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The dissociation equilibrium constant for heparin binding to antithrombin III (ATIII) is a measure of the cofactor's binding to and activation of the proteinase inhibitor, and its salt dependence indicates that ionic and non-ionic interactions contribute approximately 40 and approximately 60% of the binding free energy, respectively. We now report that phenylalanines 121 and 122 (Phe-121 and Phe-122) together contribute 43% of the total binding free energy and 77% of the energy of non-ionic binding interactions. The large contribution of these hydrophobic residues to the binding energy is mediated not by direct interactions with heparin, but indirectly, through contacts between their phenyl rings and the non-polar stems of positively charged heparin binding residues, whose terminal amino and guanidinium groups are thereby organized to form extensive and specific ionic and non-ionic contacts with the pentasaccharide. Investigation of the kinetics of heparin binding demonstrated that Phe-122 is critical for promoting a normal rate of conformational change and stabilizing AT*H, the high affinity-activated binary complex. Kinetic and structural considerations suggest that Phe-122 and Lys-114 act cooperatively through non-ionic interactions to promote P-helix formation and ATIII binding to the pentasaccharide. In summary, although hydrophobic residues Phe-122 and Phe-121 make minimal contact with the pentasaccharide, they play a critical role in heparin binding and activation of antithrombin by coordinating the P-helix-mediated conformational change and organizing an extensive network of ionic and non-ionic interactions between positively charged heparin binding site residues and the cofactor.

Protein kinase C (PKC) regulates activation of the Raf-1 signaling cascade by growth factors, but the mechanism by which this occurs has not been elucidated. Here we report that one mechanism involves dissociation of Raf kinase inhibitory protein (RKIP) from Raf-1. Classic and atypical but not novel PKC isoforms phosphorylate RKIP at serine 153 (Ser-153). RKIP Ser-153 phosphorylation by PKC either in vitro or in response to 12-O-tetradecanoylphorbol-13-acetate or epidermal growth factor causes release of RKIP from Raf-1, whereas mutant RKIP (S153V or S153E) remains bound. Increased expression of PKC can rescue inhibition of the mitogen-activated protein (MAP) kinase signaling cascade by wild-type but not mutant S153V RKIP. Taken together, these results constitute the first model showing how phosphorylation by PKC relieves a key inhibitor of the Raf/MAP kinase signaling cascade and may represent a general mechanism for the regulation of MAP kinase pathways.

Various aspects of the progression and prognosis of colorectal carcinoma have been investigated in numerous publications during recent years. An exact macroscopic and microscopic examination is still of basic importance but different factors of the molecular pathogenesis of colorectal carcinoma could be described by immunohistochemistry and molecular biology. Furthermore, they have been evaluated regarding their importance for the course of disease and prognosis and in particular, the different pathways of carcinogenesis and microsatellite instability were included. The detection of micrometastasis was investigated applying mostly molecular genetic methods. Numerous oncogenes, tumor suppressor genes and regulators of the cell cycle, markers of proliferation and apoptosis, cell adhesion antigens and angiogenetic factors were characterized with regard to their prognostic potential. In the future, so-called response predictors will presumably gain a certain relevance in the context of neoadjuvant (radiotherapy) chemotherapy. The present review summarizes these results and discusses the future clinical relevance.

Interaction of OX40 (CD134) on T cells with its ligand (OX40L) on antigen-presenting cells has been implicated in pathogenic T cell activation. This study was performed to explore the involvement of OX40/OX40L in the development of T cell-mediated chronic colitis. We evaluated both the preventive and therapeutic effects of neutralizing anti-OX40L MAb on the development of chronic colitis in SCID mice induced by adoptive transfer of CD4(+)CD45RB(high) T cells as an animal model of Crohn's disease. We also assessed the combination of anti-OX40L and anti-TNF-alpha MAbs to improve the therapeutic effect. Administration of anti-OX40L MAb markedly ameliorated the clinical and histopathological disease in preventive and therapeutic protocols. In vivo treatment with anti-OX40L MAb decreased CD4(+) T cell infiltration in the colon and suppressed IFN-gamma, IL-2, and TNF-alpha production by lamina propria CD4(+) T cells. The combination with anti-TNF-alpha MAb further improved the therapeutic effect by abolishing IFN-gamma, IL-2, and TNF-alpha production by lamina propria CD4(+) T cells. Our present results suggested a pivotal role of OX40/OX40L in the pathogenesis of T cell-mediated chronic colitis. The OX40L blockade, especially in combination with the TNF-alpha blockade, may be a promising strategy for therapeutic intervention of Crohn's disease.

OX40 (CD134) is expressed on activated T cells; its ligand, OX40 ligand (OX40L) is expressed on dendritic cells, B cells, and activated endothelial cells. To determine how OX40-OX40L interaction affects graft-versus-host disease (GVHD), we used antagonistic anti-OX40L monoclonal antibody (mAb) or OX40(-/-) donor or OX40L(-/-) recipient mice. Similar degrees of GVHD reduction were observed with each approach. Despite the fact that OX40 is up-regulated on both CD4(+) and CD8(+) T cells isolated during GVHD, the major effects of OX40 ligation were on CD4(+) and not CD8(+) T-cell-mediated alloresponses as assessed in both GVHD and engraftment model systems. GVHD inhibition by blockade of the OX40/OX40L pathway did not require CD28 signaling. Some studies have indicated OX40 is essential for inducing T-helper type 2 (Th2) responses. However, in vivo blockade of OX40-OX40L interactions reduced GVHD mortality induced by either signal transducer and activator of transcription-6(-/-) (Stat-6(-/-)) (Th2-defective) or Stat-4(-/-) (Th1-defective) major histocompatibility complex (MHC)-disparate splenocytes, indicating that the GVHD-ameliorating effects did not require Stat-4 or Stat-6 signaling. Although OX40L has been reported to be expressed on activated T cells, no effects on GVHD were observed when OX40L(-/-) versus OX40L(+/+) T cells were infused in different models. These data provide insights as to the mechanisms responsible for OX40/OX40L regulation of GVHD.

Pancreatic cancer ranks fifth as a cause of cancer-related death in the world with an overall 5-year survival rate of less than 1% and a median survival of less than a year after tumour detection. Most of these patients have already metastases at the time of diagnosis. The oncologic strategies such as chemotherapy, radiotherapy, antihormonal modalities or the systemic use of specific monoclonal antibodies have not achieved a significant improvement in the survival of pancreatic cancer patients. Recent studies suggest that alterations in molecular pathways, particularly in growth factor mediated mechanisms, that regulate cell proliferation and differentiation play a pivotal role in the pathogenesis of this cancer. The molecular knowledge regarding changes in the expression of growth factors in pancreatic cancer has the potential to improve diagnostic and therapeutic treatment strategies in the near future.

Interferon-gamma (IFN-gamma) inhibits the synthesis of the cartilage-specific extracellular matrix protein type II collagen, and suppresses the expression of the type II collagen gene ( COL2A1 ) at the transcriptional level. To further examine this mechanism, the responses of COL2A1 regulatory sequences to IFN-gamma and the role of components of the Janus kinase/signal transducer and activators of transcription (JAK/STAT) pathway were examined in the immortalized human chondrocyte cell line, C-28/I2. IFN-gamma inhibited the mRNA levels of COL2A1 and aggrecan, but not Sox9, L-Sox5 and Sox6, all of which were expressed by these cells as markers of the differentiated phenotype. IFN-gamma suppressed the expression of luciferase reporter constructs containing sequences of the COL2A1 promoter spanning -6368 to +125 bp in the absence and presence of the intronic enhancer and stimulated activity of the gamma-interferon-activated site (GAS) luciferase reporter vector, associated with induction of Stat1 alpha-binding activity in nuclear extracts. These responses to IFN-gamma were blocked by overexpression of the JAK inhibitor, JAK-binding protein (JAB), or reversed by dominant-negative Stat1 alpha Y701F containing a mutation at Tyr-701, the JAK phosphorylation site. IFN-gamma had no effect on COL2A1 promoter expression in Jak1 (U4A)-, Jak2 (gamma 2A)- and Stat1 alpha (U3A)-deficient cell lines. In the U3A cell line, the response to IFN-gamma was rescued by overexpression of Stat1 alpha, but not by either Stat1 alpha Y701F or Stat1 beta. Functional analysis using deletion constructs showed that the IFN-gamma response was retained in the COL2A1 core promoter region spanning -45 to +11 bp, containing the TATA-box and GC-rich sequences but no Stat1-binding elements. Inhibition of COL2A1 promoter activity by IFN-gamma persisted in the presence of multiple deletions within the -45/+11 bp region. Our results indicate that repression of COL2A1 gene transcription by IFN-gamma requires Jak1, Jak2 and Stat1 alpha and suggest that this response involves indirect interaction of activated Stat1 alpha with the general transcriptional machinery that drives constitutive COL2A1 expression.

A series of N,N-disubstituted piperazines were prepared and evaluated for binding to alpha4beta2(*) and alpha7(*) neuronal nicotinic acetylcholine receptors using rat striatum and whole brain membrane preparations, respectively. This series of compounds exhibited selectivity for alpha4beta2(*) nAChRs and did not interact with the alpha7(*) nAChRs subtype. The most potent analogues were compounds 8b and 8f (K(i)=32 microM). Thus, linking together a pyridine pi-system and a cyclic amine moiety via a piperazine ring affords compounds with low affinity, but good selectivity for alpha4beta2(*) nicotinic receptors.

Recently, DCC (Deleted in Colorectal Cancer) protein has been forwarded as a receptor for netrin. The Netrin/DCC complex is critical for axon guidance and cell migration. In the developing nervous system, netrin protein secreted by midline cells attracts commissural axons by activating the DCC receptor on growth cones. This attraction can be switched to repulsion or silenced completely, depending on the DCC binding partner. The potential suppressor function of DCC in prostate tumorigenesis, through a still unknown mechanism, prompted us to quantify the expression of several genes involved in this axon guidance pathway. The relative expression levels of DCC, NEO1, NTN1, NTN2L, NTN4, UNC5C, Slit1, Slit2, Slit3, Robo1 and Robo2 were simultaneous quantified in 48 tumors and 7 normal prostate tissues by using real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). A reduction in DCC, NEO1, NTN1 and NTN4 expression was observed in prostate tumors, while many of the same prostate tumors over-expressed either Slit genes or their receptors, Robo.

Ion channels formed by expanded polyglutamine tracts have been proposed to play an important role in the pathological processes leading to neurodegeneration in Huntington's disease and other CAG repeat diseases. We tested the capacity of a huntingtin fragment containing an expanded polyglutamine tract to form ion channels in two cell types. Whole cell current from Xenopus oocytes was recorded using two-electrode voltage-clamp technique, and whole cell current from CHO-K1 cells was recorded by patch-clamp technique. The fragment with an expanded polyglutamine sequence induced no change in the currents recorded in any of the two expression systems, indicating no changes in ion channel activity. The results therefore argue against the proposed hypothesis of expanded polyglutamines forming ion channels.

In a screen for Drosophila genes that interfere with transcriptional repression mediated by the Polycomb group of genes, we identified a dominant mutation affecting the Alhambra (Alh) gene, the fly homologue of the human AF10 gene. AF10 has been identified as a fusion partner of both MLL and CALM in infant leukemias. Both fusion proteins retain the leucine zipper domain of AF10 but not its PHD domain. We show here that, while the full-length ALH protein has no activity on Polycomb group-responsive elements (PREs), overexpression of the isolated ALH leucine zipper domain activates several PREs. Within the ALH full-length protein, the PHD domain inhibits the PRE deregulation mediated by the leucine zipper domain. This deregulation is conserved in the human AF10 leucine zipper domain, which confers the same activity on an oncogenic MLL-AF10 fusion protein expressed in Drosophila melanogaster. These data reveal new properties for the leucine zipper domain and thus might provide new clues to understanding the mechanisms by which AF10 fusion proteins in which the PHD domain is lost might trigger leukemias in humans.

Lactoferrin is a member of the transferrin family of iron-binding glycoproteins present in milk, mucosal secretions, and the secondary granules of neutrophils. While several physiological functions have been proposed for lactoferrin, including the regulation of intestinal iron uptake, the exact function of this protein in vivo remains to be established. To directly assess the physiological functions of lactoferrin, we have generated lactoferrin knockout (LFKO(-/-)) mice by homologous gene targeting. LFKO(-/-) mice are viable and fertile, develop normally, and display no overt abnormalities. A comparison of the iron status of suckling offspring from LFKO(-/-) intercrosses and from wild-type (WT) intercrosses showed that lactoferrin is not essential for iron delivery during the postnatal period. Further, analysis of adult mice on a basal or a high-iron diet revealed no differences in transferrin saturation or tissue iron stores between WT and LFKO(-/-) mice on either diet, although the serum iron levels were slightly elevated in LFKO-/- mice on the basal diet. Consistent with the relatively normal iron status, in situ hybridization analysis demonstrated that lactoferrin is not expressed in the postnatal or adult intestine. Collectively, these results support the conclusion that lactoferrin does not play a major role in the regulation of iron homeostasis.

Proteins of the Tol system play a key role in the maintenance of outer membrane integrity and cell morphology in gram-negative bacteria. In Pseudomonas putida, the seven genes, orf1, tolQ, tolR, tolA, tolB, oprL, and orf2, which encode the proteins of this complex, are clustered in a 5.8-kb region of chromosomal DNA. Analysis of polar mutations, reverse transcriptase PCR assays, and transcriptional fusion constructs with a promoterless lacZ gene revealed that the genes are arranged in two operons: orf1 tolQ tolR tolA tolB and oprL orf2. We were also able to find a transcript that was initiated at the orf1 promoter and covered the two operons in a single mRNA. On the basis of the OprL protein level, we surmised that this transcript contributed only about 10 to 15% of the total OprL protein. Primer extension analysis identified the oprL orf2 operon promoter within the tolB gene, and the -10 and -35 regions exhibited some similarity to those of sigma(70)-recognized promoters. The transcription start point of orf1 was located 91 bp upstream of the orf1 start codon, and the -10/-35 region also exhibited sigma(70) -10/-35 recognition sequences. The expression from both promoters in rich and minimal media was constitutive and was very little influenced by the growth phase or iron-deficient conditions. In addition, analyses of the beta-galactosidase activities of different translational fusion constructs revealed that translation of tolA and orf2 genes was dependent on the translation of their corresponding upstream genes (tolR and oprL, respectively).

Huntington's Disease belongs to the CAG repeat family of neurodegenerative diseases and is characterized by the presence of an expanded polyglutamine (polyQ) repeat in the huntingtin (htt) gene product. PolyQ-expanded htt accumulates within large aggregates that are found in various subcellular compartments, but are more often localized within the nucleus. It has been suggested that the sequestration of proteins essential to cell viability may be one mechanism that accounts for toxicity generated by polyQ-expanded proteins. Nuclear inclusions containing polyQ-expanded htt recruit the transcriptional cofactor, CREB-binding protein (CBP). PolyQ toxicity appears to involve alterations of gene transcription and reduced neuronal cell viability. In the HT22 hippocampal cell line, we find that toxicity within individual cells induced by polyQ-expanded htt, as revealed by a TUNEL assay, is associated with the localization of the mutant htt within either nuclear or perinuclear aggregates. However, in addition to CBP recruitment, we show here that CBP ubiquitylation and degradation can be selectively enhanced by polyQ-expanded htt. Thus, selected substrates may be directed to the ubiquitin/proteasome-dependent protein degradation pathway in response to polyQ-expanded htt within the nucleus.

