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Hereditary hemochromatosis (HH) is the most common genetic disease among individuals of European descent. Two mutations (845G-->A, C282Y and 187C-->G, H63D) in the hemochromatosis gene (HFE gene) are associated with HH. About 85-90% of patients of northern European descent with HH are C282Y homozygous. The prevalence of HH in the Brazilian population, which has a very high level of racial admixture, is unknown. The aims of the present study were to identify individuals with diagnostic criteria for HH among patients with a body iron overload attended at the university hospital of the Faculty of Medicine of Ribeirao Preto from 1990 to 2000, and to evaluate the prevalence of HFE mutations. We screened first-degree relatives for HFE mutations. Four of 72 patients (three men and one woman, mean age 47 years) fulfilled the criteria for HH. HFE mutations were studied in three patients [two C282Y homozygotes (patients 1 and 2) and one H63D heterozygote]. Patient 1 had four children (all C282Y heterozygotes with no iron overload) and seven brothers and sisters: two sisters (66 and 76 years old) were C282Y homozygotes and both had an iron overload (a liver biopsy in one showed severe iron deposits), one sister (79 years old) was a compound heterozygote with no iron overload, one brother (78 years old) was a C282Y heterozygote with no iron overload, two individuals were H63D heterozygotes (one brother, 49 years old, obese, with a body iron overload and abnormal liver enzymes - a biopsy showed non-alcoholic steatohepatitis, and one 70-year-old sister with no iron overload). Patient 2 had two children (22 and 24 years old who were C282Y heterozygotes with no iron overload) but no brothers or sisters. These results showed that HH was uncommon among individuals attended at our hospital, although HFE mutations were found in all patients. Familial screening is valuable for the early diagnosis of individuals at risk since it allows treatment to be initiated before the onset of the clinical manifestations of organ damage associated with HH.

Basal cell carcinoma (BCC) is one of the most common neoplasms in the world and its incidence has been increasing worldwide in recent years. BCCs are caused by an interplay between genetic and environment factors. We conducted a case-control association study in BCC patients and controls from Sweden and Finland. Fifteen single nucleotide polymorphisms (SNPs), IL-6-174G/C, -634G/C, and -597G/A; IL-10-1082G/A and -592C/A; IL-1beta-511C/T; NBS1 exon 5 Glu185Gln; XPC exon 15 Lys939Gln; XPD exon 23 Lys751Gln; XRCC1 exon 10 Arg399Gln; XRCC3 exon 7 Thr241Met; cyclin D1 exon 4 G870A; MTHFR exon 4 Ala222Val and exon 7 Glu429Ala; HFE exon 4 C282Y were performed by Pyrosequencing and RFLP techniques. Most of the genotype distributions were in accordance with the Hardy-Weinberg equilibrium (HWE), except for IL-10-1082G/A, where cases with BCC showed a significant deviation from HWE (P = 0.04). Linkage disequilibrium was observed between the -174 and -597 alleles in the IL-6 gene in the present populations. No difference between BCC and controls appeared in any of the SNPs analyzed. Only the combined distributions of TT/AA genotypes in MTHFR exon 4 (C/T) and exon 7 (A/C) showed slight increase in BCC compared to controls (P < 0.07, OR: 1.94; 95% CI: 0.96-3.89).

<h4>Background</h4>Rotavirus genotyping is performed by using reverse transcription PCR with type-specific-primers. Because the high rotavirus mutation rate generates an extensive genomic variation, different G-type-specific primer sets are applied in different geographical locations. In Bangladesh, a significant proportion (36.9%) of the rotavirus strains isolated in 2002 could not be G-typed using the routinely used primer set. To investigate the reason why the strains were untypeable, nucleotide sequencing of the VP7 genes was performed.<h4>Results</h4>Four nucleotide substitutions at the G1 primer-binding site of the VP7 gene of Bangladeshi G1 rotaviruses rendered a major proportion of circulating strains untypeable using the routine primer set. Using an alternative primer set, we could identify G1 rotaviruses as the most prevalent genotype (44.8%), followed by G9 (21.7%), G2 (15.0%) and G4 (13.8%).<h4>Conclusion</h4>Because of the natural variation in the rotaviral gene sequences, close monitoring of rotavirus genotyping methods is important.

Neuronal migration and lamina-specific primary afferent projections are crucial for establishing spinal cord circuits, but the underlying mechanisms are poorly understood. Here, we report that in mice lacking Dcc (deleted in colorectal cancer), some early-born neurons could not migrate ventrally in the spinal cord. Conversely, forced expression of Dcc caused ventral migration and prevented dorsolateral migration of late-born spinal neurons. In the superficial layer of the spinal cord of Dcc-/- mutants, mislocalized neurons are followed by proprioceptive afferents, while their presence repels nociceptive afferents through Sema3a. Thus, our study has shown that Dcc is a key molecule required for ventral migration of early-born neurons, and that appropriate neuronal migration is a prerequisite for, and coupled to, normal projections of primary afferents in the developing spinal cord.

Sry high mobility group (HMG) box (Sox) transcription factors are involved in the development of central nervous system (CNS) in all metazoans. Little is known on the molecular mechanisms that regulate their transcriptional activity. Covalent posttranslational modification by small ubiquitin-like modifier (SUMO) regulates several nuclear events, including the transcriptional activity of transcription factors. Here, we demonstrate that SoxNeuro, an HMG box-containing transcription factor involved in neuroblast formation in Drosophila, is a substrate for SUMO modification. SUMOylation assays in HeLa cells and Drosophila S2 cells reveal that lysine 439 is the major SUMO acceptor site. The sequence in SoxNeuro targeted for SUMOylation, IKSE, is part of a small inhibitory domain, able to repress in cis the activity of two adjacent transcriptional activation domains. Our data show that SUMO modification represses SoxNeuro transcriptional activity in transfected cells. Overexpression in Drosophila embryos of a SoxN form that cannot be targeted for SUMOylation strongly impairs the development of the CNS, suggesting that SUMO modification of SoxN is crucial for regulating its activity in vivo. Finally, we present evidence that SUMO modification of group B1 Sox factors was conserved during evolution, because Sox3, the human counterpart of SoxN, is also negatively regulated through SUMO modification.

<h4>Objective</h4>To analyse somatic molecular changes, clinicopathological features, family history, and germline mutations in families with colorectal cancer (CRC).<h4>Methods</h4>Molecular changes (K-ras and beta-catenin mutations, chromosome 18q allele loss (LOH), APC LOH, microsatellite instability (MSI), and expression of beta-catenin and p53) were examined in four series of CRC patients with proven or probable hereditary disease: hereditary non-polyposis colon cancer (HNPCC); MYH associated polyposis (MAP); multiple (>5) colorectal adenomas without familial adenomatous polyposis (FAP); and other families/cases referred to family cancer clinics (FCC series). HNPCC was diagnosed using a combination of germline mutation screening and tumour studies. A series of unselected CRC patients was also studied.<h4>Results</h4>There was overlap between genetic pathways followed by each type of CRC, but significant differences included: increased frequency of K-ras mutation and reduced frequency of APC LOH in cancers from MAP, but not from multiple adenoma patients; reduced frequency of LOH in HNPCC CRCs; and increased MSI in CRCs from HNPCC, but not from FCC or multiple adenoma patients. HNPCC was apparently detected efficiently by combined germline and somatic analysis. Cancers from the FCC, unselected, and multiple adenoma series shared similar molecular characteristics. In the FCC and multiple adenoma series, hierarchical cluster analysis using the molecular features of the cancers consistently identified two distinct groups, distinguished by presence or absence of K-ras mutation.<h4>Conclusions</h4>While K-ras mutation status is known to differentiate hereditary bowel cancer syndromes such as MAP and FAP, it may also distinguish groups of non-HNPCC, FCC patients whose disease has different, as yet unknown, genetic origins.

<h4>Background</h4>Differential gene expression specifies the highly diverse cell types that constitute the nervous system. With its sequenced genome and simple, well-defined neuroanatomy, the nematode C. elegans is a useful model system in which to correlate gene expression with neuron identity. The UNC-4 transcription factor is expressed in thirteen embryonic motor neurons where it specifies axonal morphology and synaptic function. These cells can be marked with an unc-4::GFP reporter transgene. Here we describe a powerful strategy, Micro-Array Profiling of C. elegans cells (MAPCeL), and confirm that this approach provides a comprehensive gene expression profile of unc-4::GFP motor neurons in vivo.<h4>Results</h4>Fluorescence Activated Cell Sorting (FACS) was used to isolate unc-4::GFP neurons from primary cultures of C. elegans embryonic cells. Microarray experiments detected 6,217 unique transcripts of which approximately 1,000 are enriched in unc-4::GFP neurons relative to the average nematode embryonic cell. The reliability of these data was validated by the detection of known cell-specific transcripts and by expression in UNC-4 motor neurons of GFP reporters derived from the enriched data set. In addition to genes involved in neurotransmitter packaging and release, the microarray data include transcripts for receptors to a remarkably wide variety of signaling molecules. The added presence of a robust array of G-protein pathway components is indicative of complex and highly integrated mechanisms for modulating motor neuron activity. Over half of the enriched genes (537) have human homologs, a finding that could reflect substantial overlap with the gene expression repertoire of mammalian motor neurons.<h4>Conclusion</h4>We have described a microarray-based method, MAPCeL, for profiling gene expression in specific C. elegans motor neurons and provide evidence that this approach can reveal candidate genes for key roles in the differentiation and function of these cells. These methods can now be applied to generate a gene expression map of the C. elegans nervous system.

<h4>Background</h4>The purpose of this study was to investigate whether rats dosed with serotonin develop changes similar to those seen in human carcinoid heart disease.<h4>Methods and results</h4>Ten Sprague-Dawley rats were given serotonin injections subcutaneously once daily for 3 months; controls were given saline. A long-lasting hyperserotoninemia with a >10-fold increase in both platelet-poor plasma and dialysate from the femoral muscles appeared. The animals developed clinical signs such as flushing and loose stools. After 3 months, 6 of 10 rats given serotonin had pathological echocardiographs. Two animals had a combination of aortic and pulmonary valve insufficiency, 1 had isolated aortic valve insufficiency, and 3 had isolated pulmonary valve insufficiency. Histopathological examination revealed shortened and thickened aortic cusps and carcinoidlike plaques characterized by a collection of myofibroblasts within an extracellular matrix of collagen ground substance. Immunostaining for Ki-67 demonstrated an increased number of proliferating subendocardial cells. In the control group, no pathological changes were seen. With the use of reverse-transcription polymerase chain reaction, normal rat aortic cusps were shown to express mRNA for serotonin receptors 5-HT1A, 5-HT2A, and 5-HT2B and the serotonin transporter 5-HTT.<h4>Conclusions</h4>For the first time, long-term serotonin administration was performed in rats. Morphological and echocardiographic changes similar to those seen in human carcinoid heart disease developed. This study demonstrates that serotonin most likely is involved in the pathogenesis of carcinoid heart disease.

