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Condensin is a protein complex associated with mitotic chromosomes that has been implicated in chromosome condensation. In vertebrates, two types of condensin complexes have recently been identified, called condensin I and II. Here, we show that in mammalian cells condensin II associates with chromatin in prophase, in contrast to condensin I which is cytoplasmic and can thus interact with chromosomes only after nuclear envelope breakdown. RNA interference experiments in conjunction with imaging of live and fixed cells revealed that condensin II is required for chromosome condensation in early prophase, whereas condensin I appears to be dispensable at this stage. By contrast, condensin I is required for the complete dissociation of cohesin from chromosome arms, for chromosome shortening and for normal timing of progression through prometaphase and metaphase, whereas normal condensin II levels are dispensable for these processes. After depletion of both condensin complexes, the onset of chromosome condensation is delayed until the end of prophase, but is then initiated rapidly before nuclear envelope breakdown. These results reveal that condensin II and I associate with chromosomes sequentially and have distinct functions in mitotic chromosome assembly.

Identifying evolutionarily conserved blocks in orthologous genomic sequences is an effective way to detect regulatory elements. In this study, with the aim of elucidating the architecture of the regulatory network, we systematically estimated the degree of conservation of the upstream sequences of 3,750 human-mouse orthologue pairs along 8-kb stretches. We found that the genes with high upstream conservation are predominantly transcription factor (TF) genes. In particular, developmental process-related TF genes showed significantly higher conservation of the upstream sequences than other TF genes. Such extreme upstream conservation of the developmental process-related TF genes suggests that the regulatory networks involved with developmental processes have been evolutionarily well conserved in both human and mouse lineages.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most worldwide frequent primary carcinomas resulting in the death of many cirrhotic patients. Unfortunately, the molecular mechanisms of this cancer are not well understood; therefore, we need a good model system to study HCC. The dog is recognized as a promising model for human medical research, namely compared with rodents. The objective of this study was to establish and characterize a spontaneous canine tumor cell line as a potential model for studies on HCC. RESULTS: Histomorphological, biochemical, molecular biological and quantitative assays were performed to characterize the canine HCC cell line that originated from a dog with a spontaneous liver tumor. Morphological investigations provided strong evidence for the hepatocytic and neoplastic nature of the cell line, while biochemical assays showed that they produced liver-specific enzymes. PCR analysis confirmed expression of ceruloplasmin, alpha-fetoprotein and serum albumin. Quantitative RT-PCR showed that the canine HCC cell line resembles human HCC based on the measurements of expression profiles of genes involved in cell proliferation and apoptosis. CONCLUSIONS: We have developed a novel, spontaneous tumor liver cell line of canine origin that has many characteristics of human HCC. Therefore, the canine HCC cell line might be an excellent model for comparative studies on the molecular pathogenesis of HCC.

CarBaBb, the class III extradiol dioxygenase involved in carbazole degradation by Pseudomonas resinovorans CA10, was crystallized at 278 K by the hanging-drop vapour-diffusion method using PEG MME 550 as a precipitant. The crystals had a transparent thin square-pillar shape and belonged to space group P2(1)2(1)2, with unit-cell parameters a = 122.8, b = 144.6, c = 49.2 A, alpha = beta = gamma = 90 degrees . The crystals diffracted to a maximum resolution of 1.9 A and gave a data set with an overall R(merge) of 5.7% and a completeness of 98.6%. The V(M) value was 2.52 A(3) Da(-1), which indicated a solvent content of 51.2%.

In this study, measures of the quality and availability of social supports were found to moderate risk for depression associated with a history of maltreatment and the presence of the short (s) allele of the serotonin transporter gene promoter polymorphism (5-HTTLPR). The present investigation (i) replicates research in adults showing that 5-HTTLPR variation moderates the development of depression after stress, (ii) extends the finding to children, and (iii) demonstrates the ability of social supports to further moderate risk for depression. Maltreated children with the s/s genotype and no positive supports had the highest depression ratings, scores that were twice as high as the non-maltreated comparison children with the same genotype. However, the presence of positive supports reduced risk associated with maltreatment and the s/s genotype, such that maltreated children with this profile had only minimal increases in their depression scores. These findings are consistent with emerging preclinical and clinical data suggesting that the negative sequelae associated with early stress are not inevitable. Risk for negative outcomes may be modified by both genetic and environmental factors, with the quality and availability of social supports among the most important environmental factors in promoting resiliency in maltreated children, even in the presence of a genotype expected to confer vulnerability for psychiatric disorder.

BACKGROUND: Alterations in carbohydrate metabolism are frequently observed in cirrhosis. We conducted this study to define the prevalence of diabetes mellitus (DM) and impaired glucose tolerance (IGT) in Iranian patients with chronic liver disease (CLD), and explore the factors associated with DM in these patients. METHODS: One hundred and eighty-five patients with CLD were enrolled into the study. Fasting plasma glucose and two-hour plasma glucose were measured in patients' sera. DM and IGT were diagnosed according to the latest American Diabetes Association criteria. RESULTS: The subjects included 42 inactive HBV carriers with a mean age of 42.2 +/- 12.0 years, 102 patients with HBV or HCV chronic hepatitis with a mean age of 41.2 +/- 10.9 years, and 41 cirrhotic patients with a mean age of 52.1 +/- 11.4 years. DM and IGT were diagnosed in 40 (21.6%) and 21 (11.4%) patients, respectively. Univariate analysis showed that age (P = 0.000), CLD status (P = 0.000), history of hypertension (P = 0.007), family history of DM (P = 0.000), and body mass index (BMI) (P = 0.009) were associated with DM. Using Multivariate analysis, age (OR = 4.7, 95%CI: 1.8-12.2), family history of DM (OR = 6.6, 95%CI: 2.6-17.6), chronic hepatitis (OR = 11.6, 95%CI: 2.9-45.4), and cirrhosis (OR = 6.5, 95%CI: 2.4-17.4) remained as the factors independently associated with DM. When patients with cirrhosis and chronic hepatitis were analyzed separately, higher Child-Pugh's score in cirrhotic patients (OR = 9.6, 95%CI: 1.0-88.4) and older age (OR = 7.2, 95%CI: 1.0-49.1), higher fibrosis score (OR = 59.5, 95%CI: 2.9-1211.3/ OR = 11.9, 95%CI: 1.0-132.2), and higher BMI (OR = 30.3, 95%CI: 3.0-306.7) in patients with chronic hepatitis were found to be associated with higher prevalence of DM. CONCLUSIONS: Our findings indicate that patients with cirrhosis and chronic hepatitis are at the increased risk of DM occurrence. Older age, severe liver disease, and obesity were associated with DM in these patients.

Experimental arthritis models are considered valuable tools for delineating mechanisms of inflammation and autoimmune phenomena. Use of microarray-based methods represents a new and challenging approach that allows molecular dissection of complex autoimmune diseases such as arthritis. In order to characterize the temporal gene expression profile in joints from the reactivation model of streptococcal cell wall (SCW)-induced arthritis in Lewis (LEW/N) rats, total RNA was extracted from ankle joints from naive, SCW injected, or phosphate buffered saline injected animals (time course study) and gene expression was analyzed using Affymetrix oligonucleotide microarray technology (RAE230A). After normalization and statistical analysis of data, 631 differentially expressed genes were sorted into clusters based on their levels and kinetics of expression using Spotfire profile search and K-mean cluster analysis. Microarray-based data for a subset of genes were validated using real-time PCR TaqMan analysis. Analysis of the microarray data identified 631 genes (441 upregulated and 190 downregulated) that were differentially expressed (Delta > 1.8, P < 0.01), showing specific levels and patterns of gene expression. The genes exhibiting the highest fold increase in expression on days -13.8, -13, or 3 were involved in chemotaxis, inflammatory response, cell adhesion and extracellular matrix remodelling. Transcriptome analysis identified 10 upregulated genes (Delta > 5), which have not previously been associated with arthritis pathology and are located in genomic regions associated with autoimmune disease. The majority of the downregulated genes were associated with metabolism, transport and regulation of muscle development. In conclusion, the present study describes the temporal expression of multiple disease-associated genes with potential pathophysiological roles in the reactivation model of SCW-induced arthritis in Lewis (LEW/N) rat. These findings improve our understanding of the molecular events that underlie the pathology in this animal model, which is potentially a valuable comparator to human rheumatoid arthritis (RA).

The purpose of this study was to characterize the binding properties of serotonin transporter (5-HTT) in the brain of the patients with generalized anxiety disorder (GAD) in comparison to healthy subjects using single photon emission computer tomography (SPECT) with the radioligand [123I]nor-beta-CIT. The subjects were 7 patients with GAD and 7 matched healthy volunteers. The regions of interest (ROI) were the midbrain and the thalamus. The comparison of the volumes of distribution did not show significant differences between the patients and controls in the binding of nor-beta-CIT to 5-HTT in the ROI. Binding of 5-HTT in the midbrain of patients was significantly and negatively correlated with their anxiety levels measured by the visual analogue scale immediately before the first scan (r=-0.79, p=0.035). This study failed to demonstrate an altered functional activity of 5-HTT in patients with GAD when compared with controls.

