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Hereditary hemochromatosis (HH) is a condition in which intestinal iron absorption is greatly elevated. Present treatment is weekly phlebotomy, affecting quality of life and leading to recurrent infections. The iron transporter divalent metal transporter-1 (DMT-1) of enterocytes is responsible for iron uptake from the intestinal lumen; iron is further extruded into the blood by the basolateral transporter ferroportin-1. A therapeutic approach for HH could start with a long-term reduction of iron transport by reduction of DMT-1 levels. We designed an AAV vector coding for a short antisense RNA (AAV-DMT-1-AS) against DMT-1, which reduced iron uptake by 50-60% in human intestinal cells (Caco-2). At low infection levels, DMT-1 mRNA virtually disappeared, suggesting RNAi-like and/or RNase H antisense effects. DMT-1 mRNA levels returned to normal at higher infection levels, indicating that an additional mechanism of mRNA occupation, able to block DMT-1 translation and to avoid feedback regulation by iron responsive elements (IRE), also exists. Cell morphology was normal in all cases and no increases in the interferon-related responses, measured by (a) 2'-5' A oligo synthetase (b) IFITM1 and (c) ISGF3gamma mRNA levels, were observed. Studies presented herein indicate that enterocyte targeting with a gene coding for a short antisense against iron transport blocks enterocyte iron uptake, which may have therapeutic value.

A total of 921 fecal specimens collected from 44 infants in a day care center (DCC) in Tokyo, Japan during June 1999 to July 2000 were tested for the presence of rotavirus, norovirus, sapovirus, astrovirus and adenovirus by reverse-transcription-multiplex polymerase chain reaction (RT-multiplex PCR) and sequence analysis. Of 88 fecal specimens from infants with acute gastroenteritis, 51.1% (45) were found to be positive for diarrheal viruses. Astrovirus was the most prevalent (15.9%, 14 of 88), followed by norovirus GII (14.8%, 13 of 88), adenovirus (12.5%, 11 of 88), and sapovirus (2.3%, 2 of 88). Viral mixed infection accounted for 5.7% (5 of 88). Interestingly, 230 of 833 (27.6%) fecal specimens collected from asymptomatic infants were also infected with diarrheal viruses. Of these, astrovirus, norovirus GII, adenovirus and sapovirus were identified in 53, 46, 96 and 22 fecal specimens (23%, 20%, 41.7%, and 9.6%, respectively). Moreover, 13 of 833 (1.6%) normal specimens showed mixed viral infections. Surprisingly, no rotavirus (known as the most common causative agent of acute gastroenteritis in DCCs) was detected in those subjects. Another interesting feature was the demonstration of five separate outbreaks of acute gastroenteritis identified in a single DCC. Outbreak A was associated with both astrovirus serotype 1 and norovirus GII/3 (known as Toronto virus cluster); Outbreak B with adenovirus 12; Outbreak C with norovirus GII/4 (Lordsdale virus cluster); Outbreak D with sapovirus GIV and Outbreak E with astrovirus serotype 1. To our knowledge, this is the first proof of multiple outbreaks of viral gastroenteritis in Japanese infants in a single DCC. Our results confirm the presence as well as the importance of these viruses and warn of the threat they pose.

The potential of neural stem cells (NSCs) for the treatment of neurodegenerative diseases makes the identification and characterization of genes involved in neural stem cell responses therapeutically important. Although technologies exist for measuring gene expression in cells, they often provide only a representative expression profile specific to a stimulus and time. We developed a complementary technology based on a retroviral-vector gene-trap approach that uses beta-lactamase-induced disruption of fluorescence resonance energy transfer in the fluorophore CCF-2/AM. A library of 'tagged' adult rat NSCs was generated by transduction with gene-trap virus produced from a single-integrant packaging cell line that allowed us to quantitatively analyze dynamic gene expression changes in real time in living NSCs. Using this library we identified previously unknown genes regulated by oxidative stress, indomethacin and factors that induce differentiation, and show that one of the trapped genes, Sox6, is sufficient to induce astrocytic differentiation when overexpressed.

Solute carrier family 11, member 2 (SLC11A2) is the only transmembrane iron transporter known to be involved in cellular iron uptake. It is widely expressed and has been postulated to play important roles in intestinal iron absorption, erythroid iron utilization, hepatic iron accumulation, placental iron transfer, and other processes. Previous studies have suggested that other transporters might exist, but their physiological significance remained uncertain. To define the activities of Slc11a2 in vivo, we inactivated the murine gene that encodes it globally and selectively. We found that fetal Slc11a2 is not needed for materno-fetal iron transfer but that Slc11a2 activity is essential for intestinal non-heme iron absorption after birth. Slc11a2 is also required for normal hemoglobin production during the development of erythroid precursors. However, hepatocytes and most other cells must have an alternative, as-yet-unknown, iron uptake mechanism. We previously showed that Slc11a2 serves as the primary portal for intestinal iron entry in hemochromatosis. However, inactivation of murine Hfe ameliorates the phenotype of animals lacking Slc11a2.

Huntington disease (HD) results from polyglutamine expansion in the huntingtin protein (htt). Despite the widespread tissue expression pattern of htt, neuronal loss is highly selective to medium spiny neurons of the striatum. Huntingtin is phosphorylated on serine-421 (S421) by the pro-survival signaling protein kinase Akt (PKB) and this has been previously shown to be protective against the toxicity of polyglutamine-expanded htt in cell culture. Using an antibody specific for htt phosphorylated on S421, we now demonstrate that htt phosphorylation is present at significant levels under normal physiological conditions in human and mouse brain. Furthermore, htt phosphorylation shows a regional distribution with the highest levels in the cerebellum, less in the cortex, and least in the striatum. In cell cultures and in YAC transgenic mice, the endogenous phosphorylation of polyglutamine-expanded htt is significantly reduced relative to wild-type htt. The presence and pattern of significant htt phosphorylation in the brain indicates that this dynamic post-translational modification is important for the regulation of htt and may contribute to the selective neurodegeneration seen in HD.

The p53 tumor suppressor protein is a master regulatory transcription factor that coordinates cellular responses to DNA damage and cellular stress. Besides mutations in p53, or in proteins involved in the p53 response pathway, genetic variation in promoter response elements (REs) of p53 target genes is expected to alter biological responses to stress. To identify SNPs in p53 REs that may modify p53-controlled gene expression, we developed an approach that combines a custom bioinformatics search to identify candidate SNPs with functional yeast and mammalian cell assays to assess their effect on p53 transactivation. Among approximately 2 million human SNPs, we identified >200 that seem to disrupt functional p53 REs. Eight of these SNPs were evaluated in functional assays to determine both the activity of the putative RE and the impact of the candidate SNPs on transactivation. All eight candidate REs were functional, and in every case the SNP pair exhibited differential transactivation capacities. Additionally, six of the eight genes adjacent to these SNPs are induced by genotoxic stress or are activated directly by transfection with p53 cDNA. Thus, this strategy efficiently identifies SNPs that may differentially affect gene expression responses in the p53 regulatory pathway.

<h4>Background and aims</h4>Colon cancer tumorigenesis is a multistep process of mutation accumulation in a number of oncogenes and tumour suppressor genes. NF1 gene protein, neurofibromin, acts as a tumour suppressor by turning the active form of Ras into an inactive form. This molecular switch has an important role in the control of the cell cycle and differentiation, and changes in Ras activity are present in many different cancers. This is the first study to investigate the role of NF1 in sporadic colon cancer.<h4>Methods</h4>We investigated loss of heterozygosity (LOH) at the NF1 locus. Real time reverse transcription-polymerase chain reaction was used to determine NF1 mRNA expression in tumours and corresponding normal tissue. Expression of neurofibromin was analysed by immunohistochemistry. Relative ratio of NF1 mRNA type I and II isoform expression was also examined.<h4>Results</h4>LOH of the NF1 gene was detected in 20.7% of heterozygous samples. NF1 mRNA expression was significantly increased in tumour tissue compared with corresponding normal tissue (p = 0.04291). There was a statistically significant increase in NF1 type I isoform expression (p = 0.0005) in tumour tissue compared with corresponding normal colon tissue. NF1 isoform type II was predominantly expressed in normal tissue while the NF1 isoform type I prevailed in tumour samples. The transition from dominant expression of isoform type II in normal mucous tissue 15 cm away from the tumour to dominant expression of isoform type I in tumour tissue itself was detected. Total neurofibromin expression increased as tumours were more advanced but expression of wild-type neurofibromin remained the same.<h4>Conclusions</h4>Our findings suggest that the NF1 gene may play a role in the development and progression of colon cancer and the NF1 gene may be a potential tumour marker and a new potential target for colon cancer therapy.