Previous studies have demonstrated the ability of bone morphogenetic proteins (BMPs) to promote chondrogenic differentiation in vitro. However, the in vivo role of BMP signaling during chondrogenesis has been unclear. We report here that BMP signaling is essential for multiple aspects of early chondrogenesis. Whereas mice deficient in type 1 receptors Bmpr1a or Bmpr1b in cartilage are able to form intact cartilaginous elements, double mutants develop a severe generalized chondrodysplasia. The majority of skeletal elements that form through endochondral ossification are absent, and the ones that form are rudimentary. The few cartilage condensations that form in double mutants are delayed in the prechondrocytic state and never form an organized growth plate. The reduced size of mutant condensations results from increased apoptosis and decreased proliferation. Moreover, the expression of cartilage-specific extracellular matrix proteins is severely reduced in mutant elements. We demonstrate that this defect in chondrocytic differentiation can be attributed to lack of Sox9, L-Sox5, and Sox6 expression in precartilaginous condensations in double mutants. In summary, our study demonstrates that BMPR1A and BMPR1B are functionally redundant during early chondrogenesis and that BMP signaling is required for chondrocyte proliferation, survival, and differentiation in vivo.

Netrins and their classical receptors--DCC and neogenin--play key roles in neuronal guidance. Recent developments identify new roles for netrins in epithelial and vascular morphogenesis. Netrins accomplish these effects, at least in part, through binding to integrins and/or activating integrin-associated kinases such as FAK and Fyn. Here we discuss these recent findings and propose that integrins and classical netrin receptors cooperate to regulate multiple aspects of development.

The L1 family of cell adhesion molecules is predominantly expressed in the nervous system. Mutations in human L1 cause neuronal diseases such as HSAS, MASA, and SPG1. Here we show that sax-7 gene encodes an L1 homologue in Caenorhabditis elegans. In sax-7 mutants, the organization of ganglia and positioning of neurons are abnormal in the adult stage, but these abnormalities are not observed in early larval stage. Misplacement of neurons in sax-7 mutants is triggered by mechanical force linked to body movement. Short and long forms of SAX-7 exhibited strong and weak homophilic adhesion activities in in vitro aggregation assay, respectively, which correlated with their different activities in vivo. SAX-7 was localized on plasma membranes of neurons in vivo. Expression of SAX-7 only in a single neuron in sax-7 mutants cell-autonomously restored its normal neuronal position. Expression of SAX-7 in two different head neurons in sax-7 mutants led to the forced attachment of these neurons. We propose that both homophilic and heterophilic interactions of SAX-7 are essential for maintenance of neuronal positions in organized ganglia.

Nuclear deposits of stainable iron in hepatocytes are a sign of liver iron overload in mice. Animals with no, partial or total knock-out of the HFE alleles, the deletion of which is responsible for hereditary haemochromatosis, were given different forms of dietary iron to measure nuclear iron deposits which were then related to cytoplasmic iron load. Wild type and heterozygous HFE-knock-out mice kept for 52 weeks on a standard diet showed no such deposits. These were, however, demonstrated in low numbers and with small diameters in homozygous HFE-knock-out mice kept on this diet. Nuclear iron deposits were most abundant in all type of mice fed carbonyl iron (2.5% w/w) for 52 weeks almost irrespective of their genetic background. The diameter of these deposits increased with the genetically conditioned extent of hepatocellular iron overload. Mice that were fed a diet containing TMH-ferrocene for 4 weeks showed amounts of hepatic iron that were comparable to those in the carbonyl iron-fed group but nuclear deposits were small and present in only 0.3% of the hepatocytes. While surrounding karyoplasm was immunostained for H- and L-ferritin, the nuclear iron deposits were not. As the nuclear iron deposits corresponded electron microscopically to aggregated ferritin molecules, they represent a non-immunoreactive form of presumably denatured ferritin.

Lung surfactant dipalmitoylphosphatidylcholine (DPPC) is endocytosed by alveolar epithelial cells and degraded by lysosomal-type phospholipase A2 (aiPLA2). This enzyme is identical to peroxiredoxin 6 (Prdx6), a bifunctional protein with PLA2 and GSH peroxidase activities. Lung phospholipid was studied in Prdx6 knockout (Prdx6-/-) mice. The normalized content of total phospholipid, phosphatidylcholine (PC), and disaturated phosphatidylcholine (DSPC) in bronchoalveolar lavage fluid, lung lamellar bodies, and lung homogenate was unchanged with age in wild-type mice but increased progressively in Prdx6-/- animals. Degradation of internalized [3H]DPPC in isolated mouse lungs after endotracheal instillation of unilamellar liposomes labeled with [3H]DPPC was significantly decreased at 2 h in Prdx6-/- mice (13.6 +/- 0.3% vs. 26.8 +/- 0.8% in the wild type), reflected by decreased dpm in the lysophosphatidylcholine and the unsaturated PC fractions. Incorporation of [14C]palmitate into DSPC at 24 h after intravenous injection was decreased by 73% in lamellar bodies and by 54% in alveolar lavage surfactant in Prdx6-/- mice, whereas incorporation of [3H]choline was decreased only slightly. Phospholipid metabolism in Prdx6-/- lungs was similar to that in wild-type lungs treated with MJ33, an inhibitor of aiPLA2 activity. These results confirm an important role for Prdx6 in lung surfactant DPPC degradation and synthesis by the reacylation pathway.

Hepcidin, originally discovered in urine as a bactericidal peptide synthesized by hepatocytes was later proved to be a key regulator of iron metabolism at the whole body level, namely, in conditions of altered iron demand such as the increased or decreased total amount of body iron, inflammation, hypoxia and anemia. The major mechanism of hepcidin function seems to be the regulation of transmembrane iron transport. Hepcidin binds to its receptor, protein ferroportin, which serves as a transmembrane iron channel enabling iron efflux from cells. The hepcidin-ferroportin complex is then degraded in lysosomes and iron is locked inside the cells (mainly enterocytes, hepatocytes and macrophages). This leads to lowering of iron absorption in the intestine and to a decrease in serum iron concentration. Utilizing this mechanism, hepcidin regulates serum iron levels during inflammation, infection and possibly also in cancer. Under these conditions iron is shifted from circulation into cellular stores in hepatocytes and macrophages, making it less available for invading microorganisms and tumor cells. In anemia and hypoxia, hepcidin regulates the availability of iron for erythropoiesis. Hepcidin or hepcidin-related therapeutics could find a place in the treatment of various diseases such as hemochromatosis and anemia of chronic disease.

It was recently postulated that the effects of general anesthetics on protein global dynamics might underlie a unitary molecular mechanism of general anesthesia. To verify that the specific dynamics effects caused by general anesthetics were not shared by nonanesthetic molecules, two parallel 8-ns all-atom molecular dynamics simulations were performed on a gramicidin A (gA) channel in a fully hydrated dimyristoylphosphatidylcholine membrane in the presence and absence of hexafluoroethane (HFE), which structurally resembles the potent anesthetic molecule halothane but produces no anesthesia. Similar to halothane, HFE had no measurable effects on the gA channel structure. In contrast to halothane, HFE produced no significant changes in the gA channel dynamics. The difference between halothane and HFE on channel dynamics can be attributed to their distinctly different distributions within the lipid bilayer and consequently to the different interactions of the anesthetic and the nonanesthetic molecules with the channel-anchoring tryptophan residues. The study further supports the notion that anesthetic-induced changes in protein global dynamics may play an important role in mediating anesthetic actions on proteins.

Dinucleoside polyphosphates act as agonists on purinergic P2Y receptors to mediate a variety of cellular processes. Symmetrical, naturally occurring purine dinucleotides are found in most living cells and their actions are generally known. Unsymmetrical purine dinucleotides and all pyrimidine containing dinucleotides, however, are not as common and therefore their actions are not well understood. To carry out a thorough examination of the activities and specificities of these dinucleotides, a robust method of synthesis was developed to allow manipulation of either nucleoside of the dinucleotide as well as the phosphate chain lengths. Adenosine containing dinucleotides exhibit some level of activity on P2Y(1) while uridine containing dinucleotides have some level of agonist response on P2Y(2) and P2Y(6). The length of the linking phosphate chain determines a different specificity; diphosphates are most accurately mimicked by dinucleoside triphosphates and triphosphates most resemble dinucleoside tetraphosphates. The pharmacological activities and relative metabolic stabilities of these dinucleotides are reported with their potential therapeutic applications being discussed.

Ath1 is a quantitative trait locus on mouse chromosome 1 that renders C57BL/6 mice susceptible and C3H/He mice resistant to diet-induced atherosclerosis. The quantitative trait locus region encompasses 11 known genes, including Tnfsf4 (also called Ox40l or Cd134l), which encodes OX40 ligand. Here we report that mice with targeted mutations of Tnfsf4 had significantly (P <or= 0.05) smaller atherosclerotic lesions than did control mice. In addition, mice overexpressing Tnfsf4 had significantly (P <or= 0.05) larger atherosclerotic lesions than did control mice. In two independent human populations, the less common allele of SNP rs3850641 in TNFSF4 was significantly more frequent (P <or= 0.05) in individuals with myocardial infarction than in controls. We therefore conclude that Tnfsf4 underlies Ath1 in mice and that polymorphisms in its human homolog TNFSF4 increase the risk of myocardial infarction in humans.

<h4>Background</h4>The exogenous administration of Insulin-like Growth Factor-I (IGF-I) induces hepatoprotective and antifibrogenic actions in experimental liver cirrhosis. To better understand the possible pathways behind the beneficial effect of IGF-I, the aim of this work was to investigate severe parameters involved in oxidative damage in hepatic tissue from cirrhotic animals treated with IGF-I (2 microg x 100 g(-1) x day(-1)). Iron and copper play an important role in oxidative mechanisms, producing the deleterious hydroxyl radical (*OH) that peroxides lipid membranes and damages DNA. Myeloperoxidase (MPO) and nitric oxide (NO) are known sources of free radicals and induce reduction of ferritin-Fe3+ into free Fe2+, contributing to oxidative damage.<h4>Methods</h4>Liver cirrhosis was induced by CCl4 inhalation in Wistar male rats for 30 weeks. Healthy controls were studied in parallel (n = 10). Fe and Cu were assessed by atomic absoption spectrometry and iron content was also evaluated by Perls' staining. MPO was measured by ELISA and transferrin and ferritin by immunoturbidimetry. iNOS expression was studied by immuno-histochemistry.<h4>Results</h4>Liver cirrhosis was histologically proven and ascites was observed in all cirrhotic rats. Compared to controls untreated cirrhotic rats showed increased hepatic levels of iron, ferritin, transferrin (p < 0.01), copper, MPO and iNOS expression (p < 0.01). However, IGF-treatment induced a significant reduction of all these parameters (p < 0.05).<h4>Conclusion</h4>the hepatoprotective and antifibrogenic effects of IGF-I in cirrhosis are associated with a diminution of the hepatic contents of several factors all of them involved in oxidative damage.