Efficient T cell priming by GM-CSF and CD40 ligand double-transduced C26 murine colon carcinoma is not sufficient to cure metastases in a therapeutic setting. To determine whether a cellular vaccine that interacts directly with both APC and T cells in vivo might be superior, we generated C26 carcinoma cells transduced with the T cell costimulatory molecule OX40 ligand (OX40L) either alone (C26/OX40L) or together with GM-CSF (C26/GM/OX40L), which is known to activate APC. Mice injected with C26/OX40L cells displayed only a delay in tumor growth, while the C26/GM/OX40L tumor regressed in 85% of mice. Tumor rejection required granulocytes, CD4+, CD8+ T cells, and APC-mediated CD40-CD40 ligand cosignaling, but not IFN-gamma or IL-12 as shown using subset-depleted and knockout (KO) mice. CD40KO mice primed with C26/GM/OX40L cells failed to mount a CTL response, and T cells infiltrating the C26/GM/OX40L tumor were OX40 negative, suggesting an impairment in APC-T cell cross-talk in CD40KO mice. Indeed, CD4+ T cell-depleted mice failed to mount any CTL activity against the C26 tumor, while treatment with agonistic mAb to CD40, which acts on APC, bypassed the requirement for CD4+ T cells and restored CTL activation. C26/GM/OX40L cells cured 83% of mice bearing lung metastases, whereas C26/OX40L or C26/GM vaccination cured only 28 and 16% of mice, respectively. These results indicate the synergistic activity of OX40L and GM-CSF in a therapeutic setting.

In these studies, we examined the effects of OX40 ligand (OX40L) deficiency on the development of Th2 cells during the Th2 immune response to the intestinal nematode parasite Heligmosomoides polygyrus. Elevations in IL-4 production and total and Ag-specific serum IgE levels were partially inhibited during both the primary and memory immune responses to H. polygyrus in OX40L(-/-) mice. The host-protective memory response was compromised in OX40L(-/-) mice, as decreased worm expulsion and increased egg production were observed compared with H. polygyrus-inoculated OX40L(+/+) mice. To further examine the nature of the IL-4 defect during priming, adoptively transferred DO11.10 T cells were analyzed in the context of the H. polygyrus response. Although Ag-specific T cell IL-4 production was reduced in the OX40L(-/-) mice following immunization with OVA peptide plus H. polygyrus, Ag-specific T cell expansion, cell cycle progression, CXCR5 expression, and migration were comparable between OX40L(+/+) and OX40L(-/-) mice inoculated with OVA and H. polygyrus. These studies suggest an important role for OX40/OX40L interactions in specifically promoting IL-4 production, as well as associated IgE elevations, in Th2 responses to H. polygyrus. However, OX40L interactions were not required for serum IgG1 elevations, increases in germinal center formation, and Ag-specific Th2 cell expansion and migration to the B cell zone.

Pal and Cpl-1, two purified bacteriophage lytic enzymes, were tested for their in vitro activity, alone and in combination, against several serotypes of Streptococcus pneumoniae, including penicillin-resistant strains. The enzymes demonstrated synergism in their ability to cleave the bacterial peptidoglycan and thus may be more efficient for the prevention and elimination of pneumococcal colonization.

<h4>Background</h4>In recent years important information has accumulated on the genetic alterations present in colorectal tumors. However, thus far few studies have analyzed the impact of numerical abnormalities of chromosomes 17 and 18, which carry the p53 and DCC plus SHAD4/DPC4 genes involved in colorectal cancer, on the clinical and biological behaviors of the disease.<h4>Methods</h4>With the use of interphase fluorescence in situ hybridization (FISH), we analyzed the incidence of numerical abnormalities of chromosomes 17 and 18 in a series of malignant colorectal tumors and explored its potential association with clinicobiological behavior and the prognosis of the disease. For this purpose, 94 consecutive patients newly diagnosed with colorectal cancer were analyzed. In all cases, FISH analyses of the number of copies and nuclei of chromosomes 17 and 18 were performed in interphase nuclei with the use of double stainings. For all patients, information on age, sex, tumor size, Dukes' stage, tumor localization, DNA ploidy status, and the proportion of S-phase tumor cells was recorded. Median follow-up was 38 months.<h4>Results</h4>Numerical abnormalities of chromosomes 17 and 18 were present in most patients with colorectal cancer (57% and 52%, respectively). Gains of chromosome 17 and monosomy 18 were found in 51% and 29% of cases, respectively, and they were the most frequent individual abnormalities for each chromosome. The simultaneous analysis of the number of copies of both chromosomes in the same cell showed that, in most cases displaying numerical abnormalities for these chromosomes, two or more different tumor cell clones were present. From a clinical point of view, numerical abnormalities of chromosome 17, especially monosomy 17, were associated with a significantly higher incidence of rectal tumors (P = 0.001) and Dukes' stage D (P = 0.02) and a lower median of disease-free survival among patients who underwent curative surgery (P = 0.05), as compared with diploid cases. In addition, cases with an altered number of copies of chromosome 17 showed a higher incidence of DNA aneuploidy (P = 0.0001) and a greater proportion of S-phase cells (P = 0.001) by flow cytometry. In contrast, no clear association was found between the presence of numerical abnormalities of chromosome 18 and clinicobiological disease characteristics, except for a higher incidence of DNA aneuploidy by flow cytometry (P = 0.001) and a lower median of disease-free survival (P = 0.06). Multivariate analysis showed that numerical abnormalities of chromosome 17, but not of chromosome 18, are an independent prognostic factor for predicting disease-free survival in patients with colorectal cancer.<h4>Conclusions</h4>Numerical abnormalities of chromosomes 17 and 18 were relatively common findings in patients with colorectal cancer, with chromosome 17 being associated with a higher incidence of tumors localized to the rectum and a worse clinical outcome. Cytometry Part B (Clin. Cytometry) 51B:14-20, 2003.

During primary infection, varicella-zoster virus (VZV) is spread via lymphocytes to skin, where it induces a rash and establishes latency in sensory ganglia. A live, attenuated varicella vaccine (vOka) was generated by using the VZV Oka strain (pOka), but the molecular basis for vOka attenuation remains unknown. Little is known concerning the effects of wild-type or attenuated VZV on cellular gene regulation in the host cells that are critical for pathogenesis. In this study, transcriptional profiles of primary human T cells and fibroblasts infected with VZV in cell culture were determined by using 40,000-spot human cDNA microarrays. Cellular gene transcription in human skin xenografts in SCID mice that were infected with VZV in vivo was also evaluated. The profiles of cellular gene transcripts that were induced or inhibited in infected human foreskin fibroblasts (HFFs), T cells, and skin in response to pOka and vOka infection were similar. However, significant alterations in cellular gene regulation were observed among the three differentiated human cell types that were examined, suggesting specific differences in the biological consequences of VZV infection related to the target cell. Changes in cellular gene transcription detected by microarray analysis were confirmed for selected genes by quantitative real-time reverse transcription-PCR analysis of VZV-infected cells. Interestingly, the transcription of caspase 8 was found to be decreased in infected T cells but not in HFFs or skin, which may signify a tissue-specific antiapoptosis mechanism. The use of microarrays to demonstrate differences in effects on host cell genes in primary, biologically relevant cell types provides background information for experiments to link these various response phenotypes with mechanisms of VZV pathogenesis that are important for the natural course of human infection.

A general, modular strategy for the first completely stereoselective synthesis of defined heparin oligosaccharides is described. Six monosaccharide building blocks (four differentially protected glucosamines, one glucuronic and one iduronic acid) were utilized to prepare di- and trisaccharide modules in a fully selective fashion. Installation of the alpha-glucosamine linkage was controlled by placing a conformational constraint on the uronic acid glycosyl acceptors thereby establishing a new concept for stereochemical control. Combination of disaccharide modules to form trans-uronic acid linkages was completely selective by virtue of C2 participating groups. Coupling reactions between disaccharide modules exhibited sequence dependence. While the union of many glucosamine uronic acid disaccharide modules did not meet any problems, certain sequences proved not accessible. Elaboration of glucosamine uronic acid disaccharide building blocks to trisaccharide modules by addition of either one additional glucosamine or uronic acid allowed for stereoselective access to oligosaccharides as demonstrated on the example of a hexasaccharide resembling the ATIII-binding sequence. Final deprotection and sulfation yielded the fully synthetic heparin oligosaccharides.

<h4>Aim</h4>To characterize the gene expression profiles in different stages of carcinogenesis of esophageal epithelium.<h4>Methods</h4>A microarray containing 588 cancer related genes was employed to study the gene expression profile at different stages of esophageal squamous cell carcinoma including basal cell hyperplasia, high-grade dysplasia, carcinoma in situ, early and late cancer. Principle component analysis was performed to search the genes which were important in carcinogenesis.<h4>Results</h4>More than 100 genes were up or down regulated in esophageal epithelial cells during the stages of basal cell hyperplasia, high-grade dysplasia, carcinoma in situ, early and late cancer. Principle component analysis identified a set of genes which may play important roles in the tumor development. Comparison of expression profiles between these stages showed that some genes, such as P160ROCK, JNK2, were activated and may play an important role in early stages of carcinogenesis.<h4>Conclusion</h4>These findings provided an esophageal cancer-specific and stage-specific expression profiles, showing that complex alterations of gene expression underlie the development of malignant phenotype of esophageal cancer cells.

<h4>Aim</h4>To clone genes that may predispose us to human gastric cancer and to analyze it's expression in gastric tissues.<h4>Methods</h4>Specimens of paired tumor, paratumor and normal gastric mucosa tissues collected from fifteen patients who suffered from stomach antrum adenocarcinoma were used for analysis. Seven out of the fifteen cases were first studied by fluorescent differential display reverse transcription polymerase chain reaction (DDTR-PCR) analysis. The differentially expressed bands of interest were cloned, analyzed by Northern blot, sequencing and RT-PCR. Through BLAST, the sequencing results were compared with GenBank database for homology analysis. In situ hybridization with DIG-labeled cRNA probes was used to analyze the expression of interesting cDNA bands in paraffin embedded paired normal gastric mucosa and cancer tissues isolated from 30 gastric adenocarcinoma patients.<h4>Results</h4>DDRT-PCR showed that one of the interesting cDNA bands, which was named W2, expressed much higher in all seven tested tumor and paratumor samples than in their normal counterparts, it was sub-cloned into a pGEM-T Easy vector. Two subclones were subsequently obtained. One of the subclone, GCRG224, was studied further. The sequencing result showed that GCRG224 consisted of 1 159 base pairs and had one open reading frame (ORF). It located at human chromosome 11q14. No homologue was found in GenBank database with GCRG224-ORF. This nucleotide sequence data were submitted to GenBank with accession No. AF438406. RT-PCR showed that GCRG224 expressed higher in 11/15 gastric cancer tissues than in non-tumor tissues. However, the result of Northern blot analysis showed a higher GCRG224 expression in the non-tumor tissue than in the tumor one. Human multiple tissue Northern blot analysis revealed that GCRG224 also expressed in human normal colon tissue, and peripheral blood leukocyte. In situ hybridization analysis showed that only 5/30 adenocarcinoma, 3/18 dysplasia and 6/18 intestinal metaplasia showed higher GCRG224 expression level than the normal gastric glands. However, GCRG224 was over-expressed predominantly in 26/30 cases of normal mucosal epithelium.<h4>Conclusion</h4>A novel gene named GCRG224 was identified from human gastric mucosal tissue. It overexpressed in almost all gastric mucosal epithelium but only a small portion of cancer and precancerous leisions. The role of GCRG224 expression in gastric epithelium needs further study.

<h4>Aim</h4>To detect the expression of PTEN encoding product in normal mucosa, intestinal metaplasia (IM), dysplasia and carcinoma of the stomach, and to investigate its clinical implication in tumorigenesis and progression of gastric carcinoma.<h4>Methods</h4>Formalin-fixed paraffin embedded specimens from 184 cases of gastric carcinoma, their adjacent normal mucosa, IM and dysplasia were evaluated for PTEN protein expression by SABC immunohistochemistry. PTEN expression was compared with tumor stage, lymph node metastasis, Lauren's and WHO's histological classification of gastric carcinoma. Expression of VEGF was also detected in 60 cases of gastric carcinoma and its correlation with PTEN was concerned.<h4>Results</h4>The positive rates of PTEN protein were 100 %(102/102), 98.5 %(65/66), 66.7 % (4/6) and 47.8 %(88/184) in normal mucosa, IM, dysplasia and carcinoma of the stomach, respectively. The positive rates in dysplasia and carcinoma were lower than in normal mucosa and IM (P<0.01). Advanced gastric cancers expressed less frequent PTEN than early gastric cancer (42.9 % vs 67.6 %, P<0.01). The positive rate of PTEN protein was lower in gastric cancer with than without lymph node metastasis (40.3 % vs 63.3 %, P<0.01). PTEN was less expressed in diffuse-type than in intestinal-type gastric cancer (41.5 % vs 57.8 %, P<0.05). Signet ring cell carcinoma showed the expression of PTEN at the lowest level (25.0 %, 7/28); less than well and moderately differentiated ones (P<0.01). Expression of PTEN was not correlated with expression of VEGF (P>0.05).<h4>Conclusion</h4>Loss or reduced expression of PTEN protein occures commonly in tumorigenesis and progression of gastric carcinoma. It is suggested that PTEN can be an objective marker for pathologically biological behaviors of gastric carcinoma.

<h4>Aim</h4>There is increasing evidence that alcohol-induced liver damage may be associated with increased oxidative stress. We aimed to investigate free-radical scavenger effect of n-acetylcysteine in rats intragastrically fed with ethanol.<h4>Methods</h4>Twenty-four rats divided into three groups were fed with ethanol (6 g/kg/day, Group 1), ethanol and n-acetylcysteine (1 g/kg, Group 2), or isocaloric dextrose (control group, Group 3) for 4 weeks. Then animals were sacrificed under ether anesthesia, intracardiac blood and liver tissues were obtained. Measurements were performed both in serum and in homogenized liver tissues. Malondialdehyde (MDA) level was measured by TBARS method. Glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) levels were studied by commercial kits. Kruskal-Wallis test was used for statistical analysis.<h4>Results</h4>ALT and AST in Group 1 (154 U/L and 302 U/L, respectively) were higher than those in Group 2 (94 U/L and 155 U/L) and Group 3 (99 U/L and 168 U/L) (P=0.001 for both). Serum and tissue levels of MDA in Group 1 (1.84 nmol/mL and 96 nmol/100 mg-protein) were higher than Group 2 (0.91 nmol/mL and 64 nmol/100 mg-protein) and Group 3 (0.94 nmol/mL and 49 nmol/100 mg-protein) (P<0.001 for both). On the other hand, serum GSH-Px level in Group 1 (8.21 U/g-Hb) was lower than Group 2 (16 U/g-Hb) and Group 3 (16 U/g-Hb) (P<0.001). Serum and liver tissue levels of SOD in Group 1 (11 U/mL and 26 U/100 mg-protein) were lower than Group 2 (18 U/mL and 60 U/100 mg-protein) and Group 3 (20 U/mL and 60 U/100 mg-protein) (P<0.001 for both).<h4>Conclusion</h4>This study demonstrated that ethanol-induced liver damage is associated with oxidative stress, and co-administration of n-acetylcysteine attenuates this damage effectively in rat model.