HFE is a protein known to be involved in iron metabolism; yet, other than its homology with major histocompatibility complex (MHC) class I molecules, it has not been described as having an immunologic function. Here we report that peripheral blood mononuclear cells (PBMCs) from patients with hereditary hemochromatosis (HH) carrying the C282Y mutation in HFE have reduced cell-surface expression of MHC class I due to an enhanced endocytosis rate of MHC class I molecules caused by premature peptide and beta2-microglobulin dissociation. This faster turnover also leads to increased expression levels of cell-surface free class I heavy chains in mutant PBMCs. Biochemical analysis indicates an earlier peptide loading and endoplasmic reticulum maturation of MHC class I molecules in C282Y mutant cells. Thermostability assays further showed that in HFE mutants the MHC class I peptide loading gives rise to low-stability heterotrimers that dissociate prematurely during its intracellular traffic. The present results suggest the existence of an intriguing cross-talk between a particular HFE mutation and the classical MHC class I route. These findings constitute the first description of peptide presentation pathway abnormalities linked to HFE and provide additional evidence for the occurrence of immunologic defects in patients with HH.

No abstract available.

<h4>Background</h4>Since the early stages of tumorigenesis involve adhesion, escape from immune surveillance, vascularization and angiogenesis, we devised a strategy to study the expression profiles of all publicly known and putative secreted and cell surface genes. We designed a custom oligonucleotide microarray containing probes for 3531 secreted and cell surface genes to study 5 diverse human transformed cell lines and their derivative xenograft tumors. The origins of these human cell lines were lung (A549), breast (MDA MB-231), colon (HCT-116), ovarian (SK-OV-3) and prostate (PC3) carcinomas.<h4>Results</h4>Three different analyses were performed: (1) A PCA-based linear discriminant analysis identified a 54 gene profile characteristic of all tumors, (2) Application of MANOVA (Pcorr < .05) to tumor data revealed a larger set of 149 differentially expressed genes. (3) After MANOVA was performed on data from individual tumors, a comparison of differential genes amongst all tumor types revealed 12 common differential genes. Seven of the 12 genes were identified by all three analytical methods. These included late angiogenic, morphogenic and extracellular matrix genes such as ANGPTL4, COL1A1, GP2, GPR57, LAMB3, PCDHB9 and PTGER3. The differential expression of ANGPTL4 and COL1A1 and other genes was confirmed by quantitative PCR.<h4>Conclusion</h4>Overall, a comparison of the three analyses revealed an expression pattern indicative of late angiogenic processes. These results show that a xenograft model using multiple cell lines of diverse tissue origin can identify common tumorigenic cell surface or secreted molecules that may be important biomarker and therapeutic discoveries.

Myosin VI plays a role in the maintenance of Golgi morphology and in exocytosis. In a yeast 2-hybrid screen we identified optineurin as a binding partner for myosin VI at the Golgi complex and confirmed this interaction in a range of protein interaction studies. Both proteins colocalize at the Golgi complex and in vesicles at the plasma membrane. When optineurin is depleted from cells using RNA interference, myosin VI is lost from the Golgi complex, the Golgi is fragmented and exocytosis of vesicular stomatitis virus G-protein to the plasma membrane is dramatically reduced. Two further binding partners for optineurin have been identified: huntingtin and Rab8. We show that myosin VI and Rab8 colocalize around the Golgi complex and in vesicles at the plasma membrane and overexpression of constitutively active Rab8-Q67L recruits myosin VI onto Rab8-positive structures. These results show that optineurin links myosin VI to the Golgi complex and plays a central role in Golgi ribbon formation and exocytosis.

Prostate-specific membrane antigen (PSMA) is highly expressed in prostate cancer cells and nonprostatic solid tumor neovasculature and is a target for anticancer imaging and therapeutic agents. PSMA acts as a glutamate carboxypeptidase (GCPII) on small molecule substrates, including folate, the anticancer drug methotrexate, and the neuropeptide N-acetyl-l-aspartyl-l-glutamate. Here we present the 3.5-A crystal structure of the PSMA ectodomain, which reveals a homodimer with structural similarity to transferrin receptor, a receptor for iron-loaded transferrin that lacks protease activity. Unlike transferrin receptor, the protease domain of PSMA contains a binuclear zinc site, catalytic residues, and a proposed substrate-binding arginine patch. Elucidation of the PSMA structure combined with docking studies and a proposed catalytic mechanism provides insight into the recognition of inhibitors and the natural substrate N-acetyl-l-aspartyl-l-glutamate. The PSMA structure will facilitate development of chemotherapeutics, cancer-imaging agents, and agents for treatment of neurological disorders.

<h4>Purpose</h4>In order to identify specific genes that may play important roles in maintaining the specialized functions of lens epithelial and fiber cells, we have analyzed the global gene expression profiles of these two cell types in the human lens. This analysis will also reveal those genes that are exclusively expressed in the epithelial and cortical fiber cells and those genes that may play important roles in the differentiation of epithelial cells to mature fiber cells.<h4>Methods</h4>Oligonucleotide microarray hybridization was used to analyze the expression profiles of 22,215 genes between adult (average age greater than 56 years) human lens epithelial and cortical fiber cells. The expression levels of selected genes were further compared by semi-quantitative RT-PCR and selected genes were functionally clustered into common categories using the EASE bioinformatics software package.<h4>Results</h4>Analysis of three separate microarray hybridizations revealed 1,196 transcripts that exhibit increased expression and 1,278 transcripts that exhibit decreased expression at the 2 fold or greater level between lens epithelial cells and cortical fiber cells on all three of the arrays analyzed. Of these, 222 transcripts exhibited increased expression and 135 transcripts exhibited decreased expression by an average of 5 fold or greater levels on all three arrays. Semi-quantitative RT-PCR analysis of 21 randomly selected genes revealed identical expression patterns as those detected by microarray hybridization indicating that the microarray data are accurate. Functional clustering of the identified gene expression patterns using the EASE program revealed a wide variety of biological pathways that exhibited altered expression patterns between the two cell types including mRNA processing, cell adhesion, cell proliferation, translation, protein folding, oxidative phosphorylation, and apoptosis, among others.<h4>Conclusions</h4>These data reveal novel and previously identified gene expression differences between lens epithelial and cortical fiber cells. The gene expression differences indicate distinct pathways and functions important for the specialization of lens epithelial and fiber cells and provide insight into potential mechanisms important for lens cell differentiation.

The Drosophila larval neuromuscular system serves as a valuable model for studying the genes required for synaptic development and function. N-Ethylmaleimide sensitive factor (NSF) is a molecule known to be important in vesicular trafficking but neural expression of a dominant negative form of NSF2 induces an unexpected overgrowth of the Drosophila larval neuromuscular synapse. We have taken a genetic approach to understanding this novel phenotype by conducting a gain-of-function modifier screen to isolate genes that interact with the overgrowth phenotype. Our approach was to directly visualize the neuromuscular junction (NMJ) using a GFP transgene and screen for suppressors of NMJ overgrowth using the Gene Search collection of P-element insertions. Of the 3000 lines screened, we identified 99 lines that can partially restore the normal phenotype. Analysis of the GS element insertion sites by inverse PCR and comparison of the flanking DNA sequence to the Drosophila genome sequence revealed nearby genes for all but 10 of the 99 lines. The recovered genes, both known and predicted, include transcription factors, cytoskeletal elements, components of the ubiquitin pathway, and several signaling molecules. This collection of genes that suppress the NSF2 neuromuscular junction overgrowth phenotype is a valuable resource in our efforts to further understand the role of NSF at the synapse.

Extracellular matrix (ECM) mineralization is a physiological process in bone and a pathological one in soft tissues. The mechanisms determining the spatial restriction of ECM mineralization to bone physiologically are poorly understood. Here we show that a normal extracellular phosphate concentration is required for bone mineralization, while lowering this concentration prevents mineralization of any ECM. However, simply raising extracellular phosphate concentration is not sufficient to induce pathological mineralization, this is because of the presence in all ECMs of pyrophosphate, an inhibitor of mineralization. ECM mineralization occurs only in bone because of the exclusive coexpression in osteoblasts of Type I collagen and Tnap, an enzyme that cleaves pyrophosphate. This dual requirement explains why Tnap ectopic expression in cells producing fibrillar collagen is sufficient to induce pathological mineralization. This study reveals that coexpression in osteoblasts of otherwise broadly expressed genes is necessary and sufficient to induce bone mineralization and provides evidence that pathological mineralization can be prevented by modulating extracellular phosphate concentration.

Ferroportin (FPN) mediates iron export from cells; FPN mutations are associated with the iron overloading disorder hemochromatosis. Previously, we found that the A77D, V162del, and G490D mutations inhibited FPN activity, but that other disease-associated FPN variants retained full iron export capability. The peptide hormone hepcidin inhibits FPN as part of a homeostatic negative feedback loop. We measured surface expression and function of wild-type FPN and fully active FPN mutants in the presence of hepcidin. We found that the Y64N and C326Y mutants of FPN are completely resistant to hepcidin inhibition and that N144D and N144H are partially resistant. Hemochromatosis-associated FPN mutations, therefore, either reduce iron export ability or produce an FPN variant that is insensitive to hepcidin. The former mutation type is associated with Kupffer-cell iron deposition and normal transferrin saturation in vivo, whereas patients with the latter category of FPN mutation have high transferrin saturation and tend to deposit iron throughout the liver parenchyma. FPN-linked hemochromatosis may have a variable pathogenesis depending on the causative FPN mutant.

