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The development of effector and memory CD4 cell populations depends upon both T cell receptor (TCR) engagement of peptide/major histocompatibility complex (MHC) class II complexes and ligation of costimulatory molecules with counter receptors on antigen-presenting cells (APCs). We showed previously that sustained interactions with APCs could be crucial for optimal expansion of CD4 cells and for development of effectors that secrete cytokines associated with Th2 cells. Using an adoptive transfer model with TCR transgenic CD4 cells, we now show that responses of CD4 cells primed in B cell-deficient mice become aborted, but are fully restored upon the transfer of activated B cells. Although B cells have the capacity to secrete multiple cytokines that could affect CD4 priming, including IL-4, we were unable to distinguish a role for cytokines that are secreted by B cells. However, B cell costimulation via the OX40L/OX40 pathway that has been implicated in CD4 cell expansion, survival, and Th2 development was required. Th2 but not Th1 responses were impaired in OX40L-deficient recipients and normal responses were restored with OX40L sufficient B cells. The results suggest that without engagement of OX40L on B cells, CD4 cell responses to many protein Ag would be dominated by Th1 cytokines. These data have important implications for strategies to achieve optimal priming of CD4 subsets.

Human hepcidin, a 25-amino acid peptide made by hepatocytes, may be a new mediator of innate immunity and the long-sought iron-regulatory hormone. The synthesis of hepcidin is greatly stimulated by inflammation or by iron overload. Evidence from transgenic mouse models indicates that hepcidin is the predominant negative regulator of iron absorption in the small intestine, iron transport across the placenta, and iron release from macrophages. The key role of hepcidin is confirmed by the presence of nonsense mutations in the hepcidin gene, homozygous in the affected members, in 2 families with severe juvenile hemochromatosis. Recent evidence shows that deficient hepcidin response to iron loading may contribute to iron overload even in the much milder common form of hemochromatosis, from mutations in the HFE gene. In anemia of inflammation, hepcidin production is increased up to 100-fold and this may account for the defining feature of this condition, sequestration of iron in macrophages. The discovery of hepcidin and its role in iron metabolism could lead to new therapies for hemochromatosis and anemia of inflammation.

X-linked sideroblastic anemia (XLSA) is caused by mutations in the erythroid-specific 5-aminolevulinate synthase gene (ALAS2). XLSA was diagnosed in a 32-year-old woman with a mild phenotype and moderately late onset. Pyridoxine therapy had no effect in the proband, but in her affected son engendered a modest increase in hemoglobin concentration and a 4-fold reduction in ferritin iron. Molecular analysis identified a C to G transversion at nucleotide -206 from the transcription start site, as defined by primer extension, in the proximal promoter region of ALAS2. No other mutations were found in the promoter region, the flanking intronic sequences, the exons, or the 3' genomic region. The same mutation was found in her affected son but not in any other of her unaffected relatives. The mutation resulted in a 94% loss of activity relative to the wild-type sequence for a luciferase reporter construct containing the proximal 293 nucleotides (nt's) of the ALAS2 promoter when transfected into human erythroid K562 cells. Confirming the mutation's deleterious effect, the ALAS2 mRNA level in the proband's erythroid precursors was reduced 87%. The mutation occurred in or near 3 different putative transcription factor binding sites of unknown erythroid importance. The dramatic decreases in reporter activity and mRNA level suggest that the region of the mutation may bind a novel and important erythroid regulatory element.

The three mammalian receptors UNC5H1, UNC5H2, and UNC5H3 (also named UNC5A, UNC5B, and UNC5C in human) that belong to the family of the netrin-1 receptors, UNC5H, were initially proposed as mediators of the chemorepulsive effect of netrin-1 on specific axons. However, they were also recently shown to act as dependence receptors. Such receptors induce apoptosis when unbound to their ligand. We show here that the expression of the human UNC5A, UNC5B, or UNC5C is down-regulated in multiple cancers including colorectal, breast, ovary, uterus, stomach, lung, or kidney cancers. In colorectal tumors, this down-regulation is associated with loss of heterozygosity occurring within UNC5A, UNC5B, and UNC5C genes but may also be partially related to epigenetic processes because histone deacetylase inhibitor increased UNC5C expression in various cancer cell lines. Moreover, sequencing of UNC5C gene in patients with colorectal tumors revealed the presence of missense mutations. The lossreduction of expression may be a crucial mechanism for tumorigenicity because the expression of UNC5H1, UNC5H2, or UNC5H3 inhibits tumor cell anchorage-independent growth and invasion. Moreover, these hallmarks of malignant transformation can be restored by netrin-1 addition or apoptosis inhibition. Hence, UNC5H1, UNC5H2, and UNC5H3 receptors may represent tumor suppressors that inhibit tumor extension outside the region of netrin-1 availability by inducing apoptosis.

No abstract available.

Experimental data suggest the antimicrobial peptide hepcidin as a central regulator in iron homeostasis. In this study, we characterized the expression of human hepcidin in experimental and clinical iron overload conditions, including hereditary hemochromatosis. Using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), we determined expression of hepcidin and the most relevant iron-related genes in liver biopsies from patients with hemochromatosis and iron-stain-negative control subjects. Regulation of hepcidin mRNA expression in response to transferrin-bound iron, non-transferrin-bound iron, and deferoxamine was analyzed in HepG2 cells. Hepcidin expression correlated significantly with serum ferritin levels in controls, whereas no significant up-regulation was observed in patients with hemochromatosis despite iron-overload conditions and high serum ferritin levels. However, patients with hemochromatosis showed an inverse correlation between hepcidin transcript levels and the serum transferrin saturation. Moreover, we found a significant correlation between hepatic transcript levels of hepcidin and transferrin receptor-2 irrespective of the iron status. In vitro data indicated that hepcidin expression is down-regulated in response to non-transferrin-bound iron. In conclusion, the presented data suggest a close relationship between the transferrin saturation and hepatic hepcidin expression in hereditary hemochromatosis. Although the causality is not yet clear, this interaction might result from a down-regulation of hepcidin expression in response to significant levels of non-transferrin-bound iron.

The Drosophila SINA (seven in absentia) protein and its mammalian orthologs (Siah, seven in absentia homolog) are RING domain proteins that function in E3 ubiquitin ligase complexes and facilitate ubiquitination and degradation of a wide range of cellular proteins, including beta-catenin. Despite these diverse targets, the means by which SINASiah recognize substrates or binding proteins has remained unknown. Here we identify a peptide motif (RPVAxVxPxxR) that mediates the interaction of Siah protein with a range of protein partners. Sequence alignment and mutagenesis scanning revealed residues that are important to this interaction. This consensus sequence correctly predicted a high-affinity interaction with a peptide from the cytoskeletal protein plectin-1 (residues 95-117). The unusually high-affinity binding obtained with a 23-residue peptide (K(Dapp) = 29 nM with SINA) suggests that it may serve as a useful dominant negative reagent for SINASiah proteins.

CD8+ cells from healthy HIV-infected individuals can suppress HIV replication in infected CD4(+) cells without killing the cells. This CD8+ cell noncytotoxic antiviral response (CNAR), observed by coculture of CD8+ cells with infected CD4+ cells, is associated with secretion of a CD8+ cell antiviral factor (CAF). In attempts to identify CAF, we discovered that certain protease inhibitors, particularly leupeptin, can block, by up to 95%, the anti-HIV activity in CD8+ cell culture fluids as well as inhibit CNAR. The effect is dose-dependent and is observed in up to 70% of the CAF and CNAR assays by using fluids and cells from several different subjects. Pretreatment of CD8+ cells with leupeptin reduces CNAR, further supporting an inhibitory effect on a CD8+ cell product. This inhibitory activity of protease inhibitors does not affect cell growth, expression of activation antigens, or viability of either CD8+ cells or the infected CD4+ cells. The results suggest that a part of the CD8+ cell noncytotoxic response involves the activity of a protease or a protein that interacts with protease inhibitors. Proteolysis of a CD8+ cell product(s) may be involved. This observation offers a promising approach for identifying the mechanism of CNARCAF activity.