<h4>Objective</h4>To determine if there is an association between radiographic osteoarthritis (OA) of the hand and the presence of hemochromatosis HFE gene mutations.<h4>Methods</h4>One hundred seventy-six patients with radiographic OA of the hand were randomly selected from an academic rheumatology practice. We measured serum transferrin saturation (TS) and ferritin levels, and genotyped for the presence of the 2 common HFE mutations, C282Y and H63D. The prevalences of HFE mutations in these patients were compared to those in a hemochromatosis screening study from the same primary care patient base.<h4>Results</h4>There was a significantly increased prevalence of the C282Y mutation in the OA population compared to the unselected controls (12.5 vs 7.8%; p = 0.029). The prevalence of C282Y in OA was higher among older patients: 15.75% in the group older than 65 years versus 4.08% in the younger group. The mean TS level was higher among OA patients who were heterozygous for C282Y compared to those who lacked both HFE mutations (35.75 vs 25.93%; p < 0.0001). This difference was also found in the general population.<h4>Conclusion</h4>This is the first report to show an increased risk of OA among individuals who are heterozygous for the C282Y HFE mutation. The increase in this mutation in patients older than 65 suggests that this is associated with a late onset subset of OA. If this association is substantiated by larger randomized controlled studies, it could have major therapeutic implications in the development of specific therapy directed at individuals heterozygous for C282Y HFE mutation.

Arthropathy is a leading clinical manifestation of hereditary hemochromatosis (HH), but involvement of the ankle and hindfoot joints is rare. We describe 3 male patients who presented with symmetrical pain and swelling of the ankles. Radiographs and magnetic resonance imaging showed severe osteoarthritic degenerative changes with a radiological triad of joint space narrowing, subchondral sclerosis, and cyst formation. In all 3 cases a homozygous C282Y mutation in the HFE gene was detected and liver biopsies confirmed the diagnosis of HH. Other differential diagnoses could be excluded. Severe arthropathy of the ankle and hindfoot in comparatively young men can be a leading presentation of HH.

Adenylate/uridylate-rich element (ARE)-mediated mRNA turnover is an important regulatory component of gene expression for innate and specific immunity, in the hematopoietic system, in cellular growth regulation, and for many other cellular processes. This diversity is reflected in the distribution of AREs in the human genome, which we have established as a database of more than 900 ARE-containing genes that may utilize AREs as a means of controlling cellular mRNA levels. The p38 mitogen-activated protein kinase (MAP kinase) pathway has been implicated in regulating the stability of nine ARE-containing transcripts. Here we explored the entire spectrum of ARE-containing genes for p38-dependent regulation of ARE-mediated mRNA turnover with a custom cDNA array containing probes for 950 ARE mRNAs. The human monocytic cell line THP-1 treated with lipopolysaccharide (LPS) was used as a reproducible cellular model system that allowed us to precisely control the conditions of mRNA induction and decay in the absence and presence of the p38 inhibitor SB203580. This approach allowed us to establish an LPS-induced ARE mRNA expression profile in human monocytes and determine the half-lives of 470 AU-rich mRNAs. Most importantly, we identified 42 AU-rich genes, previously unrecognized, that show p38-dependent mRNA stabilization. In addition to a number of cytokines, several interesting novel AU-rich transcripts likely to play a role in macrophage activation by LPS exhibited p38-dependent transcript stabilization, including macrophage-specific colony-stimulating factor 1, carbonic anhydrase 2, Bcl2, Bcl2-like 2, and nuclear factor erythroid 2-like 2. Finally, the identification of the p38-dependent upstream activator MAP kinase kinase 6 as a member of this group identifies a positive feedback loop regulating macrophage signaling via p38 MAP kinase-dependent transcript stabilization.

Probing a cerebellar expression library with TrAb sera from patients with Hodgkin's disease and paraneoplastic cerebellar degeneration resulted in the isolation of MAZ (myc-associated zinc-finger protein). Eleven of 19 TrAb sera and 16 of 131 controls reacted with MAZ, indicating a significant, although not specific, association between Tr and MAZ immunities (p < 0.001). Interestingly, 9 of 16 positive control patients also had cerebellar dysfunction. Purified MAZ antibodies reacted with Purkinje cells. In neuronal cells, MAZ interacts with DCC (Deleted in Colorectal Cancer product), the receptor for netrin-1, a neuronal survival factor. These findings suggest epitope spreading between the Tr antigen and the MAZ-DCC complex and offer a possible model of immune-mediated cerebellar disease.

CAS/CSE1L is the human homologue of the yeast gene CSE1. It was first cloned while searching for genes that rendered breast cancer cells resistant towards toxin induced apoptosis. Since depletion of CSE1 leads to cell-cycle arrest, CAS is thought to be involved in proliferation. CAS functions in the mitotic spindle checkpoint. CAS is located on chromosome 20q13, a locus often amplified in cancers of various origin, e.g. colonic or breast cancer. Since genetic instability is a hallmark of cancer, amplification or over expression of the CAS gene might interfere with or override its role in the mitotic spindle checkpoint. CAS is also implicated in the nuclear to cytoplasmic reshuffling of importin alpha, which itself is necessary for the nuclear transport of several proliferation activating proteins, transcription factors, oncogene and tumor suppressor gene products such as p53 and BRCA1. Inhibition of MEK1 mediated phosphorylation has been shown to enhance paclitaxel (Taxol) induced apoptosis in breast, ovarian, and lung tumor cell lines in-vitro. Since CAS is also phosphorylated (activated) by MEK1, and since the anti-cancer drug Taxol alters the microtubule assembly and activates pro-apoptotic signaling pathways, altering the activity/phosphorylation status of CAS via MEK1 inhibition may present a potential strategy in experimental cancer therapy.

<h4>Background</h4>Although inherited thrombophilias are more common in patients with venous thromboembolism, their influence on the development of myocardial infarction (MI) requires clarification.<h4>Methods and results</h4>To determine whether there are increased frequencies of mutations/polymorphisms in 14 genes potentially causing thrombophilia in patients with no flow-limiting stenoses after MI compared with patients with > or =1 flow-limiting stenosis of >50%, we studied 395 patients (60 with no flow-limiting stenosis) who underwent angiography at approximately 1 month. The mutations/polymorphisms studied included Factor V Leiden, prothrombin variant G20210A, beta-fibrinogen 448 (G/A), endothelial protein C receptor (23-base pair insertion), methyl tetrahydrofolate reductase 677 (C/T), platelet glycoprotein IIIa PlA1/A2, plasminogen activator inhibitor-1 4G/5G, angiotensin II type 1 receptor (A/C), hemochromatosis gene 282 (G/A), nitric oxide synthase (NOS) (3 forms: eNOS, eNOS3, eNOS4), p22 phox of NADPH oxidase C242T, and angiotensin-converting enzyme insertion/deletion polymorphism. The frequencies of Factor V Leiden and the beta-fibrinogen 448 A allele were higher in patients with no flow-limiting stenosis than in patients with > or =1 stenosis (11.7% vs 3.6%, odds ratio [OR] 3.6, 95% CI 1.3-9.4, P =.015; and 42% vs 27%, OR 2.0, 95% CI 1.1-3.5, P =.018, respectively), and there was a trend toward an increased frequency of prothrombin variant G20210A (6.7% vs 2.1%, OR 3.4, 95% CI 0.95-11.8, P =.069). However, in patients with no flow-limiting stenosis after MI the frequencies of the other gene mutations/polymorphisms were not increased. Also, there were no significant interactions between any of these 14 mutation/polymorphisms, major cardiovascular risk factors, and the absence of any flow-limiting stenosis, except for Factor V Leiden and hypertension (OR 6.34, 95% CI 2.67-100, P =.004).<h4>Conclusions</h4>Patients with no flow-limiting stenosis after MI had increased frequencies of 2 inherited thrombophilias (Factor V Leiden and beta-fibrinogen 448 A allele), and there was a trend toward an increased frequency of prothrombin variant G20210A compared with patients with > or =1 stenosis. These data suggest that polymorphisms/mutations in some gene products influencing coagulation may influence the pathogenesis of MI.

Spontaneously occurring urinary tract infection (UTI) in dogs was exploited as an experiment of nature to gain insights into UTI pathogenesis in humans. Concurrent urinary and rectal Escherichia coli isolates from 37 dogs with UTI were compared with respect to phylogenetic background, O antigens, and extended virulence genotype. In 54% of the UTI episodes, the dog's urinary and rectal isolates represented the same strain. Urinary isolates differed dramatically from rectal-only isolates in that they derived predominantly from E. coli phylogenetic group B2, expressed typical (human) UTI-associated O antigens, and possessed many virulence-associated genes, most notably pap elements (P fimbriae), papG (adhesin) allele III, sfa/foc and sfaS (S fimbriae), hly (hemolysin), fyuA (yersiniabactin), iroN (siderophore), and ompT (outer membrane protease T). The 20 urinary isolates that corresponded with the host's predominant rectal strain were no less virulent according to the markers analyzed than were the 17 urinary isolates that differed from the host's predominant rectal strain. These findings suggest that UTI pathogenesis is similar in dogs and humans, provide added support for the special-pathogenicity over the prevalence hypothesis of UTI pathogenesis, and identify numerous specific virulence-associated factors as significant correlates of urovirulence.

No abstract available.

The SNP Consortium website (http://snp.cshl.org) has undergone many changes since its initial conception three years ago. The database back end has been changed from the venerable ACeDB to the more scalable MySQL engine. Users can access the data via gene or single nucleotide polymorphism (SNP) keyword searches and browse or dump SNP data to textfiles. A graphical genome browsing interface shows SNPs mapped onto the genome assembly in the context of externally available gene predictions and other features. SNP allele frequency and genotype data are available via FTP-download and on individual SNP report web pages. SNP linkage maps are available for download and for browsing in a comparative map viewer. All software components of the data coordinating center (DCC) website (http://snp.cshl.org) are open source.

The noncoding RNAs database is a collection of currently available sequence data on RNAs, which have no protein-coding capacity and have been implicated in regulation of cellular processes. The RNAs included in the database form very heterogenous group of molecules that act on different levels of information transmission in the cell. It includes RNAs acting on the level of chromatin structure, transcriptional and translational regulation of gene expression, modulation of protein function and regulation of subcellular distribution of RNAs and proteins. Those RNAs, with potential regulatory functions have been identified in prokaryotic, animal and plant cells. The database can be accessed at http://biobases.ibch.poznan.pl/ncRNA/.

Netrins promote axon outgrowth and turning through DCC/UNC-40 receptors. To characterize Netrin signaling, we generated a gain-of-function UNC-40 molecule, MYR::UNC-40. MYR::UNC-40 causes axon guidance defects, excess axon branching, and excessive axon and cell body outgrowth. These defects are suppressed by loss-of-function mutations in ced-10 (a Rac GTPase), unc-34 (an Enabled homolog), and unc-115 (a putative actin binding protein). ced-10, unc-34, and unc-115 also function in endogenous unc-40 signaling. Our results indicate that Enabled functions in axonal attraction as well as axon repulsion. UNC-40 has two conserved cytoplasmic motifs that mediate distinct downstream pathways: CED-10, UNC-115, and the UNC-40 P2 motif act in one pathway, and UNC-34 and the UNC-40 P1 motif act in the other. Thus, UNC-40 might act as a scaffold to deliver several independent signals to the actin cytoskeleton.

Stage I non-small cell carcinoma (NSCLC) of the lung is typically treated with surgery alone, but with a 30 to 40% recurrence rate. Prognostic factors to stratify these patients into high- and low-risk groups would be of significant clinical value, but published data are conflicting. We studied 39 Stage I NSCLC treated with resection alone, followed for a minimum of 5 years, and divided into recurrent (RC) and non-recurrent (NRC) groups (n = 12 and 27, respectively). Allelic imbalance (loss of heterozygosity, LOH) involving genomic regions containing L-myc (1p32), hOGG1 (3p26), APC/MCC (5q21), c-fms (5q33.3), p53 (17p13), and DCC (18q21), and point mutational change in K-ras-2 (12p12) were studied by PCR-based microsatellite analysis and DNA sequencing. Mutations in k-ras-2 were seen in 25% and 19% of RC and NRC tumors, respectively, most frequently in adenocarcinomas. LOH in the RC and NRC respectively were 50% and 37% for L-myc, 60% and 33% for hOGG1, 60% and 50% for APC, 38% and 35% for c-fms, 78% and 75% for p53, and 17% and 45% for DCC. No statistical significance was seen comparing any of the allelic alterations with recurrence. LOH for hOGG1 and L-myc were more commonly seen in squamous cell carcinomas. Stage I NSCLC are genetically heterogeneous with respect to mutation acquisition. The approach of investigating a panel of genes for alterations can be applied to any given tumor type, and provides information on patterns of mutations/LOH that can help us better understand the molecular biology of tumorigenesis.

Cortical and striatal perinuclear cytoplasmic aggregates and intranuclear inclusions of mutant huntingtin are neuropathological hallmarks of Huntington disease (HD). Although the mechanisms involved in the formation of these aggregates are unclear, a recent hypothesis implicates cross-linking of mutant huntingtin protein into aggregates by transglutaminase. This study explores the hypothesis that transglutaminase catalyzes cross-linking of huntingtin into intranuclear inclusions. Using immunofluorescence and confocal microscopy we demonstrate 99% colocalization of transglutaminase-catalyzed epsilon-(gamma-glutamyl) lysine covalent cross-links with nuclear aggregates of huntingtin protein in the frontal cortex of postmortem HD brain tissue. Furthermore, the transglutaminase 2 isoform colocalizes with both huntingtin protein and epsilon-(gamma-glutamyl) lysine covalent cross-links in HD intranuclear inclusions. Transient transfection of N-terminally truncated huntingtin with an expanded glutamine domain (htt-N63-148Q-myc) with and without and transglutaminase 2 into HEK 293T cells resulted in an increase in cross-linked huntingtin in the insoluble formic acid-treated pellet in comparison to transfection of N-terminally truncated huntingtin with normal length glutamine domain (htt-N63-18Q-myc). Transfection with both htt-N63-148Q-myc and transglutaminase 2 resulted in high molecular weight huntingtin in the insoluble fraction. These data support the hypothesis that transglutaminase catalyzed cross-linking of mutant huntingtin is involved in the formation and/or stabilization of huntingtin protein aggregates in HD. Based on these and other studies, modulation of transglutaminase activity could be explored as a treatment for HD.