Huntington disease (HD) is an adult-onset neurodegenerative disease caused by a toxic gain of function in the huntingtin (htt) protein. The contribution of wild-type htt function to the pathogenesis of HD is currently uncertain. To assess the role of wild-type htt in HD, we generated YAC128 mice that do not express wild-type htt (YAC128-/-) but express the same amount of mutant htt as normal YAC128 mice (YAC128+/+). YAC128-/- mice perform worse than YAC128+/+ mice in the rotarod test of motor coordination (P = 0.001) and are hypoactive compared with YAC128+/+ mice at 2 months (P = 0.003). Striatal neuropathology was not clearly worse in YAC128-/- mice compared with YAC128+/+ mice. There was no significant effect of decreased wild-type htt on striatal volume, neuronal counts or DARPP-32 expression but a modest worsening of striatal neuronal atrophy was evident (6%, P = 0.03). The testis of YAC128+/+ mice showed atrophy and degeneration, which was markedly worsened in the absence of wild-type htt (P = 0.001). YAC128+/+ mice also showed a male specific deficit in survival compared with WT mice which was exacerbated by the loss of wild-type htt (12-month-male survival, P < 0.001). Overall, we demonstrate that the loss of wild-type htt influences motor dysfunction, hyperkinesia, testicular degeneration and impaired lifespan in YAC128 mice. The mild effect of wild-type htt on striatal phenotypes in YAC128 mice suggests that the characteristic striatal neuropathology in HD is caused primarily by the toxicity of mutant htt and that replacement of wild-type htt will not be an adequate treatment for HD.

Hepatocellular carcinoma (HCC) is the commonest malignancy of the liver and is usually due to cirrhosis. Early detection of HCC and the premalignant dysplastic nodules has implications on the management options of tumor ablation, liver resection and transplantation. Magnetic resonance imaging is useful for the detection and characterization of lesions, in the identification of dysplastic nodules and their malignant transformation into HCC.

No abstract available.

With its good properties of biocompatibility and bioactivity hydroxyapatite (HA) is highly used as bone substitutes and as coatings on metallic prostheses. In order to improve the bioactive properties of HA, we have elaborated Zn2+ doped hydroxyapatite. Zn2+ ions substitute for Ca2+ cations in the HA structure and four Zn concentrations (Zn/Zn+Ca) were prepared at 0.5, 1, 2 and 5 at.%. To study physico-chemical reactions at the materials periphery, we immersed the bioceramics into biological fluids for intervals from 1 day to 20 days. The surface changes were studied at the nanometer scale by scanning transmission electron microscopy associated with energy dispersive X-ray spectroscopy. After 20 days of immersion, we observed the formation of a calcium-phosphate layer at the periphery of the HA doped with 5% zinc. This layer contains magnesium and its thickness was around 200 nm. Formation of this Ca-P-Mg layer represents the bioactive properties of 5% Zn-substituted hydroxyapatite. This biologically active layer improves the properties of HA and will permit a chemical bond between the ceramic and bone.

The occurrence of MLL gene rearrangement in acute megakaryoblastic leukemia (AML-M7, acute myeloid leukemia, French-American-British type M7) is very rare and limited to pediatric age: in particular, MLL-MLLT10 fusion, previously reported as characteristic of monocytic leukemia, has been reported in only one case of pediatric megakaryoblastic leukemia. We describe the second case with this association in light of the few reported cases of AML-M7 with MLL and/or 11q23 involvement.

Complex transitions in chromatin structure produce changes in genome function during development in metazoa. Linker histones, the last component of nucleosomes to be assembled into chromatin, comprise considerably divergent subtypes as compared with core histones. In all metazoa studied, their composition changes dramatically during early embryogenesis concomitant with zygotic gene activation, leading to distinct functional changes that are still poorly understood. Here, we show that early embryonic linker histone B4, which is maternally expressed, is functionally different from somatic histone H1 in influencing chromatin structure and dynamics. We developed a chromatin assembly system with nucleosome assembly protein-1 as a linker histone chaperone. This assay system revealed that maternal histone B4 allows chromatin to be remodeled by ATP-dependent chromatin remodeling factor, whereas somatic histone H1 prevents this remodeling. Structural analysis shows that histone B4 does not significantly restrict the accessibility of linker DNA. These findings define the functional significance of developmental changes in linker histone variants. We propose a model that holds that maternally expressed linker histones are key molecules specifying nuclear dynamics with respect to embryonic totipotency.

Huntington's disease (HD) is a fatal, dominant neurogenetic disorder. HD results from polyglutamine repeat expansion (CAG codon, Q) in exon 1 of HD, conferring a toxic gain of function on the protein huntingtin (htt). Currently, no preventative treatment exists for HD. RNA interference (RNAi) has emerged as a potential therapeutic tool for treating dominant diseases by directly reducing disease gene expression. Here, we show that RNAi directed against mutant human htt reduced htt mRNA and protein expression in cell culture and in HD mouse brain. Importantly, htt gene silencing improved behavioral and neuropathological abnormalities associated with HD. Our data provide support for the further development of RNAi for HD therapy.

Once genetic linkage has been identified for a complex disease, the next step is often association analysis, in which single-nucleotide polymorphisms (SNPs) within the linkage region are genotyped and tested for association with the disease. If a SNP shows evidence of association, it is useful to know whether the linkage result can be explained, in part or in full, by the candidate SNP. We propose a novel approach that quantifies the degree of linkage disequilibrium (LD) between the candidate SNP and the putative disease locus through joint modeling of linkage and association. We describe a simple likelihood of the marker data conditional on the trait data for a sample of affected sib pairs, with disease penetrances and disease-SNP haplotype frequencies as parameters. We estimate model parameters by maximum likelihood and propose two likelihood-ratio tests to characterize the relationship of the candidate SNP and the disease locus. The first test assesses whether the candidate SNP and the disease locus are in linkage equilibrium so that the SNP plays no causal role in the linkage signal. The second test assesses whether the candidate SNP and the disease locus are in complete LD so that the SNP or a marker in complete LD with it may account fully for the linkage signal. Our method also yields a genetic model that includes parameter estimates for disease-SNP haplotype frequencies and the degree of disease-SNP LD. Our method provides a new tool for detecting linkage and association and can be extended to study designs that include unaffected family members.

No abstract available.

Huntington disease (HD) is caused by an abnormal expanded polyglutamine repeat in the huntingtin protein. Insulin-like growth factor-1 is of particular interest in HD because it strongly inhibits polyQ-huntingtin-induced neurotoxicity. This neuroprotective effect involves the phosphorylation of huntingtin at Ser(421) by the prosurvival kinase Akt (Humbert, S., Bryson, E. A., Cordelieres, F. P., Connors, N. C., Datta, S. R., Finkbeiner, S., Greenberg, M. E., and Saudou, F. (2002) Dev. Cell 2, 831-837). Here, we report that Akt inhibits polyQ-huntingtin-induced toxicity in the absence of phosphorylation of huntingtin at Ser(421), suggesting that Akt also acts on other downstream effector(s) to prevent neuronal death in HD. We show that this survival effect involves the ADP-ribosylation factor-interacting protein arfaptin 2, the levels of which are increased in HD patients. Akt phosphorylated arfaptin 2 at Ser(260). Lack of phosphorylation of arfaptin 2 at this site substantially modified its subcellular distribution and increased neuronal death and intranuclear inclusions caused by polyQ-huntingtin. In contrast, arfaptin 2 had a neuroprotective effect on striatal neurons when phosphorylated by Akt. This effect is mediated through the proteasome, as phosphorylated arfaptin 2 inhibited the blockade of the proteasome induced by polyQ-huntingtin. This study points out a new mechanism by which Akt promotes neuroprotection in HD, emphasizing the potential therapeutic interest of this pathway in the disease.

