm2 at the lumbar spine (LS) and 0.778+/-0.13 g/cm2 at the femoral neck (FN). Osteopenia (T-score<-1 SD) was observed in 78.9% of patients and osteoporosis (T-score<-2.5 SD) in 34.2%. Vitamin D levels were normal, and no 1-84 parathyroid hormone dysfunction was found. Hypogonadism was found in only 13.2% of patients. Patients with hypogonadism had lower LS BMD than eugonadal patients (0.788+/-0.16 and 0.954+/-0.14 g/cm2). Bone remodeling and parathyroid hormone levels were lower in patients with cirrhosis, but BMD values were similar to those in patients without cirrhosis. FN BMD appeared to fall with rising hepatic iron concentrations (r=-0.399). We conclude that there is significant bone loss in HFE-related hemochromatosis that cannot solely be explained by hypogonadism or cirrhosis. Further investigations are needed to determine the role of iron overload itself.

<h4>Aim</h4>To report a patient with C282Y homozygocity, depleted body iron and intestinal atrophy caused by celiac disease (CD) who experienced resolution of the enteropathy with subsequent normalization of iron metabolism upon gluten-free diet.<h4>Methods</h4>To obtain information on the tissue distribution and quantitative expression of proteins involved in duodenal iron trafficking, we determined the expression of divalent-metal transporter 1 (DMT1), ferroportin 1 (FP1) and transferrin receptor (TfR1) by means of immunohistochemistry and real-time PCR in duodenal biopsies of this patient.<h4>Results</h4>Whereas in hereditary hemochromatosis patients without CD, DMT1 expression was up-regulated leading to excessive uptake of iron, we identified a significant reduction in protein and mRNA expression of DMT1 as a compensatory mechanism in this patient with HH and CD.<h4>Conclusion</h4>Occult CD may compensate for increased DMT1 expression in a specific subset of individuals with homozygous C282Y mutations in the hemochromatosis (HFE) gene, thus contributing to the low penetrance of HH.

<h4>Background</h4>Although most patients with hereditary haemochromatosis have HFE C282Y mutations, the lifetime risk to HFE C282Y homozygotes of developing fatal diseases such as hepatocellular carcinoma is uncertain. We have carried out a cross-sectional study to determine the proportion of diagnosed hepatocellular carcinoma patients who are homozygous for the HFE C282Y mutation; and to estimate the penetrance of this genotype with respect to hepatocellular carcinoma in East Anglia.<h4>Methods</h4>Tissue biopsies were analysed from 144 cases of hepatocellular carcinoma for HFE C282Y mutations; the data produced were compared with the frequency of HFE mutations in a large sample of the local population. Data were also retrieved from the East Anglian Cancer Intelligence Unit to determine the annual incidence of hepatocellular carcinoma; and from appropriate life tables.<h4>Results</h4>Eight out of 144 of the cases were homozygous for the HFE C282Y mutation, all 8 cases were male. 6 of these 8 cases had a previous diagnosis of hereditary haemochromatosis. Male HFE C282Y homozygotes were more likely to be diagnosed with hepatocellular carcinoma (odds ratio [OR] = 14, 95% confidence interval [CI] = 5-37). For this population, we estimate that the penetrance of the HFE C282Y homozygous genotype, with respect to hepatocellular carcinoma, was between 1.31 % and 2.1% for males and was zero for females.<h4>Conclusion</h4>In this population, we found that only a very small proportion of homozygotes for the HFE C282Y mutation developed hepatocellular carcinoma. However, individuals with this genotype have a significantly increased risk of this rare disease relative to those who do not carry the mutations.