There is currently limited empirical and theoretical support for the prevailing view that adaptation typically results from the fixation of many mutations, each with small phenotypic effects. Recent theoretical work suggests that, on the contrary, most of the phenotypic change during an episode of adaptation can result from the selection of a few mutations with relatively large effects. I studied the genetics of adaptation by populations of budding yeast to a culture regime of daily hundredfold dilution and transfer in a glucose-limited minimal liquid medium. A single haploid genotype isolated after 2000 generations showed a 76% fitness increase over its ancestor. This evolved haploid was crossed with its ancestor, and tetrad dissections were used to isolate a complete series of six meiotic tetrads. The Castle-Wright estimator of the number of loci at which adaptive mutations had been selected, modified to account for linkage and variation among mutations in the size of their effect, is 4.4. The estimate for a second haploid genotype, isolated from a separate population and with a fitness gain of 60%, was 2.7 loci. Backcrosses to the ancestor with the first evolved genotype support the inference that adaptation resulted primarily from two to five mutations. These backcrosses also indicated that deleterious mutations had hitchhiked with adaptive mutations in this evolved genotype.

<h4>Background</h4>Androgen-independent prostate cancer is today an incurable disease, but increased understanding of the mechanisms for the transition into an androgen-independent state may increase the possibilities for more efficient strategies in the future.<h4>Methods</h4>An androgen-independent subline, LNCaP-19, to the androgen-dependent prostate cancer cell line LNCaP was developed in vitro under standard culture conditions. The characteristics of LNCaP-19 regarding androgen responsiveness, PSA, and VEGF secretion was studied in vitro. The growth in vivo and the microvessel density (MVD) of the tumors were studied after inoculation in nude mice.<h4>Results</h4>LNCaP-19 grows equally well in dextran-charcoal stripped FBS (DCC-FBS) as in normal FBS, and rapidly gives rise to tumors in both intact and castrated mice, indicating a true androgen-independent growth. The PSA secretion from LNCaP-19 cells was lower than from LNCaP cells, while the VEGF level was comparable to the secretion from LNCaP cells without androgen stimulation. The MVD was increased in the LNCaP-19 tumors, and the vessels also displayed a changed morphology with exclusively small microvessels without lumen.<h4>Conclusions</h4>LNCaP-19 shows characteristics resembling those of androgen-independent prostate cancer. An increased MVD and changed vessel morphology in the tumor, makes it an interesting model system for studies regarding angiogenesis in the context of the acquisition of androgen independence.

The first steps of glucose metabolism are carried out by members of the families of GLUTs (glucose transporters) and HKs (hexokinases). Previous experiments using the inhibitor of glucose transport, CB (cytochalasin B), revealed that compartmentalization of GLUTs and HKs is a major factor in the control of glucose uptake in L6 myotubes [Whitesell, Ardehali, Printz, Beechem, Knobel, Piston, Granner, Van Der Meer, Perriott and May (2003) Biochem. J. 370, 47-56]. In the present paper, we evaluate compartmentalization of GLUTs and HKs in a hepatoma cell line, H4IIE, which is characterized by excess GLUT activity, HKI in a particulate and a cytosolic fraction, and insignificant G6Pase (glucose-6-phosphatase) activity. The measured activity of glucose transport exceeded the rate of phosphorylation approx. 30-fold. Treatment with 25 microM CB (K(i) approximately 3 microM in H4IIE cells) paradoxically increased the excess of GLUTs over phosphorylation (GLUTs are inhibited 80%, while phosphorylation is inhibited 98%). The global relationships of the data could be reconciled most simply by a two-compartment model. In this model, phosphorylation of glucose is carried out by a subset of HK molecules supplied by a subset of GLUTs that are more sensitive to CB than the other GLUTs. The agent, DCC (dicyclohexylcarbodi-imide) caused HKI to translocate from the particulate compartment to the cytosolic compartment and potently inhibited glucose phosphorylation. The particulate compartment may represent the mitochondria, to which the more CB-sensitive GLUTs may control the transport of glucose.

While genetic factors clearly play a key role in colorectal cancer (CRC) pathogenesis and in determining its phenotypic features, the precise genes that involved are largely unknown. To gain insight into these genes, consecutive Israeli CRC patients were genotyped using SNPs from within candidate genes: APC, beta-Catenin, K-RAS, DCC, P16, PTEN, RB1, P15, APOE, ERCC2, P53, MTHFR and hMSH2. Genotyping of consecutive, unselected colorectal cancer patients was done mostly by utilizing the MassARRAY technology (Sequenom) and to a lesser extent DGGE, ARMS and direct DNA sequencing. Correlation of genotypes with specific phenotypic features was carried out for all patients and separately for the Ashkenazim. Overall, 456 patients were analyzed, the majority (64.25%) being of Ashkenazi origin; mean age at diagnosis was 65.6 +/- 14 (range 25-90 years), and the mean follow-up was 4.7 +/- 0.28 (range 0-30 years). Statistically significant associations were noted between SNPs in beta-catenin and APOE and a positive family history of cancer (beta-catenin: p=0.034, APOE: p=0.033); tumor location and a DCC SNP (p=0.038) and the P53 R72P mutation and survival (p=0.0336). In Ashkenazi patients, ERCC2 and MTHFR genes' SNPs were associated with age at diagnosis (ERCC2: p=0.025, MTHFR: p=0.0005); a P53 polymorphism, APOE and Rb SNPs with a family history of cancer (P53 p=0.034;APOE p=0.04, Rb p= 0.022); DCC SNP with tumor location (p=0.014); and p15 SNP with tumor grade (p=0.032). This preliminary study shows that genetic factors play a role in determining CRC phenotypic features and that a larger cohort with longer follow-up is clearly needed.

Toll-like receptor 2 (TLR2) is a pattern recognition receptor that plays an important role in enabling cells of the innate immune system to recognize conserved structural motifs on a wide array of pathogens including gram-positive bacteria. Although microglia have recently been shown to express TLR2, the functional significance of this receptor in mediating microglial activation remains unknown. To ascertain the importance of TLR2 in microglial responses to S. aureus and its cell wall product peptidoglycan (PGN), we evaluated primary microglia from TLR2 knockout (KO) and wild-type (WT) mice. TLR2 was found to play a pivotal role in PGN recognition and subsequent activation in primary microglia, as demonstrated by the attenuated expression of TNF-alpha, IL-12 p40, MIP-2, and MCP-1 in PGN-treated TLR2 KO microglia compared with WT cells. In contrast, the responses of TLR2 KO and WT microglia to S. aureus were qualitatively similar, indicating that alternative receptors are responsible for recognizing intact bacteria. Microarray analysis confirmed that TLR2 plays a central role in PGN recognition by primary microglia. The expression of MyD88, a central adapter molecule in TLR-dependent signaling, was similar in both TLR2 KO and WT microglia, suggesting that the defect in PGN recognition by the former is not due to alterations in this key signaling intermediate. These findings reveal the complex nature of gram-positive bacterial recognition by microglia, which occurs, in part, through engagement of TLR2.

Elucidation of the molecular pathways of iron transport through cells and its control is leading to an understanding of genetic iron loading conditions. The general phenotype of haemochromatosis is iron accumulation in liver parenchymal cells, a raised serum transferrin saturation and ferritin concentration. Four types have been identified: type 1 is the common form and is an autosomal recessive disorder of low penetrance strongly associated with mutations in the HFE gene on chromosome 6(p21.3); type 2 (juvenile haemochromatosis) is autosomal recessive, of high penetrance with causative mutations identified in the HFE2 gene on chromosome 1 (q21) and the HAMP gene on chromosome 19 (q13); type 3 is also autosomal recessive with mutations in the TfR2 gene on chromosome 3 (7q22); type 4 is an autosomal dominant condition with heterozygous mutations in the ferroportin 1 gene. In type 4, iron accumulates in both parenchymal and reticuloendothelial cells and the transferrin saturation may be normal. There are also inherited neurodegenerative conditions associated with iron accumulation. The current research challenges include understanding the central role of the HAMP gene (hepcidin) in controlling iron absorption and the reasons for the variable penetrance in HFE type 1.

The carbazole 1,9a-dioxygenase (CARDO) system of Pseudomonas resinovorans strain CA10 catalyzes the dioxygenation of carbazole; the 9aC carbon bonds to a nitrogen atom and its adjacent 1C carbon as the initial reaction in the mineralization pathway. The CARDO system is composed of ferredoxin reductase (CarAd), ferredoxin (CarAc), and terminal oxygenase (CarAa). CarAc acts as a mediator in the electron transfer from CarAd to CarAa. To understand the structural basis of the protein-protein interactions during electron transport in the CARDO system, the crystal structure of CarAc was determined at 1.9 A resolution by molecular replacement using the structure of BphF, the biphenyl 2,3-dioxygenase ferredoxin from Burkholderia cepacia strain LB400 as a search model. CarAc is composed of three beta-sheets, and the structure can be divided into two domains, a cluster-binding domain and a basal domain. The Rieske [2Fe-2S] cluster is located at the tip of the cluster-binding domain, where it is exposed to solvent. While the overall folding of CarAc and BphF is strongly conserved, the properties of their surfaces are very different from each other. The structure of the cluster-binding domain of CarAc is more compact and protruding than that of BphF, and the distribution of electric charge on its molecular surface is very different. Such differences are thought to explain why these ferredoxins can act as electron mediators in respective electron transport chains composed of different-featured components.

The SMC proteins form the cores of three protein complexes in eukaryotes, cohesin, condensin and the Smc5-6 complex. Cohesin holds sister chromatids together after DNA replication and is involved in both the repair of double-strand breaks by homologous recombination and the intra-S-phase checkpoint. Condensin assists in the condensation of chromosomes at mitosis and also has a role in checkpoint control pathways. The Smc5-6 complex is involved in a variety of DNA repair and damage response pathways by as yet unknown mechanisms, but is also associated with repair by homologous recombination.

In comparing purified mouse olfactory sensory neurons (OSNs) with neighboring cells, we identified 54 differentially expressed transcripts. One-third of the transcripts encode proteins with no known function, but the others have functions that correlate with challenges faced by OSNs. The OSNs expressed a diversity of signaling protein genes, including stomatin (Epb7.2), S100A5, Ddit3, Sirt2, CD81, Sdc2, Omp, and Ptpla. The elaboration of dendrites, cilia, and axons that places OSNs in contact with diverse cell types and signals presumably also requires large investments in cytoskeletal-associated proteins, lipid biosynthesis, and energy production. Several of the genes encode proteins that participate in these biological processes, including ATP5g3, Ndufa9, Sqrdl, Mdh1, Got1, beta-2 tubulin, Capza1, Bin3, Tom1, Acl6, and similar to O-MACS. Three transcripts had restricted expression patterns. Similar to O-MACS and Gstm2 had zonally restricted expression patterns in OSNs and sustentacular cells but not in Bowman's glands, suggesting that zonality can be differentially regulated by cell type. The mosaic expression pattern of S100A5 in approximately 70% of OSNs predicts that it is coexpressed with a subset of odorant receptors. We captured four abundant transcripts, Cyp2a4, similar to Cyp2g1, Gstm2, and Cbr2, that encode xenobiotic metabolizing enzymes expressed by sustentacular cells or Bowman's glands, reinforcing the interpretation that clearance of xenobiotic compounds is a major function of these cells. Within the olfactory epithelium, Cbr2 is a new anatomical marker for sustentacular cells. We also discovered that Reg3g is a marker for respiratory epithelium.