HFE, an atypical MHC class I type molecule, has a critical, yet still elusive function in the regulation of systemic iron metabolism. HFE mutations are linked to hereditary haemochromatosis type 1, a common autosomal recessive disorder of iron overload. Most patients are homozygous for a C282Y point mutation that abrogates the interaction of HFE with beta(2)-microglobulin (beta(2)M) and, thus, impairs its proper processing and expression on the cell surface. An H63D substitution is also associated with disease. To investigate the function of HFE we have generated clones of human H1299 lung cancer cells that express wild-type, C282Y or H63D HFE under the control of a tetracycline-inducible promoter. Consistent with earlier observations in other cell lines, the expression of wild-type or H63D, but not C282Y, HFE induces an apparent iron-deficient phenotype, manifested in the activation of iron-regulatory protein and concomitant increase in transferrin receptor levels and decrease in ferritin content. This phenotype persists in cells expressing wild-type HFE after transfection with a beta(2)M cDNA. Whereas endogenous beta(2)M is sufficient for the presentation of at least a fraction of chimeric HFE on the cell surface, this effect is stimulated by approx. 2.8-fold in beta(2)M transfectants. The co-expression of exogenous beta(2)M does not significantly affect the half-life of HFE. These results suggest that the apparent iron-deficient phenotype elicited by HFE is not linked to beta(2)M insufficiency.

Transferrin receptor 1 (TfR1) which mediates uptake of transferrin-bound iron, is essential for life in mammals. Recently, a close homologue of human transferrin receptor 1 was cloned and called transferrin receptor 2 (TfR2). A similar molecule has been identified in the mouse. Human transferrin receptor 2 is 45% identical with transferrin receptor 1 in the extracellular domain, but contains no iron responsive element in its mRNA and is apparently not regulated by intracellular iron concentration nor by interaction with HFE. Transferrin receptor 2, like transferrin receptor 1, binds transferrin in a pH-dependent manner (but with 25 times lower affinity) and delivers iron to cells. However, transferrin receptor 2 distribution differs from transferrin receptor 1, increasing in differentiating hepatocytes and decreasing in differentiating erythroblasts. Expression of both receptors is cell cycle dependent. Mutations in the human transferrin receptor 2 gene cause iron overload disease, suggesting it has a role in iron homeostasis.

The notochord has major roles in vertebral column formation: indirectly by inducing sclerotome cell differentiation; and directly by forming the nucleus pulposus of intervertebral discs. Sox5 and Sox6 encode Sry-related HMG box transcription factors that act redundantly to promote chondroblast differentiation in all cartilages of the mouse embryo. We show that Sox5 and Sox6 are expressed in the notochord cell lineage and required for notochord late development. In Sox5(-/-)/Sox6(-/-) embryos, the notochord formed a typical rod-like structure. It fulfilled its inductive functions, as indicated by expression of sonic hedgehog and sclerotome specification. However, the notochord failed to become surrounded with an extracellular matrix sheath. This phenotype was associated with a downregulation of extracellular matrix genes, including the genes for collagen 2, aggrecan and perlecan in both notochord cells and surrounding chondrocytic cells of presumptive inner annuli and vertebral bodies. The mutant notochord then underwent an aberrant, fatal dismantling after sclerotome cell migration. Its cells became removed first from intervertebral spaces and then from vertebral bodies, and it progressively underwent apoptosis. Meanwhile, the development of inner annuli and vertebral bodies was dramatically impaired. Consequently, the vertebral column of Sox5(-/-)/Sox6(-/-) fetuses consisted of a very deficient cartilage and was devoid of nuclei pulposi. In Sox5(-/-)/Sox6(+/-) and more severely in Sox5(+/-)/Sox6(-/-) embryos, the notochord sheath was thinner, but cells survived. By birth, nuclei pulposi were rudimentary, and its cells poorly swelled and still expressing sonic hedgehog. Hence, Sox5 and Sox6 are required for notochord extracellular matrix sheath formation, notochord cell survival and formation of nuclei pulposi. Through these roles and essential roles in cartilage formation, they are central transcriptional regulators of vertebral column development.

Human longevity may be correlated with optimal functioning of the immune system, suggesting that genetic determinants of longevity also resides in those polymorphisms for the immune system genes that regulate immune responses as histocompatibility (HLA) antigens. However, conflicting results have been obtained. Some well planned and designed association studies performed in Caucasians suggest that longevity is associated with positive selection of alleles (i.e. HLA-DR11) or haplotypes (i.e. HLA-B8,DR3) that confer resistance to infectious diseases, respectively, via peptide presentation or via antigen non-specific control of immune response. Association studies are subjected to a number of possible confounding factors, the homogeneity of the population in term of geographical origin among others. Because of the lack of large-scale heterogeneity, the Sardinians represent a suitable population for association studies addressed to dissect the complex traits as longevity. Thus, we have evaluated, by the amplification refractory mutation system/polymerase chain reaction, HLA-DR frequencies in 120 centenarians (79 women and 41 men) and 86 controls (53 women and 33 men) from Sardinia, to validate, in this very homogeneous population, the associations between HLA alleles or haplotypes and longevity observed in other Caucasoid populations. No significant differences were obtained by analysing the differences between Centenarians and controls except for HLA-DRB1*15 that was increased in centenarians. However, the significance was not maintained by multiplying P values for the number of alleles under study. Thus, in Sardinian centenarians, we were not able to confirm the findings observed in the well planned and designed studies performed in other Caucasoid populations. Besides, HLA HFE gene polymorphisms have been recently demonstrated to be associated with longevity in the Sicilian population but not in Danish one. On the whole these findings clearly show that HLA/longevity associations are population-specific, being heavily affected by the population-specific genetic and environmental history. So, in our opinion, HLA genes might be considered survival genes not longevity genes.

No abstract available.

We observed the development of phenotypic hereditary haemochromatosis in a non-hereditary haemochromatosis liver transplant recipient, following transplantation with a liver from a C282Y heterozygous donor. No cause for secondary iron overload was identified. Subsequent sequencing of the HFE gene of both donor and recipient revealed a strong candidate for a novel pathogenic HFE mutation. In the recipient, heterozygosity for a single base substitution in exon 1, g.18 G>C, resulting in the substitution of arginine by serine at codon 6 (R6S), was detected. This R6S variation is likely to represent a novel pathogenic missense mutation of the HFE gene. An interaction between R6S heterozygosity in the recipient and C282Y heterozygosity in the donor liver is the most likely explanation for the development of iron overload in this patient. The report suggests that an hepatic defect is required for expression of hereditary haemochromatosis and that the intestinal HFE genotype is not the exclusive determinant of iron status. It also raises the possibility that a hereditary haemochromatosis phenotype may result from transplantation of C282Y heterozygous donor livers into recipients with heterozygous pathogenic HFE mutations. This possibility may have significant implications for the common practice of transplanting C282Y heterozygous livers.

<h4>Background/aims</h4>The impact of heterozygous HFE mutations on the course of chronic hepatitis C and iron indices was studied.<h4>Methods</h4>Ferritin, transferrin saturation (TS), serum iron, C282Y and H63D mutations were determined in 401 patients with chronic hepatitis C virus (HCV) infection and 295 healthy controls. Liver histologies were available in 217 and HCV genotypes in 339 patients.<h4>Results</h4>Allele frequencies of the C282Y and H63D mutation did not differ between HCV patients and healthy controls (6.95 vs. 6.2%; 14.75 vs. 16.4%; n.s.). HFE heterozygous HCV patients had higher ferritin (349+/-37 vs. 193+/-15 microg/l; P<0.0005), TS (38+/-2 vs. 32+/-1%; P<0.0005), serum iron (144+/-6 vs. 121+/-3 microg/dl; P<0.0005), semiquantitative liver iron staining (0.26+/-0.07 vs. 0.09+/-0.03; P<0.006) and fibrosis scores (1.9+/-0.2 vs. 1.4+/-0.1; P<0.003) compared to HFE wildtypes. By multivariate regression analysis odds ratios for liver cirrhosis were 5.9 (confidence interval (CI) 1.6-22.6; P<0.009) for C282Y heterozygotes and 2.9 (CI 1.0-8.4; P<0.05) for H63D heterozygotes compared to HFE wildtypes. Considering all HFE heterozygous HCV patients, odds ratios of 3.6 (CI 1.4-9.3; P<0.009) for cirrhosis and 3.1 (CI 1.3-7.3; P<0.009) for fibrosis were calculated.<h4>Conclusions</h4>C282Y or H63D heterozygosity is an independent risk factor for liver fibrosis and cirrhosis in HCV infected individuals. Screening for HFE mutations should be considered in HCV infection.