<h4>Background</h4>Hereditary hemochromatosis (HH) is a common inherited disorder of iron metabolism in Northern European populations. The discovery of a candidate gene in 1996 (HFE), and of its main mutation (C282Y), has radically altered the way to diagnose this disease. The aim of this study was to assess the impact of the HFE gene discovery on the clinical presentation and epidemiology of HH.<h4>Methods</h4>We studied our cohort of 415 patients homozygous for the C282Y allele and included in a phlebotomy program in a blood centre in western Brittany, France.<h4>Results</h4>In this cohort, 56.9% of the patients were male and 21.9% began their phlebotomy program before the implementation of the genetic test. A significant decrease in the sex ratio was noticed following implementation of this DNA test, from 3.79 to 1.03 (p < 10(-5)), meaning that the proportion of diagnosed females relatives to males greatly increased. The profile of HH patients at diagnosis changed after the DNA test became available. Serum ferritin and iron values were lower and there was a reduced frequency of clinical signs displayed at diagnosis, particularly skin pigmentation (20.1 vs. 40.4%, OR = 0.37, p < 0.001) and hepatomegaly (11.0 vs. 22.7%, OR = 0.42, p = 0.006). In contrast, fatigue became a more common symptom at diagnosis (68.0 vs. 51.2%, OR = 2.03, p = 0.004).<h4>Conclusion</h4>This study highlights the importance of the HFE gene discovery, which has simplified the diagnosis of HH and modified its clinical presentation and epidemiology. This study precisely measures these changes. Enhanced diagnosis of HFE-related HH at an early stage and implementation of phlebotomy treatment are anticipated to maintain normal life expectancy for these patients.

With the completion of full genome sequences and advancement in high-throughput technologies, in silico methods have been successfully used to integrate diverse data sources toward unraveling the combinatorial nature of transcriptional regulation. So far, almost all of these studies are restricted to lower eukaryotes such as budding yeast. We describe here a computational search for functional transcription-factor (TF) combinations using phylogenetically conserved sequences and microarray-based expression data. Taking into account both orientational and positional constraints, we investigated the overrepresentation of binding sites in the vicinity of one another and whether these combinations result in more coherent expression profiles. Without any prior biological knowledge, the search led to the discovery of several experimentally established TF associations, as well as some novel ones. In particular, we identified a regulatory module controlling cell cycle-dependent transcription of G2-M genes and expanded its functional generality. We also detected many homotypic combinations, supporting the importance of binding-site density in transcriptional regulation of higher eukaryotes.

Dysfunction of dopaminergic neurons may contribute to motor impairment in Huntington's disease. Here, we study the role of brain-derived neurotrophic factor (BDNF) in alterations of the nigrostriatal system associated with transgenics carrying mutant huntingtin. Using huntingtin-BDNF+/- double-mutant mice, we analyzed the effects of reducing the levels of BDNF expression in a model of Huntington's disease (R6/1). When compared with R6/1 mice, these mice exhibit an increased number of aggregates in the substantia nigra pars compacta. In addition, reduction of BDNF expression exacerbates the dopaminergic neuronal dysfunction seen in mutant huntingtin mice, such as the decrease in retrograde labelling of dopaminergic neurons and striatal dopamine content. However, mutant huntingtin mice with normal or lowered BDNF expression show the same decrease in the anterograde transport, number of dopaminergic neurons and nigral volume. In addition, reduced BDNF expression causes decreased dopamine receptor expression in mutant huntingtin mice. Examination of changes in locomotor activity induced by dopamine receptor agonists revealed that, in comparison with R6/1 mice, the double mutant mice exhibit lower activity in response to amphetamine, but not to apomorphine. In conclusion, these findings demonstrate that the decreased BDNF expression observed in Huntington's disease exacerbates dopaminergic neuronal dysfunction, which may participate in the motor disturbances associated with this neurodegenerative disorder.

Striatal and cortical mitochondria from knock-in and transgenic mutant huntingtin mice were examined for their sensitivity to calcium induction of the permeability transition, a cause of mitochondrial depolarization and ATP loss. The permeability transition has been suggested to contribute to cell death in Huntington's Disease. Mitochondria were examined from slowly progressing knock-in mouse models with different length polyglutarnine expansions (Q20, Q50, Q92, Q111) and from the rapidly progressing transgenic R6/2 mice overexpressing exon I of human huntingtin with more than 110 polyglutamines. As previously observed in rats, striatal mitochondria from background strain CD1 and C57BL/6 control mice were more sensitive to calcium than cortical mitochondria. Between 5 and 12 months in knock-in Q92 mice and between 8 and 12 weeks in knock-in Q111 mice, striatal mitochondria developed resistance, becoming equally sensitive to calcium as cortical mitochondria, while those from Q50 mice were unchanged. Cortical mitochondrial calcium sensitivity did not change. In R6/2 mice striatal and cortical mitochondria were equally resistant to Ca2+ while striatal mitochondria from littermate controls were more susceptible. No increases in calcium sensitivity were observed in the mitochondria from Huntington's Disease (HD) mice compared to controls. Neither motor abnormalities, nor expression of cyclophilin D corresponded to the changes in mitochondrial sensitivity. Polyglutamine expansions in huntingtin produced an early increased resistance to calcium in striatal mitochondria suggesting mitochondria undergo compensatory changes in calcium sensitivity in response to the many cellular changes wrought by polyglutamine expansion.