Selective serotonin-reuptake inhibitors (SSRIs) antagonize the serotonin (5-hydroxytryptamine) transporter (5-HTT), and are frequently prescribed to children and adolescents to treat depression. However, recent findings of functional serotonergic pathways in bone cells and preliminary clinical evidence demonstrating detrimental effects of SSRIs on bone growth have raised questions regarding the effects of these drugs on the growing skeleton. The current work investigated the impact of 5-HTT inhibition on the skeleton in: 1) mice with a null mutation in the gene encoding for the 5-HTT; and 2) growing mice treated with a SSRI. In both models, 5-HTT inhibition had significant detrimental effects on bone mineral accrual. 5-HTT null mutant mice had a consistent skeletal phenotype of reduced mass, altered architecture, and inferior mechanical properties, whereas bone mineral accrual was impaired in growing mice treated with a SSRI. These phenotypes resulted from a reduction in bone formation without an increase in bone resorption and were not influenced by effects on skeletal mechanosensitivity or serum biochemistries. These findings indicate a role for the 5-HTT in the regulation of bone accrual in the growing skeleton and point to a need for further research into the prescription of SSRIs to children and adolescents.

<h4>Background</h4>Type 2 diabetes (T2DM) patients are characterized by a very high risk of cardiovascular diseases. Among the factors that are responsible for this phenomenon are abdominal obesity and hemostatic abnormalities.<h4>Aim of the study</h4>To examine the association of the markers of coagulation and fibrinolysis with the parameters of abdominal obesity and metabolic compensation in T2DM patients.<h4>Methods</h4>46 T2DM patients participated in the study: 24 men (mean age 61.1 +/- 7.9 years) and 22 postmenopausal women (mean age 62.6 +/- 8.7 years). In each patient the content and distribution of fatty tissue was measured by a dual energy X-ray absorptiometry method (DEXA). The central abdominal fat/gynoid hip fat (CAF/GF) ratio was calculated. The following hemostatic parameters were measured: fibrinogen (Fb), factor VII (fVII), antithrombin III (ATIII), C protein (pC), tissue plasminogen activator inhibitor (PAI-1) and alpha 2 antiplasmin (alpha2 AP). In addition, the biochemical indices of metabolic compensation were measured: HbA1c, glucose levels and lipids.<h4>Results</h4>Patients of both genders were divided according to median CAF/GF ratio. The activity of PAI-1 was significantly higher in women with CAF/GF ratio >or= 0.88 as compared to those with CAF/GF < 0.88 (2.64 +/- 1.28 vs. 1.61 +/- 0.27 U/ml, p < 0.05). The activity of ATIII was significantly lower in men with CAF/GF ratio >or= 1.17, as compared to those with CAF/GF < 1.17 (105.10 +/- 10.02 vs. 113.42 +/- 10.72 %, p < 0.05). There was a significant correlation between the CAF/GF ratio and the activity of PAI-1 in women (r = 0.30, p < 0.05). In addition, in men the CAF/GF ratio was negatively correlated with ATIII activity (r = -0.44, p < 0.05). Multiple stepwise regression analysis demonstrated independent association between the CAF/ GF ratio and the activity of PAI-1 (p < 0.001), and between the CAF/GF ratio and the activity of alpha2 AP (p < 0.01). There was an independent association between the concentration of HbA1c and the concentration of Fb (p < 0.001) and between triglycerides and the activity of fVII (p < 0.01).<h4>Conclusions</h4>The results of our study show that the patients with T2DM and with higher markers of abdominal obesity measured by DEXA show fibrinolysis impairment and thrombinogenesis elevation compared to those with lower abdominal obesity markers. Independent factors determining hypercoagulation also include metabolic control and lipids. Hemostatic disorders place subjects with diabetes and abdominal obesity at risk of developing vascular complications.

Clinical benefits from monoclonal antibody therapy for B-chronic lymphocytic leukemia (B-CLL) have increased interest in developing additional immunotherapies for the disease. CD40 ligand is an accessory signal for T-cell activation and can overcome T-cell anergy. The OX40-OX40 ligand pathway is involved in the subsequent expansion of memory antigen-specific T cells. We expressed both CD40L and OX40L on B-CLL cells by exploiting the phenomenon of molecular transfer from fibroblasts overexpressing these ligands. We analyzed the effects of the modified B-CLL cells on the number, phenotype, and cytotoxic function of autologous T cells in 7 B-CLL patients. Transfer of CD40L and OX40L was observed in all and was followed by the up-regulation of B7-1 and B7-2. The culture of CD40L/OX40L-expressing B-CLL cells with autologous T cells generated CD4+/CD8+ cytotoxic T-cell lines, which secreted interferon-gamma (IFN-gamma) and granzyme-B/perforin in response to autologous, but not to allogeneic, B-CLL cells or to autologous T-cell blasts. CD40L or OX40L alone was insufficient to expand tumor-reactive T cells. The combination of CD40L and OX40L on B-CLL cells may allow the generation of therapeutic immune responses to B-CLL, either by active immunization with modified tumor cells or by adoptive immunotherapy with tumor-reactive autologous T cells.

<h4>Introduction</h4>We showed in a previous study that prenylated proteins play a role in estradiol stimulation of proliferation. However, these proteins antagonize the ability of estrogen receptor (ER) alpha to stimulate estrogen response element (ERE)-dependent transcriptional activity, potentially through the formation of a co-regulator complex. The present study investigates, in further detail, how prenylated proteins modulate the transcriptional activities mediated by ERalpha and by ERbeta.<h4>Methods</h4>The ERE-beta-globin-Luc-SV-Neo plasmid was either stably transfected into MCF-7 cells or HeLa cells (MELN cells and HELN cells, respectively) or transiently transfected into MCF-7 cells using polyethylenimine. Cells deprived of estradiol were analyzed for ERE-dependent luciferase activity 16 hours after estradiol stimulation and treatment with FTI-277 (a farnesyltransferase inhibitor) or with GGTI-298 (a geranylgeranyltransferase I inhibitor). In HELN cells, the effect of prenyltransferase inhibitors on luciferase activity was compared after transient transfection of plasmids coding either the full-length ERalpha, the full-length ERbeta, the AF-1-deleted ERalpha or the AF-2-deleted ERalpha. The presence of ERalpha was then detected by immunocytochemistry in either the nuclei or the cytoplasms of MCF-7 cells. Finally, Clostridium botulinum C3 exoenzyme treatment was used to determine the involvement of Rho proteins in ERE-dependent luciferase activity.<h4>Results</h4>FTI-277 and GGTI-298 only stimulate ERE-dependent luciferase activity in stably transfected MCF-7 cells. They stimulate both ERalpha-mediated and ERbeta-mediated ERE-dependent luciferase activity in HELN cells, in the presence of and in the absence of estradiol. The roles of both AF-1 and AF-2 are significant in this effect. Nuclear ERalpha is decreased in the presence of prenyltransferase inhibitors in MCF-7 cells, again in the presence of and in the absence of estradiol. By contrast, cytoplasmic ERalpha is mainly decreased after treatment with FTI-277, in the presence of and in the absence of estradiol. The involvement of Rho proteins in ERE-dependent luciferase activity in MELN cells is clearly established.<h4>Conclusions</h4>Together, these results demonstrate that prenylated proteins (at least RhoA, RhoB and/or RhoC) antagonize the ability of ERalpha and ERbeta to stimulate ERE-dependent transcriptional activity, potentially acting through both AF-1 and AF-2 transcriptional activities.

Hereditary hemochromatosis (HH) is an autosomal recessive disorder of iron metabolism characterized by increased iron absorption and progressive storage resulting in organ damage. HFE gene mutations C282Y and H63D are responsible for the majority of HH cases. A third HFE mutation, S65C, has been associated with the development of a mild form of hemochromatosis. The beta-thalassemia trait is characterized by mild, ineffective erythropoiesis that can induce excess iron absorption and ultimately lead to iron overload. The aim of this study was to evaluate the effect of genetic markers (HFE mutations C282Y, H63D, and S65C) on the iron status of beta-thalassemia carriers. A total of 101 individuals heterozygous for beta-thalassemia and 101 normal control individuals were studied. The allelic frequencies of C282Y (1.5 versus 3.5%), H63D (15.3 versus 18.3%), and S65C (1.0 versus 1.5%) did not differ significantly between beta-thalassemia carriers and normal controls. Serum iron (P=0.029) and transferrin saturation (P=0.009) were increased in beta-thalassemia carriers heterozygous for H63D mutation. The number of subjects carrying C282Y or S65C mutations was too low to conclude their effect on the iron status. These results suggest that the beta-thalassemia trait tends to be aggravated with the coinheritance of H63D mutation, even when present in heterozygosity.

Hereditary hemochromatosis (HH) is an autosomal recessive disease that leads to parenchymal iron accumulation. The most common form of HH is caused by a single amino acid substitution in the HH protein, HFE, but the mechanism by which HFE regulates iron homeostasis is not known. In the absence of transferrin (Tf), HFE interacts with transferrin receptor 1 (TfR1) and the 2 proteins co-internalize, and in vitro studies have shown that HFE and Tf compete for TfR1 binding. Using a cell line lacking endogenous transferrin receptors (TRVb cells) transfected with different forms of HFE and TfR1, we demonstrate that even at low concentrations Tf competes effectively with HFE for binding to TfR1 on living cells. Transfection of TRVb cells or the derivative line TRVb1 (which stably expresses human TfR1) with HFE resulted in lower ferritin levels and decreased Fe2+ uptake. These data indicate that HFE can regulate intracellular iron storage independently of its interaction with TfR1. Earlier studies found that in HeLa cells, HFE expression lowers Tf-mediated iron uptake; here we show that HFE lowers non-Tf-bound iron in TRVb cells and add to a growing body of evidence that HFE may play different roles in different cell types.