Neuronal connectivity is established by the axo-dendritic polarity, correct guidance and targeting of neurons. Unlike for axons, the mechanisms responsible for directed outgrowth of dendrites are not well understood. Using single-cell labeling, we describe specific guidance defects in dendrites of identified neurons in frazzled, robo, netrin and commissureless mutant embryos of Drosophila melanogaster. We found that the cell-surface molecules Frazzled and Robo work as guidance molecules not only for axons but also for dendrites as they navigate within the CNS. Furthermore, we report that each neuron showed a cell-autonomous and independent use of guidance molecules.

The collective motions of the core and C-terminal domains of human topoisomerase I (topo I) have been analysed by molecular dynamics simulation of the protein in covalent complex with a 22 bp DNA duplex. The analysis evidenced a great number of correlated movements of core subdomain I and II residues, and a central role for helix 5 in the protein-DNA communication, in particular with the scissile strand downstream of the cleavage site. The flow of information between these core subdomains and DNA suggests that subdomains I and II play an essential role in the DNA relaxation process. In core subdomain III the majority of DNA contacting residues do not communicate with protein regions far from DNA, suggesting that they have a structural role. However, selected core subdomain III residues, involved in the orientation of the active site region, show correlated movements with residues distant from DNA, indicating that the information concerning the catalytic event is also transmitted. The flexibility of two loops formed by residues 519-520 and 580-584 seems indispensable to the dynamic participation of core subdomain III to the DNA cleavage and religation steps. The motion of specific residues has also been found to explain the effect of single point mutations that make topo I resistant to the anticancer drug camptothecin.

Expression of the type XI collagen gene Col11a2 is directed to cartilage by at least three chondrocyte-specific enhancer elements, two in the 5' region and one in the first intron of the gene. The three enhancers each contain two heptameric sites with homology to the Sox protein-binding consensus sequence. The two sites are separated by 3 or 4 bp and arranged in opposite orientation to each other. Targeted mutational analyses of these three enhancers showed that in the intronic enhancer, as in the other two enhancers, both Sox sites in a pair are essential for enhancer activity. The transcription factor Sox9 binds as a dimer at the paired sites, and the introduction of insertion mutations between the sites demonstrated that physical interactions between the adjacently bound proteins are essential for enhancer activity. Additional mutational analyses demonstrated that although Sox9 binding at the paired Sox sites is necessary for enhancer activity, it alone is not sufficient. Adjacent DNA sequences in each enhancer are also required, and mutation of those sequences can eliminate enhancer activity without preventing Sox9 binding. The data suggest a new model in which adjacently bound proteins affect the DNA bend angle produced by Sox9, which in turn determines whether an active transcriptional enhancer complex is assembled.

The prognostic significance of somatic activating codon 816 c-kit mutations in pediatric urticaria pigmentosa has not yet been established in detail. Detection of such mutations in archival paraffin-embedded biopsies is usually hampered by an abundance of surrounding normal cells. Here we describe a method for the selective amplification and specific detection of c-kit mutation Asp816-->Val in complete tissue sections cut from up to 24-year-old paraffin blocks. Peptide nucleic acid-mediated polymerase chain reaction clamping of the wild-type allele was combined with on-line mutation detection using oligonucleotide hybridization probes. In DNA extracted from HMC-1 cells heterozygously carrying the c-kit mutation Asp816-->Val, the one-tube assay allowed specific detection of this mutation in a more than 1000-fold excess of normal background DNA within 1 hour and without the need for additional analytical steps. In a series of 38 cases with pediatric urticaria pigmentosa we detected c-kit codons 815 and 816 mutations in 16 cases. Mutation detection did not correlate with clinical outcome after a mean follow-up of 11.2 years. In conclusion, the procedure described may represent an ideal screening tool for all kinds of clinical applications, using point mutations as markers of, for example, early events in carcinogenesis, circulating metastatic tumor cells, and minimal residual disease.

Serotonergic receptor binding in the arcuate nucleus, n. raphé obscurus, and other medullary regions is decreased in sudden infant death syndrome (SIDS) cases. Further, a variable tandem repeat sequence polymorphism in the promoter region of the serotonin transporter protein (5-HTT) gene has recently been associated with risk of SIDS in a Japanese cohort. This polymorphism differentially regulates 5-HTT expression, with the long allele (L), the SIDS-associated allele, being a more effective promoter than the short allele (S). We therefore investigated the 5-HTT promoter polymorphism in a cohort of 87 SIDS cases (43 African American and 44 Caucasian) and gender/ethnicity-matched controls. Significant positive associations were found between SIDS and the 5-HTT genotype distribution (P = 0.022), specifically with the L/L genotype (P = 0.048), and between SIDS and the 5-HTT L allele (P = 0.005). There was also a significant negative association between SIDS and the S/S genotype (P = 0.011). The comparisons were repeated in the African American and Caucasian subgroups. The data patterns were consistent in the subgroups, i.e., the L/L genotype and L allele were increased in the cases, but not all subgroup comparisons were statistically significant. These results indicate a relationship between SIDS and the L allele of the 5-HTT gene in African Americans and Caucasians, and if confirmed, will provide an important tool for identifying at-risk individuals and estimating the risk of recurrence.

This study was designed to analyse possible associations between DNA polymorphisms in the 5-hydroxytryptamine (5-HT; serotonin) 5-HT(2A) receptor and the 5-HT transporter (5-HTT) genes, and myocardial infarction (MI). 5-HT has been shown to be involved in cardiovascular pathophysiology. In addition to platelet aggregation and vascular contraction, 5-HT induces hyperplasia of artery smooth muscle cells. Recently, a 5-HT transporter gene polymorphism has been associated with MI. To determine the influence of genetic variation at the 5-HT(2A) receptor (T102C polymorphism) and the 5-HTT (insertion/deletion polymorphism) on the risk of developing early MI, we genotyped 210 MI patients of < 55 years old and 238 healthy control subjects for DNA polymorphisms in these genes. In addition, we genotyped 95 patients with late-onset MI (> 60 years old) to analyse the effects of these polymorphisms on the age at which the first MI episode occurred. The 5-HT(2A) receptor polymorphism was not associated with MI in our population. In addition, since the 5-HT(2A) receptor gene and genotype frequencies did not differ between patients with early and late onset of MI, this polymorphism does not appear to have an effect on age at the first MI episode. Gene and genotype frequencies for the 5-HTT promoter did not differ between patients < 55 years old and healthy controls (independent of smoking status). However, homozygotes for the deletion (the ss genotype, where s denotes the short allele) were present at a significantly higher frequency in patients >60 years old compared with patients < 55 years old (P = 0.009; P = 0.004 when only smokers were compared). According to our data, the ss genotype would seem to have a protective role against MI, delaying the age of onset of the first episode, especially among smokers. This could be a consequence of the lower 5-HTT levels linked to the s allele, so that individuals homozygous for the ss genotype may have lower 5-HT re-uptake by platelets.

Bioapatite, the main constituent of mineralized tissue in mammalian bones, is a calcium-phosphate-based mineral that is similar in structure and composition to hydroxyapatite. In this work, the crystallographic structure of bioapatite in human fetuses was investigated by synchrotron radiation x-ray diffraction (XRD) and microdiffraction ( micro -XRD) techniques. Rietveld refinement analyses of XRD and micro -XRD data allow for quantitative probing of the structural modifications of bioapatite as functions of the mineralization process and gestational age.