<h4>Background</h4>The monitoring of phlebotomies in hemochromatosis patients depends on iron status measured by ferritin and transferrin saturation (TS). However, in the presence of inflammation or liver injury, soluble transferrin receptor (sTfR) determination was proposed to replace ferritin for diagnosing iron deficiency (ID). The present study evaluated performances of sTfR for the prediction of iron deficiency in a large number of hemochromatosis patients under phlebotomy therapy.<h4>Methods</h4>We studied 52 patients undergoing therapeutic phlebotomies and obtained 2 samples from 37 patients. Biological parameters were determined before each phlebotomy began. Performances of sTfR and TS in the diagnosis of iron deficiency were compared, according to ferritin levels under 12 microg/l.<h4>Results</h4>Ferritin and TS were correlated with removed iron (r=0.473, p<0.005 and r=0.345, p<0.05, respectively) and sTfR was correlated with the decrease in hemoglobin levels induced by phlebotomies (r=-0.678, p<0.0001). Areas under Receiver Operating Characteristics (ROC) curves for sTfR and TS were not statistically different for prediction of iron deficiency and sensitivity/specificity of sTfR at 1.64 mg/l were 67/86%.<h4>Conclusions</h4>sTfR determination could be used to predict iron depletion induced by phlebotomies when ferritin is of limited interest, to avoid the appearance of anemia.

<h4>Background</h4>The measurement of soluble transferrin receptor (sTfR) has been proposed as a valuable marker of erythropoietic activity and iron status. However, the possibility that mutations in HFE and/or transferrin genes have a direct effect on this parameter has not been sufficiently investigated. The present report addresses this point in the general population.<h4>Methods</h4>Serum sTfR, ferritin, iron and transferrin, as well as the H63D and the C282Y polymorphisms of the HFE gene and the TF C1/C2 polymorphism of the transferrin gene, were analysed in 348 subjects.<h4>Results</h4>We observed significant and independent associations of serum sTfR with sex (2.68+/-1.27 mg/L in men vs. 2.25+/-1.33 in women; P=0.002), H63D polymorphism (2.61+/-1.34 in wild type homozygotes vs. 2.28+/-1.25 in carriers of one or two mutated alleles; P=0.009), and serum iron concentration (r=-0.17; P=0.002).<h4>Conclusion</h4>The H63D mutation of the HFE gene has a moderate but significant influence on sTfR concentration in the general population, the presence of one or two mutated alleles being associated with an average of 0.27 mg/L less sTfR than nonmutated homozygotes.

We compared the frequency of CpG island methylation phenotype (CIMP), inactivation of APC, p53 and DCC genes and K-ras and BRAF mutations in 39 mucinous carcinomas (MC) and 34 non-mucinous carcinomas (NMC) of the colorectum with different microsatellite instability (MSI) status. The higher incidence of MSI (36% vs. 18%) was observed in MC compared with NMC. APC inactivation and K-ras mutations occurred more frequently in NMC (APC, 88%, p<0.001; K-ras, 58%, p=0.01) than in MC (APC, 24%; K-ras, 28%) regardless of MSI status. BRAF mutation occurred at a higher frequency in MC (18%, p=0.01) than in NMC (0%). However, with respect to inactivation of p53 and DCC, MSI status did matter and in both NMC and MC, more frequent inactivation of p53 and DCC was observed in MSS tumors than in MSI tumors. MSS tumors of NMC had a higher frequency of p53 (58% by IHC, p=0.03 and 83% by LOH, p=0.02) and DCC inactivation (83%, p=0.02) compared to MSI tumors of NMC (p53, 33% by IHC and 20% by LOH; DCC, 20%). MSS tumors of MC also showed a higher frequency of p53 and DCC inactivation (p53, 45% by IHC, p=0.02 and 53% by LOH, p=0.005; DCC, 82%, p=0.001) compared to MSI tumors of MC (p53, 0% by IHC and 0% for LOH; DCC, 17%). MC showed a higher frequency of CIMP compared with NMC (41% vs. 11%, p=0.01). These results indicate that mucinous carcinomas of the colorectum exhibit distinct molecular genetic characteristics and may arise from distinct pathogenic pathways.

<h4>Purpose of review</h4>The number of newly identified genes participating in the regulation of iron homeostasis has continued to expand at a remarkable pace. The roles for many have begun to be elucidated and there is an increasing indication that hepatocytes play a central role in determining the level of intestinal iron absorption. Total body iron homeostasis is dependent upon carefully regulated absorption of dietary iron, thus these genes are of fundamental importance in understanding of pathophysiology of such common disorders as hereditary hemochromatosis (HH) and the anaemia of chronic diseases.<h4>Recent findings</h4>The hepatic peptide hepcidin plays a key role as a circulating hormone that regulates the absorption of dietary iron from the duodenum. Hepcidin expression is inappropriately decreased in hereditary hemochromatosis and is abnormally increased in the anaemia of chronic diseases. Other hepatic proteins essential for normal iron homeostasis, including HFE, transferrin receptor 2 (TfR2), and hemojuvelin, function at least in part, by modulating the expression of hepcidin.<h4>Summary</h4>New insights into the pathophysiology of hereditary hemochromatosis and the anaemia of chronic diseases have been achieved with the recognition of the central role for hepcidin as an iron regulatory hormone. Investigations into the biologic control of this hormone and its mechanism of action offer the possibility of new therapeutic approaches to disorders of iron metabolism.

PTOV1 is a mitogenic protein that shuttles between the nucleus and the cytoplasm in a cell cycle-dependent manner. It consists of two homologous domains arranged in tandem that constitute a new class of protein modules. We show here that PTOV1 interacts with the lipid raft protein flotillin-1, with which it copurifies in detergent-insoluble floating fractions. Flotillin-1 colocalized with PTOV1 not only at the plasma membrane but, unexpectedly, also in the nucleus, as demonstrated by immunocytochemistry and subcellular fractionation of endogenous and exogenous flotillin-1. Flotillin-1 entered the nucleus concomitant with PTOV1, shortly before the initiation of the S phase. Protein levels of PTOV1 and flotillin-1 oscillated during the cell cycle, with a peak in S. Depletion of PTOV1 significantly inhibited nuclear localization of flotillin-1, whereas depletion of flotillin-1 did not affect nuclear localization of PTOV1. Depletion of either protein markedly inhibited cell proliferation under basal conditions. Overexpression of PTOV1 or flotillin-1 strongly induced proliferation, which required their localization to the nucleus, and was dependent on the reciprocal protein. These observations suggest that PTOV1 assists flotillin-1 in its translocation to the nucleus and that both proteins are required for cell proliferation.

<h4>Aim</h4>To investigate the effect of atorvastatin (Lipitor), a commonly used drug for dyslipidaemia in experimental autoimmune uveitis (EAU).<h4>Methods</h4>48 B10-RIII mice were immunised with human interphotoreceptor retinoid binding protein (IRBP) peptide p161-180. They were divided into three groups of 16 each and treated orally once daily for 14 days; group one received phosphate buffered saline (control group), group two received 1 mg/kg of atorvastatin (low dose group), and group three received 10 mg/kg (high dose). On day 14 lymph nodes, spleens, and right eyes were harvested. RNA was extracted from lymph nodes for RNase protection assay (RPA) to determine proinflammatory (IL-1 alpha and IL-1 beta), Th1 (TNF-alpha, IL-2, IL-12), and Th2 (IL-4, IL-5, and IL-10) cytokine levels. Protein was extracted from spleens for western blot to detect the expression of phosphorylated signal transducer and activator of transcription (STAT) 4 and STAT6. The severity of inflammation in enucleated eyes was graded by a masked observer. Paired t test was performed for the mean difference in histological scoring between treated groups and the immunised control group.<h4>Results</h4>Surprisingly, atorvastatin did not modulate the immune response. The proinflammatory cytokines, IL-1 alpha and IL-1 beta, and Th1 cytokines, TNF-alpha and IL-2, were upregulated equally in control and atorvastatin treated groups. IL-12 and Th2 cytokines were not upregulated in all three groups. Western blot analysis showed high levels of phosphorylated STAT4, but not STAT6 protein in the control and atorvastatin treated groups. Mean differences in histological scoring between treated groups and the immunised control group were not statistically significant.<h4>Conclusions</h4>Atorvastatin treatment had no effect on Th1 and Th2 cytokine transcription. Although histological grading suggested mildly decreased inflammation in the high dose treated group, the equivalence of cytokine expression in all groups suggests that the statins may not modulate IRBP induced uveoretinitis.

The progression of colorectal cancer is a multistage process associated with specific molecular alterations. The stepwise accumulation of these multiple genetic mutations progressively results in the acquisition of neoplastic cell behavior. The genetic abnormalities associated with the expression of metastatic phenotype, therefore, may be of prognostic significance in the clinical treatment of colorectal cancer patients. In this study, the immunohistochemical expression of the deleted in colorectal cancer gene (DCC) and p27Kip1 was assessed in 168 paraffin-embedded, formalin-fixed tumors of patients with stage II and III colorectal cancer. Kaplan-Meier survival curves and log-rank statistics were used to analyze survival times after curative primary tumor resection, and Cox proportional hazards models were used to adjust the assessment of demographic and clinical covariates. Loss of DCC or p27Kip1 expression had no influence on survival in patients with stage II or III colorectal cancer. The 5-year survival rates of DCC-positive and DCC-negative tumors were 51.8% and 35.7% (P=0.40), respectively. The 5-year survival rate of patients with p27Kip1-positive tumors was 47.9%, whereas the rate for patients with p27Kip1-negative tumors was 38.8% (P=0.68). After adjustment for all evaluated variables, neither DCC or p27Kip1 was found to be a predictor of survival (risk ratio for DCC, 0.98; 95% confidence interval, 0.66-1.56; P=0.92; risk ratio for p27Kip1, 0.87; 95% confidence interval, 0.58-1.29; P=0.49). The present study demonstrated that the expression of neither DCC nor p27Kip1 was predictive in poor survival outcome in patients with stage II or III colorectal cancer.