<h4>Background</h4>Iron is a pro-oxidant that may promote carcinogenesis. Mutations in the hemochromatosis (HFE) gene are associated with increased total body iron stores in some individuals. We assessed the risk of colon cancer among individuals with and without HFE gene mutations.<h4>Methods</h4>We performed a population-based, case-control study in North Carolina. Case patients with colon cancer and control subjects provided information on multiple environmental exposures, including total iron intake and nonsteroidal anti-inflammatory drug (NSAID) use. They also provided a venous blood sample, from which DNA was extracted, amplified, and subjected to diagnostic restriction enzyme mapping to detect two major HFE gene mutations, C282Y and H63D. Data were analyzed with Fisher's exact test and logistic regression. All statistical tests were two-sided.<h4>Results</h4>Thirteen hundred and eight subjects participated (475 case patients, 833 control subjects). The allele frequencies of the H63D and C282Y mutations were greater among case patients (0.11 and 0.046, respectively) than among control subjects (0.09 and 0.044, respectively; P =.14 and P =.85, respectively). When we controlled for age, race, sex, red meat consumption, NSAID use, and total iron intake, subjects with any HFE gene mutation were more likely to have colon cancer than subjects with no HFE gene mutations (adjusted odds ratio [OR] = 1.40, 95% confidence interval [CI] = 1.07 to 1.87). The magnitude of the effect was similar for both the H63D (adjusted OR = 1.44, 95% CI = 1.04 to 1.98) and C282Y mutations (adjusted OR = 1.39, 95% CI = 0.88 to 2.19). The risk of colon cancer associated with an HFE gene mutation was similar for those who did and did not have a family history of colon cancer. Among those with HFE mutations, cancer risk increased with increasing age and total iron intake.<h4>Conclusions</h4>HFE gene mutations are associated with an increased risk of colon cancer. Cancer risk is greatest in mutation carriers who are older or consume high quantities of iron.

Chemical mutagenesis in the mouse is a powerful approach for phenotype-driven genetics, but questions remain about the efficiency with which new mutations ascertained by their phenotype can be localized and identified, and that knowledge applied to a specific biological problem. During a global screen for dominant phenotypes in about 30,000 animals, a novel class of pigmentation mutants were identified by dark skin (Dsk). We determined the genetic map location, homozygous phenotype, and histology of 10 new Dsk and 2 new dark coat (Dcc) mutations, and identified mutations in Agouti (Met1Leu, Dcc4), Sox18 (Leu220ter, Dcc1), Keratin 2e (Thr500Pro, Dsk2), and Egfr (Leu863Gln, Dsk5). Cutaneous effects of most Dsk mutations are limited to melanocytes, except for the Keratin 2e and Egfr mutations, in which hyperkeratosis and epidermal thickening precede epidermal melanocytosis by 3-6 wk. The Dsk2 mutation is likely to impair intermediate filament assembly, leading to cytolysis of suprabasal keratinocytes and secondary hyperkeratosis and melanocytosis. The Dsk5 mutation causes increased tyrosine kinase activity and a decrease in steady-state receptor levels in vivo. The Dsk mutations represent genes or map locations not implicated previously in pigmentation, and delineate a developmental pathway in which mutations can be classified on the basis of body region, microscopic site, and timing of pigment accumulation.

Antidepressants in the treatment of neuropathic pain are thought to partially exert their effect by inhibition of serotonin (5-HT) reuptake and thus activation of central antinociceptive pathways. Mice deficient for the 5-HT transporter (5-HTT-/- mice) are regarded as a model of lifelong treatment with a serotonin reuptake inhibitor. Here we investigated 5-HTT-/- mice and compared their pain-related behavior after a unilateral chronic constrictive sciatic nerve injury (CCI) with that of wild-type littermates. Wild-type mice reproducibly developed ipsilateral thermal hyperalgesia and mechanical allodynia after CCI. 5-HTT-/- mice did not develop thermal hyperalgesia, but showed bilateral mechanical allodynia after the nerve injury. 5-HT levels as measured with HPLC increased after CCI in the injured nerve in both genotypes and decreased in the lumbar spinal cord in wild-type mice. 5-HTT-/- mice had significantly lower 5-HT concentrations than wild-type mice in all tissues investigated. Thus, in 5-HTT-/- mice, reduced 5-HT levels in the injured peripheral nerves correlate with diminished behavioral signs of thermal hyperalgesia, a pain-related symptom caused by peripheral sensitization. In contrast, bilateral mechanical allodynia, a centrally mediated phenomenon, was associated with decreased spinal 5-HT concentrations in 5-HTT-/- mice and may possibly be caused by a lack of spinal inhibition.

We investigated the mechanism of CD4 T cell accumulation in B cell follicles after immunization. Follicular T cell numbers were correlated with the number of B cells, indicating B cell control of the niche that T cells occupy. Despite this, we found no role for B cells in the follicular migration of T cells. Instead, T cells are induced to migrate into B cell follicles entirely as a result of interaction with dendritic cells (DCs). Migration relies on CD40-dependent maturation of DCs, as it did not occur in CD40-deficient mice but was reconstituted with CD40(+) DCs. Restoration was not achieved by the activation of DCs with bacterial activators (e.g., lipopolysaccharide, CpG), but was by the injection of OX40L-huIgG1 fusion protein. Crucially, the up-regulation of OX40L (on antigen-presenting cells) and CXCR-5 (on T cells) are CD40-dependent events and we show that T cells do not migrate to follicles in immunized OX40-deficient mice.

Intracerebroventricular (icv) administration of betacytotoxics alters brain monoamine neurotransmission, without producing hyperglycemia. By means of in situ hybridization, we have investigated the expression of dopamine, noradrenaline and serotonin transporter (DAT, NAT, 5-HTT, respectively) mRNAs, in the brain of alloxan- and streptozotcin-icv treated rats. DAT1 mRNA expression is increased in 1-week alloxan-icv treated rats in arcuate nucleus (+51%) and ventral medial bundle /VMB/ (+32%), and decrease in VMB of 4-week alloxan- (-53%) and streptozotocin- (-9%) icv treated rats, respectively. NAT1 mRNA expression in locus coeruleus is decreased in 4-week alloxan-icv treated rats (-35%) and increased in A1 cell group of 1- (+19%) and 4- (+14%) week streptozotocin-icv treated rats. 5-HTT mRNA expression in dorsal raphe nucleus is increased in 1- (+13%) and 4- (+21%) week alloxan-icv treated rats. Observed changes may suggest altered response to antidepressants in streptozotocin-icv treated rats, used as an animal model of sporadic Alzheimer's disease.

The phenotypic screening for genetic haemochromatosis (GH) relies upon the determination of transferrin saturation (TS). In large-scale screening programs, the time of blood sampling can be uneasy to control. We studied the circadian variations of TS at 08.00 hours, 12.00 hours, 18.00 hours and 00.00 hours in 46 C282Y homozygous patients (GH) and 47 non-GH patients (NH), to determine whether the time of blood sampling influenced the results of screening. In both groups, there were significant circadian variations in TS, with the highest values at 08.00 hours and the lowest at 00.00 hours. For any given time-point, TS was significantly higher in the GH group when compared with the NH group (P < 0.0001). For both groups, there was a significant decrease in TS between 08.00 hours and 00.00 hours (P < 0.0001) but this decrease was not as significant in GH when compared with NH patients (interaction P < 0.0073). Receiver operating characteristics (ROC) curves generated for TS at 08.00 hours, 12.00 hours, 18.00 hours and 00.00 hours, presented the same efficiency of diagnosis of GH, with TS threshold varying between 64% at 08.00 hours and 36% at 00.00 hours. In conclusion, for screening studies of C282Y homozygosity, determination of transferrin saturation may be performed at any time during the day.

Effects of DRD4 and 5-HTTLPR length polymorphisms have been reported on neonatal and infant temperament as well as adult personality traits. The 7-repeat form of the DRD4 III exon VNTR polymorphism has been associated with childhood ADHD, and recently we have reported its link with attachment disorganization in a nonclinical population of infants. Here, we report associations of these polymorphisms with infant temperament at 12 months of age. Maternal accounts of temperament and observed response to novelty were investigated for 90 infants, who were independently genotyped for the DRD4 III exon, and for 5-HTT-linked promoter region length polymorphisms. Maternal rating of temperament was not affected by these polymorphisms, but we found combined genotype effects for infants' observed responses to a novel, anxiety-provoking stimulus: the appearance of, and approach by, a stranger. Infants with at least one copy of both the 7-repeat DRD4 allele and the long variant of 5-HTTLPR (7(+), l/l&l/s) responded with significantly less anxiety than infants with other genotypes. However, infants with the 7-repeat DRD4 allele and homozygous for the short form of 5-HTTLPR (7(+), s/s) showed more anxiety and resistance to the stranger's initiation of interaction. These genotype effects were not redundant with the previously reported association between the 7-repeat DRD4 allele and disorganized attachment behavior. Although both temperament and attachment behavior were affected by the DRD4 repeat polymorphism, the effect on temperament measures was modified by the infants' 5-HTTLPR genotype.

Tumour angiogenesis and the levels of plasminogen activator inhibitor type 1 (PAI-1) are both informative prognostic markers in breast cancer. In cell cultures and in animal model systems, PAI-1 has a proangiogenic effect. To evaluate the interrelationship of angiogenesis and the PAI-1 level in breast cancer, we have evaluated the prognostic value of those factors in a total of 228 patients with primary, unilateral, invasive breast cancer, evaluated at a median follow-up time of 12 years. Microvessels were immunohistochemically stained by antibodies against CD34 and quantitated by the Chalkley counting technique. The levels of PAI-1 and its target proteinase uPA in tumour extracts were analysed by ELISA. The Chalkley count was not correlated with the levels of uPA or PAI-1. High values of uPA, PAI-1, and Chalkley count were all significantly correlated with a shorter recurrence-free survival and overall survival. In the multivariate analysis, the uPA level did not show independent prognostic impact for any of the analysed end points. In contrast, the risk of recurrence was independently and significantly predicted by both the PAI-1 level and the Chalkley count, with a hazard ratio (95% CI) of 1.6 (1.01-2.69) and 1.4 (1.02-1.81), respectively. For overall survival, the Chalkley count, but not PAI-1, was of significant independent prognostic value. The risk of death was 1.7 (1.30-2.15) for Chalkley counts in the upper tertile compared to the lower one. We conclude that the PAI-1 level and the Chalkley count are independent prognostic markers for recurrence-free survival in patients with primary breast cancer, suggesting that the prognostic impact of PAI-1 is not only based on its involvement in angiogenesis.

<h4>Background and objectives</h4>serotonin transporter (5-HTT) gene allelic variants were shown to be associated with Neuroticism and Harm Avoidance but the results were not replicated in other studies. The current investigation was undertaken in a further attempt to study the relationship between 5-HTT polymorphism and personality traits.<h4>Subjects and methods</h4>to evaluate a spectrum of personality traits, MMPI was administered to a sample including patients with affective disorders (n=114), patients with schizophrenia spectrum illnesses (n=110) and psychiatrically well controls (n=124). All groups were genotyped for VNTR-17 and functional insertion-deletion (5-HTTLPR) polymorphisms.<h4>Results</h4>an association was found between 5-HTTLPR polymorphism and scores on three MMPI scales: Psychopathic deviance, Paranoia and Schizophrenia in patients with affective disorders and S chizophrenia in normal subjects. Both affected and control individuals with 'ss' genotype exhibited lower scores on these scales.<h4>Conclusion</h4>we demonstrated that functional deletion/insertion allelic variation associated with decreased expression of serotonin transporter ('s' allele or 'ss' genotype) may restrict expression of schizoid traits in normal subjects and patients with affective disorders.

The H63D mutation in the hemochromatosis gene (HFE) has recently been considered as a risk factor in Alzheimer's disease (AD) with advancing age at onset of the disease, independently of the apolipoprotein E (ApoE) epsilon4 allele effect. We examined the distribution of the H63D mutation and ApoE genotypes as a function of age at AD onset in 328 patients with sporadic AD. Our data show that the mutant H63D allele potentially interacts with the ApoE epsilon4 allele to significantly reduce age at onset of AD compared to ApoE epsilon4 carriers alone, but has no effect on age at onset in ApoE epsilon4 non-carriers.

<h4>Objective</h4>To review current knowledge of the genetics, presentation, diagnosis, and management of hereditary hemochromatosis.<h4>Quality of evidence</h4>MEDLINE was searched from January 1966 to June 2002, and references of relevant papers were reviewed. Most articles were reviews, practice guidelines, or observational studies. Several randomized controlled trials were identified but none studied primary therapy for hemochromatosis.<h4>Main message</h4>Hemochromatosis, the most common genetic disease in white populations, has a prevalence of one in 200, yet is still underrecognized. This disease of unregulated iron absorption leads to generalized iron overload that can eventually impair organ systems and lead to cirrhosis, diabetes, and cardiomyopathy. Symptoms are often nonspecific and patients are identified by mild abnormalities in routine laboratory testing. Transferrin saturation, ferritin levels, and genotyping can often establish the diagnosis. Iron depletion therapy with phlebotomy is helpful if initiated before organ damage occurs.<h4>Conclusion</h4>Family physicians should be aware that hemochromatosis can be treated effectively if diagnosed early.

Kilon (kindred of IgLON) and opioid-binding cell adhesion molecule belong to the IgLON subgroup of immunoglobulin superfamily together with the limbic system-associated membrane protein and neurotrimin. In the present study, we have analyzed biochemical and ultrastructural characterization of Kilon and opioid-binding cell adhesion molecule such as regional and developmental expression patterns, light and electron microscopic localization, and intermolecular interactions. Western blotting revealed a widespread distribution pattern of Kilon with high expression levels in the olfactory bulb, cerebral cortex, diencephalon, hippocampus, and cerebellum and low expression levels in the medulla oblongata and spinal cord. In contrast, opioid-binding cell adhesion molecule showed a regionally restricted expression pattern with high levels only in the cerebral cortex and hippocampus. Expression of Kilon and opioid-binding cell adhesion molecule was increased gradually during postnatal development and maintained until adulthood. Light microscopic immunohistochemistry demonstrated that the localization of opioid-binding cell adhesion molecule and Kilon coincided well with that of vesicle-associated membrane protein 2, a synaptic marker protein, in the cerebral cortex and hippocampus of adult brain. In the cerebellum, Kilon-immunoreactive puncta were observed to colocalize well with that of vesicle-associated membrane protein 2, while opioid-binding cell adhesion molecule immunoreactivity was observed only at part of synaptic glomeruli in the granular layer and rare in the molecular layer. Electron microscopic analysis revealed that Kilon and opioid-binding cell adhesion molecule immunoreactivity was observed mainly at postsynaptic sites of dendritic and somatic synapses in adult cerebral cortex and hippocampus. Only trace levels of Kilon and opioid-binding cell adhesion molecule were detected in the soluble fraction of a cortical homogenate, although a substantial amount of F3 was present in the soluble fraction. A binding analysis using a cross-linker and the immunoprecipitation technique demonstrated that Kilon and opioid-binding cell adhesion molecule interacted heterophilically and homophilically. These findings show that Kilon and opioid-binding cell adhesion molecule are clearly distinguishable from each other in regional expression and localization, and binding patterns. These differences possibly represent diverse functions of each IgLON molecule.

2-Hydroxy-6-oxo-6-(2'-aminophenyl)-hexa-2,4dienoic acid [6-(2'-aminophenyl)-HODA] hydrolase, involved in carbazole degradation by Pseudomonas resinovorans strain CA10, was purified to near homogeneity from an overexpressing Escherichia coli strain. The enzyme was dimeric, and its optimum pH was 7.0-7.5. Phylogenetic analysis showed the close relationship of this enzyme to other hydrolases involved in the degradation of monocyclic aromatic compounds, and this enzyme was specific for 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (6-phenyl-HODA), having little activity toward 2-hydroxy-6-oxohepta-2,4-dienoic acid and 2-hydroxymuconic semialdehyde. The enzyme had a Km of 2.51 microM and k(cat) of 2.14 (s(-1)) for 6-phenyl-HODA (50 mM sodium phosphate, pH 7.5, 25 degrees C). The effect of the presence of an amino group or hydroxyl group at the 2'-position of phenyl moiety of 6-phenyl-HODA on the enzyme activity was found to be small; the activity decreased only in the order of 6-(2'-aminophenyl)-HODA (2.44 U/mg) > 6-phenyl-HODA (1.99 U / mg) > 2-hydroxy-6-oxo-6-(2'-hydroxyphenyl)-hexa-2,4-dienoic acid (1.05 U/mg). The effects of 2'-substitution on the activity were in accordance with the predicted reactivity based on the calculated lowest unoccupied molecular orbital energy for these substrates.