High frequencies of the C282Y and H63D mutations of the HFE gene occur in European populations, even though homozygous and compound heterozygous states are associated with hereditary haemochromatosis, which is a disease that decreases fitness. This suggests that heterozygotes may possess a selective advantage. HFE mutations increase iron absorption in patients with haemochromatosis, and the mean transferrin saturations and ferritin levels are mildly increased in heterozygotes, suggesting that HFE mutations may protect against iron depletion and iron deficiency anaemia. In this study of 23,681 Caucasian adults, mean transferrin saturation, serum ferritin and haemoglobin levels were significantly higher in subjects carrying HFE mutations compared with wild types. Analysed by ethnicity, mean haemoglobin and mean erythrocyte volume (MCV) were significantly lower in those with a southern versus northern European ancestry. C282Y mutation carriers had an increased mean haemoglobin level in both ethnic groups. Prevalence of non-anaemic iron deficiency was significantly lower among female carriers of the C282Y mutation compared with HFE wild types. However, prevalence of frank iron deficiency anaemia did not differ significantly among genotypes. Quantile:quantile plots showed a small but significant upward shift in the mid-range of the haemoglobin distribution among C282Y mutation carriers that was consistent with an increased mean haemoglobin level without significant changes in the anaemic range.

The aim of the present studies was to determine the effects of reduced or absent serotonin (5-HT) transporters (5-HTTs) on 5-HT2A and 5-HT2C receptors. The density of 5-HT2C receptors was significantly increased in the amygdala and choroid plexus of 5-HTT knockout mice. On the other hand, the density of 5-HT2A receptors was significantly increased in the hypothalamus and septum, but reduced in the striatum, of 5-HTT knockout mice. However, 5-HT2A mRNA was not changed in any brain region measured. 5-HT2C mRNA was significantly reduced in the choroid plexus and lateral habenula nucleus of these mice. The function of 5-HT2A receptors was evaluated by hormonal responses to (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI). Oxytocin, but not adrenocorticotrophic hormone or corticosterone, responses to DOI were significantly greater in 5-HTT knockout mice. In addition, Gq and G11 proteins were not significantly changed in any brain region measured. The present results suggest that the constitutive alteration in the function of 5-HTTs changes the density of 5-HT2A and 5-HT2C receptors in a brain region-specific manner. These changes may not be mediated by alterations in their gene expression or in the level of Gq/11 proteins. The alterations in these receptors may be related to the altered behaviors of 5-HTT knockout mice.

Several 3'-fluoro analogues, 1a, 1b, and 1c of selective and potent adenosine A(3) receptor agonist, Cl-IB-MECA were synthesized from D-xylose via highly regioselective opening of lyxo-epoxides, 8a and 8b with fluoride anion. Compared to the high binding affinity of Cl-IB-MECA to the A(3) adenosine receptor, the corresponding 3'-fluoro derivative showed remarkably decreased binding affinity, indicating that 3'-hydroxyl group acts as hydrogen bonding acceptor, not hydrogen bonding donor like fluorine atom in binding to the A(3) adenosine receptor.

Huntingtin is the protein whose mutation leads to Huntington's disease (HD). The protein is heterogeneously distributed in the telencephalon, and not consistently correlated with cell vulnerability in HD [Fusco, F.R., Chen, Q., Lamoreaux, W.J., Figueredo-Cardenas, G., Jiao, Y., Coffman, J.A., Surmeier, D.J., Honig, M.G., Carlock, L.R., and Reiner, A., J. Neurosci., 19 (1999) 1189-1202]. The aim of our study was to investigate a possible preferential distribution of huntingtin among the two main striatal output pathways, namely, the striatonigral and the striatopallidal circuit. Dual label immunofluorescence by means of confocal microscopy was used to detect the presence of huntingtin among striatal projection neurons identified by their cellular content of Substance P, Enkephalin, CB1 receptor, and D1a dopamine receptor. Our data showed that striatopallidal neurons co-containing SP and D1a [Surmeier, D.J., Song, W.J., and Yan, Z., J. Neurosci., 16 (1996) 6579-6591] co-localized with huntingtin in a higher proportion than striatonigral neurons.

Silicon-based chips with discrete, independently temperature-controlled islands have been developed for use in DNA microarray hybridization studies. Each island, containing a heater made of a diffusion layer and a temperature sensor based on a p-n junction, is created on a silicon dioxide/nitride surface by anisotropic etching. Different reactive groups are subsequently added to the surface of the islands, and allele-specific oligonucleotide probes are attached to discrete spots on the chip. Hybridization is performed with Cy5-tagged single-stranded targets derived by PCR from genomic DNA. Results are assessed by measuring fluorescence of bound dye-tagged targets after hybridization and washing. Temperatures at each island can be set at different values to obtain optimal distinction between perfect matches and mismatches. This approach facilitates definition of optimal temperatures for probe/target annealing and for distinction between perfectly matched versus mismatched solution-phase targets. The thermal gradient DNA chips were then tested for genotyping, and the results for four different loci in two genes are presented. Unambiguous typing was achieved for clinically relevant loci within the factor VII and hemochromatosis genes.

A novel glycoprotein, dysadherin, has an anti-cell - cell adhesion function through downregulating E-cadherin. In this study, we investigated the expressions of dysadherin and E-cadherin in 82 patients with stage II and III colorectal carcinomas to determine the correlation between the two molecules and the clinicopathologic features of each tumour. Dysadherin was not expressed in normal colorectal epithelium. Fifty-one per cent of tumours showed dysadherin immunopositivity in over 50% of cancer cells. Thirty-eight per cent of tumours showed reduced E-cadherin immunopositivity. The increased expression of dysadherin was significantly associated with lung metastasis (P=0.003). The increased expression of dysadherin had a significant impact on patient survival (P=0.0099 and 0.0036, log-rank test for overall and recurrence-free survival rate, respectively). Furthermore, tumour with increased expression of dysadherin and reduced expression of E-cadherin showed the worst prognosis (P=0.0043 and 0.0028, log-rank test for overall and recurrence-free survival rate, respectively). These results suggest that increased dysadherin expression is a significant indicator of poor prognosis for patients with advanced colorectal carcinoma.

Primary hepatocellular carcinoma (HCC) is one of the most common malignancies and has the fourth highest mortality rate worldwide. The major risk factors, including chronic infections with the hepatitis B or C virus, are exposure to dietary aflatoxin B1(AFB1), vinyl chloride, or alcohol consumption. Southern China and sub-Saharan Africa have the highest dietary AFB1 exposure, making it and hepatitis B virus (HBV) the major causes of cancer mortality in these geographic areas. Recent studies have discovered genetic and epigenetic changes involved in the molecular pathogenesis of HCC, including somatic mutations in the p53 tumor suppressor gene (TP53). AFB1 induces typical G:C to T:A transversions at the third base in codon 249 of p53. Chronic active hepatitis B and C (HCV) infection, and further inflammatory and oxyradical disorders including Wilson disease (WD) or hemochromatosis, generate reactive oxygen/nitrogen species that can damage DNA and mutate the p53 gene. The X gene of HBV (HBx) is the most common open reading frame integrated into the host genome in HCC. The integrated HBx is frequently mutated and has a diminished ability to function as a transcriptional cotransactivator and to activate the NF-kappa B pathway. However, the mutant HBx proteins still retain their ability to bind to and abrogate p53-mediated apoptosis. In summary, both viruses and chemicals are implicated in the etiology and molecular pathogenesis of HCC. The resultant molecular changes in the ras and Wnt signal-transduction pathways, and the p53 and Rb tumor suppressor pathways significantly contribute to liver carcinogenesis