<h4>Background</h4>The serotonin transporter (5-HTT) and tryptophan hydroxylase (TPH) gene are important candidate genes for the psychiatric disorders. Many studies of patients with anxiety disorders have found abnormalities of serotonin metabolism and dysfunction of regulation in the transporter itself. In this study, we hypothesize that genetic variation in the 5-HTT and TPH gene may have an effect on the etiology of generalized anxiety disorder (GAD).<h4>Methods</h4>Using a polymerase chain reaction-based technique, the allele and genotype frequencies of three polymorphisms in the serotonin transporter gene (a deletion/insertion polymorphism in the transcriptional control region and a variable number of tandem repeats in intron 2) and TPH gene (A218C in intron 7) were analyzed in 138 patients with GAD and 90 healthy controls. These two groups were matched for ethnic and geographic origin.<h4>Results</h4>The frequencies of 5-HTT gene-linked functional polymorphic region (5-HTTLPR) SS (short/short) genotype were significantly higher in GAD patients than in control subjects (68% versus 49%, chi = 12.274, df = 2, P = 0.002), and the frequencies of S (short) allele observed in the GAD patients were higher than those in healthy subjects (79 versus 71%, chi = 4.063, df = 1, P = 0.044). The odds ratio for the SS genotype versus the other two genotypes was 2.33 (95% confidence interval, 1.29-3.86). Similarly, the odds ratio for the S allele versus L allele was 1.56 (95% confidence interval, 1.01-2.41). The genotypic and allelic distribution of 5-HTT VNTR and TPH A218C polymorphisms did not show statistically significant differences between patients and controls.<h4>Conclusion</h4>Our findings support that the presence of 5-HTTLPR-SS genotype may increase the risk of GAD.

<h4>Purpose of review</h4>The anemia of inflammation has been associated for nearly two decades with elevated cytokine levels, but the primary mediator of this condition was unknown. Recently hepcidin antimicrobial peptide has emerged as the hormone that links the type II acute phase response to iron handling and erythropoiesis.<h4>Recent findings</h4>Hepcidin antimicrobial peptide likely modulates iron transport from macrophages and enterocytes to red blood cell precursors as a consequence of its interaction with SLC40A1/ferroportin, the only known transporter that facilitates iron egress. Insights into the regulation of hepcidin antimicrobial peptide expression by known iron metabolic proteins such as HFE, hemojuvelin, and transferrin receptor 2 are expanding the understanding of the genetic circuitry that controls iron absorption and utilization.<h4>Summary</h4>Increasingly, experiments suggest the hepatocyte is not just the iron storage depot but is the 'command central' for the maintenance of iron homeostasis. It receives multiple signals related to iron balance and responds via transcriptional control of hepcidin antimicrobial peptide.

Hepcidin, the iron hormone, is produced by the liver in response to iron and inflammation. Its synthesis during inflammation is triggered by cytokines, but the details of iron activation are obscure. We tested the role of Kupffer cells and macrophages by studying iron-loaded or inflamed mice with selective inactivation of Kupffer cells or the in vitro effect of conditioned human macrophages on hepcidin expression. Hepcidin messenger RNA (mRNA) expression was studied by Northern blot and reverse transcriptase polymerase chain reaction analysis in mice that were treated with 40 mg/kg gadolinium (III) chloride (GdCl(3)) as a Kupffer cell inactivating agent and subjected to inflammatory challenges with either lipopolysaccharide (LPS) and turpentine or iron overload by iron-dextran administration. Similar analyses were performed in human hepatoma cells (HepG2) cultured with medium from LPS- or iron-conditioned macrophages from blood donors or patients with HFE-linked hereditary hemochromatosis (HH). In vivo, LPS and particularly turpentine stimulated hepcidin mRNA expression, and this effect was prevented by the inactivation of Kupffer cells. Also, iron overload markedly upregulated hepatic hepcidin mRNA, but this activity persisted in spite of Kupffer cell blockade. In vitro, the medium of LPS-treated normal or hemocromatotic macrophages turned on hepcidin expression. On the contrary, medium of iron-manipulated macrophages, regardless of their HFE status, did not affect hepcidin mRNA steady-state levels. In conclusion, Kupffer cells are required for the activation of hepcidin synthesis during inflammation, and HH inflamed macrophages are capable of mounting a normal response, eventually leading to hepcidin stimulation. However, both Kupffer cells and human macrophages are dispensable for the regulatory activity exerted by iron on hepatic hepcidin.

Hereditary hemochromatosis is a common disorder of iron metabolism most frequently associated with mutations in the HFE gene. Hereditary hemochromatosis may be caused by other less common genetic mutations including those in the ferroportin gene. Whereas hereditary hemochromatosis associated with HFE mutations is an autosomal recessive disorder, essentially all cases of hereditary hemochromatosis associated with ferroportin mutations follow an autosomal dominant pattern of inheritance, and most cases are notable for the lack of an elevated transferrin saturation and presence of iron deposition in Kupffer cells. This report describes the clinical and laboratory features of a family with hereditary hemochromatosis associated with a previously unrecognized ferroportin mutation (Cys326Ser). Three generations of the family are described. The disease in this family is notable for young age at onset, elevated transferrin saturation values, and hepatocyte iron deposition. The distinct molecular and clinical features reflect the heterogeneous nature of this disease.

<h4>Objective</h4>OX40, a member of the TNF receptor superfamily, and its ligand (OX40L) play crucial roles in induction and maintenance of integrated T cell immune response. Engagement of OX40L delivers a costimulatory signal to T cells. In this study, we investigated whether inoculation of OX40L-transfected EL4, a murine T cell lymphoma cell line, could induce anti-lymphoma immunity in mice.<h4>Materials and methods</h4>Female C57BL/6 mice were inoculated with 1 x 10(5) cells of parental EL4, OX40L-transfected EL4 (EL4-OX40L), or mock control vector-transfected EL4 (EL4-mock), and then the tumor size, overall survival, CTL activity of spleen cells, and the immunohistochemistry were compared.<h4>Results</h4>While both parental EL4 and EL4-mock grew rapidly, EL4-OX40L was rejected or grew slower than parental EL4 or EL4-mock. Pretreatment of mice with either anti-CD4 or anti-CD8 mAb accelerated the growth of EL4-OX40L, suggesting that both CD4+ and CD8+ T cells were involved in anti-lymphoma immunity. The immunohistochemical study revealed the infiltration of CD8+ T cells into the tumor of EL4-OX40L. In vitro CTL assay demonstrated that spleen cells of mice that had rejected EL4-OX40L had significant cytotoxic activity against parental EL4.<h4>Conclusion</h4>The gene transfer of OX40L into lymphoma cells is an eligible and efficient modality to induce anti-lymphoma immunity.

<h4>Aims</h4>To investigate the incidence of genetic aberrations in the DNA repair genes in a cohort of oesophageal cancers.<h4>Methods</h4>One hundred oesophagectomy samples of squamous cell carcinoma were studied. Normal and tumour DNA were isolated using a standard phenol/chloroform extraction procedure. Six recommended microsatellite loci with high informativity were analysed. The following markers were used: D2S123 (2p), D3S659 (3p), D3S1255 (3p), Bat 25 (4q), Bat 26 (2p), and Bat 40 (1p). The results were analysed using software attached to an automated DNA sequencer. The molecular data were then correlated with clinicopathological parameters.<h4>Results</h4>The incidence of microsatellite instability and loss of heterozygosity was very low. There was no significant correlation between the clinicopathological and molecular data. However, D2S123 genetic abnormalities were seen more frequently in both moderately and well differentiated tumours than in poorly differentiated tumours (p = 0.033). Follow up data were available for only 67 of the 100 patients. Fifty patients were alive and 17 patients had died.<h4>Conclusion</h4>Low frequencies of genetic aberrations in these mismatch repair loci are found in squamous carcinomas of the oesophagus from a high incidence area in South Africa.

<h4>Background</h4>Altered serotonergic (5-HT) neurotransmission is implicated in the antidepressant and anxiolytic properties of physical activity. In the current study, we investigated whether physical activity alters factors involved in the regulation of central 5-HT neural activity.<h4>Methods</h4>In situ hybridization was used to quantify levels of 5-HT transporter (5-HTT), 5-HT(1A), 5-HT(1B), and alpha(1b)-adrenergic receptor (alpha(1b) ADR) messenger ribonucleic acids (mRNAs) in the dorsal (DRN) and median raphe (MR) nuclei of male Fischer rats after either sedentary housing or 3 days, 3 weeks, or 6 weeks of wheel running.<h4>Results</h4>Wheel running produced a rapid and lasting reduction of 5-HT(1B) mRNA in the ventral DRN. Three weeks of wheel running decreased 5-HTT mRNA in the DRN and MR and increased alpha(1b) ADR mRNA in the DRN. After 6 weeks of wheel running, 5-HTT mRNA remained reduced, but alpha(1b) ADR mRNA returned to sedentary levels. Serotonin(1A) mRNA was increased in the MR and certain DRN subregions after 6 weeks only.<h4>Conclusions</h4>Data suggest that the central 5-HT system is sensitive to wheel running in a time-dependent manner. The observed changes in mRNA regulation in a subset of raphe nuclei might contribute to the stress resistance produced by wheel running and the antidepressant and anxiolytic effects of physical activity.

Using RT-PCR, in situ hybridization, Western blotting, and immunofluorescence, we have analyzed the expression of neuropilin 1 (Np1) in two models of spinal cord injury (spinal cord hemisection and dorsal column crush) and following dorsal root rhizotomy in adult rats. Our results show that Np1 RNA and protein are up-regulated in the spinal cord after all these lesions but remain unaltered in the adjacent dorsal root ganglia. In control animals, Np1 levels in the spinal cord are low and appear to be localized mainly in blood vessels, motoneurons, and in the superficial layers of the dorsal horn. After DCC and rhizotomy, Np1 is expressed de novo around the injury and in the deafferentated dorsal horn, respectively, mainly by OX42-positive microglial cells. Both lesions affect the sensory projections, and interestingly a consistent increase of Np1 signal is additionally seen in the dorsal horn where these projections terminate. Unexpectedly, this increase is bilateral after unilateral rhizotomy.

We investigated the roles of netrin-1 and slit-2 in regulation and navigation of dopamine (DA) axon growth using an explant culture preparation of embryonic ventral midbrain (embryonic day 14) and a co-culture system. We found that netrin-1 protein significantly enhanced DA axonal outgrowth and promoted DA axonal outgrowth in a co-culture system of netrin-1 expressing cells. Such effects were mediated by the receptor DCC as demonstrated by antibody perturbation of the DCC receptor. In contrast, slit-2 inhibited DA neuron extensions and repelled DA neurite growth. These slit-2 activities required robo receptors since the reduced neurite extension was abolished by addition of excess robo receptors. In this system, netrin-1 stimulated and slit-2 opposed DA neurite growth. Such regulation may be important for DA axonal maintenance, regeneration, and phenotypic target recognition.