The centromere sequence parC of Escherichia coli low-copy-number plasmid R1 consists of two sets of 11 bp iterated sequences. Here we analysed the intrinsic sequence-directed curvature of parC by its migration anomaly in polyacrylamide gels. The 159 bp long parC is strongly curved with anomaly values (k-factors) close to 2. The properties of the parC curvature agree with those of other curved DNA sequences. parC contains two regions of 5-fold repeated iterons separated by 39 bp. We modified 4 bp within this intermediate sequence so that we could analyse the two 5-fold repeated regions independently. The analysis shows that the two repeat regions are not independently curved parts of parC but that the overall curvature is a property of the whole fragment. Since the centromere sequence of an E.coli plasmid as well as eukaryotic centromere sequences show DNA curvature, we speculate that curvature might be a general property of centromeres.

Expression rates of long (L) and short (S) alleles of the serotonin (5-HT) transporter (5-HTT) gene have been shown to differ under various circumstances. We compared 5-HTT uptake (function) level and paroxetine binding (density) in platelets of alcoholics as indices of 5-HTT expression rate among LL, LS, and SS genotypes. Concentration curves of [3H]5-HT and [3H]paroxetine were used to quantify the equilibrium constant (Km) and maximum 5-HT uptake rate (Vmax) for 5-HTT uptake into intact platelets and the dissociation constant (Kd) and maximum specific binding density (Bmax) for paroxetine binding to platelet membranes, respectively. Genotypes were determined using electrophoresis with fluorescent markers. Vmax for 5-HTT uptake did not correlate with Bmax for paroxetine binding (r=-0.095, P=0.415). Means of Vmax and Bmax did not differ in a statistically significant manner among LL, LS, and SS genotypes in these alcoholic subjects. However, Vmax for LL and SS appeared to have a bimodal distribution, so the percentage of subjects with Vmax <200 fmol/min-10(7) platelets was statistically significantly higher in LL than in SS (51.5% vs. 22.7%, respectively), with an odds ratio of 3.6 (P<0.05). The percentage of Vmax <200 fmol/min-10(7) platelets for LS was 39.3% (not significant vs. LL or SS). Previous studies of healthy human controls have shown that 5-HTT density in raphe nuclei and 5-HTT uptake in platelets are higher in the LL genotype than in S carriers. Our findings in currently drinking alcoholics support the hypothesis that those with the LL genotype of the 5'-HTTLPR region of the 5-HTT gene have reduced 5-HTT function.

<h4>Purpose</h4>Recent discoveries in molecular mechanisms of iron metabolism have changed the classical view of hereditary iron overload conditions. We present natural mutations in newly discovered genes and related phenotypes observed in patients with different form of haemochromatosis.<h4>Current knowledge and key points</h4>Most haemochromatosis patients are homozygous for the C282Y mutation in the HFE gene. Ferroportin, TFR2, hemojuvelin and hepcidin mutations also cause iron overload. Recent data support the hypothesis that haemochromatosis should no longer be considered a monogenic disease but rather an oligogenic disorder. Several results suggest that haemochromatosis could result from digenic inheritance of mutations in HFE and HAMP.<h4>Future prospects and projects</h4>Other modifier genes probably influence penetrance in C282Y homozygous patients. Such genes could enhance or reduce the phenotypic expression in various iron overload conditions.

Staufen is a conserved double-stranded RNA-binding protein required for mRNA localization in Drosophila oocytes and embryos. The mammalian homologues Staufen 1 and Staufen 2 have been implicated in dendritic RNA targeting in neurons. Here we show that in rodent oligodendrocytes, these two proteins are present in two independent sets of RNA granules located at the distal myelinating processes. A third kind of RNA granules lacks Staufen and contains major myelin mRNAs. Myelin Staufen granules associate with microfilaments and microtubules, and their subcellular distribution is affected by polysome-disrupting drugs. Under oxidative stress, both Staufen 1 and Staufen 2 are recruited into stress granules (SGs), which are stress-induced organelles containing transiently silenced messengers. Staufen SGs contain the poly(A)-binding protein (PABP), the RNA-binding proteins HuR and TIAR, and small but not large ribosomal subunits. Staufen recruitment into perinuclear SGs is paralleled by a similar change in the overall localization of polyadenylated RNA. Under the same conditions, the distribution of recently transcribed and exported mRNAs is not affected. Our results indicate that Staufen 1 and Staufen 2 are novel and ubiquitous SG components and suggest that Staufen RNPs are involved in repositioning of most polysomal mRNAs, but not of recently synthesized transcripts, during the stress response.

With the discovery that transferrin serves as the iron source for hemoglobin-synthesizing immature red blood cells came the demonstration that a cell surface receptor, now known as transferrin receptor 1, is required for iron delivery from transferrin to cells. (A recently described second transferrin receptor, with as yet poorly understood function, will not be discussed in this brief review.) In succeeding years transferrin receptor 1 was established as a gatekeeper for regulating iron uptake by most cells, and the transferrin-to-cell endocytic pathway characterized in detail. HFE, the protein incriminated in the pathogenesis of hereditary hemochromatosis, a disorder of progressive and toxic iron overload, competes with transferrin for binding to receptor, thereby impeding the uptake of iron from transferrin. Mutation of HFE destroys this competition, thus facilitating access of transferrin and its iron to cells. Availability of the crystal structure of transferrin receptor 1, along with those of transferrin and HFE, opened research on molecular mapping of the transferrin-HFE- transferrin receptor interfaces by correlated synchrotron-generated hydroxyl radical footprinting and cryo-electron microscopy. The emerging challenge is to relate structure to the functional effects of receptor binding on the iron-binding and iron-releasing properties of transferrin within the iron-dependent cell.

Inherited antithrombin deficiency generally causes a predisposition toward vascular thrombus above the age of 15 years. A 1-year-old boy developed renal hypertension caused by renal artery obstruction due to thrombus formation. This thrombus formation was attributed to antithrombin deficiency caused by a novel SERPINCI gene mutation (AT III Akita, M352R). This suggests that antithrombin deficiency can cause renal artery obstruction, inducing renal hypertension through vascular thrombosis even in children.

Peroxiredoxins (PRDXs) form a family of peroxidases involved in antioxidant protection and cell signaling. Due to their peroxide reductase activity, these enzymes might be involved in fine-tuning peroxide levels in embryos during in vitro production. In this study, RT-PCR was used to examine the expression of the six PRDX isoforms (PRDX1 to PRDX6) in bovine oocytes and embryos. PRDXs were detected in oocytes both before and after in vitro maturation. Besides, PRDX6 was up-regulated after maturation. Single embryos were analyzed from the two-cell to the blastocyst stages. PRDX1 and PRDX5 transcripts were detected throughout development. PRDX2, PRDX3, and PRDX6 were not expressed around the 9- to 16-cell stage. PRDX4 transcripts were weakly detected in pools of embryos from the 9- to 16-cell stage onwards. In situ immunodetection of PRDX5, which was previously reported to exhibit the widest subcellular distribution among PRDXs in adult mammalian cells, showed a mitochondrial distribution pattern in the bovine embryo. Finally, the potential modulation by oxidative stress of PRDX expression around the major embryonic genome activation was evaluated by culturing embryos under 20% O2 instead of 5%. No significant difference in the pattern of PRDX expression was observed under 20% O2. In conclusion, our data show for the first time that PRDXs are expressed in mammalian oocytes and early embryos. Moreover, the bovine transcripts exhibit various patterns of expression that might be related to the potential role of PRDXs in oocyte maturation and embryo development.

Autoreactive T cells play a major role in the development of insulin-dependent diabetes mellitus, suggesting that costimulatory molecules that regulate T cell responses might be essential for disease progression. In NOD mice, CD28/B7 and CD40/CD40 ligand (L) interactions control the onset of diabetes from 2 to 4 weeks of age, but blocking these molecules has little effect after this time. Hence, it is possible that other ligand/receptor pairs control a later phase of disease. We now show that OX40 is expressed on CD4 and CD8 T cells several weeks prior to islet destruction, which is initiated around weeks 12-14, and that OX40L is present on dendritic cells in both secondary lymphoid organs and the pancreas from 11 to 13 weeks of age. Blocking OX40L at 6, 9, or 15 weeks after birth had little effect on disease; however, inhibiting OX40/OX40L interactions at week 12, or continuous treatment from week 12 onwards, significantly reduced the incidence of diabetes. Histological examination showed that islet destruction was prevented and insulitis reduced by targeting OX40L. These studies show that OX40/OX40L interactions form a late checkpoint in diabetes development and suggest that these molecules are realistic targets for therapeutic intervention.

Intrahepatic iron overload is commonly seen in chronic hepatitis C infection. High levels of intrahepatic iron may lead to accelerated liver injury and development of fibrosis and cirrhosis. This is frequently seen in hereditary hemochromatosis, which in most of the cases is caused by homozygous mutations in the HFE gene. In patients suffering from chronic hepatitis C, the presence of heterozygous HFE mutations associates with higher hepatic iron scores and advanced stages of fibrosis. HFE mutations must therefore be considered as important comorbidity factors in chronic hepatitis C infection.