<h4>Objective</h4>To determine whether pulmonary vascular bed contributes to the development of in situ thrombosis and vascular remodelling in secondary pulmonary hypertension (SPH) via changes in its local secretory activities.<h4>Methods</h4>Seventy-one patients with the diagnosis of secondary pulmonary hypertension (38 females, mean age 40.36+/-1.05 years) were included in the study. Selective right and left heart catheterization was performed to each patient for diagnostic purposes. Blood samples obtained from left ventricle (LV) and pulmonary artery (PA) of each patient were analyzed for levels of plasminogen activator inhibitor-1 (PAI-1), platelet derived growth factor (PDGF), vascular endothelial growth factor (VEGF), D-dimer, von Willebrand factor (vWF), protein-C, antithrombin-III, fibrinogen, and plasminogen. Results were compared between LV and PA. Correlation analysis between each parameter and mean pulmonary artery pressure (MPAP) was performed.<h4>Results</h4>Although mean level of VEGF in LV and PA were found to be in normal range, it was significantly higher in LV than in PA (p<0.001). Mean PDGF and D-dimer levels, which remained in normal range were also higher in LV (p<0.001 and p<0.001, respectively) than in PA;.vWF showed similar degree of elevation in both LV and PA. Only one parameter, PAI-1, was found to be significantly higher in PA than in LV (p=0.012). Antithrombin-III, protein C, plasminogen, and fibrinogen levels showed no significant differences between two chambers. They also remained in normal range, except for fibrinogen, which was slightly elevated in both LV and PA. Correlation analysis revealed strong positive correlation between D-dimer level in both LV and PA and MPAP (r=0.775, p<0.001 and r=0.649, p<0.001, respectively).<h4>Conclusion</h4>In SPH, pulmonary vascular bed shows increased thrombotic, hypofibrinolytic, and proliferative activities, which are partially related to the severity of illness.

Pericentric heterochromatin plays an important role in epigenetic gene regulation. We show that pericentric heterochromatin aggregates during myogenic differentiation. This clustering leads to the formation of large chromocenters and correlates with increased levels of the methyl CpG-binding protein MeCP2 and pericentric DNA methylation. Ectopic expression of fluorescently tagged MeCP2 mimicked this effect, causing a dose-dependent clustering of chromocenters in the absence of differentiation. MeCP2-induced rearrangement of heterochromatin occurred throughout interphase, did not depend on the H3K9 histone methylation pathway, and required the methyl CpG-binding domain (MBD) only. Similar to MeCP2, another methyl CpG-binding protein, MBD2, also increased during myogenic differentiation and could induce clustering of pericentric regions, arguing for functional redundancy. This MeCP2- and MBD2-mediated chromatin reorganization may thus represent a molecular link between nuclear genome topology and the epigenetic maintenance of cellular differentiation.

Whereas a recent study reported an increased risk of colorectal cancer associated with any HFE germ line mutation (C282Y or H63D), other investigators have concluded there is no increased risk, or that any increase is dependent on polymorphisms in HFE-interacting genes such as the transferrin receptor (TFR). We have established the frequency of HFE mutations in colorectal cancer patients (n = 327) with a family history of the disease and randomly selected controls (n = 322); this design increases greatly the study's power. Genotyping for the TRF S142G polymorphism was also conducted on a large proportion of the study group. Using PCR, restriction enzyme mapping, sequencing followed by data analysis with Fisher's exact test and logistic regression, we show that the presence of any HFE mutation (Y282 or D63) was not associated with colorectal cancer risk (P = 0.57). In contrast, individuals compound heterozygous for both mutations (15 cases versus 5 controls) had thrice the odds of developing colorectal cancer (odds ratio, 3.03; 95% confidence interval, 1.06-8.61) compared with those with a single mutation. This finding did not quite reach statistical significance after allowing for multiple post hoc testing (P(observed) = 0.038 versus P = 0.025, with Bonferonni correction). Overall, our data indicate that individuals with a single HFE mutation, C282Y or H63D, are unlikely predisposed to develop colorectal cancer. However, risk of colorectal cancer might be increased by compound heterozygosity for the HFE mutations in the small number of subjects studied. TFR gene polymorphism was not an independent risk factor and did not modify the disease risk associated with HFE mutation.