There is growing evidence that serotonin (5-hydroxtryptamine, 5-HT) has major influences on brain development in mammals. Genetic and pharmacological disruption of 5-HT signaling during early postnatal development in rodents causes neuroanatomical cortical abnormalities, including malformations in the somatosensory cortex. Possible functional consequences of this developmental perturbation by 5-HT are not yet understood. We have examined the effects of deletion of the 5-HT transporter (5-HTT) gene on somatosensory responses to sensory stimulation in mice. Local cerebral glucose utilization (lCMR(glc)) was measured by the quantitative 2-deoxy[(14)C]glucose method during unilateral whisker stimulation in awake adult mice. lCMR(glc) was increased by stimulation but to a markedly lesser extent in 5-HTT(-/-) mice than in 5-HTT(+/+) controls in each of four major stations in the whisker-to-barrel cortex pathway (the spinal and principal sensory trigeminal nuclei, the ventral posteromedial thalamic nucleus, and the barrel region of the somatosensory cortex). Lowering brain 5-HT levels by administration of the selective tryptophan hydroxylase inhibitor p-chlorophenylalanine on postnatal days 0 and 1 restored the metabolic responses to functional activation in the whisker-to-barrel cortex pathway in adult 5-HTT(-/-) mice. These results indicate that functional deficits in this pathway in 5-HTT(-/-) mice may be due to excessive postnatal 5-HT activity. With or without postnatal p-chlorophenylalanine treatment, 5-HTT(-/-) mice exhibited lower resting (unstimulated) lCMR(glc) than did 5-HTT(+/+) controls in the whisker-to-barrel cortex pathway and throughout the brain. These findings have implications for understanding the potential long-term consequences of genetic and pharmacological disruption of 5-HT neurotransmission on cerebral functions during critical periods of postnatal development.

Huntington disease is a fatal neurodegenerative disorder caused by expansion of a polyglutamine tract in the protein huntingtin (Htt), which leads to its aggregation in nuclear and cytoplasmic inclusion bodies. We recently identified 52 loss-of-function mutations in yeast genes that enhance the toxicity of a mutant Htt fragment. Here we report the results from a genome-wide loss-of-function suppressor screen in which we identified 28 gene deletions that suppress toxicity of a mutant Htt fragment. The suppressors are known or predicted to have roles in vesicle transport, vacuolar degradation, transcription and prion-like aggregation. Among the most potent suppressors was Bna4 (kynurenine 3-monooxygenase), an enzyme in the kynurenine pathway of tryptophan degradation that has been linked directly to the pathophysiology of Huntington disease in humans by a mechanism that may involve reactive oxygen species. This finding is suggestive of a conserved mechanism of polyglutamine toxicity from yeast to humans and identifies new candidate therapeutic targets for the treatment of Huntington disease.

Brain-2 (Brn-2), a Class III POU transcription factor, plays an important role in the development of the neocortex and the establishment of neural cell lineage. Here we performed a yeast two-hybrid screening to identify the Brn-2 binding proteins. We obtained Jun-activation-domain-binding protein 1 (Jab1) as a new Brn-2 binding protein. Direct interaction between Brn-2 and Jab1 was confirmed by using a surface plasmon resonance biosensor. Considering the involvement of Jab1 in the onset of the neurodegenerative disorders such as Parkinson's disease and Alzheimer's disease, the interaction between Brn-2 and Jab1 may provide some insights into the understanding of neuronal development and neurodegenerative diseases.

Loss of chromosome arm 18q is a common event in human pancreatic, colon, and breast cancers and is often interpreted as representing loss of one or more tumor-suppressor genes. In this article, we describe two novel biallelic deletions at chromosome band 18q21.1 in a recently characterized human breast cancer cell line, HCC-1428. One lesion deletes a fragment of approximately 300 kb between SMAD4 and DCC that encodes no known genes. The second lesion is an in-frame SMAD4 deletion (amino acids 49-51) that affects the level of SMAD4 protein but not the SMAD4 message. This change accelerates 26S proteasome-mediated degradation of both endogenous and exogenous mutant SMAD4. Examination of normal DNA from the same patient demonstrated that both lesions are somatic and associated with loss of both normal alleles. These data support the concept that two independent tumor-suppressor loci exist at chromosome segment 18q21.1, one at SMAD4 and the other potentially at an enhancer of DCC or an unrelated novel gene.

Huntington disease (HD) is a devastating neurologic disorder that is characterized by abnormal expansion of a CAG nt repeat in the first exon of the huntingtin (htt) gene, producing a mutant protein with an elongated polyglutamine stretch. The presence of this mutant protein is correlated with the characteristic loss of striatal neurons and the clinical manifestation of HD. Currently there is no effective treatment for the associated cell death. The aim of this study was to evaluate an innovative strategy combining RNA interference (RNAi) and gene transfer via the nonviral Sleeping Beauty (SB) transposon system to down-regulate Htt expression. siRNA expression vectors were designed to target exons 1, 4, 6, and 62 of the human htt gene. Real-time RT-PCR and Western blot analysis were used to quantify Htt mRNA and protein levels, respectively, in human cell lines. The results indicated that selected siRNA constructs significantly decreased Htt mRNA and protein levels relative to controls. In addition, SB transposition of the siRNA constructs into the genome reduced long-term protein expression of Htt by approximately 90%. The combination of siRNA, the SB transposon, and an accurate transgenic mouse model may permit evaluation of this approach in preventing the pathogenesis associated with expression of mutant Htt.

The structural requirements for high affinity at the serotonin transporter (5-HTT) have been investigated through the preparation of rigid paroxetine analogs. Tropane-derived analogs (4a-i) of paroxetine (2) were designed and synthesized as potential inhibitors of serotonin reuptake based on the structural and biological similarity between the two compound classes. Overall, the affinity of tropane-derived analogs at the 5-HTT was found to be at least an order of magnitude lower than that of paroxetine and ranged from 2-400nM. The reduced affinity at the 5-HTT may be attributed to the inability of the rigid tropane-derived analogs to adopt conformations favored by the 5-HTT. Within the series of tropane analogs, the 2beta,3beta- and 2beta,3alpha-isomers, 4a and 4d, were the most potent at the DAT and NET and are also significantly more potent than paroxetine (2) suggesting that their reduced conformational flexibility maximizes residence time in conformations favored by these transporters. Examination of the previously published preparation and structural assignment of 4a by additional NMR and X-ray crystallographic data has established that nucleophilic addition to the intermediate 2beta-methanesulfonyloxymethyl-3beta-(4-fluorophenyl)tropane unexpectedly provided the aza-bicyclo[3.2.2]nonane derivative 10a.

High-speed chronoamperometry was used to determine the kinetics of clearance of exogenously applied serotonin (5-HT) in the dorsal raphe nucleus (DRN), dentate gyrus, CA3 region of the hippocampus or corpus callosum of anesthetized rats. Maximal velocity (Vmax) for 5-HT clearance was greatest in the DRN > dentate gyrus > CA3 > corpus callosum. Apparent affinity (K(T)) of the serotonin transporter (5-HTT) was similar in DRN and CA3 but greater in dentate gyrus and corpus callosum. A 90% loss of norepinephrine transporters (NET) produced by 6-hydroxydopamine pretreatment, resulted in a two-fold reduction in Vmax and a 30% decrease in K(T) in the dentate gyrus, but no change in kinetic parameters in the CA3 region. Pretreatment with 5,7-dihydroxytryptamine that resulted in a 90% reduction in 5-HTT density, modestly reduced Vmax in dentate gyrus but not in CA3. The same treatment had no effect on K(T) in the dentate gyrus but increased K(T) two-fold in the CA3. Neurotoxin treatments had no effect on 5-HT clearance in the corpus callosum. In hippocampal regions of intact rats, local application of the selective serotonin reuptake inhibitor, fluvoxamine, inhibited 5-HT clearance most robustly when the extracellular concentration of 5-HT was less than the K(T) value. By contrast, the NET antagonist, desipramine, significantly inhibited 5-HT clearance when extracellular concentrations of 5-HT were greater than the K(T) value. These data indicate that hippocampal uptake of 5-HT may be mediated by two processes, one with high affinity but low capacity (i.e. the 5-HTT) and the other with low affinity but a high capacity (i.e. the NET). These data show for the first time in the whole animal that 5-HT clearance in brain is regionally distinct with regard to rate and affinity.

<h4>Objectives</h4>To determine the allele frequency in the north Indian population of the two mutations in the HFE gene, the C282Y and H63D, which are responsible for causing hereditary haemochromatosis particularly in Caucasians of north European descent. We also wanted to correlate these mutations with the iron status in beta thalassemia traits.<h4>Patients and methods</h4>Sixty normal subjects and 215 individuals with beta thalassemia trait from north India were screened for the C282Y and H63D by polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP). We studied the iron status in these subjects and correlated the same with the HFE gene mutations.<h4>Results</h4>On screening for the C282Y gene mutation, all individuals were detected to be of the wild-type. The overall allele frequency of H63D was 9.09% with three individuals being homozygous for 63D. No statistically significant difference in the iron status was detected between the individuals of the wild-type and mutant for H63D. Haplotyping of the homozygous 63D alleles revealed the pattern to be identical to the Europeans.<h4>Conclusions</h4>Our study shows that H63D is prevalent and C282Y is rare in north Indians and the presence of 63D mutation does not increase body iron as measured by serum ferritin in beta thalassemia traits. Haplotype of H63D gene mutation is of an European haplotype, indicating a common origin.