<h4>Aim</h4>To investigate the expression of CD34 and c-kit (receptor of stem cell factor) in cholangiocarcinoma.<h4>Methods</h4>Fifteen cases of intrahepatic cholangiocarcinoma and 17 cases of extrahepatic cholangiocarcinoma were studied in this experiment. Using Envision detection system, paraffin-embedded sections of the resected cholangiocarcinoma tissue were stained with antibodies against CD34 and c-kit, respectively. The sections were counterstained with hematoxylin, and the results were examined under light microscope. Normal tonsil and mammary tissues were used as positive controls for CD34 and c-kit, respectively.<h4>Results</h4>CD34 was positive in all sections, but only in capillary endothelial cells of tumor tissue. No cholangiocarcinoma cells were positive for CD34. In one case of extrahepatic cholangiocarcinoma, a few tumor cells (about 5%) were immunoreactive with c-kit.<h4>Conclusion</h4>CD34 or c-kit positive cells in liver tissue may represent liver stem cells, as they can differentiate into mature biliary cells in vitro. The expression of c-kit by some cholangiocarcinoma cells suggests that cholangiocarcinoma might originate from liver stem cells. However, other mechanisms of hepatocarcinogenesis, such as de-differentiation of mature cholangiocytes, may also exist.

Peroxiredoxin 6 (Prdx6; also called antioxidant protein 2, or Aop2) is a candidate gene for Ath1, a locus responsible for the respective susceptibility and resistance of mouse strains C57BL/6J (B6) and C3H/HeJ (C3H) to diet-induced atherosclerosis. To evaluate if Prdx6 underlies Ath1, we compared the diet-induced atherosclerotic lesions in Prdx6 targeted mutant (Prdx6-/-) mice of different genetic backgrounds: B6, 129, and B6;129. PRDX6 protein and mRNA were expressed in normal and atherosclerotic aortas. B6;129 Prdx6-/- macrophages oxidized LDL significantly more than did controls. Plasma lipid hydroperoxide levels were higher in atherogenic diet-fed Prdx6-/- mice with B6;129 and B6 backgrounds than in controls. Prdx6-/- and controls in a 129 genetic background were equally lesion-resistant, and Prdx6-/- and controls in a B6 background were equally lesion-susceptible. In contrast, Prdx6-/- mice in a B6;129 background had significantly larger aortic root lesions than did littermate wild type controls. Therefore, although PRDX6 protein did not affect atherosclerosis susceptibility in either the resistant 129 background or the susceptible B6 background, it may inhibit atherosclerosis in backgrounds with mixed pro- and anti-atherogenic genes. Thus, genetic background plays an important role in modulating atherogenesis in targeted mutant mice. However, we think it is unlikely that Prdx6 underlies Ath1.

<h4>Objective</h4>Although DCC has been considered as a candidate tumor suppressor, the roles it plays in the uterine endometrium and in the carcinogenic process remains unclear. To define these roles more clearly, we examined the expression of DCC and its ligand, netrin-1, in the normal endometrium and in endometrial cancer.<h4>Methods</h4>The expression of DCC and netrin-1 in normal endometrial glands and in cancer cell lines was examined by RT-PCR and immunohistochemistry. The effects of exogenous DCC and netrin-1 expression were observed together with the respective expression vector transfection.<h4>Results</h4>Endometrial glands in the proliferative and early secretory phase expressed both DCC and netrin-1, but glands in the late-secretory phase tended to silence DCC expression. In addition, all of the endometrial cancer cell lines lost normal DCC expression. Restored DCC expression in the cancer cell lines in the absence of netrin-1 induced apoptosis. However, no changes were observed in the presence of netrin-1.<h4>Conclusion</h4>Our observations suggest that DCC/netrin-1 signaling may commit cells to the transition of endometrial gland architecture or function from a proliferating to a secretory phase. In addition, the silencing of DCC expression may contribute to the escape of endometrial cancer cells from a DCC-regulated apoptotic program, thereby promoting malignant phenotypes.

Localization of mRNA is an important way of generating early asymmetries in the developing embryo. In Drosophila, Staufen is intimately involved in the localization of maternally inherited mRNAs critical for cell fate determination in the embryo. We show that double-stranded RNA-binding Staufen proteins are present in the oocytes of a vertebrate, Xenopus, and are localized to the vegetal cytoplasm, a region where important mRNAs including VegT and Vg1 mRNA become localized. We identified two Staufen isoforms named XStau1 and XStau2, where XStau1 was found to be the principal Staufen protein in oocytes, eggs, and embryos, the levels of both proteins peaking during mid-oogenesis. In adults, Xenopus Staufens are principally expressed in ovary and testis. XStau1 was detectable throughout the oocyte cytoplasm by immunofluorescence and was concentrated in the vegetal cortical region from stage II onward. It showed partial codistribution with subcortical endoplasmic reticulum (ER), raising the possibility that Staufen may anchor mRNAs to specific ER-rich domains. We further showed that XStau proteins are transiently phosphorylated by the MAPK pathway during meiotic maturation, a period during which RNAs such as Vg1 RNA are released from their tight localization at the vegetal cortex. These findings provide evidence that Staufen proteins are involved in targeting and/or anchoring of maternal determinants to the vegetal cortex of the oocyte in Xenopus. The Xenopus oocyte should thus provide a valuable system to dissect the role of Staufen proteins in RNA localization and vertebrate development.

The past few years have seen remarkable progress in our understanding of embryonic stem cell (ES cell) biology. The necessity of examining human ES cells in culture, coupled with the wealth of genomic data and the multiplicity of cell lines available, has enabled researchers to identify critical conserved pathways regulating self-renewal and identify markers that tightly correlate with the ES cell state. Comparison across species has suggested additional pathways likely to be important in long-term self-renewal of ES cells including heterochronic genes, microRNAs, genes involved in telomeric regulation, and polycomb repressors. In this review, we have discussed information on molecules known to be important in ES cell self-renewal or blastocyst development and highlighted known differences between mouse and human ES cells. We suggest that several additional pathways required for self-renewal remain to be discovered and these likely include genes involved in antisense regulation, microRNAs, as well as additional global repressive pathways and novel genes. We suggest that cross species comparisons using large-scale genomic analysis tools are likely to reveal conserved and divergent paths required for ES cell self-renewal and will allow us to derive ES lines from species and strains where this has been difficult.

Hereditary hemochromatosis (HH) is associated with an increased risk for hepatocellular carcinoma (HCC). The risk previously had been estimated to be as high as 200-fold increased. Recent studies suggest that the risk for HCC in HFE -associated HH may be much lower and occurs predominantly in patients with cirrhosis at the time of diagnosis. The risk for HCC also is increased among patients with African iron overload and possibly in other iron-loading disorders such as homozygous beta thalassemia. The greatly increased iron stores in the liver observed in these disorders can stimulate carcinogenesis via both direct and indirect pathways. The prevalence of HCC also appears to be higher among patients with end-stage liver disease undergoing liver transplantation. It is not clear whether mildly to moderately increased hepatic iron stores or HFE mutations are associated independently with an increased risk for HCC among patients with other types of liver disease. In this article, the incidence and prevalence of HCC in patients with HH and other liver diseases associated with iron overload are discussed as well as the possible mechanisms for the increased risk for hepatic carcinogenesis in these disorders.

The pathogenesis of hepatitis C virus (HCV)-associated insulin resistance remains unclear. Therefore, we investigated mechanisms for HCV-associated insulin resistance. Homeostasis model assessment for insulin resistance was increased in patients with HCV infection. An increase in fasting insulin levels was associated with the presence of serum HCV core, the severity of hepatic fibrosis and a decrease in expression of insulin receptor substrate (IRS) 1 and IRS2, central molecules of the insulin-signaling cascade, in patients with HCV infection. Down-regulation of IRS1 and IRS2 was also seen in HCV core-transgenic mice livers and HCV core-transfected human hepatoma cells. Carbobenzoxy-l-leucyl-l-leucyl-l-leucinal, a potent proteosomal proteolysis inhibitor, blocked down-regulation of IRS1 and IRS2 in HCV core-transfected hepatoma cells. In human hepatoma cells, HCV core up-regulated suppressor of cytokine signaling (SOCS) 3 and caused ubiquitination of IRS1 and IRS2. HCV core-induced down-regulation of IRS1 and IRS2 was not seen in SOCS3(-/-) mouse embryonic fibroblast cells. Furthermore, HCV core suppressed insulin-induced phosphorylation of p85 subunit of phosphatidylinositol 3-kinase and Akt, activation of 6-phosphofructo-2-kinase, and glucose uptake. In conclusion, HCV infection changes a subset of hepatic molecules regulating glucose metabolism. A possible mechanism is that HCV core-induced SOCS3 promotes proteosomal degradation of IRS1 and IRS2 through ubiquitination.