The 2-nitrobenzoic acid degradation pathway of Pseudomonas fluorescens strain KU-7 proceeds via a novel 3-hydroxyanthranilate intermediate. In this study, we cloned and sequenced a 19-kb DNA locus of strain KU-7 that encompasses the 3-hydroxyanthranilate meta-cleavage pathway genes. The gene cluster, designated nbaEXHJIGFCDR, is organized tightly and in the same direction. The nbaC and nbaD gene products were found to be novel homologs of the eukaryotic 3-hydroxyanthranilate 3,4-dioxygenase and 2-amino-3-carboxymuconate-6-semialdehyde decarboxylase, respectively. The NbaC enzyme carries out the oxidation of 3-hydroxyanthranilate to 2-amino-3-carboxymuconate-6-semialdehyde, while the NbaD enzyme catalyzes the decarboxylation of the latter compound to 2-aminomuconate-6-semialdehyde. The NbaC and NbaD proteins were overexpressed in Escherichia coli and characterized. The substrate specificity of the 23.8-kDa NbaC protein was found to be restricted to 3-hydroxyanthranilate. In E. coli, this enzyme oxidizes 3-hydroxyanthranilate with a specific activity of 8 U/mg of protein. Site-directed mutagenesis experiments revealed the essential role of two conserved histidine residues (His52 and His96) in the NbaC sequence. The NbaC activity is also dependent on the presence of Fe(2+) but is inhibited by other metal ions, such as Zn(2+), Cu(2+), and Cd(2+). The NbaD protein was overproduced as a 38.7-kDa protein, and its specific activity towards 2-amino-3-carboxymuconate-6-semialdehyde was 195 U/mg of protein. Further processing of 2-aminomuconate-6-semialdehyde to pyruvic acid and acetyl coenzyme A was predicted to proceed via the activities of NbaE, NbaF, NbaG, NbaH, NbaI, and NbaJ. The predicted amino acid sequences of these proteins are highly homologous to those of the corresponding proteins involved in the metabolism of 2-aminophenol (e.g., AmnCDEFGH in Pseudomonas sp. strain AP-3). The NbaR-encoding gene is predicted to have a regulatory function of the LysR family type. The function of the product of the small open reading frame, NbaX, like the homologous sequences in the nitrobenzene or 2-aminophenol metabolic pathway, remains elusive.

<h4>Objective</h4>To examine the role of hemochromatosis (HFE) gene mutations, which are associated with porphyria cutanea tarda (PCT), in the therapeutic response to chloroquine.<h4>Design</h4>We retrospectively analyzed a database (Excel version 2001 [Microsoft Excel, Redmond, Wash]; date range of search, 1985-1999) of chloroquine-treated patients with PCT on whether HFE mutations (C282Y and H63D) might have influenced the clinical response, urinary porphyrin excretion, liver enzyme activities, and serum iron markers. Serum samples and corresponding complete sets of data before and after therapy were available in 62 of 207 patients with PCT who were treated exclusively with chloroquine.<h4>Settings</h4>Academic teaching hospital.<h4>Intervention</h4>For treatment, low-dose chloroquine diphosphate, 125 to 250 mg twice weekly, was used during a median time of 16 months (range, 12-26 months).<h4>Results</h4>Of the 62 German patients with PCT, 37 (60%) carries HFE mutations. Chloroquine therapy was accompanied by clinical remission and reduced urinary porphyrin excretion (P<.001) in the 24 patients (39%) with HFE wild type as well as in 35 HFE heterozygous patients with PCT (56%). Decreases of serum iron markers following chloroquine therapy were limited to patients with PCT and HFE wild type. All patients homozygous for the C282Y mutation (3 [5%] of 62) had high serum iron, ferritin, and transferrin saturation and failed to respond to chloroquine treatment.<h4>Conclusions</h4>The therapeutic response to chloroquine was not compromised by C282Y heterozygosity and compound heterozygosity of HFE mutations. Because HFE C282Y homozygotes (+/+) did not respond to chloroquine and a decrease in serum iron concentration was limited to patients with PCT and HFE wild type, phlebotomy should be first-line therapy in patients with PCT and HFE mutations.

The CD134-CD134 ligand (CD134L) costimulatory pathway has been shown to be critical for both T and B cell activation; however, its role in regulating the alloimmune response remains unexplored. Furthermore, its interactions with other costimulatory pathways and immunosuppressive agents are unclear. We investigated the effect of CD134-CD134L pathway blockade on allograft rejection in fully MHC-mismatched rat cardiac and skin transplantation models. CD134L blockade alone did not prolong graft survival compared with that of untreated recipients, and in combination with donor-specific transfusion, cyclosporine, or rapamycin, was less effective than B7 blockade in prolonging allograft survival. However, in combination with B7 blockade, long-term allograft survival was achieved in all recipients (>200 days). Moreover, this was synergistic in reducing the frequency of IFN-gamma-producing alloreactive lymphocytes and inhibiting the generation of activated/effector lymphocytes. Most impressively, this combination prevented rejection in a presensitized model using adoptive transfer of primed lymphocytes into athymic heart transplant recipients. In comparison to untreated recipients (mean survival time (MST): 5.3 +/- 0.5 days), anti-CD134L mAb alone modestly prolonged allograft survival (MST: 14 +/- 2.8 days) as did CTLA4Ig (MST: 21.5 +/- 1.7 days), but all grafts were rejected within 24 days. Importantly, combined blockade further and significantly prolonged allograft survival (MST: 75.3 +/- 12.7 days) and prevented the expansion and/or persistence of primed/effector alloreactive T cells. Our data suggest that CD134-CD134L is a critical pathway in alloimmune responses, especially recall/primed responses, and is synergistic with CD28-B7 in mediating T cell effector responses during allograft rejection. Understanding the mechanisms of collaboration between these different pathways is important for the development of novel strategies to promote long-term allograft survival.

Serotonin (5-HT) is known to reduce apoptosis and in rodent models of brain ischemia. Modulation of programmed cell death during neural development was assessed in early postnatal brains of serotonin transporter (5-HTT) knockout mice, characterized by elevated extracellular 5-HT levels. The number of apoptotic cells visualized at postnatal day-1 (P1) by ISEL+ or TUNEL staining was significantly reduced in the striatum, thalamus/hypothalamus, cerebral cortex and hippocampus of 5-HTT knockout mice, compared to wild type and heterozygote mice, with differences displaying an increasing fronto-caudal gradient and regional specificity. These findings underscore 5-HT roles in the regulation of programmed cell death during brain development, and spur interest into pharmacological interventions aimed at relieving pathological apoptosis by potentiating serotoninergic neurotransmission.

The extracellular matrix protein fibulin-1 suppresses the motility and invasiveness of a variety of tumour cell types in vitro as well as the growth of fibrosarcoma tumours in nude mice. In this study, fibulin-1 protein expression in breast carcinoma specimens and normal breast tissue was evaluated immunohistologically. Fibulin-1 protein expression was also semiquantitatively assessed by immunoblot analysis in a collection of normal breast tissues (n=18), benign tumours (n=5) and breast carcinomas (n=39). In normal breast tissue, fibulin-1 protein expression predominated in the ductal epithelium and underlying myoepithelium, with weaker staining evident in the loose connective surrounding the ducts. Examination of breast carcinomas revealed that the tumour cells also expressed fibulin-1 protein. The level of mature fibulin-1 polypeptide (100 kDa) was higher in the breast carcinoma specimens as compared to normal breast tissue (Mann-Whitney U-test, P=0.0005). In addition to the mature fibulin-1 polypeptide, several smaller sized polypeptides of 55, 50 and 25 kDa were detected using monoclonal antibodies reactive towards an epitope located at the N-terminus of fibulin-1. The immunoreactive 50 kDa polypeptide was detected more frequently in breast carcinoma specimens than in normal breast tissue (chi(2)=17.22, P<0.0001). Furthermore, the ratio of the 50 kDa fragment to the mature fibulin-1 polypeptide correlated with the level of oestrogen receptor alpha (Spearman correlation coefficient, rs=0.49, P<0.003, n=36) and progesterone receptor (rs=0.43, P=0.008, n=36) expression in the tumour specimens. Taken together, these findings indicate that elevated expression and altered processing of fibulin-1 is associated with human breast cancer.