<h4>Background and aims</h4>It has been postulated that the HFE C282Y mutation (linked to human leukocyte antigen [HLA]-A3-B7 haplotype) is not only responsible for hereditary hemochromatosis; HLA class I alleles would also contribute to the disease pathogenesis. In addition, H63D mutation linked to HLA-A29-B44 would also be pathogenetic, particularly in the Mediterranean Basin and throughout the world. However, sporadic porphyria cutanea tarda (s-PCT) has also been linked to these HFE mutations. In the present work, we have studied HFE mutations and HLA genes to test these hypotheses.<h4>Methods</h4>C282Y and H63D mutations together with HLA genetic typing have been performed in Spanish hereditary hemochromatosis (n = 98) and PCT (n = 63) patients. The etiologic fraction (delta) has been used to determine the absolute strongest gene linkage to both diseases.<h4>Results</h4>The Spanish frequent HLA-A29-B44 haplotype is not significantly associated to the H63D mutations in hereditary hemochromatosis patients (although it is found more frequently in patients than in controls). Sporadic porphyria cutanea tarda patients do not show a significant association to H63D mutations, although it is also more frequent than in controls; however, compound H63D/C282Y subjects seem to bear a significant risk to s-PCT. Allelic C282Y (and not H63D) frequencies show a significant association with s-PCT.<h4>Conclusions</h4>The postulated additional risk of hereditary hemochromatosis given by class I HLA antigens may be secondary to the HFE gene linkage disequilibrium with certain class I alleles or to the existence of other neighboring genetic pathogenetic factors in our Spanish sample.

<h4>Objective</h4>The long-term effects of the use of 3,4-methylenedioxymethamphetamine (MDMA, or Ecstasy) in humans are controversial and unclear. The authors' goal was to assess the contribution of a functional polymorphism in the gene encoding serotonin transporter to changes in emotional processing following chronic Ecstasy use.<h4>Method</h4>They investigated Beck Depression Inventory scores and performance on the Affective Go/No-Go test, a computerized neuropsychological test sensitive to emotional processing, in Ecstasy users and comparison subjects, stratifying the results by serotonin transporter genotype.<h4>Results</h4>Ecstasy use was associated with higher Beck Depression Inventory score and abnormalities in the Affective Go/No-Go test in individuals with the ss and ls genotype but not those with the ll genotype.<h4>Conclusions</h4>Ecstasy users carrying the s allele, but not comparison subjects carrying the s allele, showed abnormal emotional processing. On the basis of a comparison with acute tryptophan depletion, the authors hypothesize that chronic Ecstasy use may cause long-term changes to the serotonin system, and that Ecstasy users carrying the s allele may be at particular risk for emotional dysfunction.

Estrogenic activity in environmental samples could be mediated through a wide variety of compounds and by various mechanisms. High-affinity compounds for estrogen receptors (ERs), such as natural or synthetic estrogens, as well as low-affinity compounds such as alkylphenols, phthalates, and polychlorinated biphenyls are present in water and sediment samples. Furthermore, compounds such as polycyclic aromatic hydrocarbons, which do not bind ERs, modulate estrogen activity by means of the aryl hydrocarbon receptor (AhR). In order to characterize compounds that mediate estrogenic activity in river water and sediment samples, we developed a tool based on the ER-alphaligand-binding domain, which permitted us to estimate contaminating estrogenic compound affinities. We designed a simple transactivation assay in which compounds of high affinity were captured by limited amounts of recombinant ER-alpha and whose capture led to a selective inhibition of transactivation. This approach allowed us to bring to light that water samples contain estrogenic compounds that display a high affinity for ERs but are present at low concentrations. In sediment samples, on the contrary, we showed that estrogenic compounds possess a low affinity and are present at high concentration. Finally, we used immobilized recombinant ER-alpha to separate ligands for ER and AhR that are present in river sediments. Immobilized ER-alpha, which does not retain dioxin-like compounds, enabled us to isolate and concentrate ER ligands to facilitate their further analysis.

Cytoplasmic Ca2+ elevation and changes in Rho GTPase activity are both known to mediate axon guidance by extracellular factors, but the causal relationship between these two events has been unclear. Here we show that direct elevation of cytoplasmic Ca2+ by extracellular application of a low concentration of ryanodine, which activated Ca2+ release from intracellular stores, upregulated Cdc42/Rac, but downregulated RhoA, in cultured cerebellar granule cells and human embryonic kidney 293T cells. Chemoattractive turning of the growth cone triggered by a gradient of ryanodine was blocked by overexpression of mutant forms of Cdc42 but not of RhoA in Xenopus spinal cord neurons. Furthermore, Ca2+-induced GTPase activity correlated with activation of protein kinase C and required a basal activity of Ca2+/calmodulin-dependent protein kinase II. Thus, Rho GTPases may mediate axon guidance by linking upstream Ca2+ signals triggered by guidance factors to downstream cytoskeletal rearrangements.

<h4>Background and objectives</h4>The low prevalence of the C282Y mutation of the HFE gene in Japan means that the genetic background of hemochromatosis in Japanese patients remains unclear. In a previous report, we showed that 3 patients from one family had an AVAQ 594-597 deletion of the transferrin receptor (TfR2) gene. This suggests that the TfR2 gene is involved in hemochromatosis in Japanese patients.<h4>Design and methods</h4>Nine patients clinically diagnosed with hemochromatosis were included in the study. DNA was extracted from whole blood samples collected with informed consent. The HFE and TfR2 genes were analyzed by sequencing the coding region and splicing sites.<h4>Results</h4>There were no mutations in the HFE gene. In the TfR2 gene, 2 novel mutations, 1469T->G (L490R) and 1665delC (V561X), were found in 2 patients. A known variation, 714C-> (I238M), was also found in the patient with L490R. The patient homozygous for both L490R and I238M presented with a mild manifestation of hemochromatosis at the age of 41 years. His liver was cirrhotic with parenchymal iron deposits and the result of a glucose tolerance test was compatible with diabetes mellitus. The patient homozygous for V561X had severe iron overload with the triad of cirrhosis, diabetes mellitus and skin pigmentation at the age of 58 years.<h4>Interpretation and conclusions</h4>Taken together with the previous report, 5 of our 12 patients with hemochromatosis manifesting in middle age had mutations in the TfR2 gene. Thus, TfR2 plays a role in the pathogenesis of hemochromatosis in Japan.

Of the nasopharyngeal cultures recovered from 942 day care center (DCC) attendees in Lisbon, Portugal, 591 (62%) yielded Streptococcus pneumoniae during a surveillance performed in February and March of 1999. Forty percent of the isolates were resistant to one or more antimicrobial agents. In particular, 2% were penicillin resistant and 20% had intermediate penicillin resistance. Multidrug resistance to macrolides, lincosamides, and tetracycline was the most frequent antibiotype (17% of all isolates). Serotyping and molecular typing by pulsed-field gel electrophoresis were performed for 202 out of 237 drug-resistant pneumococci (DRPn). The most frequent serotypes were 6B (26%), 14 (22%), 19F (16%), 23F (10%), and nontypeable (12%). The majority (67%) of the DRPn strains were representatives of nine international clones included in the Pneumococcal Molecular Epidemiology Network; eight of them had been detected in previous studies. Fourteen novel clones were identified, corresponding to 26% of the DRPn strains. The remaining 7% of the strains were local clones detected in our previous studies. Comparison with studies conducted since 1996 in Portuguese DCCs identified several trends: (i) the rate of DRPn frequency has fluctuated between 40 and 50%; (ii) the serotypes most frequently recovered have remained the same; (iii) nontypeable strains appear to be increasing in frequency; and (iv) a clone of serotype 33F emerged in 1999. Together, our observations highlight that the nasopharynxes of children in DCCs are a melting pot of successful DRPn clones that are important to study and monitor if we aim to gain a better understanding on the epidemiology of this pathogen.

In humans, 5-HT1A receptors are implicated in anxiety and depressive disorders and their treatment. However, the physiological and genetic factors controlling 5-HT1A receptor expression are undetermined in health and disease. In this study, the influence of two genetic factors on 5-HT1A receptor expression in the living human brain was assessed using the 5-HT1A-selective positron emission tomography (PET) ligand [11C]WAY 100635. After the genotyping of 140 healthy volunteers to study population frequencies of known single nucleotide polymorphisms (SNPs) in the 5-HT1A receptor gene, the influence of the common SNP [(-1018) C>G] on 5-HT1A receptor expression was examined in a group of 35 healthy individuals scanned with [11C]WAY 100635. In the PET group, we also studied the influence of a common variable number tandem repeat polymorphism [short (S) and long (L) alleles] of the 5-HT transporter (5-HTT) gene on 5-HT1A receptor density. Whereas, the 5-HT1A receptor genotype did not show any significant effects on [11C]WAY 100635 binding, 5-HT1A receptor binding potential values were lower in all brain regions in subjects with 5-HTTLPR short (SS or SL) genotypes than those with long (LL) genotypes. Although the PET groups are necessarily a small sample size for a genetic association study, our results demonstrate for the first time that a functional polymorphism in the 5-HTT gene, but not the 5-HT1A receptor gene, affects 5-HT1A receptor availability in man. The results may offer a plausible physiological mechanism underlying the association between 5-HTTLPR genotype, behavioral traits, and mood states.

Aceruloplasminemia is a rare autosomal recessive disorder. The lack of ceruloplasmin ferroxidase activity leads to parenchymal and reticuloendothelial iron overload, resulting in diabetes and progressive neurodegeneration with extrapyramidal disorders, ataxia, and dementia. We describe the MR imaging findings in a 40-year-old woman with hereditary aceruloplasminemia. The abnormal T2 hypointensities were more marked than those seen in any other condition, including degenerative disorders of the basal ganglia and Wilson disease, and they may be typical of aceruloplasminemia. To our knowledge, involvement of the cortex has not been described and suggests that brain iron accumulation in aceruloplasminemia is more extensive than previously believed, even in asymptomatic patients.

<h4>Background</h4>Inherited and environmental factors are implicated in the expression of porphyria cutanea tarda (PCT); the contribution of each factor depends on the population.<h4>Objective</h4>To provide a review of PCT cases diagnosed in Argentina over 24 years and evaluate the role of different precipitating factors in its pathogenesis. Methods Plasma and urinary porphyrin levels and erythrocyte uroporphyrinogen decarboxylase (URO-D) activity were determined. Potential precipitating factors were identified in each patient. Additional tests for hepatitis C virus (HCV) and hemochromatosis gene mutations were carried out.<h4>Results</h4>Several factors (mainly alcohol abuse in men and estrogen ingestion in women), alone or combined were identified in our patients. Prevalence of HCV infection was 35.2%. Inherited URO-D deficiency occurs in 25.0% of cases. H63D was the most common hemochromatosis gene mutation. High incidence of PCT associated with HIV infection was found.<h4>Conclusions</h4>PCT is multifactorial. Therefore, knowledge of all risk factors in each patient is important for the management of the disease.