Genotypic testing of nonselected patients with the myelodysplastic syndrome (MDS) for the C282Y and H63D mutations of the HFE gene responsible for hereditary hemochromatosis revealed a significantly increased frequency of these mutations when compared to healthy blood donors reflecting the average population. Among the 50 patients examined [26 refractory anemia (RA), 9 refractory anemia with ring sideroblasts (RARS), 2 refractory anemia with excess of blasts (RAEB) and 13 refractory anemia with excess of blasts in transformation (RAEB-t)] there were 24 heterozygotes (20 for H63D and 4 for C282Y), 1 homozygote for H63D and 1 compound heterozygote. The difference between the HFE-positive and HFE-negative MDS patients as regards initial serum iron and transferrin saturation was not significant. Inevitably the iron overload syndrome eventually develops in MDS patients due to intrinsic characteristics of the disease as well as an escalating need for blood transfusion therapy in the course of the disease. The high incidence rate of HFE gene mutations among MDS patients may also contribute to this vicious circle.

Comprised mainly of monocytes and tissue macrophages, the reticuloendothelial system (RES) plays two major roles in iron metabolism: it recycles iron from senescent red blood cells and it serves as a large storage depot for excess iron. Although iron recycling by the RES represents the largest pathway of iron efflux in the body, the precise mechanisms involved have remained elusive. However, studies characterizing the function and regulation of Nramp1, DMT1, HFE, FPN1, CD163, and hepcidin are rapidly expanding our knowledge of the molecular aspects of RE iron handling. This review summarizes fundamental physiological and biochemical aspects of iron metabolism in the RES and focuses on how recent studies have advanced our understanding of these areas. Also discussed are novel insights into the molecular mechanisms contributing to the abnormal RE iron metabolism characteristic of hereditary hemochromatosis and the anemia of chronic disease.

Apoptotic cell death is executed by the caspase-mediated cleavage of various vital proteins. Elucidating the consequences of this endoproteolytic cleavage is crucial for our understanding of cell death and other biological processes. Many caspase substrates are just cleaved as bystanders, because they happen to contain a caspase cleavage site in their sequence. Several targets, however, have a discrete function in propagation of the cell death process. Many structural and regulatory proteins are inactivated by caspases, while other substrates can be activated. In most cases, the consequences of this gain-of-function are poorly understood. Caspase substrates can regulate the key morphological changes in apoptosis. Several caspase substrates also act as transducers and amplifiers that determine the apoptotic threshold and cell fate. This review summarizes the known caspase substrates comprising a bewildering list of more than 280 different proteins. We highlight some recent aspects inferred by the cleavage of certain proteins in apoptosis. We also discuss emerging themes of caspase cleavage in other forms of cell death and, in particular, in apparently unrelated processes, such as cell cycle regulation and cellular differentiation.

We have isolated a selectively deglycosylated form of vitamin D binding protein (DBP-maf) generated from systemically available DBP by a human pancreatic cancer cell line. DBP-maf is antiproliferative for endothelial cells and antiangiogenic in the chorioallantoic membrane assay. DBP-maf administered daily was able to potently inhibit the growth of human pancreatic cancer in immune compromised mice (T/C=0.09). At higher doses, DBP-maf caused tumor regression. Histological examination revealed that treated tumors had a higher number of infiltrating macrophages as well as reduced microvessel density, and increased levels of apoptosis relative to untreated tumors. Taken together, these data suggest that DBP-maf is an antiangiogenic molecule that can act directly on endothelium as well as stimulate macrophages to attack both the endothelial and tumor cell compartment of a growing malignancy.

Iron overload was found to be the major cause of disability in Chinese HbH disease patients although they were not on regular blood transfusion. The transferrin receptor 2 (TFR2) and hereditary hemochromatosis (HFE) genes were examined to see if inheritance of these gene defects may be a possible cause of iron overload in 45 HbH patients. A novel intronic (IVS6 (+6) T-->A) mutation of the TFR2 gene was identified in one patient, and six others were found to carry a known missense mutation (exon 5, I238M) that was also present in one normal control subject. One HbH patient and one normal control carried the H63D mutation of the HFE gene. Since only eight out of 45 iron-overloaded HbH patients carry a defect in the TFR2 or HFE gene in the heterozygote state and their iron loading status was comparable to the matched controls without such defects, it would appear that the accumulation of excess iron in HbH disease is more likely a result of increase dietary absorption secondary to ineffective erythropoiesis.

The effects of angiotensin-II, angiotensin-III, and [des-Asp1-angiotensin-I on drinking and attendant behaviors of rats were studied. Special behavioral patterns were observed after intracerebroventricular administration of angiotensins. The nature and possible mechanisms of behavioral effects of peptidergic brain stimulation are discussed.

<h4>Background</h4>To evaluate the role of genetic factors in the pathogenesis of porphyria cutanea tarda (PCT) and their association with chronic hepatitis C.<h4>Objective</h4>To investigate the relations between hemochromatosis gene (HFE) mutations and PCT in the south of France and their links with chronic hepatitis C virus (HCV) infection.<h4>Methods</h4>The genotype for the C282Y, H63D and S65C mutations of HFE was determined in 33 patients with PCT, 46 patients with HCV infection but without PCT and 58 controls. Iron status and HCV, HBV and HIV serologies were studied in all patients.<h4>Results</h4>A statistically significant increase in the C282Y mutation was found in PCT patients. No difference was found for H63D or S65C mutations. The prevalence of HCV infection was higher in PCT patients than controls.<h4>Conclusions</h4>C282Y mutations and HCV infection but not H63D or S65C mutations are PCT-triggering or associated factors in the south of France.

Immunofluorescence labeling was performed to study the expression of high voltage-activated Ca(2+) channel subunits on rat retinal cholinergic and dopaminergic amacrine cells, which were double labeled with antibodies against choline acetyltransferase and tyrosine hydroxylase, respectively. The alpha(1A) subunit was predominantly expressed on the processes but not on the somata of cholinergic amacrine cells, whereas staining for alpha(1B) and alpha(1E) was observed in both structures of the cells. Immunoreactivity of alpha(1C) and alpha(1D) was not found in the cholinergic amacrine cells. Dopaminergic amacrine cells, on the other hand, exhibited a differential expression pattern of the Ca(2+) channel subunits, with alpha(1A), alpha(1C) and alpha(1E) being expressed on both somata and processes and alpha(1B) predominantly on the processes of the cells. No alpha(1D) labeling was seen. These results suggest that Ca(2+) channel subunits differentially expressed on the cholinergic and dopaminergic amacrine cells may endow these two cell types with different physiological properties.

The frequencies of the hereditary hemochromatosis gene (HFE) mutations C282Y and H63D vary between different populations. There are a limited number of reports regarding the frequency of these mutations in populations of southeastern Europe. Two hundred and sixty-four adult individuals of Greek origin were examined for the C282Y and H63D mutations to determine the allele and genotype frequencies. The HFE gene region of DNA samples extracted from peripheral leukocytes was amplified by the polymerase chain reaction. Restriction enzyme analysis was performed using RSAI for C282Y and MBOI for H63D. None of the 264 individuals carried the mutation C282Y. Forty-three individuals (16.2%) were heterozygous carriers of the H63D allele and 2 were homozygous for this mutation (0.75%). The overall H63D allele prevalence is thus estimated at 8.9%. HFE mutation frequencies were low in the population studied and this may explain, in part, the relative rarity of clinical cases of hereditary hemochromatosis in Greece.

The genes involved in the serotonin system are major candidates in association studies on affective disorders and responses to antidepressants. We studied a functional polymorphism of the serotonin transporter (5-HTT) gene (a 44 bp insertion/deletion in the 5-HTT-linked polymorphic region (5-HTTLPR)) and lifetime history of antidepressant-induced mania (AIM) in a population of 305 patients with bipolar affective disorder. AIM was defined using a broad definition and a restrictive definition. No association was found between the "s" allele of the 5-HTTLPR and AIM for either definition. However, we found an association between the 5-HTTLPR and lifetime history of rapid cycling in a subsample of patients (for allele and genotype distributions: exact probability, p=0.0009 and chi(2)=9.4; df=1; p=0.002, respectively). These results may help to explain the conflicting association results obtained with the 5-HTT gene polymorphism, in particular with AIM. Indeed, the precise phenotype associated with the 5-HTT gene is unclear. The association between the "s" allele and rapid cycling may provide further evidence for an association between the 5-HTTLPR "s" allele and a pattern of affective instability.

The prognosis of patients with colorectal cancer is impacted by various factors at the time of diagnosis, including location of the tumor, gender, age and overall performance status of the patient. Optimal postoperative management of patients who have undergone successful tumor resection involves the utilization of reliable determninants of prognosis to help select patients who would benefit from adjuvant treatment, while sparing others from drug-related adverse effects. Tailoring chemotherapy for patients with disseminated cancer, or for patients who receive adjuvant chemotherapy, is also critical. Interpatient differences in tumor response and drug toxicity are common during chemotherapy. Genomic variability of key metabolic enzyme complexes, drug targets, and drug transport molecules is an important contributing factor. The identification of genetic markers of response and prognosis will aid in the development of more individualized chemotherapuetic strategies for cancer patients. Potential prognostic indicators in colorectal cancer include oncogenes, tumor suppressor genes, genes involved in angiogenic and apoptotic pathways and cell proliferation, and those encoding targets of chemotherapy. Specifically, molecular markers such as deletion of 18q (DCC), p27 and microsatellite instability are promising as indicators of good or poor prognosis. Molecular determinants of efficacy and host toxicity of the most commonly used drugs in colorectal cancer, fluoracil, irinotecan and oxaliplatin, are being investigated. Alterations in gene expression, protein expression and polymorphic variants in genes encoding thymidylate synthase, dihydropyrimidine dehydrogenase, dUTP nucleotidehydrolase and thymidine phosphorylase (for fluoropyrimidine-based chemotherapy), uridine diphosphate glucosyltransferase (UGT) 1A1 and carboxylesterase (for irinotecan therapy), and excision repair cross-complementing genes (ERCC1 and ERCC2) and glutathione-S-transferase P1 (for oxalilplatin-based regimens) may be useful as markers for clinical drug response, survival and host toxicity.

<h4>Background</h4>Americans of African ancestry are less likely to receive a selective serotonin reuptake inhibitor (SSRI) for treatment of major depressive disorder than Americans of European ancestry. A functional insertion/deletion polymorphism in the promoter of the serotonin transporter (5-HTT) gene SLC6A4 has been shown to modulate SLC6A4 transcription, affecting response to SSRIs. Several studies in populations of predominantly European ancestry have consistently found that the SLC6A4 promoter polymorphism (referred to as the 5-HTT-linked polymorphic region; 5-HTTLPR) long (L) allele is associated with better response to SSRI treatment than the short (S) allele.<h4>Objective</h4>The frequency of SLC6A4 (5-HTTLPR) alleles in 865 black Americans and Afro-Caribbeans was examined to assess possible implications for treatment.<h4>Study design and methods</h4>SLC6A4 (5-HTTLPR) genotypes were determined in individuals with self-identified African ancestry from South Carolina (n = 489), western Pennsylvania (n = 207), and Tobago (n = 169). Frequencies were compared using chi-square analyses.<h4>Results</h4>It was verified that the L allele is highly prevalent in Americans of African ancestry, ranging from 77% in western Pennsylvania to 87% in South Carolina. The frequency of the SLC6A4-(L) allele is significantly higher in African-Americans than has been reported for European-Americans (typically 56-60%). There are both statistically significant geographic differences and slight deviations from Hardy-Weinberg equilibrium.<h4>Conclusions</h4>Given the potential influence on treatment response, these findings have implications for the use of SSRIs in this population. The results suggest that additional studies to examine the impact of these alleles on treatment response in African-Americans are warranted.

Different cellular pathways can lead to apoptosis. Apaf1 is the molecular core of the apoptosome, a multiproteic complex mediating the so-called mitochondrial pathway of cell death. The importance of this pathway during development has been clearly demonstrated by knocking out key genes. Also, the relevance of Apaf1 dosage during development has been recently underlined. Moreover, a growing body of evidences seems to point out a possible role of the mitochondria-dependent apoptosis in different pathologies. In particular, we discuss here some recent evidences regarding the putative role of the apoptosome in neurodegeneration and cancer.

<h4>Purpose</h4>To elucidate the intratumoral distribution of genetic changes, surgically resected colorectal carcinomas were investigated regarding their loss of heterozygosity in the regions of tumor suppressor genes and the mutation of ras genes.<h4>Methods</h4>Full-thickness fresh tumor slices from 23 colorectal carcinomas were removed and divided into multiple specimens, which were then submitted separately for DNA and histopathological analyses. The loss of heterozygosity was analyzed in 23 primary carcinomas and 9 metastasized carcinomas by the use of restriction fragment-length polymorphism markers on chromosome 1p, 5q, 17p, 18q, and 22q.<h4>Results</h4>Intratumoral heterogeneity was identified in 11 of 23 primary carcinomas (47.8%) and we could successfully map the regional genetic variation. In both stages I and II, 1 of 5 cases (20%); in stage III, 3 of 6 cases (50%); and in stage IV, 6 of 7 cases (85.7%) were heterogeneous. With respect to venous invasion positive primary carcinomas, hepatic metastasis occurred in 75% (6/8) of the heterogeneous carcinomas, whereas hepatic metastasis occurred in only 12.5% (1/8) of homogeneous carcinomas.<h4>Conclusion</h4>The present results suggest that the existence of intratumoral heterogeneity, which may reflect genetic instability, may thus play a role in the enhancement of aggressive progression and the metastasis of colorectal carcimomas.

No abstract available.

The polymorphism at codon 201 of the "deleted in colorectal carcinoma" (DCC) gene has been liked to susceptibility to colorectal cancer. However, its clinicopathological significance has not been reported. We examined the codon 201 polymorphism and loss of heterozygosity (LOH) by PCR-restriction fragment length polymorphism (PCR-RFLP) in 59 colorectal cancers, 48 samples from transitional mucosa and 67 samples from normal mucosa. The frequencies of the polymorphism did not significantly differ from normal to transitional mucosa and to tumor, but LOH was increased from transitional mucosa to tumor. Almost all of the LOH cases showed the polymorphism. The polymorphism was increased from well/moderately to poorly differentiated and to mucinous carcinoma (P=0.03). The polymorphism was more frequently seen in advanced stages than in earlier stages (P=0.02), and further predicted worse survival (P=0.04). The data suggest that the codon 201 polymorphism of the DCC gene was a target of LOH, and predicted prognosis in colorectal cancer patients.