Gain-of-function mutations of the Cu/Zn superoxide dismutase (SOD1) gene cause dominantly inherited familial amyotrophic lateral sclerosis. The identification of differentially regulated proteins in spinal cords of paralyzed mice expressing SOD1(G93A) may contribute to understanding mechanisms of toxicity by mutant SOD1. Protein profiling showed dysregulation of Stathmin with a marked decrease of its most acidic and phosphorylated isoform, and up-regulation of heat shock proteins 25 and 27, peroxiredoxin 6, phosphatidylinositol transfer protein-alpha, apolipoprotein E, and ferritin heavy chain. Stathmin accumulated in the cytoplasm of 30% of spinal cord motor neurons with fragmented Golgi apparatus. Overexpression of Stathmin in HeLa cells was associated with collapse of microtubule networks and Golgi fragmentation. These results, together with the decrease of one Stathmin isoform, suggest a role of the protein in Golgi fragmentation. Mutant SOD1 co-precipitated and co-localized with Hsp25 in neurons and astrocytes. Mutant SOD1 may thus deprive cells of the anti-apoptotic and other protective activities of Hsp25. Astrocytes contained peroxiredoxin 6, a unique nonredundant antioxidant. The up-regulation of peroxiredoxin 6 probably constitutes a defense to oxidative stress induced by SOD1(G93A). Direct effects of SOD1(G93A) or sequential reactions triggered by the mutant may cause the protein changes.

Spinal muscular atrophy (SMA) is characterized by degeneration of lower motor neurons caused by mutations of the survival motor neuron 1 gene (SMN1). SMN is involved in various processes including the formation of the spliceosome, pre-mRNA splicing and transcription. To know whether SMN has an essential role in all mammalian cell types or an as yet unknown specific function in the neuromuscular system, deletion of murine Smn exon 7, the most frequent mutation found among SMA patients, has been restricted to liver. Homozygous mutation results in severe impairment of liver development associated with iron overload and lack of regeneration leading to dramatic liver atrophy and late embryonic lethality of mutant mice. These data strongly suggest an ubiquitous and essential role of full-length SMN protein in various mammalian cell types. In SMA patients, the residual amount of SMN allows normal function of various organs except motor neurons. However, data from mouse and human suggest that other tissues might be involved in severe form of SMA or during prolonged disease course which reinforce the need of therapeutic approaches targeted to all tissues. In addition, liver function of patients should be carefully investigated and followed up before and during therapeutic trials.

Stimulation of dendritic cells (DCs) by the egg stage of the helminth parasite Schistosoma mansoni activates a signaling pathway resulting in type I interferon (IFN) and IFN-stimulated gene (ISG) expression. Here, we demonstrate that S. mansoni eggs disjointedly activate myeloid differentiation factor 88 (MyD88)-dependent and MyD88-independent pathways in DCs. Inflammatory cytokine expression and NF-kappa B activation in DCs from MyD88-deficient mice were impaired, whereas signaling transducer activator of transcription (STAT) 1(Tyr701) phosphorylation and ISG expression were intact in MyD88 or Toll-like receptor (TLR)4-deficient counterparts. Accordingly, we analyzed distinct TLR members for their ability to respond to schistosome eggs and established that TLR3 resulted in the activation of NF-kappa B and the positive regulatory domain III-I site from IFN-beta promoter. Unexpectedly, egg-derived RNA possessed RNase A-resistant and RNase III-sensitive structures capable of triggering TLR3 activation, suggesting the involvement of double-stranded (ds) structures. Moreover, DCs from TLR3-deficient mice displayed a complete loss of signaling transducer activator of transcription 1 phosphorylation and ISG expression in response to egg-derived dsRNA. Finally, TLR3-deficient DCs showed a reduced response to schistosome eggs relative to wild-type cells. Collectively, our data suggest for the first time that dsRNA from a non-viral pathogen may act as an inducer of the innate immune system through TLR3.

Differentiation of skeletal myoblasts into multinucleated myotubes is a multistep process orchestrated by several families of transcription factors, including myogenic bHLH and NFAT proteins. The activities of these factors and formation of myotubes are regulated by signal transduction pathways, but few extracellular factors that might initiate such signals have been identified. One exception is a cell surface complex containing promyogenic Ig superfamily members (CDO and BOC) and cadherins. Netrins and their receptors are established regulators of axon guidance, but little is known of their function outside the nervous system. We report here that myoblasts express the secreted factor netrin-3 and its receptor, neogenin. These proteins stimulate myotube formation and enhance myogenic bHLH- and NFAT-dependent transcription. Furthermore, neogenin binds to CDO in a cis fashion, and myoblasts lacking CDO are defective in responding to recombinant netrin. It is proposed that netrin-3 and neogenin may promote myogenic differentiation by an autocrine mechanism as components of a higher order complex of several promyogenic cell surface proteins.

No abstract available.

Despite recent advances in linear whole genome amplification of intact DNA/RNA, amplification of degraded nucleic acids in an unbiased fashion remains a serious challenge for genetic diagnosis. We describe a new whole genome amplification procedure, RCA-RCA (Restriction and Circularization-Aided Rolling Circle Amplification), which retains the allelic differences among degraded amplified genomes while achieving almost complete genome coverage. RCA-RCA utilizes restriction digestion and whole genome circularization to generate genomic sequences amenable to rolling circle amplification. When intact genomic DNA is used, RCA-RCA retains gene-amplification differences (twofold or higher) between complex genomes on a genome-wide scale providing highly improved concordance with unamplified material as compared with other amplification methodologies including multiple displacement amplification. Using RCA-RCA, formalin-fixed samples of modest or substantial DNA degradation were successfully amplified and screened via array-CGH or Taqman PCR that displayed retention of the principal gene amplification features of the original material. Microsatellite analysis revealed that RCA-RCA amplified genomic DNA is representative of the original material at the nucleotide level. Amplification of cDNA is successfully performed via RCA-RCA and results to unbiased gene expression analysis (R(2) = 0.99). The simplicity and universal applicability of RCA-RCA make it a powerful new tool for genome analysis with unique advantages over previous amplification technologies.

Blood vessels and nerves often follow parallel trajectories, suggesting that distal targets use common cues that induce vascularization and innervation. Netrins are secreted by the floor plate and attract commissural axons toward the midline of the neural tube. Here, we show that Netrin-1 is also a potent vascular mitogen. Netrin-1 stimulates proliferation, induces migration, and promotes adhesion of endothelial cells and vascular smooth muscle cells with a specific activity comparable to vascular endothelial growth factor and platelet-derived growth factor. Our evidence indicates that the netrin receptor, Neogenin, mediates netrin signaling in vascular smooth muscle cells, but suggests that an unidentified receptor mediates the proangiogenic effects of Netrin-1 on endothelial cells. Netrin-1 also stimulates angiogenesis in vivo and augments the response to vascular endothelial growth factor. Thus, we demonstrate that Netrin-1 is a secreted neural guidance cue with the unique ability to attract both blood vessels and axons, and suggest that other cues may also function as vascular endothelial growth factors.

No abstract available.

We identified six genes of Paramecium caudatum, which differentially expressed in Holospora obtusa-bearing and H. obtusa-free cells using differential display reverse transcribed PCR (DDRT-PCR). Northern blot analyses revealed that two of the genes, CA10-3 and CA20-2, were expressed extensively in the H. obtusa-free cell, while the other four, AS16-1, CS14, CS21 and CA17-1, were expressed more in the H. obtusa-bearing cell. Putative amino acid sequences of CA10-3, AS16-1 and CA17-1 showed high homologies with known genes related to intracellular signaling, transcription and aerobic metabolism. CS14 and CS21 also showed homologies with some genes whose products are still functionally unknown, but CA20-2 encoded a novel protein. We show in this study that H. obtusa alters multiple gene expression of the host after establishing endosymbiosis.

The Sox6 gene is a member of the Sox gene family, which encodes transcription factors, and previous studies have suggested that it plays an important role in the development of the central nervous system. Aggregation of embryonic carcinoma P19 cells with retinoic acid (RA) results in the development of neurons, glia, and fibroblast-like cells. Sox6 mRNA increases rapidly in P19 cells during RA induction and then decreases during differentiation into neuronal cells. To investigate whether Sox6 expression is essential for neuronal differentiation, we established Sox6-suppressed P19 (P19[anti-Sox6]) cells by transfection of antisense-Sox6 cDNA. Most of the P19[anti-Sox6] cells showed no neurites and were not stained by the anti-MAP 2 antibody, while the suppression of Sox6 expression nearly totally blocked neuronal differentiation in P19 cells. Further, Sox6 suppression caused RA-dependent apoptosis by P19[anti-Sox6] cells: RA-treated P19[anti-Sox6] cells showed chromatin condensation, DNA fragmentation, and an increase in caspase-3-like activity. Thus, Sox6 is considered essential for neuronal differentiation and may play an important role in the early stages of neuronal differentiation or apoptosis.

It has been claimed that a noncoding mitochondrial polymorphism at nt 16189 is correlated with the penetrance of the homozygous state for the C282Y mutation of the HFE gene. We have genotyped homozygotes for the C282Y mutation and find no relationship between the ferritin levels and the inheritance of the mitochondrial polymorphism. Indeed, the small difference found is in the opposite direction of that reported previously.

Mice deficient in OX40 or 4-1BB costimulatory pathways show defects in T cell recall responses, with predominant effects on CD4 vs CD8 T cells, respectively. However, OX40L can also stimulate CD8 T cells and 4-1BBL can influence CD4 T cells, raising the possibility of redundancy between the two TNFR family costimulators. To test this possibility, we generated mice deficient in both 4-1BBL and OX40L. In an adoptive transfer model, CD4 T cells expressed 4-1BB and OX40 sequentially in response to immunization, with little or no overlap in the timing of their expression. Under the same conditions, CD8 T cells expressed 4-1BB, but no detectable OX40. Thus, in vivo expression of 4-1BB and OX40 can be temporally and spatially segregated. In the absence of OX40L, there were decreased CD4 T cells late in the primary response and no detectable secondary expansion of adoptively transferred CD4 T cells under conditions in which primary expansion was unaffected. The 4-1BBL had a minor effect on the primary response of CD4 T cells in this model, but showed larger effects on the secondary response, although 4-1BBL(-/-) mice show less impairment in CD4 secondary responses than OX40L(-/-) mice. The 4-1BBL(-/-) and double knockout mice were similarly impaired in the CD8 T cell response, whereas OX40L(-/-) and double knockout mice were similarly impaired in the CD4 T cell response to both protein Ag and influenza virus. Thus, 4-1BB and OX40 act independently and nonredundantly to facilitate robust CD4 and CD8 recall responses.