Hyalinizing trabecular tumour (HTT) of the thyroid is a neoplasm of follicular derivation that shares several morphological similarities with papillary thyroid carcinoma (PTC). In this study, we investigated the prevalence of B-raf point mutations, RET/PTC rearrangements and N-ras point mutations in a large HTT series (28 samples). Twenty benign thyroid lesions and 10 PTC served as control cases. A high (47%) prevalence of RET/PTC rearrangements was found in HTT. By contrast, neither B-raf nor N-ras mutations were found in HTT. These findings suggest that, although RET/PTC, N-ras, and B-raf proteins may act along the same signalling cascade, the biological and morphological outcome of their oncogenic activation is not completely overlapping. Thus, in clinical practice, the detection of B-raf mutations in a thyroid follicular tumour may prove to be a valuable tool, supplementing histological examination, and allowing a differential diagnosis between PTC and HTT.

To seek for a candidate gene that would regulate tumour progression and metastasis in gastric cancer, we investigated gene expression profiles by using DNA microarray. Tumour tissue and adjacent normal tissue were obtained from 21 patients with gastric cancer and then examined for their gene expression profiles by the Gene Chip Human U95Av2 array, which includes 12 000 human genes and EST sequences. A total of 25 genes were upregulated and two genes were downregulated by at least four-fold in the tumour tissue. In a further analysis according to lymph node metastasis, the expressed levels of maspin, as well as carcinoembryonic antigen and nonspecific crossreacting antigen were significantly higher in tumours with lymph node metastasis than in those without it. Maspin expression in 85 gastric cancer patients was further investigated by using immunohistochemistry. Maspin expression was not observed in normal gastric epithelia without intestinal metaplasia. In contrast, maspin was expressed in 74 of 85 tumour tissues. There was a significant correlation between the incidence of maspin-positive tumour staining and lymph node metastasis. These results suggest that maspin has a potential role for tumour metastasis in gastric cancer.

The synthesis and pharmacology of a new series of 3-piperazinylmethyl-3a,4-dihydro-3H-[1]benzopyrano[4,3-c]isoxazoles that combine central serotonin (5-HT) reuptake inhibition with alpha(2)-adrenoceptor blocking activity is described as potential antidepressants. Four compounds were selected for further evaluation, and the combination of both activities was found to be stereoselective, residing mainly in one enantiomer. Reversal of the loss of righting induced by the alpha(2)-agonist medetomidine in rats confirmed the alpha(2)-adrenoceptor blocking activity in vivo and also demonstrated CNS penetration. Antagonism of p-chloroamphetamine (pCA)-induced excitation as well as blockade of the neuronal 5-HT depletion induced by p-CA administration in rats confirmed their ability to block the central 5-HTT, even after oral administration. Replacement of the oxygen atom at the 5-position of the tricyclic scaffold by a nitrogen or a carbon atom, as well as O-substitution at position 7, led also to active compounds, both in vitro and in vivo.

The hereditary hemochromatosis (HHC) gene, HFE on chromosome 6p21.3, encodes a protein involved in iron homeostasis. HFE mutations have low penetrance with a mild effect on serum iron levels. Animal, twin, and population studies have shown that carrier state for C282Y can increase iron levels. A proportion of heterozygotes show slightly elevated serum iron levels. Increased serum iron has been suggested to increase the risk for oxidative damage to DNA. Epidemiologic studies established a correlation between iron levels and cancer risk. Case-control studies have reported associations between HFE mutations C282Y/H63D and several cancers, some of which in interaction with the transferrin receptor gene TFRC or dietary iron intake. Increased cancer risk in C282Y carriers is likely due to higher iron levels in a multifactorial setting. In childhood acute lymphoblastic leukemia (ALL), there is an association of C282Y with a gender effect in two British populations. No association has been found in acute myeloblastic leukemia and Hodgkin disease in adults. The childhood leukemia association possibly results from elevated intracellular iron in lymphoid cells increasing the vulnerability to DNA damage at a critical time window during lymphoid cell development. Interactions of HFE with environmental and genetic factors, most of which are recognized, may play a role in modification of susceptibility to leukemia conferred by C282Y. Given the population frequency of C282Y and the connection between iron and cancer, clarification of the mechanism of HFE associations in leukemia and cancer will have strong implications in public health.

<h4>Aim</h4>Both development and progression of malignancies occur as a multistep process, requiring the activation of oncogenes and the inactivation of several tumor suppressor genes. The loss of heterozygosity (LOH) of tumor suppressor genes is believed to play a key role in carcinogenesis of colorectal cancer (CRC). In this study, we analyzed the LOH of seven loci on chromosome 22q13 in an effort to identify candidate tumor suppressor genes involved in colorectal carcinogenesis.<h4>Methods</h4>Matched tumor and normal tissue DNA were analyzed by PCR using fluorescence-labeled polymorphic microsatellite markers in 83 CRC patients. PCR products were eletrophoresed and LOH was determined by calculating the peak height acquired through computer software. Comparisons between LOH frequency and clinicopathological features were performed by chi2 test. P<0.05 was considered as statistical significance.<h4>Results</h4>The average LOH frequency of chromosome 22q13 was 28.38%. The highest LOH frequency was 64.71% on D22S1160 locus, and the lowest was 21.43% on D22S1141 locus. We detected two obvious minimal deletion regions: one between markers D22S1171 and D22S274, the other flanked by markers D22S1160 and D22S1149, each about 2.7 and 1.8 cm, respectively. None had lost in all informative loci. LOH frequency on D22S1171 is 50% on distal colon, which was higher than that on proximal one (P = 0.020); on D22S114 locus, none LOH event occurred in patients with liver metastasis, whilst 46.94% occurred in patients without liver metastasis (P = 0.008); on D22S1160 locus, LOH frequency in lymph nodes metastasis patients was 83.33%, which was much higher than 43.75% without lymph nodes metastasis ones (P = 0.016). There was no statistical significance between clinicopathological features and other loci.<h4>Conclusion</h4>This study provides evidence of two minimal deletion regions, which may harbor putative tumor suppressor genes related to progression and metastasis in sporadic colorectal carcinoma on chromosome 22q13.

Extracellular cues direct axon extension by regulating growth cone morphology. The netrin-1 receptor deleted in colorectal cancer (DCC) is required for commissural axon extension to the floor plate in the embryonic spinal cord. Here we demonstrate that challenging embryonic rat spinal commissural neurons with netrin-1, either in solution or as a substrate, causes DCC-dependent increases in growth cone surface area and filopodia number, which we term growth cone expansion. We provide evidence that DCC influences growth cone morphology by at least two mechanisms. First, DCC mediates an adhesive interaction with substrate-bound netrin-1. Second, netrin-1 binding to DCC recruits an intracellular signaling complex that directs the organization of actin. We show that netrin-1-induced growth cone expansion requires Cdc42 (cell division cycle 42), Rac1 (Ras-related C3 botulinum toxin substrate 1), Pak1 (p21-activated kinase), and N-WASP (neuronal Wiskott-Aldrich syndrome protein) and that the application of netrin-1 rapidly activates Cdc42, Rac1, and Pak1. Furthermore, netrin-1 recruits Cdc42, Rac1, Pak1, and N-WASP into a complex with the intracellular domain of DCC and Nck1. These findings suggest that DCC influences growth cone morphology by acting both as a transmembrane bridge that links extracellular netrin-1 to the actin cytoskeleton and as the core of a protein complex that directs the organization of actin.

Nonalcoholic fatty liver disease is emerging as the most common chronic liver condition in the Western world. It is associated with insulin resistance and frequently occurs with features of the metabolic syndrome. Disease presentation ranges from asymptomatic elevated liver enzyme levels to cirrhosis with complications of liver failure and hepatocellular carcinoma. Current treatment recommendations are limited to weight loss and exercise, although several promising medications are on the horizon. In this article we discuss the etiology, pathogenesis and diagnosis of nonalcoholic fatty liver disease as well as approaches to its management.

<h4>Purpose</h4>Transferrin saturation of more than 60% has been identified as a cancer risk factor. It is unclear whether dietary iron intake increases the risk of cancer among individuals with transferrin saturation of less than 60%. The purpose of this study was to examine the association of dietary iron intake and the risk of cancer among adults with increased transferrin saturation.<h4>Methods</h4>Analysis of a cohort study, the National Health and Nutrition Examination Survey I Epidemiologic Follow-Up Study, was performed. US adults (aged 25 to 74 years at baseline) were followed up from baseline in 1971-1974 to 1992 (N = 6,309).<h4>Results</h4>A total of 7.3% of the US population had a serum transferrin saturation of more than 45% at baseline. Intake of dietary iron was essentially uncorrelated with transferrin saturation (r = 0.04). Compared with individuals who had normal serum transferrin saturation and low dietary iron intake, individuals whose serum transferrin saturation was more than 45% and who had high dietary iron intake also had an increased adjusted relative risk of cancer (2.24; 95% confidence interval [CI], 1.02-4.89). Increased risk was not found for individuals with a transferrin saturation of more than 45% but a normal dietary iron intake (hazard ratio, 1.02; 95% CI, 0.69-1.49). Transferrin saturation levels could be set as low as 41%, and the individuals with high transferrin saturation and high dietary iron intake would still have an increased adjusted relative risk of cancer (hazard ratio, 2.00; 95% CI, 1.04-3.82).<h4>Conclusions</h4>Among persons with increased transferrin saturation, a daily intake of dietary iron more than 18 mg is associated with an increased risk of cancer. Future research might focus on the benefits of dietary changes in those individuals with increased serum transferrin saturation.

The stereotypical projection of retinal ganglion cell (RGC) axons to the optic disc has served as a good model system for studying axon guidance. By both in vitro and in vivo experiments, we show that a secreted molecule, Sonic hedgehog (Shh), may play a critical role in the process. It is expressed in a dynamic pattern in the ganglion cell layer with a relatively higher expression in the center of the retina. Through gel culture and stripe assays, we show that Shh has a dual effect on RGC axonal growth, acting as a positive factor at low concentrations and a negative factor at high concentrations. Results from time-lapse video microscopic and stripe assay experiments further suggest that the effects of Shh on axons are not likely attributable to indirect transcriptional regulation by Shh. Overexpression of Shh protein or inhibition of Shh function inside the retina resulted in a complete loss of centrally directed projection of RGC axons, suggesting that precise regulation of Shh level inside the retina is critical for the projection of RGC axons to the optic disc.

At age of 3.2 years routine blood analysis showed the presence of a beta-thalassemic trait with unexpected high level of serum iron and high transferrin saturation. Hematological follow-up confirmed the moderate degree of anemia and persisting high levels of iron indices throughout the years with a progressive increase of serum ferritin. At the age of 19 years the patient was diagnosed homozygous for HC63D HFE. The patient referred by us confirm the possibility of precocious alteration of iron indices in patients with heterozygosity for beta-thalassemia inherited together with HFE mutations. This observation suggests that any children with thalassemic trait with increased transferrin saturation and/or serum ferritin might be investigated for the presence of the hemocromatosis genes in order to detect the disease before any clinical manifestation and even before organ iron loading.