<h4>Objectives</h4>Homozygosity for the C282Y mutation of the HFE gene is the main cause of iron overload in hereditary haemochromatosis. This study calculated the number of hepatocellular carcinoma cases among a cohort of white males that could be attributed to C282Y homozygosity. A better understanding of the extent of potentially preventable mortality arising from this cancer might help with decision making about the feasibility of population screening.<h4>Methods</h4>We combined information from published life tables, age-specific cancer rates and DNA studies of archived liver biopsy specimens to calculate the number of cases of hepatocellular carcinoma that might occur during the lifetime of a cohort of 1,000,000 men, including a subgroup of 5000 C282Y homozygotes.<h4>Results</h4>Hepatocellular carcinoma was estimated to occur in 2673 men in the cohort (1:374); 267 of these cases were in the subgroup of 5000 C282Y homozygotes (1:17). If these 267 cases were prevented, the remaining lifetime risk among all males would be 1:416. The relative risk for this cancer in C282Y homozygotes is 23.<h4>Conclusions</h4>There continues to be uncertainty about the efficacy of screening for haemochromatosis. Hepatocellular carcinoma is the most readily quantifiable serious health problem attributable to this source. Further confirmatory DNA (C282Y) studies would be helpful in larger, unbiased sets of archived biopsy specimens, as a way to confirm the present estimate. Any strategy designed to prevent attributable liver cancer is likely to prevent other serious problems from haemochromatasis as well.

Antioxidant protein 2 is a unique member of the thiol-specific antioxidant family of proteins known to reduce reactive oxygen species in the presence of thiol-containing electron donors. It is also a candidate atherosclerosis susceptibility gene in mice. In the present study, we sought to characterize the transcripts of this gene, and determine which, if any, are regulated by conditions associated with oxidative stress. We have identified multiple Aop2 transcripts by Northern blot, each exhibiting a unique tissue distribution. These include the previously reported 1.47 kb major transcript, two alternative Aop2 transcripts found exclusively in liver, and a testis-specific transcript believed to be the highly related intronless gene Aop2-rs1. Treatment of a murine hepatocyte cell line with glucose oxidase led to the specific and transient induction of the 1.47 kb transcript, while the 3.1 kb transcript was regulated by serum deprivation and re-stimulation with either keratinocyte growth factor or serum in a time-dependent manner. Since these ROS-inducing stimuli involve different mechanisms of action and cellular responses, our data suggest that alternative Aop2 transcripts may play distinct roles in different oxidative stress responses, and possibly in atherosclerosis.

Each year, one million people die of suicide. Among the different identified risk factors, genetic factors seem to be part of a multidimensional behavior, including psychiatric, psychosocial, biological factors and physical illness. Family studies have provided evidence for familial transmission in suicide, confirmed in twin and adoption studies. At a molecular level, serotonin seems to be one of the key neurotransmitters implicated in suicidal behavior. Therefore, genes coding for proteins involved in serotonergic neurotransmission have been extensively studied in case-control association studies on suicide. Major findings concern Tryptophan hydroxylase (TPH) gene, particularly in violent suicidal behavior. Though they may seem contradictory, studies on Serotonin transporter (5-HTT), Monaomine oxidase (MAOA), Serotonin 2A and 2C receptors (5-HT2A and 5-HT2C) and Tyrosine hydroxylase (TH) genes are promising. In spite of those observations having some limitations, it appears that genetic factors are a serious risk factor, besides environmental aspects of suicidal behavior.

Friedreich ataxia (FRDA), the most common autosomal recessive inherited ataxic disorder, is the consequence of deficiency of the mitochondrial protein frataxin, typically caused by homozygous intronic GAA expansions in the corresponding gene. The yeast frataxin homologue (yfh1p) is required for cellular respiration. Yfh1p appears to regulate mitochondrial iron homeostasis and protect from free radical toxicity. Complete loss of frataxin in knockout mice leads to early embryonic lethality, indicating an important role for frataxin during development. Heterozygous littermates with partial frataxin deficiency are apparently healthy and have no obvious phenotype. Here we evaluate iron metabolism and sensitivity to dietary and parenteral iron loading in heterozygote frataxin knockout mice (Fx(+/-)). Iron concentrations in the liver, heart, pancreas and spleen, and cellular iron distribution patterns were compared between wild type and Fx(+/-) mice. Response to parenteral iron challenge was not different between Fx(+/-) mice and wild type littermates, while sporadic iron deposits were observed in the hearts of dietary iron-loaded Fx(+/-) mice. Finally, we evaluated the effect of partial frataxin deficiency on susceptibility to cardiac damage in the mouse model of hereditary hemochromatosis (HH), the Hfe knockout mice. HH, an iron overload disease, is one of the most frequent genetic diseases in populations of European origin. By breeding Hfe(-/-) with Fx(+/-) mice, we obtained compound mutant mice lacking both Hfe and one frataxin allele. Sparse iron deposits in areas of mild to moderate cardiac fibrosis were found in the majority of these mice. However, they did not develop any neurological symptoms. Our studies indicate an association between frataxin deficiency, iron deposits and cardiac fibrosis, but no obvious association between iron accumulation and neurodegeneration similar to FRDA could be detected in our model. In addition, these results suggest that frataxin mutations may have a modifier role in HH, that predisposes to cardiomyopathy.

The variable clinical manifestations of cystic fibrosis (CF) suggest the influence of modifier genes. Genetic and environmental factors that determine whether an individual will develop associated complications are still being determined. It has been proposed that the gene for hemochromatosis, HFE, may be a modifier locus for CF disease phenotype. Recent research has suggested a relationship between mutations to the HFE gene and the development of meconium ileus (MI) and liver disease in CF. This study aims to expand our knowledge of the HFE mutations C282Y and H63D carrier rate in an Irish population of CF allele carriers. PCR restriction enzyme analysis was performed on blood samples from CF patients to identify the C282Y and H63D mutations. HFE status of CF allele carriers and CF patients (Delta F508) homozygotes with and without meconium ileus was determined. The carrier frequency for C282Y was 30.8% for the Delta F508 homozygote MI positive group, as compared to 12.5% for the non-Delta F508 MI positive group but did not reach statistical significance (p = 0.27). Interestingly, no Delta F508 homozygote patients were homozygous for the C282Y mutation.

The human serotonin transporter (5-HTT) gene is an important candidate for the pathogenesis of mood disorders. Associations have been reported between a variable-number tandem-repeat polymorphism in intron 2 of the serotonin transporter gene (5-HTTVNTR) and mood disorders in a number of studies of Western and Chinese populations. However, no such relationships have been determined in other analogous research. To replicate these positive findings in a Chinese population and to determine the association between onset age of bipolar disorder and 5-HTTVNTR, we investigated the prevalence of this polymorphism in an independent Chinese population (83 bipolar disorder patients, 77 major depressive disorder patients and 169 controls), demonstrating no significant association between the 5-HTTVNTR polymorphism and mood disorders or age at onset. Further, no association was demonstrated between this polymorphism and suicidal history in mood disorder patients. These negative findings suggest that 5-HTTVNTR does not play a major role in the pathogenesis of mood disorder in Chinese populations.

Loss of heterozygosity (LOH) and/or inactivation of tumor suppressor genes are implicated in the initiation and progression of many malignancies, including colorectal cancer. Although accumulating evidence suggests a chemopreventive role for folate in colorectal cancer, regulatory mechanisms are poorly understood. The primary objective of the current investigation was to determine whether folic acid would prevent LOH of the three tumor suppressor genes, deleted in colorectal cancer (DCC), adenomatous polyposis coli (APC) and p53 in macroscopically normal appearing rectal mucosa of patients with adenomatous polyps. In addition, the effect of folic acid on rectal mucosal proliferation was determined. Twenty patients were randomized in a double-blind study to receive either folic acid 5mg once daily or identical placebo tablets for 1 year. Genomic DNA and total protein were extracted from the rectal mucosa at baseline and after 1 year of treatment and analyzed for LOH and protein levels of APC, DCC and p53 genes. In addition, paraffin-embedded mucosal specimens were analyzed for proliferating cell nuclear antigen (PCNA) immunoreactivity, as a measure of cellular proliferative activity. Folate supplementation prevented LOH of DCC gene in five out of five (100%) patients who demonstrated baseline heterozygosity, whereas two out of four (50%) placebo-treated patients with baseline heterozygosity demonstrated allelic loss. Mucosal protein levels of DCC were also reduced in 7 of 10 (70%) placebo-treated patients compared to only 2 of 10 (20%) of patients treated with folate. Levels increased, however, in eight and three patients in the folic acid and placebo groups, respectively (P<0.02). Folic acid caused no change in allelic status of either APC or p53 gene. Folate supplementation caused a small, but not statistically significant, 16% reduction in mucosal proliferation, whereas placebo treatment resulted in a 88% (P<0.05) increase in this parameter, when compared with the corresponding baseline values. Our results indicate that folic acid prevents an increase in proliferation and arrests LOH of DCC gene and also stabilizes its protein in normal appearing rectal mucosa of patients with colorectal adenomas. Taken together, our data suggest that one of the ways folate may exert its chemopreventive effect is by stabilizing certain tumor suppressor gene(s) and preventing further increases in proliferation.

Glutamate-mediated excitotoxicity might contribute to the pathogenesis of Huntington's disease and other polyglutamine repeat disorders. We used murine neocortical cultures derived from transgenic and knock-in mice to test the effect of expression of expanded polyglutamine-containing huntingtin on neuronal vulnerability to excitotoxins or other insults. Neurons cultured from mice expressing either a normal length (Hdh(Q20)) or expanded (Hdh(Q111)) CAG repeat as a knock-in genetic alteration in exon one of the mouse Hdh gene [Hum Mol Genet 8 (1999) 115] had similar vulnerability to N-methyl-D-aspartate (NMDA) and kainate-mediated excitotoxicity. These neurons also exhibited similar vulnerability to oxidative stress (24 h exposure to 10-100 microM paraquat or 1-10 microM menadione), apoptosis (48 h exposure to 30-100 nM staurosporine or 1 microM dizocilpine maleate (MK-801) and proteasome inhibition (48 h exposure to 0.3-3 microM MG-132). Neocortical neurons cultured from mice transgenic for an expanded CAG repeat-containing exon 1 of the human HD gene (Mangiarini et al., 1996, R6/2 line) and non-transgenic littermate controls also had similar vulnerability to NMDA and kainate-mediated excitotoxicity. These observations suggest that expression of expanded polyglutamine-containing huntingtin does not acutely alter the vulnerability of cortical neurons to excitotoxic, oxidative or apoptotic insults.

Ethanol toxicity on liver is a function of duration of alcoholism, amount of daily intake of alcohol and patient's nutrition. The threshold of alcohol toxicity on the liver is about 40 g of ethanol daily in men and 20-30 g in women, however liver cirrhosis develops in no more than 8-20% of patients exceeding this values. Ethanol is oxidized in the liver to acetaldehyde--a compound considerably more toxic than ethanol itself. Despite small amount of alcohol dehydrogenase (ADH) found in gastric mucosa, the metabolism of ethanol in this site may have an important hepatoprotective effect. The oxidation of ethanol is associated with a change of hepatocyte redox homeostasis, which leads to a number of metabolic disorders such as lactic acidosis, hyperlipidaemia and hyperuricaemia. Chronic ethanol consumption does not influence ADH activity, but has a profound stimulatory effect on microsomal enzymes, in particular cytochrome CYP2E1. This fact is responsible for development in alcoholic liver associated with rise of oxygen consumption, excessive production of free radicals and increased metabolism of ethanol, vitamin A and testosterone. Ethanol and acetaldehyde have a deleterious effect, both the direct and indirect, on hepatocytes e.g., generating radical oxygen species and damaging intestinal mucosal barrier. Cellular oxidative stress that is caused by both an excess of free radicals and the antioxidatives' deficiency (glutathion, vitamin E, phosphatidylcholine), may be the principal factor responsible for progression of alcoholic liver disease. Among other factors accelerating alcohol-related liver lesion there are certain drugs, high fat diet, infection with HCV and genetic factors (female sex, enzymatic polymorphic forms of ADH and ALDH, hemochromatosis). Great importance in pathogenesis of necrotic and inflammatory hepatic events is being attributed to portal endotoxaemia and cytokines induced within the liver, in particular TNF-alpha and interleukin 8. These cytokines play a key role in development of alcoholic hepatitis, which clinical severity ranges from subclinical to fatal forms. Apart from abstinence, the treatment of alcohol liver disease is based on hyperalimentation, since alcoholism is generally associated with protein malnutrition. In severe forms of alcohol hepatitis corticosteroids are recommended.

This report describes a case of sporadic porphyria cutanea tarda involving a 38-year-old Moroccan man. Clinical diagnosis was based on characteristic features, i.e., facial hypertrichosis and bullous lesions lasting four months during the summer of 2000 followed by macular scarring on the dorsal surfaces on the hands. Three well-known precipitating factors were noted, i.e., sun, ethanol and hepatitis C virus infection. Laboratory diagnosis was based on dark red urine and elevated serum and urine uroporphyrin levels. Enhanced uroporphyrin production was due to urodecarboxylase deficiency in the liver. Urodecarboxylase activity in red blood cells and serum ferritin level were normal. The patient is heterozygous for the His63Asp HFE gene mutation associated with hereditary hemochromatosis. The photoprotective effect of melanin in this dark-skinned patient failed to offset uroporphyrin-induced photosensitivity. Avoidance of sun, ethanol and phlebotomy have prevented recurrences.

In this study the mRNA differential display method was applied to identify mastitis-associated expressed DNA sequences based on different expression patterns in mammary gland samples of non-infected and infected udder quarters of a cow. In total, 704 different cDNA bands were displayed in both udder samples. Five hundred-and-thirty two bands, (75.6%) were differentially displayed. Ninety prominent cDNA bands were isolated, re-amplified, cloned and sequenced resulting in 87 different sequences. Amongst the 19 expressed sequence tags showing a similarity with previously described genes, the majority of these sequences exhibited homology to protein kinase encoding genes (26.3%), to genes involved in the regulation of gene expression (26.3%), to growth and differentiation factor encoding genes (21.0%) and to immune response or inflammation marker encoding genes (21.0%). These sequences were shown to have mastitis-associated expression in the udder samples of animals with and without clinical mastitis by quantitative RT-PCR. They were mapped physically using a bovine-hamster somatic cell hybrid panel and a 5000 rad bovine whole genome radiation hybrid panel. According to their localization in QTL regions based on an established integrated marker/gene-map and their disease-associated expression, four genes (AHCY, PRKDC, HNRPU, OSTF1) were suggested as potentially involved in mastitis defense.