Biochemical and genetic studies implicate synaptotagmin (Syt 1) as a Ca2+ sensor for neuronal and neuroendocrine neurosecretion. Calcium binding to Syt 1 occurs through two cytoplasmic repeats termed the C2A and C2B domains. In addition, the C2A domain of Syt 1 has calcium-independent properties required for neurotransmitter release. For example, mutation of a polylysine motif (residues 189-192) reverses the inhibitory effect of injected recombinant Syt 1 C2A fragment on neurotransmitter release from PC12 cells. Here we examined the requirement of the C2A polylysine motif for Syt 1 interaction with the cardiac Cav1.2 (L-type) and the neuronal Cav2.3 (R-type) voltage-gated Ca2+ channels, two channels required for neurotransmission. We find that the C2A polylysine motif presents a critical interaction surface with Cav1.2 and Cav2.3 since truncated Syt 1 containing a mutated motif (Syt 1*1-264) was ineffective at modifying the channel kinetics. Mutating the polylysine motif also abolished C2A binding to Lc753-893, the cytosolic interacting domain of Syt 1 at Cav1.2 1 subunit. Syt 1 and Syt 1* harboring the mutation at the KKKK motif modified channel activation, while Syt 1* only partially reversed the syntaxin 1A effects on channel activity. This mutation would interfere with the assembly of Syt 1/channel/syntaxin into an exocytotic unit. The functional interaction of the C2A polylysine domain with Cav1.2 and Cav2.3 is consistent with tethering of the secretory vesicle to the Ca2+ channel. It indicates that calcium-independent properties of Syt 1 regulate voltage-gated Ca2+ channels and contribute to the molecular events underlying transmitter release.

Disabled-1 (Dab1) is a cytosolic adaptor protein that plays critical roles in cortical development. However, a detailed mechanism of action has not yet been clearly defined. Through yeast two-hybrid screening, we observed that mouse Siah-1A, an E3 ubiquitin ligase containing a RING finger motif, interacts with Dab1. Co-immunoprecipitation experiments and in vitro binding experiments both indicated direct interaction between Siah and Dab1. Steady-state expression of Siah was enhanced by the presence of Dab1 or lactacystin, a representative proteasomal inhibitor. Auto-ubiquitination of Siah was inhibited by the presence of Dab1, suggesting inhibition of Siah activity and subsequent increase of Siah expression by Dab1. Both Dab1-induced increase of steady-state expression of Deleted in colorectal cancer (DCC), one of the well-known substrates of Siah, and its inhibition by Siah Delta R suggest that Dab1 increases expression of DCC through inhibiting the activity of endogenous Siah. Our results suggest that Dab1 inhibits the activity of Siah.

<h4>Background and objectives</h4>In Italy, the prevalence of C282Y is lower than in Northern European countries. We hypothesized a higher prevalence of C282Y in Northern than in Southern Italian populations. We previously identified a nonsense mutation (E168X) in hemochromatosis probands originating from a region in the north-west of Italy. We aimed to define the prevalence of C282Y and E168X in that region and the origin of the E168X mutation by haplotype analysis.<h4>Design and methods</h4>Six-hundred and six blood donors were investigated for C282Y, H63D, S65C and E168X mutations by polymerase chain reaction (PCR)-restriction assays. Three hundred were also tested for rare HFE and TFR2 mutations by reverse-hybridization test strips. D6S265, D6S105 and D6S1281 microsatellites were analyzed to define E168X 6p-associated haplotypes.<h4>Results</h4>One C282Y homozygote, thirteen C282Y/ H63D compound heterozygotes, four E168X heterozygotes and three E168X/H63D compound heterozygotes were found. The allele frequencies of C282Y, H63D, S65C, and E168X were 4.7%, 14.9%, 0.74% and 0.58%, respectively.<h4>Interpretation and conclusions</h4>The prevalence of C282Y in the region investigated was much higher than that previously reported in Italy. This finding is probably due to the heavy Celtic component of this north-western population and suggests that in populations of Northern Italian descent screening studies for hemochromatosis could be cost-effective. The prevalence of E168X in this region, although low, suggests that the mutation probably originated here many years ago and its frequency increased as a result of a local founder effect. Given its severity, we suggest that the E168X mutation should be searched for in all hemochromatosis patients of Northern ancestry with an incomplete HFE genotype.

The HFE gene, a member of the class-I transplantation antigen gene family, is responsible for hereditary hemochromatosis, one of the most common inherited diseases in individuals of European descent. Patients exhibit predictable changes in iron homeostasis, including elevations in both transferrin saturation and serum ferritin levels. A subset of patients progress to overt clinical sequelae, resulting from iron overload. A hallmark of the disease is increased absorption of iron by the intestine. Although the HFE protein appears to modulate the function of the transferrin receptor in vitro, its precise role in vivo remains obscure. With multiple cell types involved in iron metabolism, the function of HFE is likely to be complex.

Commissural neurons in the spinal cord project their axons through the floor plate using a number of molecular interactions, such as netrins and their receptor DCC (deleted in colorectal cancer). However, the molecular cascades that control differentiation of commissural neurons are less characterized. A homeobox gene, MBH1 (mammalian BarH1) was expressed specifically in a subset of dorsal cells in the developing spinal cord. Transgenic mice that carried lacZ and MBH1-flanking genome sequences demonstrated that MBH1 was expressed by commissural neurons. To analyze the function of MBH1, we established an in vivo electroporation method for the transfer of DNA into the mouse spinal cord. Ectopic expression of MBH1 drove dorsal cells into the fate of commissural neurons with concomitant expression of TAG-1 (transiently expressed axonal surface glycoprotein 1) and DCC. Cells ectopically expressing MBH1 migrated to the deep dorsal horn, in which endogenous MBH1-positive cells accumulated. These results suggest that MBH1 functions upstream of TAG-1 and DCC and is involved in the fate determination of commissural neurons in the spinal cord.

Huntington's disease (HD) mouse models that express N-terminal huntingtin fragments show rapid disease progression and have been used for developing therapeutics. However, light microscopy reveals no significant neurodegeneration in these mice. It remains unclear how mutant huntingtin induces neurodegeneration. Using caspase staining, terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling, and electron microscopy, we observed that N171-82Q mice, which express the first 171 aa of mutant huntingtin, displayed more degenerated neurons than did other HD mouse models. The neurodegeneration was also evidenced by increased immunostaining for glial fibrillary acidic protein and ultrastructural features of apoptosis. R6/2 mice, which express exon 1 of mutant huntingtin, showed dark, nonapoptotic neurons and degenerated mitochondria associated with mutant huntingtin. In HD repeat knock-in mice (HdhCAG150), which express full-length mutant huntingtin, degenerated cytoplasmic organelles were found in both axons and neuronal cell bodies in association with mutant huntingtin that was not labeled by an antibody to huntingtin amino acids 342-456. Transfection of cultured cells with mutant huntingtin revealed that an N-terminal huntingtin fragment (amino acids 1-208 plus a 120 glutamine repeat) caused a greater increase in caspase activity than did exon 1 huntingtin and longer huntingtin fragments. These results suggest that context-dependent neurodegeneration in HD may be mediated by different N-terminal huntingtin fragments. In addition, this study has identified neurodegenerative markers for the evaluation of therapeutic treatments in HD mouse models.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by the abnormal expansion of a polyglutamine tract in the huntingtin protein. We have developed PC12 cell lines in which the expression of an N-terminal truncation of huntingtin (N63) with either wild type (23Q) or expanded polyglutamine (148Q) can be induced by the removal of doxycycline. Differentiated PC12 cells induced to express N63-148Q showed cellular toxicity reaching up to 50% at 6 days post-induction. Histone acetyltransferase (HAT) activity and global histone acetylation was significantly decreased in cells expressing truncated huntingtin with mutant but not normal huntingtin. These data suggest that altered chromatin modification via reduction in coactivator activity may cause neuronal transcriptional dysregulation and contribute to cellular toxicity.