A real-time PCR assay for detecting thermophilic Campylobacter spp. directly in chicken feces has been developed. DNA was isolated from fecal material by using magnetic beads followed by PCR with a prealiquoted PCR mixture, which had been stored at -18 degrees C. Campylobacter could be detected in less than 4 h, with a detection limit of 100 to 150 CFU/ml, in a fecal suspension. A bacterial internal control was added before DNA extraction to control both DNA isolation and the presence of PCR inhibitors in the samples. The assay was performed on 111 swab samples from a Danish surveillance program and compared to conventional culturing using selective enrichment. There was no statistically significant difference in performance between real-time PCR and culture by selective enrichment, and the diagnostic specificity was 0.96 with an agreement of 0.92. Therefore, the assay should be useful for screening poultry flocks for the presence of Campylobacter.

<h4>Objective</h4>To regenerate permanent cartilage, it is crucial to know not only the necessary conditions for chondrogenesis, but also the sufficient conditions. The objective of this study was to determine the signal sufficient for chondrogenesis.<h4>Methods</h4>Embryonic stem cells that had been engineered to fluoresce upon chondrocyte differentiation were treated with combinations of factors necessary for chondrogenesis, and chondrocyte differentiation was detected as fluorescence. We screened for the combination that could induce fluorescence within 3 days. Then, primary mesenchymal stem cells, nonchondrogenic immortalized cell lines, and primary dermal fibroblasts were treated with the combination, and the induction of chondrocyte differentiation was assessed by detecting the expression of the cartilage marker genes and the accumulation of proteoglycan-rich matrix. The effects of monolayer, spheroid, and 3-dimensional culture systems on induction by combinations of transcription factors were compared. The effects of the combination on hypertrophic and osteoblastic differentiation were evaluated by detecting the expression of the characteristic marker genes.<h4>Results</h4>No single factor induced fluorescence. Among various combinations examined, only the SOX5, SOX6, and SOX9 combination (the SOX trio) induced fluorescence within 3 days. The SOX trio successfully induced chondrocyte differentiation in all cell types tested, including nonchondrogenic types, and the induction occurred regardless of the culture system used. Contrary to the conventional chondrogenic techniques, the SOX trio suppressed hypertrophic and osteogenic differentiation at the same time.<h4>Conclusion</h4>These data strongly suggest that the SOX trio provides signals sufficient for the induction of permanent cartilage.

Strong evidence indicates that central serotoninergic system dysfunction is associated with suicidal behavior. The relationship between the serotonin transporter protein (5-HTT) gene of psychotic patients in Han Chinese with suicidal behavior was studied since the 5-HTT plays an important role in serotoninergic transmission. Two hundred and seventy-two psychotic inpatients with suicidal behavior (suicide group) and 275 psychotic inpatients without suicidal behavior (patient control group) were recruited from six mental health facilities in Shanghai, China. Another 628 healthy adults (healthy control group) without suicidal behavior and mental disorder history were enrolled from Shanghai, China. All subjects were Han Chinese. The genotypes and the alleles of 5-HTT genes of the three groups were examined by PCR amplification. Neither the intron 2 variable number tandem repeat (VNTR) polymorphism and 5-HTTLPR (5-HTT gene-linked polymorphic region), nor the haplotype frequencies of this gene have significant differences between the suicide group and the two control groups. However, there was a significant difference in 5-HTTLPR polymorphism between inpatients with a first episode of suicidal behavior and those with recurrent suicidal behavior. Except for the possible heterogeneity between inpatients with a first episode of suicidal behavior and those with more than one, the 5-HTT gene was unlikely to be associated with suicidal behavior of psychotic patient in Han Chinese.

<h4>Background and objectives</h4>Iron deficiency anemia is a common manifestation of celiac disease, which may be due to genetic and environmental factors. HFE mutations, frequent in Caucasian populations, can cause increased intestinal iron absorption and thus could protect against the development of iron deficiency. The aim of this study was to evaluate the prevalence of HFE mutations and their effect on iron metabolism in Italian celiac patients at diagnosis and after a gluten-free diet.<h4>Design and methods</h4>C282Y and H63D mutations were assessed by polymerase chain reaction (PCR) and restriction enzyme digestion in 203 patients with celiac disease and in 206 controls. HLA alleles were determined by sequence-specific primers and PCR. Duodenal histology was graded using Marsh's classification, and iron parameters measured by standard techniques.<h4>Results</h4>The frequency of the C282Y mutation was similar in celiac patients and controls (0.034 vs. 0.031); comparable frequencies were detected also for the H63D allele (0.170 vs. 0.136 in celiac patients and controls, respectively). Neither of the two HFE mutations affected iron indices in celiac patients at diagnosis, whereas a significant inverse correlation was detected between hemoglobin or ferritin and severity of histological damage (Marsh 3C or 3B vs. 3A, p<0.05 for both parameters). After a gluten-free diet, a slight increase in hemoglobin levels was observed in C282Y carriers as compared to controls, but only in female patients (p=0.044).<h4>Interpretation and conclusions</h4>In Italian patients with untreated celiac disease, HFE mutations do not constitute a protective factor against the development of iron deficiency, which seems to be mainly determined by the severity of the intestinal lesions.

The kinetics and thermodynamics of Bi(III) exchange between bismuth mononitrilotriacetate (BiL) and human serum transferrin as well as those of the interaction between bismuth-loaded transferrin and transferrin receptor 1 (TFR) were investigated at pH 7.4-8.9. Bismuth is rapidly exchanged between BiL and the C-site of human serum apotransferrin in interaction with bicarbonate to yield an intermediate complex with an effective equilibrium constant K(1) of 6 +/- 4, a direct second-order rate constant k(1) of (2.45 +/- 0.20) x 10(5) M(-1) s(-1), and a reverse second-order rate constant k(-1) of (1.5 +/- 0.5) x 10(6) M(-1) s(-1). The intermediate complex loses a single proton with a proton dissociation constant K(1a) of 2.4 +/- 1 nM to yield a first kinetic product. This product then undergoes a modification in its conformation followed by two proton losses with a first-order rate constant k(2) = 25 +/- 1.5 s(-1) to produce a second kinetic intermediate, which in turn undergoes a last modification in the conformation to yield the bismuth-saturated transferrin in its final state. This last process rate-controls Bi(III) uptake by the N-site of the protein and is independent of the experimental parameters with a constant reciprocal relaxation time tau(3)(-1) of (3 +/- 1) x 10(-2) s(-1). The mechanism of bismuth uptake differs from that of iron and probably does not involve the same transition in conformation from open to closed upon iron uptake. The interaction of bismuth-loaded transferrin with TFR occurs in a single very fast kinetic step with a dissociation constant K(d) of 4 +/- 0.4 microM, a second-order rate constant k(d) of (2.2 +/- 1.5) x 10(8) M(-1) s(-1), and a first-order rate constant k(-d) of 900 +/- 400 s(-1). This mechanism is different from that observed with the ferric holotransferrin and implies that the interaction between TFR and bismuth-loaded transferrin probably takes place on the helical domain of the receptor which is specific for the C-site of transferrin and HFE. The relevance of bismuth incorporation by the transferrin receptor-mediated iron acquisition pathway is discussed.

<h4>Objective</h4>To determine the etiology of increased ferritin concentrations and/or transferrin saturation in patients in whom classical causes were ruled out.<h4>Patients and method</h4>We studied 43 patients (35 males and 8 females) who were referred for ferritinemia greater than 300 ng/ml and or a transferrin saturation index (TSI) greater than 40%. In all patients, glycemia, cholesterol, triglycerides, uric acid, total and fractionated bilirubin, transaminase, gammaglutamyltranspeptidase, sideremia, TSI, ferritin, HFE gene mutations, ceruloplasmin and total 24-hour urine porphyrin were evaluated and abdominal ultrasonography was performed. In 14 patients liver biopsy was performed.<h4>Results</h4>Fifty-three percent was overweight and 19% was obese. Alterations in carbohydrate metabolism were detected in 33%, hypercholesterolemia was found in 14%, hypertriglyceridemia in 35%, and hyperlipemia type IIb in 16%. Thirty-two percent showed isolated elevated ferritin, 12% had elevated TSI and 56% showed elevation of both. Transaminase levels were normal in 61%. No mutation in the HFE gene was found in 10 patients, the H63D/wt mutation was found in 18, C262Y/wt in 1, C282Y/H63D in 5, C282Y/C282Y in 4, H63D/H63D in 3 and Ser65cys/wt in 1. Ultrasonography revealed steatosis in 19 patients (44%). Definitive diagnoses were HFE-linked hemochromatosis (4 patients), juvenile hemochromatosis (1 patient), hepaticocutaneous porphyria (1 patient), and non-alcoholic fatty liver disease (22 patients; 51%). Most of the remaining patients could be included under insulin resistance syndrome. Phlebotomy was performed in 25 patients, with improvement in clinical and laboratory parameters.<h4>Conclusions</h4>Non-alcoholic fatty acid disease is frequently detected in patients with iron metabolism disorders. These patients should undergo investigations for metabolic alterations and liver ultrasonography and, if necessary, biopsy. Phlebotomy can be useful in the treatment of these patients.