<h4>Objective</h4>To investigate the diagnostic value of coagulation factors in assessing the severity degree of liver cirrhosis caused by hepatitis B.<h4>Methods</h4>Fifty-eight patients with liver cirrhosis and twenty healthy persons as control were enrolled. Prothrombin time activity percentage (PTA), activated partial thromboplastin time, coagulation activity of factor II, V, VII, VIII, IX and X were detected with clotting assay. Antithrombin-III (AT-III) was detected with colorimetric assay. The biochemical markers were also detected.<h4>Results</h4>The differences of PTA, factor II, VII and AT-III among Child-Pugh A, B, C in patients with liver cirrhosis were statistically significant (P < 0.01). Through receiver operating characteristic curve analysis, when 64% and 50% were used as cut-off values for PTA and factor VII in diagnosing Child-Pugh B, the area under the curve (AUC) was 0.689 and 0.610, the sensitivity was 76.9% and 61.5%, the specificity was 62.2% and 55.6%; when 54% and 39% were used as cut-off values for PTA and factor VII in diagnosing Child-Pugh C, the AUC was 0.924 and 0.942, the sensitivity was 80.0% and 86.7%, the specificity was 88.4% and 90.7%. Stepwise linear regression was done between Child-Pugh grade and coagulation factors. PTA, cholinesterase (Che), total bilirubin (TBil), albumin (Alb), factor VII were included in regression equation, Y = 15.008 - 0.018 x PTA - 0.288 x Che + 0.264 x TBil - 0.988 x Alb - 0.034 x VII, R(2) = 0.871. Patients whose Y was less than 8 were classified as grade "a", between 8 - 10 as grade "b", more than 10 as grade "c", the diagnostic accuracy was 84.5%.<h4>Conclusion</h4>Coagulation factor VII may serve as a helpful marker in diagnosing the severity degree of liver cirrhosis.

Ca2+ influx into excitable cells is a prerequisite for neurotransmitter release and regulated exocytosis. Within the group of ten cloned voltage-gated Ca2+ channels, the Ca(v)2.3-containing E-type Ca2+ channels are involved in various physiological processes, such as neurotransmitter release and exocytosis together with other voltage-gated Ca2+ channels of the Ca(v)1, Ca(v)2 and Ca(v)3 subfamily. However, E-type Ca2+ channels also exhibit several subunit-specific features, most of which still remain poorly understood. Ca(v)2.3-containing R-type channels (here called 'E-type channels') are also located in presynaptic terminals and interact with some synaptic vesicle proteins, the so-called SNARE proteins, although lacking the classical synprint interaction site. E-type channels trigger exocytosis and are also involved in long-term potentiation. Recently, it was shown that the interaction of Ca(v)2.3 with the EF-hand motif containing protein EFHC1 is involved in the aetiology and pathogenesis of juvenile myoclonic epilepsy.

The hepatitis C virus (HCV) is a major cause of chronic liver disease worldwide, with approximately 170-200 million people infected. The HCV virus is transmitted by blood and blood products and such transmission occurs primarily through drug use by injection, sex with an infected partner and occupational exposure. The severity of the disease varies widely from mild chronic hepatitis to cirrhosis and hepatocellular carcinoma (HCC). Nowadays, the reference treatment is combination therapy of pegylated interferon and ribavirin, which is an inosine monophosphate dehydrogenase inhibitor and immunomodulator. Efficacy of treatment in our clinical trials is 87% in patients infected by HCV genotypes 2 or 3, whereas in patients infected by HCV genotype 1 response to treatment is 66%. The current combination treatment has significant side-effects and sometimes is poorly tolerated. HCV genotypes 2 or 3 can be treated with a lower dose of ribavirin and a shorter course of therapy, 24 weeks vs 48 weeks for patients with genotype 1. There is a growing consensus that acute control of HCV infection is associated with a vigorous intrahepatic antiviral CD4+ and CD8+ T-cell response, enhanced Th1 and natural killer activity. Pretreatment genotype and response to therapy measured at weeks 12 and 24 of treatment have been identified as key determinants in decisions about continuing treatment. Elevated serum ferritin levels and hepatic iron deposition as well as hepatic steatosis and high ALT levels with chronic hepatitis C are risk factors for HCC development. Heterozygosityfor the C282Y mutation in HFE contributes to iron accumulation and fibrosis progression in chronic hepatitis C. Ribavirin could cause dose-dependent reversible haemolytic anaemia, which can be managed with dose reductions or with administration of epoetin alpha at 40,000 IU once weekly without sacrificing the optimal dosing of ribavarin. Among patients who received ribavirin alone, serum ALT levels and necroinflammatory features of liver histology were improved, whereas symptoms, HCV RNA levels and hepatic fibrosis scores were not changed significantly from baseline. For HCV-HIV co-infected patients, treatment is given when blood CD4 counts are above 350/ml and before antiretroviral (ART) treatment is needed.

The Ca2+ channel alpha1B subunit is a pore-forming component capable of generating N-type Ca2+ channel activity. Although N-type Ca2+ channel plays a role in a variety of neuronal functions, alpha1B-deficient mice exhibit normal life span without apparent abnormalities of behavior, histology or plasma norepinephrine level, presumably owing to compensation by some other Ca2+ channel alpha1 or beta subunit. In this study, we studied the levels of alpha1A, alpha1C, alpha1D, C1E, beta1, beta2, beta3 and beta4 mRNAs in adrenal gland of alpha1B-deficient mice. The alpha1A mRNA in homozygous mice was expressed at higher level than in wild or heterozygous mice, but no difference in the expression levels of alpha1c, alpha1D, alpha1E, beta1, beta2, beta3 and beta4 was found among wild, heterozygous and homozygous mice. The protein level of alpha1A in homozygous mice was also expressed at higher level than in wild or heterozygous mice. To examine whether increased expression is induced by cis-regulatory element within 5'-upstream region of alpha1A gene, we examined lacZ expression in alpha1B-deficient x alpha1A6.3-lacZ mice (carrying a 6.3-kb 5'-upstream fragment of alpha1A gene fused to E. coli lacZ reporter gene), which express lacZ in medullar chromaffin cells, but not in cortex. The levels of lacZ expression in homozygous alpha1B-deficient x alpha1A6.3-lacZ mice were higher than in wild or heterozygous mice. Therefore, a possible explanation of the normal behavior and plasma norepinephrine level of alpha1B-deficient mice is that compensation by alpha1A subunit occurs and that 6.3-kb 5'-upstream region of alpha1A gene contains enhancer cis-element(s) for compensation in adrenal medulla chromaffin cells.

We now have firm evidence that the basic mechanism of chromosome segregation is similar among diverse eukaryotes as the same genes are employed. Even in prokaryotes, the very basic feature of chromosome segregation has similarities to that of eukaryotes. Many aspects of chromosome segregation are closely related to a cell cycle control that includes stage-specific protein modification and proteolysis. Destruction of mitotic cyclin and securin leads to mitotic exit and separase activation, respectively. Key players in chromosome segregation are SMC-containing cohesin and condensin, DNA topoisomerase II, APC/C ubiquitin ligase, securin-separase complex, aurora passengers, and kinetochore microtubule destabilizers or regulators. In addition, the formation of mitotic kinetochore and spindle apparatus is absolutely essential. The roles of principal players in basic chromosome segregation are discussed: most players have interphase as well as mitotic functions. A view on how the centromere/kinetochore is formed is described.

Although patients undergoing cardiac surgery often present with diverse comorbidities, those with coagulation derangements are especially challenging. The present report describes the management of a patient who presented with a Factor V Leiden mutation, lupus anticoagulant, and acquired activated protein C resistance. A 42-year-old female presented with acute shortness of breath and chest pain. She was otherwise healthy 1 month prior to admission when she presented with dysfunctional uterine bleeding, resulting in the transfusion of three units of packed red blood cells. Coagulation evaluation revealed that the patient had lupus anticoagulant, factor V Leiden mutation and an activated protein C resistance. The patient presented with an acute myocardial infarction and was found to have 90% stenosis of her left main coronary artery, moderate mitral and tricuspid regurgitation, and a left ventricular ejection fraction of 25%. An emergent off-pump coronary artery bypass procedure with placement of a vein graft to the left anterior descending artery was completed. Intraoperative thrombophilia was encountered as evidenced by both an elevated thromboelastograph coagulation index (+3.6) and an acquired antithrombin-III deficiency. Postoperatively, the patient was placed on low molecular weight heparin, but developed heparin-induced thrombocytopenia and was switched to a direct thrombin inhibitor, argatroban. The following case report describes the coagulation management of this patient from the time of admission to discharge 43 days later, and the unique challenges this combination of hemostatic defects present to the clinicians.

Ferroportin (SLC40A1) is an iron transporter postulated to play roles in intestinal iron absorption and cellular iron release. Hepcidin, a regulatory peptide, binds to ferroportin and causes it to be internalized and degraded. If ferroportin is the major cellular iron exporter, ineffective hepcidin function could explain manifestations of human hemochromatosis disorders. To investigate this, we inactivated the murine ferroportin (Fpn) gene globally and selectively. Embryonic lethality of Fpn(null/null) animals indicated that ferroportin is essential early in development. Rescue of embryonic lethality through selective inactivation of ferroportin in the embryo proper suggested that ferroportin has an important function in the extraembryonic visceral endoderm. Ferroportin-deficient animals accumulated iron in enterocytes, macrophages, and hepatocytes, consistent with a key role for ferroportin in those cell types. Intestine-specific inactivation of ferroportin confirmed that it is critical for intestinal iron absorption. These observations define the major sites of ferroportin activity and give insight into hemochromatosis.

<h4>Background</h4>Huntington's disease is an autosomal dominant progressive neurodegenerative disease associated with dramatic expansion of a polyglutamine sequence in exon 1 of the huntingtin protein htt that leads to cytoplasmic, and even nuclear aggregation of fibrils.<h4>Methods</h4>We have studied the in vitro fibril formation of mutant exon 1, and the shorter wild-type exon 1, with use of atomic force microscopy (AFM).<h4>Results</h4>Large aggregates are formed spontaneously after cleavage of the glutathione-S-transferase fusion protein of the mutant exon 1 protein. The AFM data showed that, unlike fibrils assembled by such proteins as amyloid beta-peptide and alpha-synuclein, htt forms fibrils with extensive branched morphologic features. Branching can be observed even at earlier stages of the htt self-assembly, but the effect is much more pronounced at late stages of aggregation. We also found that fusing of htt with green fluorescent protein does not change the branched-type morphologic features of the aggregates.<h4>Conclusions</h4>On the basis of the results obtained, we propose a model for htt fibrillization that explains branched morphologic features of the aggregates.

No abstract available.