<h4>History</h4>A 28 year young female presented to our hospital for further evaluation with recently diagnosed diabetes mellitus, hyperpigmentation of the skin, hepato- and splenomegaly, thrombocytopenia and an elevated transferrin saturation (96 %), but a negative test for HFE gene mutations such as C282Y and H63D.<h4>Findings</h4>Using the mini-laparascopic technique we diagnosed a smallnodular liver cirrhosis with an iron overload.<h4>Diagnosis and treatment</h4>This is the clinical presentation of one subtype of the so called Non-HFE-hemochromatosis, the juvenile hemochromatosis (HFE2). Other causes of primary and secondary iron overload have been ruled out. Different from the HFE-positive hemochromatosis (HFE 1) in which the gene defect is located on chromosome 6, the defect in HFE 2 is located on chromosome 1. The underlying genetic defect has been localized within recently identified HJV gene. Phlebotomy is the treatment of choice, to be performed until the ferritin level is lower than 50 microg/l.<h4>Conclusions</h4>If liver cirrhosis is present in hemochromatosis, the overall risk of developing heptatocellular carcinoma is 20 times higher than in the normal population. Therefore it is suggested to perform an ultrasound examination of the liver and an AFP-test every 6 months, whereas an MRI-scan should be performed once a year, as a basis for further treatment options.

<h4>Aim</h4>To determine serum leptin levels and investigate their correlations with anthropometric and metabolic parameters and biochemical liver function in patients with chronic hepatitis C virus (HCV) infection and their potential clinical implications.<h4>Methods</h4>Forty-two chronic HCV-infected patients without anti-viral treatment were enrolled in this study, 30 patients had chronic hepatitis C, 10 had cirrhosis, and 2 had hepatocellular carcinoma (HCC). Thirty age- and sex-matched healthy individuals served as controls. Serum leptin levels were determined by ELISA. The biochemical liver function and serum lipids were determined at the same time. The height and body weight of patients and controls were measured, and body mass index (BMI) and body fat were calculated simultaneously. The correlations of serum leptin levels with anthropometric and metabolic parameters and biochemical liver function were assessed statistically.<h4>Results</h4>The mean of serum leptin levels in patients with chronic hepatitis C, HCV-associated cirrhosis, HCV-associated HCC and control groups was (6.13+/-3.94), (5.25+/-4.21), (4.17+/-0.28), and (3.59+/-3.44) ng/mL, respectively. The serum leptin level in patients with chronic hepatitis C was significantly higher than that in controls. The serum leptin levels between cirrhotic patients and controls and between male and female cirrhotic patients had no significant difference. Serum leptin levels were positively-correlated with body fat, BMI, and apolipoprotein B (Apo B) in patients with chronic HCV infection. The serum alanine aminotransferase (ALT) levels were closely-correlated with BMI in patients with chronic hepatitis C.<h4>Conclusion</h4>HCV infection interferes with fat and lipid metabolism in patients with chronic HCV infection and leptin may play a role in hepatosteatosis.