<h4>Objective</h4>To investigate the 5-HTT-gene-linked polymorphic region (5-HTTLPR) genotypes and harm avoidance (HA) dimension in chronic tension-type headache (CTH).<h4>Background</h4>Serotonin transporter protein (5-HTT) is a key modulating protein in synaptic serotonergic neurotransmission. Among serotonin gene-linked polymorphism, promoter located in the regulatory region of the 5-HTTLPR has two alleles (short and long) with different transcriptional efficiencies. The HA personality trait may be heritable and associated with serotonergic neurotransmitter activity.<h4>Design</h4>We amplified the 5-HTTLPR by means of polymerase chain reaction and performed genotype polymorphism analyses and we investigated the serotonin-related personality trait by evaluating the HA dimension in tridimensional personality questionnaire (TPQ) in 107 patients with CTH and in 100 healthy controls.<h4>Results</h4>We found an excess frequency of the short allele and a different genotype distribution in patients with CTH. S/S genotype frequency was significantly higher in patients with CTH (76%) than in those with controls (59%; P=.02). Patients with CTH had significantly higher HA scores (21.4+/6.3) than controls (16.3+/-6.1).<h4>Conclusions</h4>This suggests a serotonergic activity might be involved in the development of CTH and 5-HTTLPR might be one of the genetically contributing factors.

Huntington's disease (HD) is a fully penetrant autosomal-dominant inherited neurological disorder caused by expanded CAG repeats in the Huntingtin gene. Transcriptional dysfunction, excitotoxicity, and oxidative stress have all been proposed to play important roles in the pathogenesis of HD. This study was designed to explore the therapeutic potential of mithramycin, a clinically approved guanosine-cytosine-rich DNA binding antitumor antibiotic. Pharmacological treatment of a transgenic mouse model of HD (R6/2) with mithramycin extended survival by 29.1%, greater than any single agent reported to date. Increased survival was accompanied by improved motor performance and markedly delayed neuropathological sequelae. To identify the functional mechanism for the salubrious effects of mithramycin, we examined transcriptional dysfunction in R6/2 mice. Consistent with transcriptional repression playing a role in the pathogenesis of HD, we found increased methylation of lysine 9 in histone H3, a well established mechanism of gene silencing. Mithramycin treatment prevented the increase in H3 methylation observed in R6/2 mice, suggesting that the enhanced survival and neuroprotection might be attributable to the alleviation of repressed gene expression vital to neuronal function and survival. Because it is Food and Drug Administration-approved, mithramycin is a promising drug for the treatment of HD.

Alterations in histones, chromatin-related proteins, and DNA methylation contribute to transcriptional silencing in cancer, but the sequence of these molecular events is not well understood. Here we demonstrate that on disruption of estrogen receptor (ER) alpha signaling by small interfering RNA, polycomb repressors and histone deacetylases are recruited to initiate stable repression of the progesterone receptor (PR) gene, a known ERalpha target, in breast cancer cells. The event is accompanied by acquired DNA methylation of the PR promoter, leaving a stable mark that can be inherited by cancer cell progeny. Reestablishing ERalpha signaling alone was not sufficient to reactivate the PR gene; reactivation of the PR gene also requires DNA demethylation. Methylation microarray analysis further showed that progressive DNA methylation occurs in multiple ERalpha targets in breast cancer genomes. The results imply, for the first time, the significance of epigenetic regulation on ERalpha target genes, providing new direction for research in this classical signaling pathway.

The production of haploid gametes from diploid germ cells requires two rounds of meiotic chromosome segregation after one round of replication. Accurate meiotic chromosome segregation involves the remodeling of each pair of homologous chromosomes around the site of crossover into a highly condensed and ordered structure. We showed that condensin, the protein complex needed for mitotic chromosome compaction, restructures chromosomes during meiosis in Caenorhabditis elegans. In particular, condensin promotes both meiotic chromosome condensation after crossover recombination and the remodeling of sister chromatids. Condensin helps resolve cohesin-independent linkages between sister chromatids and alleviates recombination-independent linkages between homologues. The safeguarding of chromosome resolution by condensin permits chromosome segregation and is crucial for the formation of discrete, individualized bivalent chromosomes.

Netrin-1 acts as a chemoattractant molecule to guide commissural neurons (CN) toward the floor plate by interacting with the receptor deleted in colorectal cancer (DCC). The molecular mechanisms underlying Netrin-1-DCC signaling are still poorly characterized. Here, we show that DCC is phosphorylated in vivo on tyrosine residues in response to Netrin-1 stimulation of CN and that the Src family kinase inhibitors PP2 and SU6656 block both Netrin-1-dependent phosphorylation of DCC and axon outgrowth. PP2 also blocks the reorientation of Xenopus laevis retinal ganglion cells that occurs in response to Netrin-1, which suggests an essential role of the Src kinases in Netrin-1-dependent orientation. Fyn, but not Src, is able to phosphorylate the intracellular domain of DCC in vitro, and we demonstrate that Y1418 is crucial for DCC axon outgrowth function. Both DCC phosphorylation and Netrin-1-induced axon outgrowth are impaired in Fyn(-/-) CN and spinal cord explants. We propose that DCC is regulated by tyrosine phosphorylation and that Fyn is essential for the response of axons to Netrin-1.

<h4>Background and purpose</h4>Functional MR imaging (fMRI) has been used to probe basal ganglia function in people with presymptomatic Huntington's disease (pre-HD). A previous fMRI study in healthy individuals demonstrated activation of the basal ganglia during a time-discrimination task. The current study was designed to examine the relative sensitivity of fMRI compared with that of behavioral testing and morphometric measurements in detecting early neurodegenerative changes related to Huntington's disease (HD).<h4>Methods</h4>Pre-HD participants were assigned to two groups based on estimated years to diagnosis of manifest disease: close <12 years and far >or=12 years. Age at disease onset was estimated using a regression equation based on the number of trinucleotide CAG repeats. The time-discrimination task required participants to determine whether a specified interval was shorter or longer than a standard interval of 1200 milliseconds.<h4>Results</h4>Participants in the close group performed more poorly on the time-task discrimination than did control subjects; however, no differences were observed between far participants and control subjects. Similarly, close participants had reduced bilateral caudate volume relative to that of control subjects, whereas far participants did not. On functional imaging, close participants had significantly less activation in subcortical regions (caudate, thalamus) than control subjects; far participants had an intermediate degree of activation. In contrast, far participants had hyperactivation in medial hemispheric structures (anterior cingulate, pre-supplementary motor area) relative to close and control subjects.<h4>Conclusion</h4>Hyperactivation of medial prefrontal regions compensated for reduced subcortical participation during time discrimination in pre-HD. This pattern of brain activation may represent an early neurobiologic marker of neuronal dysfunction.

<h4>Introduction</h4>Nearly 50 % of subjects with continuing symptoms of attention-deficit hyperactivity disorder (ADHD) in adulthood show a comorbid substance use disorder. Both, ADHD and alcohol dependence have a high genetic load and might even share overlapping sources of genetic liability.<h4>Method</h4>We investigated phenotype and 5-HTT/5-HT2c allelic characteristics in 314 alcoholics of German descent.<h4>Result</h4>21 % of the alcoholics fulfilled DSM-IV-criteria of ADHD with ongoing symptoms in adulthood. There was no significant difference in 5-HTT- or 5-HT2c-allele distribution between alcoholics and matched controls or between alcoholics with or without ADHD.<h4>Conclusion</h4>In our sample the functional relevant 5-HTT-promoter and the 5-HT2c-receptor Cys23Ser polymorphism do not contribute to the supposed common genetic predisposition of ADHD and alcohol dependence.

Heterochromatin is a specialized chromatin structure in chromosomal regions associated with repeated DNA sequences and low concentrations of genes. Formation of heterochromatin is determined in large part by enzymes that modify histones and structural proteins that bind to these modified histones in a cooperative fashion. In Drosophila, mutations in genes that encode heterochromatic proteins are often dominant and increase expression of genes placed into heterochromatic positions. To find components of telomeric heterochromatin in Drosophila, we screened a collection of autosomal deficiencies for dominant suppressors of silencing of a transgene at the telomere of chromosome 2L. While many deficiency chromosomes are associated with dominant suppressors, in the cases tested on chromosome 2 the suppressor mapped to the 2L telomere, rather than the deficiency. We infer that background effects may hamper the search for genes that play a role in telomeric heterochromatin formation and that either very few genes participate in this pathway or mutations in these genes are not dominant suppressors of telomeric position effect. The data also suggest that the 2L telomere region plays a major role in telomeric silencing.

We report a case of an 80-year-old woman with congenital spherocytosis who presented with massive iron overload. Iatrogenic iron overload could be ruled out. Familial history was suggestive of hereditary hemochromatosis; however, molecular genetic testing for the most common HFE mutations remained negative. The patient was treated successfully with phlebotomies. The hypothesis that this patient suffered from hereditary hemochromatosis is discussed on the basis of a brief review of the literature.