Recently, we reported that a CD4(+)CD3(-)CD11c(-) accessory cell provided OX40-dependent survival signals to follicular T cells. These accessory cells express both OX40 ligand and CD30 ligand, and the receptors, OX40 and CD30, are both expressed on Th2-primed CD4 T cells. OX40 and CD30 signals share common signaling pathways, suggesting that CD30 signals might substantially compensate in OX40-deficient mice. In this report we have dissected the signaling roles of CD30 alone and in combination with OX40. CD30-deficient mice showed an impaired capacity to sustain follicular germinal center responses, and recall memory Ab responses were substantially reduced. Deficiencies in OX40 and CD30 signals were additive; secondary Ab responses were ablated in double-deficient mice. Although the initial proliferation of OX40/CD30 double-knockout OTII transgenic T cells was comparable to that of their normal counterparts, they failed to survive in vivo, and this was associated with reduced T cell numbers associated with CD4(+)CD3(-) cells in B follicles. Finally, we show that OX40/CD30 double-knockout OTII transgenic T cells fail to survive compared with normal T cells when cocultured with CD4(+)CD3(-) cells in vitro.

<h4>Background/aims</h4>The implication of hemochromatosis (HFE) gene mutations in chronic viral hepatitis remains controversial. We therefore studied the prevalence of HFE mutations and their impact on the progression of chronic viral hepatitis in Taiwan.<h4>Patients & methods</h4>H63D and C282Y mutations were screened by using polymerase chain reaction followed by restriction fragment length polymorphism in 152 chronic hepatitis B patients with various stages of liver disease, 87 chronic hepatitis C patients with various stages of liver disease, and 49 healthy controls. The distribution of each allele frequency was then compared among different groups of patients and in various stages of liver disease.<h4>Results</h4>All three groups of patients were C282Y wild type and the majority of H63D mutations were heterozygotes. Although statistically not significant, allele frequencies of H63D mutation in hepatitis B-related liver cirrhosis (6%) and hepatitis C-related liver cirrhosis (9.1%) were higher than those in healthy control (2%). After adjustment for age and sex, hepatitis B patients with H63D heterozygosity had a higher likelihood of cirrhosis than those with H63D wild type (odds ratios (OR): 3.2, confidence interval (CI): 0.49-20.5, P = 0.22). Similarly, hepatitis C patients with H63D homozygosity had a higher likelihood of cirrhosis compared with those with H63D wild type (OR: 2.35, CI : 0.19-28.5, P = 0.52).<h4>Conclusions</h4>Almost all Taiwanese are C282Y wild type. H63D heterozygote and homozygote, occurring in less than 5% of the subjects, tended to be associated with the development of liver cirrhosis, irrespective of viral etiology. Screening for H63D mutation might be considered in patients with chronic viral hepatitis in Taiwan.

Apheresis may be performed with many different techniques. The basis for different therapeutic approaches lies in the pathophysiological processes present in the diseases that have to be treated. Over the years more sophisticated devices have been developed. The most frequent treatment is plasma exchange (plasmapheresis) using centrifugation or single filtration techniques. In addition cascade filtration and subsequent adsorption from plasma is done. Thereby removal is done by adsorption of molecules such as bilirubin, immunoglobulins (immunoadsorption), circulating immune complexes, various antibodies including those against blood types. Such adsorption technologies have also been developed to allow adsorption directly from a column perfused by whole blood (hemoperfusion). By combining various techniques, systems are available that allow bridging of patients with hepatic failure to transplantation (MARS, Prometheus). By adding e.g., hepatic cells to such systems, besides dialysis and adsorption, cells will help to degrade toxic molecules. Such bioreactors are in clinical use. Apheresis includes also the removal or retrieval of cells from blood for e.g., stemcell transplantation, polycythaemia or hemochromatosis. Removal of leukocytes from blood using leukocyte filters is indicated in inflammatory bowel diseases. By specifically irradiating lymphocytes and monocytes with UV light using the technique of extra corporeal photochemotherapy (ECTP) various immunological diseases are treated. On the other hand, various alternative techniques may be used for the same disorder. Thus for patients with high plasma LDL-cholesterol not responding to other lipid lowering strategic treatment, alternative therapy may be done either by cascade filtration, adsorption technology from plasma, heparin precipitation (HELP-system) or hemoperfusion. This article describes various techniques in clinical use.

Uterine serous papillary cancer (USPC) represents a rare but highly aggressive variant of endometrial cancer, the most common gynecologic tumour in women. We used oligonucleotide microarrays that interrogate the expression of some 10 000 known genes to profile 10 highly purified primary USPC cultures and five normal endometrial cells (NEC). We report that unsupervised analysis of mRNA fingerprints readily distinguished USPC from normal endometrial epithelial cells and identified 139 and 390 genes that exhibited >5-fold upregulation and downregulation, respectively, in primary USPC when compared to NEC. Many of the genes upregulated in USPC were found to represent adhesion molecules, secreted proteins and oncogenes, such as L1 cell adhesion molecule, claudin-3 and claudin-4, kallikrein 6 (protease M) and kallikrein 10 (NES1), interleukin-6 and c-erbB2. Downregulated genes in USPC included SEMACAP3, ras homolog gene family, member I (ARHI), and differentially downregulated in ovarian carcinoma gene 1. Quantitative RT-PCR was used to validate differences in gene expression between USPC and NEC for several of these genes. Owing to its potential as a novel therapeutic marker, expression of the high-affinity epithelial receptor for Clostridium perfringens enterotoxin (CPE) claudin-4 was further validated through immunohistochemical analysis of formalin-fixed paraffin-embedded specimens from which the primary USPC cultures were obtained, as well as an independent set of archival USPC specimens. Finally, the sensitivity of primary USPC to the administration of scalar doses of CPE in vitro was also demonstrated. Our results highlight the novel molecular features of USPC and provide a foundation for the development of new type-specific therapies against this highly aggressive variant of endometrial cancer.

Oesophageal carcinoma remains a disease of poor prognosis. Surgical cure rates are compromised by the fact that most patients are diagnosed at a late stage of disease because of the delayed onset of symptoms, by which time metastases and organ infiltration may have already occurred. Thus, invasion and metastases play a key role in influencing patient survival, and the search for novel treatments may therefore hinge on gaining insight into the mechanisms controlling these processes. It has been established that the initial step in the metastatic cascade is the detachment of tumour cells from the primary tumour via dysregulation of normal cell-cell and cell-matrix interactions. Distinct proteins known as cell adhesion molecules (CAMs) mediate these interactions. In recent years, a plethora of information has contributed to the in depth understanding of these molecules. This review provides a brief description of five families of CAMs (cadherins, integrins, CD44, immunoglobulin superfamily, and selectins) and highlights their altered expression in relation both to prognosis and tumour behaviour in squamous cell carcinoma and adenocarcinoma of the oesophagus.

Haptoglobin is the plasma protein with the highest binding affinity for hemoglobin. The strength of hemoglobin binding and the existence of a specific receptor for the haptoglobin-hemoglobin complex in the monocyte/macrophage system clearly suggest that haptoglobin may have a crucial role in heme-iron recovery. We used haptoglobin-null mice to evaluate the impact of haptoglobin gene inactivation on iron metabolism. Haptoglobin deficiency led to increased deposition of hemoglobin in proximal tubules of the kidney instead of the liver and the spleen as occurred in wild-type mice. This difference in organ distribution of hemoglobin in haptoglobin-deficient mice resulted in abnormal iron deposits in proximal tubules during aging. Moreover, iron also accumulated in proximal tubules after renal ischemia-reperfusion injury or after an acute plasma heme-protein overload caused by muscle injury, without affecting morphological and functional parameters of renal damage. These data demonstrate that haptoglobin crucially prevents glomerular filtration of hemoglobin and, consequently, renal iron loading during aging and following acute plasma heme-protein overload.

BALB/c Fech(m1Pas) mice have a mutated ferrochelatase gene resulting in protoporphyria that models the hepatic injury occurring sporadically in human erythropoietic protoporphyria. We used this mouse model to study the development of the injury and to compare the dysfunction of heme synthesis with hepatic gene expression of liver metabolism, oxidative stress, and cellular injury/inflammation. From an early age expression of total cytochrome P450 and many of its isoforms was significantly lower than in wild-type mice. However, despite massive accumulation of protoporphyrin in the liver, expression of the main genes controlling heme synthesis and catabolism (Alas1 and Hmox1, respectively) were only modestly affected even in the presence of the cytochrome P450-inducing CAR agonist 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene. In contrast, in BALB/c mice exhibiting griseofulvin-induced hepatic protoporphyria with induction and destruction of cytochrome P450, both Alas1 and Hmox1 genes were markedly up-regulated. Other expression profiles in BALB/c Fech(m1Pas) mice identified roles for oxidative mechanisms in liver injury while modulated gene expression of hepatocyte transport proteins and cholesterol and bile acid synthesis illustrated the development of cholestasis. Subsequent inflammation and cirrhosis were also shown by the up-regulation of cytokine, cell cycling, and procollagen genes. Thus, gene expression profiles studied in Fech(m1Pas) mice may provide candidates for human polymorphisms that explain the sporadic hepatic consequences of erythropoietic protoporphyria.