The C282Y and H63D mutations in the HFE gene are important causes of hemochromatosis. In the elderly, these mutations might be associated with increased morbidity because of the lifelong accumulation of iron. In a population-based sample of the elderly, we determined the value of genotyping for HFE mutations to screen for subclinical hemochromatosis. HFE genotype frequencies were determined in a random group of 2095 subjects (55 years and over). In this random group, we selected within the six genotype groups a total of 342 individuals and measured their serum transferrin saturation, iron and ferritin levels. We also estimated the heritability and parameters needed to evaluate screening, including the sensitivity, specificity, positive and negative predictive values (PPV, NPV) of HFE genotypes. Iron parameters were significantly increased in subjects homozygous, heterozygous or compound heterozygous. The effect of the mutations was more pronounced in men than in women. For the H63D mutation, an allele dose effect was observed. The HFE gene explained about 5% of the variability in serum iron indices. The PPV for hemochromatosis for the C282Y homozygous was 100% in men and 67% in women. The NPV of the wild-type allele was 97% for both men and women. The sensitivity of both mutations was 70% for men and 52% for women and the specificity was 62% for men and 64% for women. Our study shows that the HFE C282Y and H63D are determinants of iron parameters in the elderly and will be effective in detecting individuals at high risk of hemochromatosis. However, when screening based on these two mutations, some individuals with subclinical hemochromatosis will be missed.

Precipitation with a compressed or supercritical fluid antisolvent (PCA) has been used to produce microparticles of biologically active proteins, pharmaceuticals, and polymers. However, the application of PCA to a wider range of proteins is limited by the low mutual solubility of water (necessary to dissolve most proteins) and CO(2) (traditionally used as the compressed antisolvent). This investigation extends PCA to proteins in aqueous solutions by utilizing ethanol as a cosolvent to enhance the antisolvent properties of CO(2) toward aqueous systems. alpha-Chymotrypsin, a model protein, was precipitated from both compressed CO(2) and a liquid fluorinated antisolvent, a hydrofluoroether (HFE). The equilibrium phase behavior of the antisolvent/ethanol/water systems was examined to identify a one-phase region suitable for protein precipitation. Spherical protein microparticles with a primary particle size of approximately 0.2-0.6 microm were recovered using both the compressed CO(2) and fluorinated antisolvents. Although the proteins retained significant activity using both antisolvent systems, the HFE-precipitated chymotrypsin retained higher activity than the CO(2)-precipitated protein.

This case study reviews cardiopulmonary bypass (CPB) management in a Protein C deficient patient undergoing reoperation for an atrioventricular (AV) valve replacement with the use of aprotinin. Protein C inhibits factors Va and VIIIa in the coagulation cascade and inactivates tissue plasminogen activator inhibitor, thus maintaining hemostasis. Protein C deficiency can cause hypercoagulability and may result in thrombotic episodes, especially in areas of low blood flow or during activation of the coagulation cascade. A 17-year-old male presented with a functional single ventricle and AV valve regurgitation. The patient had a history of three previous AV valve replacements. Protein C deficiency was first diagnosed after thrombosis of the first valve prosthesis. Other case studies in protein C deficient patients suggested the use of fresh frozen plasma (FFP) before bypass to restore protein C levels, ATIII replacement before heparin administration, and avoidance of aprotinin because of its known competitive inhibition of activated protein C. Two units of FFP were given by anesthesia before the administration of aprotinin, and two units of FFP were added to the pump prime. The full Hammersmith loading dose of aprotinin was administered just before initiation of CPB. The same dose of aprotinin was added to the pump prime just before initiation of CPB. Additional heparin (100 U/kg) was administered every hour during bypass. Activated clotting time tests (ACTs) were performed every 15 min, and thromboelastographs (TEGs) were performed every hour. The patient recovered from surgery without major complications, and there were no perioperative thrombotic events. The patient was discharged on day 41 and is doing well. Postoperative atrial arrhythmias were a contributing factor to his delayed discharge. The use of aprotinin in a protein C deficient patient undergoing open-heart surgery may be safe if protein C levels are restored before administration of aprotinin, and anticoagulation is carefully monitored.

Huntington's disease is due to an expansion of CAG repeats in the huntingtin gene. Huntingtin interacts with several proteins including glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We performed immunohistochemical analysis of GAPDH expression in the brains of transgenic mice carrying the huntingtin gene with 89 CAG repeats. In all wild-type animals examined, GAPDH was evenly distributed among the different cell types throughout the brain. In contrast, the majority of transgenic mice showed GAPDH overexpression, with the most prominent GAPDH changes observed in the caudate putamen, globus pallidus, neocortex, and hippocampal formation. Double staining for NeuN and GFAP revealed that GAPDH overexpression occurred exclusively in neurons. Nissl staining analysis of the neocortex and caudate putamen indicated 24 and 27% of cell loss in transgenic mice, respectively. Subcellular fluorescence analysis revealed a predominant increase in GAPDH immunostaining in the nucleus. Thus, we conclude that mutation of huntingtin is associated with GAPDH overexpression and nuclear translocation in discrete populations of brain neurons.

<h4>Background</h4>Hemostatic abnormalities have been reported in various hepatocellular diseases. We evaluated the hemostatic functions in patients with Budd-Chiari syndrome.<h4>Methods</h4>Biochemical liver function tests, and measurement of prothrombin time, activated partial thromboplastin time, and plasma levels of anti-thrombin III (antigen) and activity of protein C were done in 36 patients with Budd-Chiari syndrome.<h4>Results</h4>Liver biochemistry was abnormal in 34 patients. Plasma prothrombin time and activated partial thromboplastin time were prolonged in 17 (47%) and 23 (64%) patients, respectively. Antithrombin III antigen levels and protein C activity were reduced in 15 (50%) and 25 (83%) patients, respectively, among the 30 patients studied. Albumin levels showed significant correlation with coagulation test results, levels of anti-thrombin-III, and protein C activity.<h4>Conclusion</h4>Hepatic synthesis of coagulation factors and anticoagulants is reduced in Budd-Chiari syndrome; this may play a role in recurrence of thrombosis.

<h4>Purpose</h4>Estrogen has been highly evaluated as one of the most potent endocrine agents for the treatment of prostate cancer. Unfortunately, a high risk of cardiovascular complications is a clinically important adverse effect of estrogen therapy, and occasionally the complications are fatal. In recent years a high incidence (55%) of thrombotic events has been reported in patients with congenital protein S (PS) deficiency. The aim of this study is to determine the relationship between cardiovascular complications of estrogen therapy and anticoagulant factor levels in the serum of patients with prostate cancer.<h4>Materials and methods</h4>Study 1 employed 99 patients with prostate cancer: 39 were untreated, 25 were treated with LH-RH agonist therapy alone, and 35 were treated with oral diethylstilbestrol diphosphate (DESdP) 300 mg per day. We measured the serum levels of anticoagulant factors, parameters antithrombin III (ATIII), protein C (PC), PS, coagulant and fibrinolytic factors in all patients. In study 2, the adverse effects of DESdP therapy on the serum levels of anticoagulant factors were examined in 8 patients with advanced prostate cancer.<h4>Results</h4>In study 1, the ATIII and PS levels of the patients treated with estrogen therapy were significantly lower than those in either the untreated patients or the patients treated with an LHRH agonist alone. Especially, both PS antigen (51.5 +/- 16.0%) and PS activity (42.9 +/- 16.0%) were markedly lower in estrogen-treated patients than in the untreated patients (102 +/- 20.8%, 100.6 +/- 20.7%, respectively) or the patients treated with an LH-RH agonist alone (97.9 +/- 16.8%, 91.5 +/- 17.7%, respectively, both p < 0.0001). PS was decreased to below the normal lower limit of normal in 82% (24/35) of the patients on estrogen therapy. In study 2, all 8 cases showed a significant decrease in PS after DESdP therapy.<h4>Conclusions</h4>Our results showed that the PS levels in the oral DESdP group were almost the same as in patients with congenital PS deficiency. We conclude that decreased PS may play a role in the development of cardiovascular complications in prostate cancer patients on estrogen therapy.