The influence of anticoagulation components of hemostasis system and lupus anticoagulants (LA) upon the course of multiple sclerosis (MS) was studied. 57 patients with MS diagnosed according to McDonald's criteria (McDonald WI et al., 2001) were examined: 19 males and 38 females aged 16-62 (mean age 36.8 +/- 11.2 yrs) with 1-25 yrs history of MS (mean duration 8.1 +/- 5.7 yrs). 42 patients suffered from relapsing-remitting MS, 17 patients were affected by secondary progressive MS. The degree of neurological deficit was estimated in remission according to Expanded Disability Status Scale (EDSS), mean score was 3.8 +/- 1.7. The progression of MS was estimated as EDSS score per year ratio. In patients with relapsing-remitting MS the mean number of exacerbations per year was calculated. The following laboratory findings were evaluated: thrombin clotting time, antithrombin-III activity, plasminogen reserve by the level of streptokinase-induced euglobulin fibrinolysis, protein C activity, factor Va resistance to activated protein C, lupus anticoagulants (LA). It was revealed that LA could worsen relapsing-remitting MS: in LA-positive patients there were 1.41 +/- 0.69 exacerbations per year and progression score reached 0.82 +/- 0.30, whereas in LA-negative patients the figures were 0.74 +/- 0.58 (p < 0.001) and 0.48 +/- 0.33 (p < 0.01) respectively. All the patients showed decrease of protein C activity (p < 0.001) with no relation to LA activity or factor Va resistance to activated protein C. Normal level of plasminogen and antithrombin-III activity was supposed to be the marker of benign variant of MS. The course of MS was mild in patients with high antithrombin-III activity even if they were hypercoagulative--this suggests neuroprotective activity of antithrombin-III which may relate not only to its anticoagulation effect.

Hereditary hemochromatosis (HH) is an autosomal recessive trait regarding iron metabolism frequently found in Caucasian populations. The C282Y mutation of the HFE gene, held responsible for HH, has been identified as the major genetic basis for the phenotypic expression of HH whereas two additional mutations of the HFE H63D and S65C gene appear to be associated with a milder form of HH. A high allele frequency of C282Y and H63D has been reported in Northern European populations. In Italy, the overall allele frequency was 0.5% for the C282Y mutation, 12.6% for the H63D mutation and 1.1% for the S65C mutation. In this study, we evaluated the allele frequency of the three principal HFE mutations (C282Y, H63D, S65C) together with eight additional mutations (V53M, H63H, Q127H, E168Q, E168stop, W169stop, V59M, Q238P) in 500 healthy Apulian subjects. No subject homozygous for the C282Y mutation was found while 3% of subjects were heterozygous for this mutation. Heterozygosity and homozygosity for the H63D mutation were 26 and 1%, respectively. Only five subjects were heterozygous for the S65C mutation. Overall, the allele frequency was 1.5% for C282Y, 14% for H63D, 0.5% for S65C and 0% for the other mutations. The transferrin saturation (TS) was significantly higher in subjects heterozygous for the H63D mutations with respect to subjects with a normal genotype, though all were within the normal range. No statistically significant difference in the allele frequency was noted in the Apulian population compared to that in Northern and Southern Italy.

Interaction between cell and extracellular matrix (ECM) plays a crucial role in tumor invasiveness and metastasis. Using an immortalized human bronchial epithelial (BEP2D) cell model, we showed previously that expression of a list of genes including Betaig-h3 (induced by transforming growth factor-beta), DCC (deleted in colorectal cancer), p21(cipl), c-fos, Heat shock protein (HSP27) and cytokeratin 14 were differentially expressed in several independently generated, radiation-induced tumor cell lines (TL1-TL5) relative to parental BEP2D cells. Our previous data further demonstrated that loss of tumor suppressor gene(s) as a likely mechanism of radiation carcinogenesis. In the present study, we chose Betaig-h3 and DCC that were downregulated in tumorigenic cells for further study. Restored expression of Betaig-h3 gene, not DCC gene, by transfecting cDNA into tumor cells resulted in a significant reduction in tumor growth. While integrin receptor alpha 5 beta 1 was overexpressed in tumor cells, its expression was corrected to the level found in control BEP2D cells after Betaig-h3 transfection. These data suggest that Betaig-h3 gene is involved in tumor progression by regulating integrin alpha 5 beta 1 receptor. Furthermore, exogenous TGF- beta 1 induced expression of Betaig-h3 gene and inhibited the growth of both control and tumorigenic BEP2D cells. Therefore, downregulation of Betaig-h3 gene may results from the decreased expression of upstream mediators such as TGF-beta. The findings provide strong evidence that the Betaig-h3 gene has tumor suppressor function in radiation-induced tumorigenic human bronchial epithelial cells and suggest a potential target for interventional therapy.

Huntington's disease (HD) is a genetically dominant neurodegenerative condition caused by an unique mutation in the disease gene huntingtin. Although the Huntington protein (Htt) is ubiquitously expressed, expansion of the polyglutamine tract in Htt leads to the progressive loss of specific neuronal subpopulations in HD brains. In this article, we will summarize the current understanding on mechanisms of how mutant Htt can elicit cytotoxicity, as well as how the selective sets of neuronal cell death occur in HD brains.

Huntington's disease (HD) research is aimed at understanding the root cause of the disorder, for the thrill of uncovering new biology, and for the serious purpose of finding effective therapeutic agents. Molecular genetics has revealed the disease trigger, an inherited unstable CAG expansion in a novel 4p16.3 gene (HD), that lengthens a polyglutamine segment in huntingtin. Now studies with HD patients and model systems that are genetic HD replicas are homing in on the trigger mechanism and the first formative steps that cast HD as a distinct clinical entity. At the same time, assays at the biochemical, cellular, and whole organism levels are starting to yield potential disease modifying genes and candidate drugs. These can be prioritized by testing in a panel of genetic and phenotypic HD mouse models to yield analytical tools for dissecting the early and late stages of the disease process and to maximize the chance of success in trials with HD patients.

<h4>Background/aims</h4>Hereditary hemochromatosis (HH), the most common autosomal recessive disease in the white population, is characterized by excessive gastrointestinal absorption of iron and loading of parenchymal organs. HFE mutations of C282Y and H63D are largely responsible for HH in populations of Celtic ancestry. Although many screening studies related to HH have been done in Northern Europe, the USA and Australia, as yet, no such study has been published on Turkey. In this study we aimed to screen the Turkish population for iron overload.<h4>Methods</h4>Random samples were obtained from 4,633 healthy adults (3,827 male, 806 female, mean age +/- SD 35 +/- 8 years, range 14-76) for the measurement of transferrin saturation (TS). Measurements were repeated after an overnight fast in the subjects whose initial TS was > or =50%. Serum ferritin levels and C282Y and H63D gene mutations were studied in cases when fasting TS was > or =50%. In cases where the serum ferritin level was >200 ng/ml with or without HFE mutations, liver biopsy was performed for histological evaluation and determination of iron content.<h4>Results</h4>In 158 subjects, TS was > or =50% in the non-fasting state. A second determination of TS after an overnight fast was performed in 135 subjects. In 26 subjects, the TS was > or =50% in the fasting state. HFE mutation and serum ferritin levels were measured in these 26 subjects. Eleven subject (10 male, 1 female) were heterozygote and 1 male subject was homozygote in reference to H63D. C282Y mutation was not found. Four of these 26 subjects (all males, aged 23, 24, 40, 49) had increased serum ferritin levels and liver biopsy was performed. In 1 male (aged 49) who was heterozygote for H63D genotype with a serum ferritin level of 645 ng/ml, iron overload in liver tissue was shown by histology as well as atomic absorption spectrophotometry.<h4>Conclusion</h4>The prevalence of hemochromatosis in the Turkish population is much lower in comparison to populations of Celtic ancestry and C282Y mutation is non-existent.

Brain-derived neurotrophic factor (BDNF) appears to be both regulated by and a regulator of epileptogenesis. In the flurothyl (HFE) model of kindling mice exposed to successive flurothyl trials over 8 days express a rapid, long-lasting reduction in generalized seizure threshold and a more slowly evolving change in seizure phenotype in response to subsequent flurothyl exposure. The BDNF genotype of particular mouse strains appears to influence the epileptogenic progression in this model. Thus, we hypothesized that BDNF signaling pathways are altered by flurothyl-induced seizures. Following HFE kindling, fully kindled (eight seizures) adult male C57BI/6J mice had significantly elevated whole brain BDNF levels through at least 28 days after their final seizure. Mice that received only four HFE seizures (not kindled) had elevated BDNF levels, but only at 1 day post-seizure (DPSz), while BDNF levels were not significantly altered in mice receiving just one HFE seizure at any time point studied. Regional expression patterns of BDNF in the hippocampus, hypothalamus, and frontal cortex were also elevated by one DPSz and returned to control values by 14 DPSz in mice that received four HFE seizures. No changes were seen in the cerebellum, striatum, or piriform cortex. In contrast, fully kindled mice had significantly elevated BDNF levels within the hippocampus, hypothalamus, neocortex, and striatum that remained elevated through at least 14 DPSz, while levels were unchanged in the cerebellum and piriform cortex. Regional results were confirmed using anti-BDNF immunohistochemistry (IHC). Despite changes in BDNF levels following HFE kindling, we were unable to demonstrate alterations either in full-length tyrosine kinase receptor B (TrkB) expression (Western blot and IHC) or in truncated TrkB (IHC) expression levels. Together, these data suggest a model of a positive feedback loop involving seizure activity and seizure number and persistently modified BDNF signaling pathways that influences seizure phenotypes within the HFE kindling paradigm. Thus, long-term elevations in BDNF may be responsible in part for epileptogenic processes and the development of human refractory epilepsies.

Hereditary hemochromatosis is an autosomal recessive disorder resulting in iron overload. A progressive increase in total body iron stores leads to deposition of iron in parenchymal organs and to subsequent damage to these organs. The identification of the HFE gene and its mutations has stimulated much additional interest in this under-recognized but evidently treatable chronic disease. The goal for diagnosing the disease is to identify presymptomatic patients who will respond more favorably to phleobotomy which may prevent future chronic disease.

Mitotic chromosome condensation is an essential cellular function ensuring proper compaction and segregation of sister chromatids during cell division. Condensin, a five-subunit complex, conserved among eukaryotes, is the key molecular machine of chromosome condensation. Recent advances in the structural biology and functional analysis of condensin demonstrate the unique nature and indispensable biological role of this complex. Condensin functions span chromosome dynamics during mitotic cell division, cell-cycle feedback control mechanisms, as well as formation and maintenance of interphase chromosome structure. Being at the intersection of several cell-cycle regulatory networks condensin is a promising therapeutic target for control over cell proliferation.

Opioid-binding cell adhesion molecule (OBCAM) is a member of the immunoglobulin superfamily containing limbic system-associated membrane protein (IgLON) subgroup of glycosylphosphatidylinositol-anchored immunoglobulin cell adhesion molecules. We have previously found that OBCAM is localized preferentially to dendrites compared with somata and terminals of hypothalamic vasopressin-secreting magnocellular neurons. This localization indicates that OBCAM is one of the dendrite-associated cell adhesion molecules. In the present study, we further characterized the localization and the sorting mechanism, and activity-dependent changes of this molecule in vasopressin-secreting magnocellular dendrites. Confocal microscopic observation revealed the preferential localization of OBCAM at the neurosecretory granules in the vasopressin-positive dendrites. Electron microscopic observation using chromogen-intensified and gold-conjugated methods also demonstrated the OBCAM labeling at most of the neurosecretory granules within the dendrites, while the labeling within the somata was observed at only a few neurosecretory granules. I.c.v. colchicine administration resulted in the disappearance of OBCAM immunoreactivity from the dendrites and in its concomitant accumulation at the somata, suggesting that OBCAM is synthesized at the somata and transported to the dendrites by dendrite-associated neurosecretory granules. During the postnatal development, OBCAM immunoreactivity targeted to vasopressin-positive dendrites became clear from at least 3 weeks after birth, although it appeared at only a few somata 2 weeks after birth. Phosphatidylinositol specific phospholipase C treatment of the membrane fraction of the supraoptic homogenate solubilized OBCAM. Kilon, another IgLON member, was also shown to localize at the neurosecretory granules of vasopressin-positive dendrites via the glycosylphosphatidylinositol anchor. High K(+)-stimulation appeared to cause the diffusion of OBCAM-labeled gold particles from neurosecretory granules together with the exocytosis. These findings indicate that OBCAM is synthesized within the somata, attached to vasopressin neurosecretory granules via the glycosylphosphatidylinositol anchor, and transported to the dendrites. Moreover, the subcellular localization of OBCAM is changed in an activity-dependent manner.

We examined the effects of methamphetamine (METH) on monoaminergic (i.e. dopamine and serotonin) axonal markers and glial cell activation in the rat brain. Our findings indicate that the loss of dopamine transporters (DAT), serotonin transporters (5-HTT), vesicular monoamine transporter type-2 (VMAT-2) and glial cell activation induced by METH in the striatum and in the central gray are consistent with a degenerative process. Our novel finding of METH effects on monoaminergic neurons in the central gray may have important implications on METH-induced hyperthermia. In other brain regions examined, DAT and 5-HTT deficits after METH administration were present in the absence of lasting changes in VMAT-2 levels or glial cell activation. Brain regions exhibiting protracted deficits in DAT and/or 5-HTT and VMAT-2 levels also expressed increased levels of [(3)H]-R-PK11195 binding to peripheral benzodiazepine receptors, a quantitative marker of glial cell activation. Immunohistochemical assessment of microglia and astrocytes confirmed the PBR results. Microglia activation was more pronounced than astrocytosis in affected regions in most METH-exposed brains with the exception of a small number of rats that were most severely affected by METH based on loss of body weight. In these rats, both microglia and astrocytes were highly activated and expressed a distinct regional pattern suggestive of widespread brain injury. The reason for the pattern of glial cell activation in this group of rats is not currently known but it may be associated with METH-induced hyperthermia. In summary, our findings suggest two neurotoxic endpoints in the brain of METH-exposed animals. Brain regions exhibiting DAT and 5-HTT deficits that co-localize with decreased VMAT-2 levels and glial cell activation may represent monoaminergic terminal degeneration. However, the DAT and 5-HTT deficits in brain regions lacking a deficit in VMAT-2 and glial cell activation may reflect drug-induced modulation of these plasma membrane proteins.

In the past seven years numerous genes that influence iron homeostasis have been discovered. Dr. Beutler provides a brief overview of these genes, genes that encode HFE, DMT-1, ferroportin, transferrin receptor 2, hephaestin, and hepcidin to lay the groundwork for a discussion of the various clinical forms of iron storage disease and how they differ from one another. In Section I, Dr. Beutler also discusses the types of hemochromatosis that exist as acquired and as hereditary forms. Acquired hemochromatosis occurs in patients with marrow failure, particularly when there is active ineffective erythropoiesis. Hereditary hemochromatosis is most commonly due to mutations in the HLA-linked HFE gene, and hemochromatosis clinically indistinguishable from HFE hemochromatosis is the consequence of mutations in three transferrin receptor-2 gene. A more severe, juvenile form of iron storage disease results from mutations of the gene encoding hepcidin or of a not-yet-identified gene on chromosome 1q. Autosomal dominant iron storage disease is a consequence of ferroportin mutations, and a polymorphism in the ferroportin gene appears to be involved in the African iron overload syndrome. Evidence regarding the biochemical and clinical penetrance of hemochromatosis due to mutations of the HFE gene is rapidly accumulating. These studies, emanating from several centers in Europe and the United States, all agree that the penetrance of hemochromatosis is much lower than had previously been thought. Probably only 1% of homozygotes develop clinical findings. The implications of these new findings for the management of hemochromatosis will be discussed. In Section II, Dr. Victor Hoffbrand discusses the management of iron storage disease by chelation therapy, treatment that is usually reserved for patients with secondary hemochromatosis such as occurs in the thalassemias and in patients with transfusion requirements due to myelodysplasia and other marrow failure states. Tissue iron can be estimated by determining serum ferritin levels, measuring liver iron, and by measuring cardiac iron using the MRI-T2* technique. The standard form of chelation therapy is the slow intravenous or subcutaneous infusion of desferoxamine. An orally active bidentate iron chelator, deferiprone, is now licensed in 25 countries for treatment of patients with thalassemia major. Possibly because of the ability of this compound to cross membranes, it appears to have superior cardioprotective properties. Agranulocytosis is the most serious complication of deferiprone therapy and occurs in about 1% of treated patients. Deferiprone and desferoxamine can be given together or on alternating schedules. A new orally active chelating agent ICL 670 seems promising in early clinical studies. In Section III, Dr. James Cook discusses the most common disorder of iron homeostasis, iron deficiency. He will compare some of the standard methods for identifying iron deficiency, the hemoglobin level, transferrin saturation, and mean corpuscular hemoglobin and compare these with some of the newer methods that have been introduced, specifically the percentage of hypochromic erythrocytes and reticulocyte hemoglobin content. The measurement of storage iron is achieved by measuring serum ferritin levels. The soluble transferrin receptor is a truncated form of the cellular transferrin receptor and the possible value of this measurement in the diagnosis of iron deficiency will be discussed. Until recently iron dextran was the only parental iron preparation available in the US. Sodium ferric gluconate, which has been used extensively in Europe for many years, is now available in the United States. It seems to have a distinct advantage over iron dextran in that anaphylactic reactions are much less common with the latter preparation.