We describe a patient with hereditary hemochromatosis (homozygous for C282Y mutation) and neutropenia who was found to have underlying T-cell large granular lymphocytic (T-LGL) leukemia. The diagnosis was confirmed by demonstrating T-cell receptor (TCR) gene rearrangement by polymerase chain reaction (PCR). Multiple quantitative and qualitative defects have been described for the T cells of patients with hemochromatosis. Although the association between the two may be fortuitous, this case report raises the possibility that the T cells in these patients may be susceptible to leukemic transformation as well.

No abstract available.

Twenty-seven carbazole-utilizing bacterial strains were isolated from environmental samples, and were classified into 14 groups by amplified ribosomal DNA restriction analysis. Southern hybridization analyses showed that 3 and 17 isolates possessed the car gene homologs of Pseudomonas resinovorans CA10 and Sphingomonas sp. strain KA1, respectively. Of the 17 isolates, 2 isolates also have the homolog of the carAa gene of Sphingomonas sp. strain CB3. While the genome of one isolate, a Gram-positive Nocardioides sp. strain IC177, showed no hybridization to any car gene probes, PCR and sequence analyses indicated that strain IC177 had tandemly linked carAa and carC gene homologs whose deduced amino acid sequences showed 51% and 36% identities with those of strain KA1.

Estrogen is an immunoregulatory agent, in that hormone deprivation increases while 17beta-estradiol (E2) administration blocks the inflammatory response; however, the underlying mechanism is still unknown. The transcription factor p65/relA, a member of the nuclear factor kappaB (NF-kappaB) family, plays a major role in inflammation and drives the expression of proinflammatory mediators. Here we report a novel mechanism of action of E2 in inflammation. We observe that in macrophages E2 blocks lipopolysaccharide-induced DNA binding and transcriptional activity of p65 by preventing its nuclear translocation. This effect is selectively activated in macrophages to prevent p65 activation by inflammatory agents and extends to other members of the NF-kappaB family, including c-Rel and p50. We observe that E2 activates a rapid and persistent response that involves the activation of phosphatidylinositol 3-kinase, without requiring de novo protein synthesis or modifying Ikappa-Balpha degradation and mitogen-activated protein kinase activation. Using a time course experiment and the microtubule-disrupting agent nocodazole, we observe that the hormone inhibits p65 intracellular transport to the nucleus. This activity is selectively mediated by estrogen receptor alpha (ERalpha) and not ERbeta and is not shared by conventional anti-inflammatory drugs. These results unravel a novel and unique mechanism for E2 anti-inflammatory activity, which may be useful for identifying more selective ligands for the prevention of the inflammatory response.

No abstract available.

<h4>Aim</h4>Microsatellites are the repeated DNA sequences scattered widely within the genomes and closely linked with many important genes. This study was designed to characterize the changes of microsatellite DNA loss of heterozygosity (LOH) in esophageal carcinogenesis.<h4>Methods</h4>Allelic deletions in 32 cases of matched precancerous, cancerous and normal tissues were examined by syringe microdissection under an anatomic microscope and microsatellite polymorphism analysis using 15 polymorphic markers on chromosomes 3p, 5q, 6p, 9p, 13q, 17p, 17q and 18q.<h4>Results</h4>Microsatellite DNA LOH was observed in precancerous and cancerous tissues, except D9S1752. The rate of LOH increased remarkably with the lesions progressed from basal cell hyperplasia (BCH) to squamous cell carcinoma (SCC) (P<0.05). Three markers, D9S171, D13S260 and TP53, showed the highest incidence of LOH (>60%). LOH loci were different in precancerous and cancerous tissues. LOH in D3S1234 and TP53 was the common event in different lesions from the same patients.<h4>Conclusion</h4>Microsatellite DNA LOH occurs in early stage of human esophageal carcinogenesis, even in BCH. With the lesion progressed, gene instability increases, the accumulation of this change may be one of the important mechanisms driving precancerous lesions to cancer.

mRNA localisation, as a mechanism for directing localised protein synthesis, plays a vital role in the functioning of certain cells, such as neurons and oocytes. Potentially this mechanism may also occur in polarised intestinal epithelial cells. Here we show that Staufen1(55), a protein involved in mRNA localisation and transport, is asymmetrically distributed in differentiated Caco-2 intestinal epithelial cells and partly co-localised with calnexin, a marker of the endoplasmic reticulum. The localisation to the apical region of the cell indicates that Staufen may be involved in localisation of transcripts to this domain.

During development, axons migrate long distances in responses to attractive or repulsive signals that are detected by their growth cones. One of these signals is mediated by netrin-1, a diffusible laminin-related molecule that both attracts and repels growth cones via interaction with its receptor DCC (deleted in colorectal cancer). Here we show that DCC in both commissural neurons and immortalized cells, is partially associated with cholesterol- and sphingolipid-enriched membrane domains named lipid rafts. This localization of DCC in lipid rafts is mediated by the palmitoylation within its transmembrane region. Moreover, this raft localization of DCC is required for netrin-1-induced DCC-dependent ERK activation, and netrin-1-mediated axon outgrowth requires lipid raft integrity. Thus, the presence of axon guidance-related receptors in lipid rafts appears to be a crucial pre-requisite for growth cone response to chemo-attractive or repulsive cues.

This study was undertaken to provide first-time estimates for the seroprevalence of parvovirus B19 infection among daycare educators in Montréal, Canada, and to identify factors associated with seropositivity. A cross-sectional design was used. Directors and educators from 81 daycare centres (DCCs) were surveyed about DCC and personal characteristics respectively, and serum samples from 477 female educators were tested for parvovirus B19 IgG antibodies. The seroprevalence of parvovirus B19 was 70%. Parvovirus B19 seropositivity was significantly associated with age and with working experience in DCCs, but the latter association was restricted to educators aged less than 40 years. In conclusion, working as a daycare educator appears to be associated with increased risk of acquiring parvovirus B19 infection, but this finding will require further investigation. Because of the large proportion of educators susceptible to acquiring parvovirus B19 infection, our findings also highlight the need for preventive measures.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by a CAG trinucleotide expansion in exon 1 of the Huntingtin (Htt) gene. We show herein that in an HD transgenic mouse model (R6/2), daily administration of CGS21680 (CGS), an A(2A) adenosine receptor (A(2A)-R)-selective agonist, delayed the progressive deterioration of motor performance and prevented a reduction in brain weight. 3D-microMRI analysis revealed that CGS reversed the enlarged ventricle-to-brain ratio of R6/2 mice, with particular improvements in the left and right ventricles. (1)H-MRS showed that CGS significantly reduced the increased choline levels in the striatum. Immunohistochemical analyses further demonstrated that CGS reduced the size of ubiquitin-positive neuronal intranuclear inclusions (NIIs) in the striatum of R6/2 mice and ameliorated mutant Htt aggregation in a striatal progenitor cell line overexpressing mutant Htt with expanded polyQ. Moreover, chronic CGS treatment normalized the elevated blood glucose levels and reduced the overactivation of a major metabolic sensor [5'AMP-activated protein kinase (AMPK)] in the striatum of R6/2 mice. Since AMPK is a master switch for energy metabolism, modulation of energy dysfunction caused by the mutant Htt might contribute to the beneficial effects of CGS. Collectively, CGS is a potential drug candidate for the treatment of HD.

<h4>Background</h4>The suggestion that carriers of the HFE C282Y mutation absorb nonheme iron more efficiently than do carriers of the wild type has public health implications for countries where the C282Y mutation is common and foods are fortified with iron.<h4>Objective</h4>We investigated the effect of C282Y heterozygosity on nonheme-iron absorption from a diet high in bioavailable iron and from iron-fortified cereals.<h4>Design</h4>The subjects were recruited from a parallel study investigating the relation between HFE mutations, habitual diet, and iron status. Iron absorption was measured in 15 wild-type carriers and 15 C282Y heterozygotes aged >/=40 y. Each subject consumed 3 meals of high iron bioavailability (labeled with Fe-57) for 2 d and 2 meals with fortified cereal products (labeled with Fe-54) for the next 3 d. Iron absorption was measured from isotope incorporation into red blood cells 14 d after the last labeled meal and was corrected for utilization of absorbed iron by means of an intravenous infusion of Fe-58.<h4>Results</h4>Absorption of Fe-57 with the high-iron-bioavailability diet was 6.8 +/- 6.8% (0.6 +/- 0.6 mg/d) in the wild-type carriers and 7.6 +/- 3.2% (0.7 +/- 0.3 mg/d) in the C282Y heterozygotes. Absorption of Fe-54 with cereal products was 4.9 +/- 2.0% (0.7 +/- 0.3 mg/d) in the wild-type carriers and 5.3 +/- 1.3% (0.8 +/- 0.2 mg/d) in the C282Y heterozygotes.<h4>Conclusions</h4>There was no overall significant difference between C282Y heterozygotes and wild-type men in iron absorption from either dietary nonheme iron or fortified cereal products.