<h4>Background</h4>The pathophysiology of tardive dyskinesia (TD) is not completely understood.Aim. - To assess the relationship of TD with 5-HT2A receptor gene, serotonin transporter gene (5 HTT), and catechol-o-methyltransferase (COMT) gene polymorphisms.<h4>Method</h4>Our study comprised 111 unrelated subjects who strictly met DSM-IV criteria for schizophrenia and 32 TD, and 79 healthy unrelated controls; all the subjects were of Turkish origin. The analyses of 5-HT2A receptor gene, 5 HTT gene, and COMT gene polymorphisms were performed using polymerase chain reaction (PCR) technique.<h4>Results</h4>The polymorphisms of these genes were not significantly different between the schizophrenic patients, TD and control subjects.<h4>Conclusion</h4>Our findings indicated that 5-HT2A receptor gene, 5 HTT gene, and COMT gene polymorphisms were similar in schizophrenia with non-TD, schizophrenia with TD, and healthy controls. These polymorphisms, though, do not help to evaluate the susceptibility to TD.

<h4>Objective</h4>Dysmetabolic hepatosiderosis is a recently described entity, which reunites iron overloads associated with polymetabolic syndromes, steatosis and steato-hepatitis. The aim of this study was to specify the relationship between iron overload, polymetabolic syndrome, HFE mutations of primary hemochromatosis and steatosis.<h4>Method</h4>This was a 5-year retrospective study of 51 patients hospitalised and/or seen in consultation, non-alcoholic, presenting with hyperferritinemia associated with a polymetabolic syndrome and/or hepatic steatosis.<h4>Results</h4>Patients mean age was of 53 +/- 12 years with a sex ratio M/F of 5.4. Metabolic disorders were found in 97% of the non-steatosis patients (overweight: 40%, perturbed sugar metabolism: 47%, dyslipidemia: 79.5%). Hyperferritinemia was constant but moderate (513.4 +/- 280.3 ng/ml). Transferrin saturation was predominantly normal (0.44 +/- 0.17). The hepatic enzymes were normal or only slightly perturbed. Steatosis was revealed by sonography in 62% of cases. The hepatic iron overload, documented in 6 patients by hepatic needle biopsy, was discreet (concentration/age ratio of 1.64 +/- 0.19). Among the 59% patients screened for the HFE gene, 40% were positive for C282Y and/or H63D. There was no correlation between ferritin levels and iron parameters, polymetabolic syndrome parameters and hepatic enzymes. The HFE mutations had no influence on the iron parameters.<h4>Conclusion</h4>Dysmetabolic hepatosiderosis must be know by hospital practitioners because of their prevalence in cases of hyperferritinemia and their therapeutic incidence.

<h4>Background</h4>Porphyria cutanea tarda (PCT) is sometimes associated with hepatitis C virus chronic infection. The aim of this study was to describe the effect of interferon alfa (IFN-alpha) in the treatment of patients with chronic hepatitis C and PCT.<h4>Methods</h4>We treated a total of 66 patients with chronic hepatitis C with IFN-alpha 2b (5 MU t.i.w.) for 12 months. Twenty-two of these patients suffered from PCT as well. These patients differed from patients without PCT in that they were men, past history of alcohol abuse and HFE gene mutations were more common and the source of infection was almost always unknown.<h4>Results</h4>Sustained virologic response was obtained in 19.7% of the 66 treated patients, 27.3% in the non-PCT group and 4.5% in the PCT group (P < 0.05). This difference could not be ascribed to the difference in sex of patients, history of alcohol abuse, HCV genotype or iron status.<h4>Conclusion</h4>Multivariate logistic regression analysis revealed that PCT is independently and significantly associated with non-sustained response to IFNalpha therapy. In conclusion, patients with chronic hepatitis C and PCT rarely responded to IFNalpha treatment.

<h4>Background</h4>Insulin resistance is a key feature of nonalcoholic fatty liver disease. Patients with hereditary hemochromatosis, a disease characterized by progressive iron overload due, in most cases, to homozygosity for C282Y mutation in the HFE gene, have often decreased insulin sensitivity and release.<h4>Aims</h4>To determine whether increased iron parameters/heterozygosity for the mutations of the HFE gene confer susceptibility to nonalcoholic fatty liver disease.<h4>Patients</h4>One hundred and thirty-four consecutive Italian patients with clinical and ultrasonographic diagnosis of nonalcoholic fatty liver disease (82 with hyperferritinemia), half confirmed by liver biopsy.<h4>Methods</h4>Insulin was determined by radioimmunoassay. HFE gene mutations were determined by polymerase chain reaction and restriction fragment length polymorphism analysis.<h4>Results</h4>(1) Prevalence of C282Y HFE mutation was significantly higher in patients with nonalcoholic fatty liver disease compared to controls, the difference being more striking in patients with hyperferritinemia than in those without. (2) The presence of mild iron overload was associated with a lower insulin release. (3) Carriers of C282Y mutation developed nonalcoholic fatty liver disease despite lower body mass index and triglycerides.<h4>Conclusion</h4>The mild iron overload associated with heterozygosity for C282Y HFE mutation confers susceptibility to nonalcoholic fatty liver disease, causing relative insulin deficiency.

Iron is a vital metal for the proliferation of all cells including those of the immune system. Iron deficiency causes several defects in both the humoral and cellular arms of immunity. One of the most profound changes is a reduction in peripheral T cells and atrophy of the thymus. The presence of transferrin receptor on immature, proliferating thymocytes and the inhibition of thymocyte proliferation and differentiation by anti-transferrin receptor antibody highlight the importance of iron to T cell development. Growing evidence suggests that T cells may in turn, regulate iron metabolism perhaps through interactions with the non-classical major histocompatibility complex gene HFE. The association of the iron transporter NRAMP1 with several autoimmune disorders along with evidence that iron can catalyze the production of cryptic epitopes of several autoantigens, establishes a potential role for iron in the development of autoimmunity.

<h4>Summary</h4>Thrombus organization in canine carotid arteries occluded with platinum microcoils was studied to determine if endothelial injury created with a Xenon Chloride Excimer Laser (XEL) could acclerate endovascular fibrosis. Ten common carotid artery stumps were created in ten dogs. Each of four stumps were schematically divided into four longitudinally contiguous injury zones (thermal ablation injury, non-ablative injury, proximal and distal non-injury zones) to test the effects of ablative and non-ablative injury and to establish a set of internal controls that would account for proximity to circulating blood at the ostium of the occluded artery. Following XEL irradiation of the endothelium through an arteriotomy, each stump was embolized with microcoils. Four control stumps were subjected to sham laser procedures, and embolized in an identical fashion. Two additional stumps were embolized in the absence of sham surgery. Angiographic, gross and histologic analysis was performed after four weeks. Specimens of freshly clotted whole blood mixed with microcoils were used as an additional control. In irradiated stumps and non-irradiated stumps (sham and embolization only), angiography revealed no evidence of coil compaction or recanalization. In all irradiated stumps the thermal ablation zone contained fibrous tissue and neovascularity without unorganized thrombus. The other zones in the irradiated stumps were indistingnishable from each other and from all zones in the non-irradiated sham stumps, containing primarily unorganized thrombus. Stumps embolized in the absence of sham surgery were filled with material that was grossly and microscopically identical to specimens of freshly clotted whole blood containing microcoils. The results indicate that thermal ablation injury of the endothelium accelerates thrombus organization in canine carotid arteries occluded with platinum microcoils.

<h4>Background</h4>Porphyria cutanea tarda (PCT) is sometimes associated with hepatitis C virus chronic infection. The aim of this study was to describe the effect of interferon alfa (IFN-a) in the treatment of patients with chronic hepatitis C and PCT.<h4>Methods</h4>We treated a total of 66 patients with chronic hepatitis C with IFN-a 2b (5 MU t.i.w.) for 12 months. Twenty-two of these patients suffered from PCT as well. These patients differed from patients without PCT in that they were men, past history of alcohol abuse and HFE gene mutations were more common and the source of infection was almost always unknown.<h4>Results</h4>Sustained virologie response was obtained in 19.7% of the 66 treated patients, 27.3% in the non-PCT group and 4.5% in the PCT group (P < 0.05). This difference could not be ascribed to the difference in sex of patients, history of alcohol abuse, HCV genotype or iron status.<h4>Conclusion</h4>Multivariate logistic regression analysis revealed that PCT is independently and significantly associated with non-sustained response to IFNa therapy. In conclusion, patients with chronic hepatitis C and PCT rarely responded to IFNa treatment.