<h4>Background</h4>Human genome dynamic mutations are a new class of gene mutations represented by an unstable number of trinucleotide repeats and causing severe human hereditary neuromuscular and neurodegenerative diseases. The identification of pathological expanded alleles on the molecular level is important for clinical diagnostics.<h4>Methods and results</h4>For the molecular diagnostics of expanded tandem repeat trinucleotide sequences we have introduced a fast and efficient TP-PCR fluorescent method according to Warner et al. (1996). We have modified this TP-PCR method for a rapid detection of expanded CTG alleles of the DMPK gene (myotonic dystrophy, MD) into a two-level protocol; first, the heterozygote sample DNAs were selected using P1/P2 primers flanking repeat tracts and, second, the TP-PCR protocol used was focused above all on the identification of a pathological allele. A fluorescent-labelled specific primer in TP-PCR was used for the exact determination of the number of CAG repeats of the gene IT-15 (Huntington's disease--HD) in the diagnostically important region of the grey zone (35 to 39 CAG). The reproducibility of the PCR results was demonstrated on control DNA samples with the known genotype and, in the case of MD, also by Southern blot analysis. We have especially shown the possibility of a cheaper PCR-P1/P2 and TP-PCR protocol, which can be used, with silver staining of separated PCR products on polyacrylamide gels.<h4>Conclusions</h4>Our experience with introducing the above-mentioned PCR methods into laboratory practice clearly documents the possibilities of their general applicability in the molecular diagnostics of hereditary diseases characterised by instability of the trinucleotide repeat tracts.

The occurrence of the C282Y and H63D mutations of the HFE gene, responsible for toxic iron overload in the liver (hereditary hemochromatosis), was still unknown in Tunisia. We report the screening of 194 chromosomes from 97 randomly collected cord blood samples. The mutations were analyzed by PCR followed by DNA sequencing. The mild H63D and the severe C282Y mutations were found in 17.5+/-5.34% and 0.5+/-0.97% of the alleles, respectively. The allele frequency of the IVS 2+4 T --> C polymorphism is high (46.4+/-7.01%) in this population. Risk for homozygosity for the severe C282Y mutation is present in the Tunisian population at a low theoretical incidence. However, due to the relatively high rate of consanguinity in the country, liver pathology due to HH is not to be disregarded.

The aim of this study was to analyze the role of HFE mutations in blood donors with iron parameters suggesting iron overload, taking into account the regional distribution of HFE mutations in Italy. We studied 5880 subjects undergoing evaluation for blood donation eligibility, from different areas of Italy. Abnormal iron parameters were defined as transferrin saturation (TS) >50% or >45% and serum ferritin (SF) >300 or >250 microg/ml in males and females, respectively. Subjects with increased TS and/or SF were re-tested and typed for HFE mutations C282Y and H63D. A total of 548 individuals had increased iron parameters at first testing. In total, 179/548 were available for retesting, and in 109 increased TS and/or SF were confirmed. Increased TS was confirmed in 25 individuals, among whom three were C282Y homozygotes and six were compound heterozygotes for C282Y and H63D. Increased TS was more frequent in northern Italy than in southern regions. In individuals with increased TS and/or SF, the frequency of C282Y and H63D was 0.13 and 0.21 in northern-Italy versus 0.05 and 0.45 in southern Italy (P=0.004 for H63D). Nine out of 10 individuals carrying hemochromatosis-associated genotypes (including compound heterozygosity for C282Y and H63D) originated from northern regions. Among controls, the allelic frequencies of C282Y and H63D were 0.037 and 0.16 in the northern regions and 0.015 and 0.16 in the southern regions. In conclusion, over one-third of individuals with persistently altered TS carried hemochromatosis-associated genotypes, confirming that a diagnostic approach based on TS and genotyping of selected cases may represent a viable screening procedure.

The aim of this prospective study was to investigate the diagnostic value of plasma D-dimer levels and antithrombin-III activity in predicting prosthetic valve thrombus. The study group comprised 97 consecutive patients with prosthetic heart valves (59 with mitral, 21 with aortic, and 17 with both mitral and aortic prostheses) and 35 healthy control subjects. Six patients presented with symptoms of obstruction; the remaining 91 were asymptomatic. Patients were evaluated by both transthoracic and transesophageal echocardiography. Asymptomatic nonobstructive thrombus was detected in 13 patients (13%), whereas obstructive thrombus was demonstrated in all symptomatic patients. Plasma antithrombin-III levels of patients with prosthetic valve thrombi were slightly lower than those of patients without thrombus and of the control group, but the difference was not statistically significant. However, significantly higher plasma D-dimer levels were observed in patients with prosthetic valve thrombi, compared with patients without thrombus and the control group (735 +/- 633 microg/L, 372 +/- 342 microg/L, and 228 +/- 219 microg/L, respectively). Valve thrombus, the prosthetic heart valve itself, and INR levels were identified as major determinants of plasma D-dimer levels. A plasma D-dimer level of >445 microg/L predicted the presence of a prosthetic valve thrombus with 57.8% sensitivity and 83.3% specificity (positive predictive value, 47.8%; negative predictive value, 87.8%). Current data suggest that increased plasma D-dimer levels can be clinically helpful in predicting the presence of prosthetic valve thrombus. Plasma antithrombin-III activity does not seem to have a diagnostic value in predicting prosthetic valve thrombi.

Hereditary hemocromatosis (HH) is a genetic disease with a recessive autosomic pattern, in which inadequate iron (Fe) absorption is made by the intestinal cell. As consequence of that process, takes place a progressive accumulation of metal in different organs, predominantly in the liver. This leads to an alteration of liver structure and function: cirrhosis and hepatocarcinoma (1). The gene implied in this pathology was identified (HFE) in 1996. This codes a similar molecule to the mayor histocompatibility complex type 1(MHC-T1 like) that can modulate the transport of PE binding the transferrin receptor. This progress allows a deep understanding of the molecular and cellular biology of the homeostasis of the Fe and its alterations in the NH. The diagnosis of disease by means of a genetic test let to carry out a familiar screening and to detect asymptomatic carriers. This makes possible to begin the appropriate treatment at early stages of the disease in order to avoid its consequences and offering a better quality of life to these patients.

Genetic aberrations in radiation-associated colorectal cancer have not been studied in detail. We analyzed genetic aberrations in five rectal cancers that developed long after radiotherapy had been performed for cervical cancer. Microsatellite instability (MSI) in tumors was examined at five loci: D2S123, D3S966, TP53, DCC, and BAT26. Mutation of simple repeat sequences within the hMSH3, BAX, and transforming growth factor Beta type II receptor ( TGFBetaRII) genes was examined by polymerase chain reaction and single-strand conformation polymorphism (PCR-SSCP). Mutation of p53 exons 5-8 was examined by PCR-SSP and direct sequencing. Mutations of the K- ras gene were analyzed by two-step PCR. No MSI was found in tumor specimens at any of the loci examined, and no mutations in the target genes were observed. K- ras mutation was detected in two carcinomas, but not in their irradiated normal mucosa, while p53 mutation was observed in another two carcinomas, but not in their irradiated normal mucosa. Our results suggest that the radiation-associated rectal carcinomas examined in this study did not develop through the mutator phenotype pathway; rather, tumorigenesis was probably mediated through the multistep carcinogenesis pathway.

Ovarian cancer has a high mortality rate largely due to the limited number of ovarian carcinomas detected at an early stage. Understanding the molecular changes occurring during the progression of ovarian carcinoma would aid in the development of therapies that may inhibit or target metastasis. Primary and metastatic lesions from 54 and 40 patients with advanced ovarian carcinoma, respectively (including matched primary and metastatic lesions from 30 patients) were evaluated for nuclear accumulation of p53 (clone BP53-12-1) and cytoplasmic and membranous immunostaining of p185 erbB-2 (clone 3B5) by immunohistochemistry. No differences in the immunostaining of p53 and p185erbB-2 (cytoplasm or membrane) were observed between primary and metastatic lesions of the matched cases. Similarly, no differences in the proportion of positive cases of p53 between primary and metastatic lesions of the matched cases was observed. Thus, novel therapies that target p53 or p185erbB-2 can utilize specimens from either primary or metastatic lesions to characterize these targets prior to therapy. Spearman correlations between p53 and p185erbB-2 (cytoplasm or membrane) immunohistochemistry scores were insignificant for the matched cases, all primary lesions, and all metastatic lesions. Also, no significant associations occurred between nuclear accumulation of p53 (positive versus negative) and phenotypic expression of p185erbB-2 (cytoplasm or membrane) immunostaining scores for the matched cases, all primary lesions, and all metastatic lesions. Thus, the nuclear accumulation of p53 and immunostaining of p185erbB-2 in the cytoplasm or on the cellular membranes are independent.

Conserved segments have been identified by ZOO-FISH between pig chromosome 9 (SSC9) and human chromosomes 1, 7 and 11. To assist in the identification of positional candidate genes for QTL on SSC9, the comparative map was further developed. Primers were designed from porcine EST sequence homologous to genes in regions of human chromosomes 1, 7 and 11. Porcine ESTs were then physically assigned using the INRA somatic cell hybrid panel (INRASCHP) and the high-resolution radiation hybrid panel (IMpRH). Seventeen genes (PEPP3, RAB7L1, FNBP2, MAPKAPK2, GNAI1, ABCB1, STEAP, AKAP9, CYP51A1, SGCE, ROBO4, SIAT4C, GLUL, CACNA1E, PTGS2, C1orf16 and ETS1) were mapped to SSC9, while GUSB, CPSF4 and THG-1 were assigned to SSC3.

Primary or secondary pulmonary hypertension (PH) is an unexplained condition whose severe forms in adults or neonates are fatal and for which no satisfactory treatment is available. Considerable progress has been achieved over the last few years in our understanding of this disease, both through genetic studies that have identified mutations in the gene for BMP-RII (bone morphogenetic protein receptor II) in patients with familial primary PH (PPH) and through pathophysiological investigations that have elucidated the molecular mechanisms involved in hypertrophic arterial wall remodelling, most notably the role for the serotonin transporter (5-HTT) in hyperplasia of pulmonary artery smooth muscle cells (PA-SMCs). Identification of the BMP (bone morphogenetic protein) pathway as relevant to the aetiology of PPH now raises many questions about the link between the BMP-RII mutant genotype and the PPH phenotype. That PPH does not develop in all subjects with BMP-RII mutations suggests a crucial role for environmental or associated genetic factors. Simultaneously, mechanistic studies investigating the biological processes that underlie the complex vascular changes associated with PPH have identified major molecular pathways involved in constriction and proliferation of pulmonary vascular smooth muscle cells, dysfunction of endothelial cells, and remodelling of extracellular matrix. Such mechanisms may be involved either in initiating or in perpetuating the disease. The finding that genetic polymorphism of some of the candidate genes related to these processes is closely associated with PPH suggests a causal relationship between the expression, or function, of these genes and the PPH phenotype. The association between PPH and the L allelic variant of the serotonin transporter (5-HTT) gene indicates that 5-HTT, which controls smooth muscle hyperplasia, probably contributes to susceptibility to PPH or is an important modifier of the PPH phenotype. Recognition of these molecular pathways should provide insight into the pathogenesis not only of primary PH, but also of secondary forms of PH. This should lead soon to the development of new and more selective therapeutic approaches to pulmonary hypertension.

In an attempt to integrally investigate the loss of tumor suppressor genes and search for putative suppressor loci associated with tumor occurrence and progression, we conducted a genome-wide loss of hetero zygosity (LOH) study of 83 tumor samples obtained from Chinese patients with sporadic colorectal cancer. We employed 400 fluorescence-labeled microsatellite marker primers to amplify the corresponding loci of the genomic DNA and then electrophoresed the polymerase chain reaction products and analyzed the fluorescent signals. The LOH frequencies were high (>35%) but were not associated with the tumor stage and progression in 20 loci, including the regions where TP53, E-cadherin, deleted in colorectal carcinoma (DCC), phosphatase and tensin homolog deleted on chromosome 10 (PTEN), mothers against decapentaplegic, Drosophila, homolog of 2 (MADH2) and mothers against decapentaplegic, Drosophila, homolog of 4 (MADH4) reside. Loss of other loci, including two narrow regions on chromosome 2, was found to relate to the tumor stage, suggesting that this genomic instability may contribute to tumor progression.

Genetic issues are important in the primary care of adolescents. A genetic diagnosis may not be made until adolescence, when the teenager presents with the first signs or symptoms of the condition. The physician's knowledge of the natural history of a genetic disease will aid in the anticipatory guidance for teens and their parents. The physician may be called upon to advise the patient regarding hormone therapy or contraception. The paediatrician may initiate topics such as sexuality and sex education for the developmentally delayed patient. The paediatrician is also the advocate for the teenager, who must gain independence from the family in medical as well as other aspects of life. This article examines some of these issues, using cases to illustrate the genetic problems and approaches in the care of the teenaged patient.

This chapter focuses on non-HIV antiviral agents. The development of antiviral agents to treat non-HIV infections is largely focused on therapies for the treatment of chronic hepatitis infections B and C. Nucleoside analog continue to be the mainstay of Hepatitis B Virus (HBV) therapeutics. The first small molecule inhibitor of Hepatitis C Virus (HCV), the NS3 protease inhibitor BILN-2061, entered phase 2 clinical trials, producing a striking reduction in viral load in treated individuals. The development of the HCV replicon system and its application to screening for antiviral agents provided tangible benefit with the disclosure of mechanistically and structurally diverse HCV inhibitors. Adefovir dipivoxil has been approved in the United States and the European Union for the treatment of HBV, providing a second small molecule antiviral to add to lamivudine (3TC) and the injectable protein IFNα as the only approved agents for treating HBV infection. The chapter also provides details of the inhibitors of hepatitis B and C virus, the inhibitors of simplex virus and human cytomegalovirus, the inhibitors of respiratory viruses and the inhibitors of West Nile virus and Papilloma virus.

No abstract available.

No abstract available.