The incidence of colorectal carcinomas has been increasing over the last 50 years in Japan. In order to determine whether adenoma-carcinoma sequence (ACS) or de novo cancer development, generally considered to be two separate genetic pathways, might be responsible, K-ras codon 12 mutations, good markers for ACS, were examined in 59 and 84 cases of advanced colorectal cancer surgically resected in Cancer Institute Hospital of the Japanese Foundation for Cancer Research in 1960-1969 and in 1990-1999, respectively. There was no significant difference of K-ras codon 12 mutation between the 25.4% (15/59) in the 1960s and 36.9% (31/84) in the 1990s (P = 0.148), and the reference of distal colon cancer also showed no significant difference between 24.4% (11/45) and 36.4% (20/55). Yet elderly males showed a significant difference: 27.3% (6/22) in the 1960s and 59.3% (16/27) in the 1990s. The references of males, elderly patients (over 75 years old) and distal colon cancer in the 1990s were significantly more likely to demonstrate mutations than their counterparts in the 1960s. There was no variation with the tumor location. The results suggest that the ACS pathway might have primarily contributed to the increased incidence of colorectal cancer in elderly males in Japan.

Condensins are multi-subunit protein complexes that play a central role in mitotic chromosome assembly and segregation. The complexes contain 'structural maintenance of chromosomes' (SMC) ATPase subunits, and induce DNA supercoiling and looping in an ATP-hydrolysis-dependent manner in vitro. Vertebrate cells have two different condensin complexes, condensins I and II, each containing a unique set of regulatory subunits. Condensin II participates in an early stage of chromosome condensation within the prophase nucleus. Condensin I gains access to chromosomes only after the nuclear envelope breaks down, and collaborates with condensin II to assemble metaphase chromosomes with fully resolved sister chromatids. The complexes also play critical roles in meiotic chromosome segregation and in interphase processes such as gene repression and checkpoint responses. In bacterial cells, ancestral forms of condensins control chromosome dynamics. Dissecting the diverse functions of condensins is likely to be central to our understanding of genome organization, stability and evolution.

3beta-(3'-Methyl-4'-chlorophenyl)tropane-2-carboxylic acid methyl ester (3b, RTI-112) is a 3-phenyltropane analogue that has high affinity for both the dopamine and serotonin transporters (DAT and 5-HTT, respectively). Compound 3b shows significant reduction of cocaine self-administration in rhesus monkeys, yet fails to maintain robust drug self-administration. PET studies revealed that unlike more DAT selective analogues such as GBR 12 909 and 3-(4-chlorophenyl)tropane-2-carboxylic acid phenyl ester (RTI-113), 3b shows no detectible DAT occupancy when dosed at its ED(50) for reduction of cocaine self-administration. In contrast, it highly occupies the 5-HTT at this dose. In this study, we report the synthesis and monoamine transporter binding potency of several new 3-(3',4'-disubstituted phenyl)tropane-2-carboxylic acid methyl esters (3c-k), which have binding properties very similar to 3b. With the exception of the 3',4'-dimethyl analogue 3k, all of the compounds possess subnanomolar IC(50) and K(i) values at the DAT and 5-HTT, respectively. The 3'-chloro-4'-bromo analogue 3e (IC(50) = 0.12 nM) and the 3'-bromo-4'-iodo analogue 3i (K(i) = 0.14 nM) are the most potent analogues at the DAT and 5-HTT, respectively. These compounds will be useful to further characterize the highly interesting behavioral profile of 3b.

<h4>Background</h4>That the extent to which a particular individual will engage in problematic behaviors such as delinquency, violence, or drug abuse is determined by the way psychosocial, situational, and hereditary factors interact is widely accepted. However, only recently have researchers begun to investigate the interactions between specific genotypes and psychosocial factors in relation to behavior. The purpose of the present study was to investigate possible interactions between a polymorphism in the promoter region of the serotonin transporter (5-HTT) gene and family relations on adolescent alcohol consumption.<h4>Methods</h4>A cross-sectional study with a randomized sample from a total population of 16- and 19-year-old adolescents from a Swedish county was conducted. Eighty-one male and 119 female adolescents, who volunteered to participate after having answered a questionnaire, were randomly selected from quartiles of volunteers representing various degrees of psychosocial risk behavior.<h4>Results</h4>5-HTT genotype (p=0.029) and family relations (p=0.022) predicted alcohol consumption independently as well as through an interaction with one another (p=0.05). The model explained 11% of the variance in alcohol consumption. In a binary logistic model, we found that adolescents with the LS variant of the 5-HTT gene and with family relations being "neutral" or "bad" had a 12- to 14-fold increased risk for high intoxication frequency.<h4>Conclusions</h4>In sum, our results show that a functional polymorphism of the 5-HTT genotype, family relations, and interactions between these variables predict adolescent alcohol consumption in a randomized sample of adolescents.

Huntington's disease (HD) is an adult-onset neurodegenerative disorder involving motor dysfunction, cognitive deficits, and psychiatric disturbances that result from underlying striatal and cortical dysfunction and neuropathology. The YAC128 mouse model of HD reproduces both the motor deficits and selective degeneration observed in the human disease. However, the presence of cognitive impairment in this model has not been determined. Here, we report mild cognitive deficits in YAC128 mice that precede motor onset and progressively worsen with age. Rotarod testing revealed a motor learning deficit at 2 months of age that progresses such that by 12 months of age, untrained YAC128 mice are unable to learn the rotarod task. Additional support for cognitive dysfunction is evident in a simple swimming test in which YAC128 mice take longer to find the platform than wild-type (WT) controls beginning at 8 months of age. YAC128 mice also have deficits in open-field habituation and in a swimming T-maze test at this age. Strikingly, in the reversal phase of the swimming T-maze test, YAC128 mice take twice as long as WT mice to locate the platform, indicating a difficulty in changing strategy. At 12 months of age, YAC128 mice show decreased prepulse inhibition and habituation to acoustic startle. The clear pattern of cognitive dysfunction in YAC128 mice is similar to the symptoms and progression of cognitive deficits in human HD and provides both the opportunity to examine the relationship between cognitive dysfunction, motor impairment, and neuropathology in HD and to assess whether potential therapies for HD can restore cognitive function.

Epigenetic modifications play an important role in human cancer. One such modification, histone methylation, contributes to human cancer through deregulation of cancer-relevant genes. The yeast Dot1 and its human counterpart, hDOT1L, methylate lysine 79 located within the globular domain of histone H3. Here we report that hDOT1L interacts with AF10, an MLL (mixed lineage leukemia) fusion partner involved in acute myeloid leukemia, through the OM-LZ region of AF10 required for MLL-AF10-mediated leukemogenesis. We demonstrate that direct fusion of hDOT1L to MLL results in leukemic transformation in an hDOT1L methyltransferase activity-dependent manner. Transformation by MLL-hDOT1L and MLL-AF10 results in upregulation of a number of leukemia-relevant genes, such as Hoxa9, concomitant with hypermethylation of H3-K79. Our studies thus establish that mistargeting of hDOT1L to Hoxa9 plays an important role in MLL-AF10-mediated leukemogenesis and suggests that the enzymatic activity of hDOT1L may provide a potential target for therapeutic intervention.

Certain members of the TNF-receptor family have shown proinflammatory function during immune activation and can be directly involved with the pathogenic effects observed during an autoimmune episode. The TNF-R family members summarized in this review includes: TNF-RI + II, OX40, and 4-1BB and they are expressed on a variety of leukocytes within the body. Studies within the last decade suggest that all of these proteins or their natural ligands can be targeted with various agents designed to diminish clinical signs of disease in autoimmune models. The data from the preclinical models specifically involving TNF-blockade have led to the development of clinical trials for patients with multiple sclerosis and rheumatoid arthritis. This review will chronicle the preclinical development of agents designed to inhibit OX40 and 4-1BB functions in autoimmunity and discuss relevant preclinical and clinical data associated with TNF-blockade.

Local translation in neuronal dendrites is an important basis for long-term synaptic plasticity, and RNA granules in the dendrites are involved in the local translation. Here, we identify RNG105 (RNA granule protein 105), a novel RNA-binding protein, as a component of the RNA granules in dendrites of hippocampal neurons. The RNG105-localizing RNA granules contain mRNAs, the translational products of which play key roles in synaptic plasticity. RNG105 has an ability to repress translation both in vitro and in vivo, consistent with the finding that the RNA granule is translationally arrested in the basal conditions. Dissociation of RNG105 from the RNA granules is induced by BDNF, a growth factor responsible for synaptic plasticity. The RNG105 dissociation is coincident with the induction of local translation near the granules. These findings suggest that RNG105 is a translational repressor in the RNA granules and provide insight into the link between RNG105 dynamics and local translational regulation.