<h4>Background</h4>Some experimental evidence suggests that iron may play a role in colorectal carcinogenesis, but human data for this role have been conflicting, possibly because of problems related to study design or measurement of iron exposure. We assessed dietary iron intake and genetic and biochemical markers of iron status in a prospective, nested case-control study of women enrolled in the Nurses' Health Study.<h4>Methods</h4>Among 32 826 women who provided a blood specimen, we identified 527 women with colorectal adenoma and 527 matched control subjects who underwent endoscopy but were not diagnosed with adenoma after blood collection. We assessed iron intake, mutations in the HFE gene that are associated with hereditary hemochromatosis (i.e., H63D and C282Y), and plasma biochemical measures of total body iron, including transferrin saturation and the ratio of the concentrations of transferrin receptors to ferritin. Logistic regression models were used to estimate relative risks (RR) and 95% confidence intervals (CI). All statistical tests were two-sided.<h4>Results</h4>Women with any HFE gene mutation had higher total body iron stores, as reflected by higher transferrin saturations (P < .001) and lower levels of the ratio of transferrin receptors to ferritin (P = .02), than women with no HFE gene mutation. However, HFE gene mutations were not associated with risk of adenoma (multivariable RR = 1.08, 95% CI = 0.83 to 1.39; P = .58). Moreover, comparison of extreme categories showed no associations between adenoma and the extent of transferrin saturation (multivariable RR = 0.96, 95% CI = 0.63 to 1.47; Ptrend = .66), the ratio of transferrin receptors to ferritin (multivariable RR = 0.98, 95% CI, 0.60 to 1.60; Ptrend = .99), or dietary iron intake (multivariable RR = 1.04, 95% CI = 0.68 to 1.57; Ptrend = .94).<h4>Conclusions</h4>Although our study used several distinct measures of iron status (i.e., genetic mutations, biochemical markers, and dietary intake) and a nested case-control design, we did not observe a role for iron in the pathogenesis of colorectal neoplasia in women.

The aim of this study was to evaluate, in a prospective study, the predictive role of p53 status analysed at four different levels in identifying the response to preoperative radiotherapy in rectal adenocarcinoma. Before treatment, 70 patients were staged and endoscopic forceps biopsies from the tumour area were taken. p53 status was assessed by total cDNA sequencing, allelic loss analysis, immunohistochemistry, and p53 antibodies. Neoadjuvant treatment was based on preoperative radiotherapy or radiochemotherapy. Response to therapy was evaluated after surgery by both pathologic downstaging and histologic tumour regression grade. In all, 35 patients (50.0%) had p53 gene mutations; 44.4% of patients had an allelic loss; nuclear p53 overexpression was observed in 39 patients (55.7%); and p53 antibodies were detected in 11 patients (16.7%). In the multilevel analysis of p53 status, gene mutations correlated with both nuclear protein overexpression (P < 0.0001) and loss of heterozygosity (P = 0.013). In all, 29 patients (41.4%) were downstaged by pathologic analysis, and 19 patients (29.2%) were classified as tumour regression grade 1. Whatever the method of evaluation of treatment response, no correlation between p53 alterations and response to radiotherapy was observed. Our results do not support the use of p53 alterations alone as a predictive marker for response to radiotherapy in rectal carcinoma.

During rodent cortex development, cells born in the medial ganglionic eminence (MGE) of the basal telencephalon reach the embryonic cortex by tangential migration and differentiate as interneurons. Migrating MGE cells exhibit a saltatory progression of the nucleus and continuously extend and retract branches in their neuritic arbor. We have analyzed the migration cycle of these neurons using in vitro models. We show that the nucleokinesis in MGE cells comprises two phases. First, cytoplasmic organelles migrate forward, and second, the nucleus translocates toward these organelles. During the first phase, a large swelling that contains the centrosome and the Golgi apparatus separates from the perinuclear compartment and moves rostrally into the leading neurite, up to 30 mum from the waiting nucleus. This long-distance migration is associated with a splitting of the centrioles that line up along a linear Golgi apparatus. It is followed by the second, dynamic phase of nuclear translocation toward the displaced centrosome and Golgi apparatus. The forward movement of the nucleus is blocked by blebbistatin, a specific inhibitor of nonmuscle myosin II. Because myosin II accumulates at the rear of migrating MGE cells, actomyosin contraction likely plays a prominent role to drive forward translocations of the nucleus toward the centrosome. During this phase of nuclear translocation, the leading growth cone either stops migrating or divides, showing a tight correlation between leading edge movements and nuclear movements.