<h4>Background</h4>Iron absorption is decreased in some individuals who have undergone bariatric surgery.<h4>Methods</h4>We evaluated measures of iron metabolism and therapeutic phlebotomy in 3 adults with hemochromatosis and HFE C282Y homozygosity who underwent bariatric surgery.<h4>Results</h4>1 male and 1 female had surgery before diagnosis of hemochromatosis (jejuno-ileal bypass and Roux-en-Y gastric bypass, respectively); neither had iron overload. Another man was treated with serial phlebotomy to induce iron depletion; later, he underwent Roux-en-Y gastric bypass. His maintenance phlebotomy requirement for hemochromatosis decreased substantially (on average approximately 1 unit each 71 days before surgery, and approximately 1 unit each 173 days after surgery). None of these patients developed iron deficiency, and none took supplemental iron.<h4>Conclusions</h4>Iron absorption is decreased in some patients with hemochromatosis and HFE C282Y homozygosity after bariatric surgery, but their risk of developing iron deficiency may be diminished.

We have studied the recombination rate behaviour of a set of 140 genes which were investigated for their potential importance in inflammatory disease. Each gene was extensively sequenced in 24 individuals of African descent and 23 individuals of European descent, and the recombination process was studied separately in the two population samples. The results obtained from the two populations were highly correlated, suggesting that demographic bias does not affect our population genetic estimation procedure. We found evidence that levels of recombination correlate with levels of nucleotide diversity. High marker density allowed us to study recombination rate variation on a very fine spatial scale. We found that about 40 per cent of genes showed evidence of uniform recombination, while approximately 12 per cent of genes carried distinct signatures of recombination hotspots. On studying the locations of these hotspots, we found that they are not always confined to introns but can also stretch across exons. An investigation of the protein products of these genes suggested that recombination hotspots can sometimes separate exons belonging to different protein domains; however, this occurs much less frequently than might be expected based on evolutionary studies into the origins of recombination. This suggests that evolutionary analysis of the recombination process is greatly aided by considering nucleotide sequences and protein products jointly.

Diagnosing disorders of iron metabolism the concentration of the iron storing protein ferritin reflects the body's iron reserves much better than does serum iron concentrations or transferring saturation. Merely in the event of acute phase reactions is the validity of the ferretin level compromised. This applies in particular to the redistribution of iron in anemia caused by inflammatory conditions or malignancies, as also, though less markedly, to functional iron deficiency in renal anemia. Here, an additional diagnostic work-up, in particular when EPO/iron therapy is applied. Iron overload should be recognized already in the latent state before organ damage occurs. Clinically and chemically confirmed iron overload that cannot be ascribed to hematological disease, iron replacement of transfusions, should prompt a molecular-biological analysis of hemochromatosis-associated genetic defects.

No abstract available.

Various TATA-binding protein (TBP)-associated proteins are involved in the regulation of gene expression through control of basal transcription directed by RNA polymerase (Pol) II. We recently identified a novel nuclear protein, activator of basal transcription 1 (ABT1), which binds TBP and DNA, and enhances Pol II-directed basal transcription. To better understand regulatory mechanisms for ABT1, we searched for ABT1-binding proteins using a yeast two-hybrid screening and isolated a cDNA clone encoding a novel protein termed ABT1-associated protein (ABTAP). ABTAP formed a complex with ABT1 and suppressed the ABT1-induced activation of Pol II-directed transcription in mammalian cells. Furthermore, ABTAP directly bound to ABT1, disrupted the interaction between ABT1 and TBP, and suppressed the ABT1-induced activation of Pol II-directed basal transcription in vitro. These two proteins colocalized in the nucleolus and nucleoplasm and were concomitantly relocalized into discrete nuclear bodies at higher expression of ABTAP. Taken together, these results suggest that ABTAP binds and negatively regulates ABT1. The ABT1/ABTAP complex is evolutionarily conserved and may constitute a novel regulatory system for basal transcription.

The content of D-dimer in peripheral blood was studied in thrombohemorrhagic lesions of the retina. 60 patients with retinal pathology of the retina, i.e. thrombosis of retinal veins, exudative-hemorrhagic stage of central chorioretinal dystrophy and preproliferative diaichbetic retinopathy with pronounced exudative hemorrhagic maculopathy, were examined. A higher concentration of D-dimer in peripheral blood was shown to be indicative of affected general hemostasis. The below main parameters, which matter in progression of thrombohemorrhagic conditions should be determined, apart from D-dimer, for choosing an adequate medicamental therapy: aggregation ability of platelets, activated partial thromboplastin time, antithrombin-III, protein-C, prothrombin time, soluble complexes of fibrinmonomer, thrombin time, plasma fibrinogen and fibrinolytic plasma activity.

<h4>Background</h4>Molecular predictors for the effectiveness of adjuvant chemotherapy in colorectal cancer are of considerable clinical interest. To this aim, we analyzed the serine threonine receptor-associated protein (STRAP), an inhibitor of TGF-beta signaling, with regard to prognosis and prediction of adjuvant 5-FU chemotherapy benefit.<h4>Methods</h4>The gene copy status of STRAP was determined using quantitative real-time polymerase chain reaction in 166 colorectal tumor biopsies, which had been collected from a randomized multicenter trial of 5-fluorouracil (5-FU)/mitomycin C (MMC) adjuvant chemotherapy of the Swiss Group for Clinical Cancer Research (SAKK).<h4>Results</h4>Amplification of STRAP was found in 22.8% of the tumors. When left without adjuvant chemotherapy, patients bearing tumors with a STRAP amplification had a significantly better prognosis (hazard ratio for death: 0.26; P=.004). Interestingly, these patients, when receiving adjuvant treatment, had a worse survival (hazard ratio for death: 3.48; P=.019) than without chemotherapy, whereas patients carrying tumors with diploidy or deletion of STRAP benefited from the treatment (hazard ratio for death: 0.44; P=.052). This suggests the amplification of STRAP as a strong predictor of an unfavorable effect of 5-FU-based adjuvant chemotherapy.<h4>Conclusion</h4>If confirmed, the STRAP gene copy status might provide a parameter to decide about the use of 5-FU-based adjuvant chemotherapy.

<h4>Objective</h4>To investigate the expression of the estrogen receptors(ER) in breast hypertrophy and micromastia.<h4>Methods</h4>The quantity of ER within breast tissue in 13 patients with mammary hypertrophy strictly elected was determined using DCC single dot test. The results were compared with that measured in 13 patients with micromastia.<h4>Results</h4>The average of the quantity of the ER was respectively (5.33 +/- 5.70) fmol/mg and (1.17 +/- 0.86) fmol/mg in mammary hypertrophy ground and in the micromastia group. There was obvious significant between the two groups in a statistics basis (P < 0.05) .<h4>Conclusions</h4>The breast hypertrophy is related to the excessive expression of ER within breast tissue,while the micromastia and breast hypotrophy are related to the lower level or the expression inhibition of ER.

Psychiatric patients demonstrate varied responses to treatment. Consequently, treatment strategies are trial-and-error, which has a negative effect on prognosis and compliance. The aim of pharmacogenomic research is to enable customised drug treatment by identifying variations within multiple candidate genes (those encoding drug-targeted neurotransmitter receptors, transporters and metabolic enzymes) that are likely to confer the inter-individual differences in drug response and development of drug-induced side effects. Pharmacogenetic and pharmacogenomic research to date has identified genetic polymorphisms of dopamine (DA) and serotonin (5-HT) receptor subtypes, the serotonin transporter (5-HTT) and metabolic enzymes (cytochrome P450 [CYP] family) as important contributors to the variability in response to psychiatric drugs and the development of drug-induced side effects such as tardive dyskinesia and weight gain. It is anticipated that technological and methodological advances will provide further candidate genes and refine association analyses of existing candidates, enabling pharmacogenomic research to move towards future treatment regimes that are catered to the individual.

The defensin-like circulatory peptide hepcidin is the iron-regulatory hormone that links innate immunity and iron metabolism. In response to inflammatory stimuli, the liver produces hepcidin: this antimicrobial peptide then limits the iron that is vital to invading pathogens, by decreasing iron release/transfer from enterocytes and macrophages and causing secondary hypoferremia. This may lead, however, to reduced iron availability for erythropoiesis and therefore to anemia (and anemia of chronic disease). When iron is scarce, the rate at which it is released into the bloodstream must be enhanced: indeed, iron starvation and hypoxia readily abrogate hepcidin expression. Conversely, if excess iron enters the circulation, hepcidin transcription is turned on and iron release from the intestine and macrophages abrogated. Circumstantial evidence indicates that the effect of circulatory iron on hepcidin requires functional HFE and hemojuvelin, two proteins of unknown function that have recently been linked to human hereditary hemochromatosis. In this disease it is likely that inadequate levels of circulating hepcidin lead to the uncontrolled release of iron from the intestine and macrophages, followed by tissue iron overload and organ damage. Given its role as the iron-regulatory hormone, the modulation of hepcidin activity using agonists or antagonists might offer new treatment opportunities in different human iron-dependent disorders.

The eating disorders anorexia nervosa and bulimia nervosa traditionally have been viewed as sociocultural in origin. However, recent behavioral genetic findings suggest substantial genetic influence on these disorders. Molecular genetic research of these disorders is in its infancy, but initial results are promising. This article reviews findings from family, twin, and molecular genetic studies that support substantial genetic influences on disordered eating and highlights additional areas for future research.