<h4>Background</h4>Iron overload and hemochromatosis are common, treatable conditions. HFE genotypes, levels of serum ferritin, transferrin saturation values, and self-reported medical history were studied in a multiethnic primary care population.<h4>Methods</h4>Participants were recruited from primary care practices and blood-drawing laboratories. Blood samples were tested for transferrin saturation, serum ferritin, and C282Y and H63D mutations of the HFE gene. Before genetic screening, participants were asked whether they had a history of medical conditions related to iron overload.<h4>Results</h4>Of the 99,711 participants, 299 were homozygous for the C282Y mutation. The estimated prevalence of C282Y homozygotes was higher in non-Hispanic whites (0.44 percent) than in Native Americans (0.11 percent), Hispanics (0.027 percent), blacks (0.014 percent), Pacific Islanders (0.012 percent), or Asians (0.000039 percent). Among participants who were homozygous for the C282Y mutation but in whom iron overload had not been diagnosed (227 participants), serum ferritin levels were greater than 300 mug per liter in 78 of 89 men (88 percent) and greater than 200 microg per liter in 79 of 138 women (57 percent). Pacific Islanders and Asians had the highest geometric mean levels of serum ferritin and mean transferrin saturation despite having the lowest prevalence of C282Y homozygotes. There were 364 participants in whom iron overload had not been diagnosed (29 C282Y homozygotes) who had a serum ferritin level greater than 1000 microg per liter. Among men, C282Y homozygotes and compound heterozygotes were more likely to report a history of liver disease than were participants without HFE mutations.<h4>Conclusions</h4>The C282Y mutation is most common in whites, and most C282Y homozygotes have elevations in serum ferritin levels and transferrin saturation. The C282Y mutation does not account for high mean serum ferritin levels and transferrin saturation values in nonwhites.

<h4>Background</h4>Hereditary hyperferritinemia cataract syndrome is an autosomal dominant condition that is characterized by a high serum ferritin level and bilateral early-onset cataracts in the absence of iron overload. The genetic abnormality is identified as a mutation in the 5' regulatory region of the L-ferritin messenger RNA known as the iron-responsive element (IRE). The IRE controls ferritin synthesis in response to cytoplasmic iron pools by interacting with regulatory proteins called iron responsive proteins. Mutations in the IRE decrease its affinity for iron responsive proteins, leading to the constitutive synthesis of L-ferritin which results in hyperferritinemia and the intracellular accumulation of ferritin in the lens and eventual cataract formation.<h4>Patients and methods</h4>A 22-year-old woman who was being investigated for hyperferritinemia was diagnosed with hereditary hyperferritinemia cataract syndrome after an extensive workup, including genetic testing for hemochromatosis and a liver biopsy to rule out iron overload. She developed anemia with phlebotomy treatments and subsequently developed symptomatic cataracts. The pedigree of her family affected with cataracts was consistent with an autosomal dominant transmission pattern. DNA was extracted from peripheral leukocytes of eight family members, four of whom were affected by cataracts. Polymerase chain reaction amplification of the 5' region of the L-ferritin gene was performed and a heterozygous point mutation (G32T) was identified in the bulge region of the IRE.<h4>Conclusion</h4>The combination of early-onset cataracts and an elevated ferritin level should suggest this genetic syndrome.

Regulatory T cells (Tregs) have been shown to be critical in the balance between autoimmunity and tolerance and have been implicated in several human autoimmune diseases. However, the small number of Tregs in peripheral blood limits their therapeutic potential. Therefore, we developed a protocol that would allow for the expansion of Tregs while retaining their suppressive activity. We isolated CD4+CD25 hi cells from human peripheral blood and expanded them in vitro in the presence of anti-CD3 and anti-CD28 magnetic Xcyte Dynabeads and high concentrations of exogenous Interleukin (IL)-2. Tregs were effectively expanded up to 200-fold while maintaining surface expression of CD25 and other markers of Tregs: CD62L, HLA-DR, CCR6, and FOXP3. The expanded Tregs suppressed proliferation and cytokine secretion of responder PBMCs in co-cultures stimulated with anti-CD3 or alloantigen. Treg expansion is a critical first step before consideration of Tregs as a therapeutic intervention in patients with autoimmune or graft-versus-host disease.

The HFE, H ferritin, TFR2, and ferroportin 1 genes of a Japanese patient diagnosed as having hemochromatosis were amplified by PCR and sequenced. A novel mutation in the ferroportin 1 was found in the patient. It was located in the noncoding region of the ferroportin 1; nucleotide 117 adenine was changed to guanine, 7 nucleotides downstream the iron responsive element (IRE) region. This mutation was not found in the patient's son or daughter, or in 50 healthy individuals. It was suggested that the mutation in the ferroportin 1 may be related to hemochromatosis of this patient.

In this study, we investigated the hypotheses that in human intervertebral disc (IVD) degeneration there is local production of the cytokine IL-1, and that this locally produced cytokine can induce the cellular and matrix changes of IVD degeneration. Immunohistochemistry was used to localize five members of the IL-1 family (IL-1alpha, IL-1beta, IL-1Ra (IL-1 receptor antagonist), IL-1RI (IL-1 receptor, type I), and ICE (IL-1beta-converting enzyme)) in non-degenerate and degenerate human IVDs. In addition, cells derived from non-degenerate and degenerate human IVDs were challenged with IL-1 agonists and the response was investigated using real-time PCR for a number of matrix-degrading enzymes, matrix proteins, and members of the IL-1 family. This study has shown that native disc cells from non-degenerate and degenerate discs produced the IL-1 agonists, antagonist, the active receptor, and IL-1beta-converting enzyme. In addition, immunopositivity for these proteins, with the exception of IL-1Ra, increased with severity of degeneration. We have also shown that IL-1 treatment of human IVD cells resulted in increased gene expression for the matrix-degrading enzymes (MMP 3 (matrix metalloproteinase 3), MMP 13 (matrix metalloproteinase 13), and ADAMTS-4 (a disintegrin and metalloproteinase with thrombospondin motifs)) and a decrease in the gene expression for matrix genes (aggrecan, collagen II, collagen I, and SOX6). In conclusion we have shown that IL-1 is produced in the degenerate IVD. It is synthesized by native disc cells, and treatment of human disc cells with IL-1 induces an imbalance between catabolic and anabolic events, responses that represent the changes seen during disc degeneration. Therefore, inhibiting IL-1 could be an important therapeutic target for preventing and reversing disc degeneration.

<h4>Objective</h4>Core-binding factor a1, Cbfa1, which belongs to the runt-domain gene family, is an essential transcription factor for osteoblastic differentiation and osteogenesis. To examine the effects of Cbfa1 exhibit on gene expression which involving in the chondrogenesis.<h4>Methods</h4>On the first, using RT-PCR method, we directly cloned Cbfa1/Runx2 full length cDNA from E13.5 days mouse embryos limb buds. Consequently Cbfa1/Runx2 was cloned into pcDNA3.1 plasmid to make a eukaryotic expression vector. All the clones were proved to be correct by enzyme cutting and sequencing analysis. On the second, we used pcDNA3.1-Cbfa1 to tansfect fibroblast. 48 hours later, we contrasted the gene transcription of Sox9, Sox5 and Sox6 between tansfected and untransfected. 72 hours later, type II collagen have also been examined by Western blotting.<h4>Results</h4>Cbfa1 overexpression in fibroblast can upregulate the expression of Sox9 and type II collagen, but it have no effect on the expression of Sox5 and Sox6.<h4>Conclusion</h4>Cbfa1 may involve in the regulation of chondrogenesis.

BACKGROUND: A previous clinical trial showed that selenium supplementation significantly reduced the incidence of prostate cancer. We report here a bioinformatics approach to gain new insights into selenium molecular targets that might be relevant to prostate cancer chemoprevention. MATERIALS AND METHODS: We first performed data mining analysis to identify genes which are consistently dysregulated in prostate cancer using published datasets from gene expression profiling of clinical prostate specimens. We then devised a method to systematically analyze three selenium microarray datasets from the LNCaP human prostate cancer cells, and to match the analysis to the cohort of genes implicated in prostate carcinogenesis. Moreover, we compared the selenium datasets with two datasets obtained from expression profiling of androgen-stimulated LNCaP cells. RESULTS: We found that selenium reverses the expression of genes implicated in prostate carcinogenesis. In addition, we found that selenium could counteract the effect of androgen on the expression of a subset obtained from androgen-regulated genes. CONCLUSIONS: The above information provides us with a treasure of new clues to investigate the mechanism of selenium chemoprevention of prostate cancer. Furthermore, these selenium target genes could also serve as biomarkers in future clinical trials to gauge the efficacy of selenium intervention.

No abstract available.