<h4>Aim</h4>To investigate the bile acid pool size after cholecystectomy whether or not correlated to the gastrointestinal migrating myoelectric complex (MMC) in guinea pigs.<h4>Methods</h4>Gallbladder motilities were assessed before cholecystectomy. Furthermore, we continuously monitored interdigestive gastrointestinal motilities using bipolar electrodes in conscious guinea pigs before and after surgery at 4 wk in standard diet group and high cholesterol diet (cholesterol gallstone) group. Total bile acid pool sizes were measured by isotope dilution method at meantime.<h4>Results</h4>After cholecystectomy, there were parallel falls in duration of phase I, II, III and MMC cycle duration but increase in amplitude in the guinea pigs with normal gallbladder function, and in the guinea pigs with cholesterol stones. However, There were not significantly differences. On the other hand, the bile acid pool was definitely small in the GS guinea pigs compared to normal guinea pigs and became slightly smaller after cholecystectomy. Similarly, bile acid in gallbladder bile, fecal bile acid was slightly increased in GS guinea pigs after cholecystectomy, to the same degree as normal. These differences, however, were not significant.<h4>Conclusion</h4>It is concluded that in the guinea pigs with normal gallbladder function, and in the guinea pigs with cholesterol stones: (1) Cholecystectomy produce a similar but less marked trend in bile acid pool; and (2) MMC are linked to enterohepatic circulation of bile acids, rather than surgery, which is consistent with changes of the bile acid pool size. As a result, gastrointestinal dyskinesia is not involved in occurrence of postcholecystectomy syndrome.

We here report a recent, rare case of Budd-Chiari syndrome, associated with a combination of hepatic vein and superior vena cava occlusion. A young female, who had been in good health, was admitted to our hospital because of massive ascites. The patient had used no oral contraceptives. Tests for coagulation disorders, hematological disorders, and antiphospholipid syndrome were all negative. Budd-Chiari syndrome was diagnosed by radiographic examination. The patient was suffering from a combination of hepatic vein and superior vena cava occlusion. In particular, the venous flow returned from the liver mainly through a right accessory hepatic vein, and stenosis was recognized at the orifice of this collateral vein into the vena cava. Subsequently, the patient underwent percutaneous balloon dilatation therapy for this stenosis. After this treatment, the massive ascites was gradually reduced, and she was discharged from our hospital. It has now been one year since discharge, and the patient has been doing well. If deteriorating liver function or intractable ascites occur again, a liver transplantation may be anticipated. This is the first case report of Budd-Chiari syndrome associated with a superior vena cava occlusion.

To investigate the effects of complex danshen solution and heparin on the changes of blood coagulation factors in rats with hemorrhagic shock, and to explore the therapy of coagulopathy by compound danshen solution, the rat model of hemorrhagic shock was set up, 40 SD rats were randomized into four groups: sham operation, shock, compound danshen solution and heparin groups, each group was composed of 10 SD rats. Plasma SFMC, TM, ATIII, D-D, t-PA, PAI levels and APTT were detected, incidences of bleeding complications between heparin and danshen group were compared. The results showed that plasma SFMC, D-D levels in shock group were higher but ATIII level in shock group was lower than that in sham operation group, compound danshen solution group and heparin group (P < 0.001), TM levels obviously increased in shock group and heparin group (P < 0.001). There was no significant difference between compound danshen solution and sham-operation groups. Plasma t-PA, D-D levels obviously increased after shock for 2 hours, PAI level reached the peak after shock for 4 hours, but t-PA decreased. After shock for 6 hours, plasma PAI descended, t-PA continually drop in, but PAI and D-D remained in higher levels. Plasma D-D level in heparin group was lower than that in shock group, t-PA level was higher than that in shock group, but there was no significant difference between in heparin and shock groups. Plasma t-PA, PAI and D-D levels in compound danshen solution group were lower than that in shock group. APTT of danshen group was lower than that of shock group and heparin group. Bleeding incidences was 30% in heparin group and 0% in danshen group, respectively. It is concluded that compound danshen solution may used to treat hypercoagulation and hyperfibrinolysis. In comparsion with heparin, danshen posses-ses advantages of safety with less bleeding complication and needs not tight monitor.

Hepcidin, which has been recently identified both by biochemical and genomic approaches, is a 25 amino acid polypeptide synthesized mainly by hepatocytes and secreted into the plasma. Besides its potential activity in antimicrobial defense, hepcidin plays a major role in iron metabolism. It controls two key steps of iron bioavailability, likely through a hormonal action: digestive iron absorption by enterocytes and iron recycling by macrophages. In humans, this could explain that low levels of hepcidin found during juvenile haemochromatosis and HFE-1 genetic haemochromatosis are associated with an iron overload phenotype. Conversely, an increase of hepcidin expression is suspected to play a major role in the development of anemia of chronic inflammatory diseases. However, the regulatory mechanisms of hepcidin expression are multiple, including iron-related parameters, anemia, hypoxia, inflammation and hepatocyte function. Therefore, many physiological and pathological situations may modulate hepcidin expression and subsequently iron metabolism. A better knowledge of the biological effects of hepcidin and of its expression regulatory mechanisms will clarify the place of hepcidin in the diagnosis and treatment of iron-related diseases.

Neuronal intranuclear inclusion disease (NIID) is a rare and heterogeneous group of slowly progressive neurodegenerative disorders characterized by the widespread presence of eosinophilic neuronal intranuclear inclusions (NII) accompanied by a more restricted pattern of neuronal loss. We report here the pathologic findings in a 13-year-old boy who died after a 6-year clinical history of progressive ataxia, extrapyramidal manifestations, and lower motor neuron abnormalities. Histological evaluation of the brain revealed widespread NII in most neurons. Marked loss of cerebellar Purkinje cells and neurons in the dentate nucleus, red nucleus, and spinal cord anterior horns was accompanied by a modest astrocytosis. Because of the abundance of NII and the absence of a relationship between NII and neuronal loss or microglial activation, we conclude that loss of cerebellar, brainstem, and spinal cord neurons reflects selective neuronal vulnerability. NII were immunoreactive for ubiquitin, glucocorticoid receptor, and SUMO-1, a small, ubiquitin-like protein purportedly involved in protein transport and gene transcription. NII were non-reactive for polyglutamine (1C2), TATA binding protein, promyelocytic leukemia protein, heat shock protein 90, tau, alpha-synuclein, neurofilament, and beta amyloid. The moderate ubiquitin and strong SUMO-1 staining of NII in juvenile cases is the reverse of the pattern noted in adult diseases, suggesting the two age groups are pathogenically distinct. We suggest that juvenile NIID is a spinocerebellar brainstem ataxic disease possibly related to an abnormality in SUMOylation.

<h4>Background</h4>In 1998 a clinical guideline for the targeted, accurate and early detection and treatment of HFE-related hereditary haemochromatosis (HH), which comprises a test for the causative HFE-gene mutations, was introduced in our outpatient department.<h4>Methods</h4>The impact of this guideline was evaluated retrospectively. Data were acquired from medical records of patients with discharge diagnosis codes suggestive of HH (n=878 patients), obtained from a period before (n=422) and after guideline introduction (n=456).<h4>Results</h4>Combined measurements of serum transferrin saturation and serum ferritin rose from 12.2% (n=53) to 29.5% (n=138, p<0.001), leaving 70% of the patients eligible for HH not tested for iron parameters. The HFE-gene mutation detection test was correctly used in II (40.7%) of 27 tested patients and improperly interpreted in six (22.2%) of these 27 patients. Five new HH patients were diagnosed before and 13 after introduction. Seven of these 13 patients appeared to be incorrectly diagnosed, due to misinterpretation of laboratory results. Diagnostic costs of case detection for each accurately diagnosed patient were euro 2380 before and euro 2600 after introduction of the guideline.<h4>Conclusion</h4>Evaluation of the introduction of a practical guideline for targeted HH detection reveals a low compliance with the guideline, resulting in both a small percentage of patients tested for HH and overdiagnosis of HH. Therefore, the introduction of the guideline should be combined with a more appropriate implementation strategy which includes education on its most critical points, i.e. the indication and interpretation of the iron parameters and the HFE genotype.

The evolution of cooperation by direct reciprocity requires that individuals recognize their present partner and remember the outcome of their last encounter with that specific partner. Direct reciprocity thus requires advanced cognitive abilities. Here, we demonstrate that if individuals repeatedly interact within small groups with different partners in a two person Prisoner's Dilemma, cooperation can emerge and also be maintained in the absence of such cognitive capabilities. It is sufficient for an individual to base their decision of whether or not to cooperate on the outcome of their last encounter--even if it was with a different partner.

Pancreatic cancer, the fourth leading cause of cancer death in the United States, is frequently associated with the amplification and deletion of specific oncogenes and tumor-suppressor genes (TSGs), respectively. To identify such novel alterations and to discover the underlying genes, we performed comparative genomic hybridization on a set of 22 human pancreatic cancer cell lines, using cDNA microarrays measuring approximately 26,000 human genes (thereby providing an average mapping resolution of <60 kb). To define the subset of amplified and deleted genes with correspondingly altered expression, we also profiled mRNA levels in parallel using the same cDNA microarray platform. In total, we identified 14 high-level amplifications (38-4934 kb in size) and 15 homozygous deletions (46-725 kb). We discovered novel localized amplicons, suggesting previously unrecognized candidate oncogenes at 6p21, 7q21 (SMURF1, TRRAP), 11q22 (BIRC2, BIRC3), 12p12, 14q24 (TGFB3), 17q12, and 19q13. Likewise, we identified novel polymerase chain reaction-validated homozygous deletions indicating new candidate TSGs at 6q25, 8p23, 8p22 (TUSC3), 9q33 (TNC, TNFSF15), 10q22, 10q24 (CHUK), 11p15 (DKK3), 16q23, 18q23, 21q22 (PRDM15, ANKRD3), and Xp11. Our findings suggest candidate genes and pathways, which may contribute to the development or progression of pancreatic cancer.

<h4>Objective</h4>To provide the chosen scaffold materials for experiment and application of tissue engineering and to detect the properties of the collagen bio-derived bone scaffold material loading WO-1.<h4>Methods</h4>The pure bio-derived bone scaffold material, bio-derived bone scaffold material loading collagen, collagen bio-derived bone scaffold material loading WO-1 were made by use of allograft bone, and type-I collagen, and WO-1. The morphological features, constitute components and mechanical properties were examined by scanning electron microscopy, X- rays diffraction and mechanical assay.<h4>Results</h4>The bio-derived bone scaffold material maintained natural network pore system; the bio-derived bone scaffold material loading collagen maintained natural network pore system, the surface of network pore system was coated by collagen membrane; the collagen bio-derived bone scaffold material loading WO-1 maintained natural network pore system, the surface of network pore system was coated by collagen membrane. The pore sizes of the 3 materials were 90-700 microm, 75-600 microm and 80-600 microm, respectively, and the porosities were 87.96%, 80.47%, 84.2%. There was no significant difference between them (P>0.05). The collagen bio-derived bone scaffold material loading WO-1 consisted of [HA,Ca10 (OH)2 (PO4)6. There was no significant difference in the mechanical strength of the three scaffold materials.<h4>Conclusion</h4>The bio-derived bone scaffold material loading WO-1 is as good as bio-derived bone scaffold material and collagen bio-derived bone scaffold material, and it is an effective scaffold material for tissue engineering bone.

<h4>Background</h4>Current screening methods of colorectal carcinoma are based on examination of occult bleeding in the stool, and further on endoscopic and irrigographical (barium enema) examinations. Population-based non-invasive screening method having high sensitivity and specificity is needed.<h4>Methods</h4>Detection of molecular alterations in colonocytes from the stool may be a promising new diagnostic tool for such screening. Determination of mutations in APC, K-ras, DCC, p53 genes and "long" DNA may serve for early detection of colorectal cancer from stool samples. Multi-target DNA-assays employing all these markers suggest high sensitivity and specificity, unfortunately also expensiveness. Therefore finding a marker characteristic for all tumor cells would be desirable. Nuclear faktor-kappaB (NF-kappaB) could be such marker suitable for determination in colonocytes shed into the stool.<h4>Conclusion</h4>Molecular testing of stool for early detection of colorectal cancer may be a promising screening method for this disease. Large multicenter trials are required to validate results obtained from preliminary clinical studies.

Carbazole 1,9a-dioxygenase, which consists of an oxygenase component (CARDO-O) and the electron-transport components ferredoxin (CARDO-F) and ferredoxin reductase (CARDO-R), catalyzes dihydroxylation at the C1 and C9a positions of carbazole. The electron-transport complex between CARDO-O and CARDO-F crystallizes at 293 K using hanging-drop vapour diffusion with the precipitant PEG MME 2000 (type I crystals) or PEG 3350 (type II). Blossom-shaped crystals form from a pile of triangular plate-shaped crystals. The type I crystal diffracts to a maximum resolution of 1.90 A and belongs to space group P2(1), with unit-cell parameters a = 97.1, b = 89.8, c = 104.9 A, alpha = gamma = 90, beta = 103.8 degrees. Diffraction data for the type I crystal gave an overall Rmerge of 8.0% and a completeness of 100%. Its VM value is 2.63 A3 Da(-1), indicating a solvent content of 53.2%.

No abstract available.