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Both genetic and epigenetic events have been implicated in the stepwise histological progression involving adenoma-carcinoma and hyperplastic polyp/serrated adenoma-carcinoma sequences in the development of colorectal cancer. Genetic changes have been observed at each step in the initiation and progression of polyps to adenocarcinomas. Epigenetic changes also occur at each step in the pathogenesis of colorectal cancers and include CpG island DNA hypermethylation in the promoter region of genes resulting in transcriptional silencing through associated changes in chromatin structure and effects on binding of transcription factors, and DNA global hypomethylation which leads to chromosomal instability. Recent studies on MLH1 and APC genes indicate that epigenetic and genetic changes cooperate to facilitate tumor initiation and progression. Since aberrant CGI DNA promoter hypermethylation can be detected not only in colorectal polyps and cancers, but also in sera and stool, hypermethylated genes may serve as molecular markers for early detection, risk assessment and diagnosis. In addition, silenced genes caused by CGI DNA promoter hypermethylation can be reactivated by demethylating agents and also by both the inhibitors of DNA methyltransferases and histone deacetylases. Therefore, these epigenetically acting drugs should be evaluated for their chemopreventive and therapeutic potential for colorectal cancers.

For production of proteins that are encoded by the mitochondrial genome, mitochondria rely on their own mitochondrial translation system, with the mitoribosome as its central component. Using extensive homology searches, we have reconstructed the evolutionary history of the mitoribosomal proteome that is encoded by a diverse subset of eukaryotic genomes, revealing an ancestral ribosome of alpha-proteobacterial descent that more than doubled its protein content in most eukaryotic lineages. We observe large variations in the protein content of mitoribosomes between different eukaryotes, with mammalian mitoribosomes sharing only 74 and 43% of its proteins with yeast and Leishmania mitoribosomes, respectively. We detected many previously unidentified mitochondrial ribosomal proteins (MRPs) and found that several have increased in size compared to their bacterial ancestral counterparts by addition of functional domains. Several new MRPs have originated via duplication of existing MRPs as well as by recruitment from outside of the mitoribosomal proteome. Using sensitive profile-profile homology searches, we found hitherto undetected homology between bacterial and eukaryotic ribosomal proteins, as well as between fungal and mammalian ribosomal proteins, detecting two novel human MRPs. These newly detected MRPs constitute, along with evolutionary conserved MRPs, excellent new screening targets for human patients with unresolved mitochondrial oxidative phosphorylation disorders.

<h4>Background</h4>The function and significance of the widespread expression of natural antisense transcripts (NATs) is largely unknown. The ability to quantitatively assess changes in NAT expression for many different transcripts in multiple samples would facilitate our understanding of this relatively new class of RNA molecules.<h4>Results</h4>Here, we demonstrate that standard expression analysis Affymetrix MOE430 and HG-U133 GeneChips contain hundreds of probe sets that detect NATs. Probe sets carrying a "Negative Strand Matching Probes" annotation in NetAffx were validated using Ensembl by manual and automated approaches. More than 50 % of the 1,113 probe sets with "Negative Strand Matching Probes" on the MOE430 2.0 GeneChip were confirmed as detecting NATs. Expression of selected antisense transcripts as indicated by Affymetrix data was confirmed using strand-specific RT-PCR. Thus, Affymetrix datasets can be mined to reveal information about the regulated expression of a considerable number of NATs. In a correlation analysis of 179 sense-antisense (SAS) probe set pairs using publicly available data from 1637 MOE430 2.0 GeneChips a significant number of SAS transcript pairs were found to be positively correlated.<h4>Conclusion</h4>Standard expression analysis Affymetrix GeneChips can be used to measure many different NATs. The large amount of samples deposited in microarray databases represents a valuable resource for a quantitative analysis of NAT expression and regulation in different cells, tissues and biological conditions.

The canonical transient receptor potential (TRPC) channels are a family of non-selective cation channels that are activated by increases in intracellular Ca(2+) and G(q)/phospholipase C-coupled receptors. We used quantitative real-time PCR, in situ hybridization, immunoblots and patch-clamp recording from several brain regions to examine the expression of the predominant TRPC channels in the rodent brain. Quantitative real-time PCR of the seven TRPC channels in the rodent brain revealed that TRPC4 and TRPC5 channels were the predominant TRPC subtypes in the adult rat brain. In situ hybridization histochemistry and immunoblotting further resolved a dense corticolimbic expression of the TRPC4 and TRPC5 channels. Total protein expression of HIP TRPC4 and 5 proteins increased throughout development and peaked late in adulthood (6-9 weeks). In adults, TRPC4 expression was high throughout the frontal cortex, lateral septum (LS), pyramidal cell layer of the hippocampus (HIP), dentate gyrus (DG), and ventral subiculum (vSUB). TRPC5 was highly expressed in the frontal cortex, pyramidal cell layer of the HIP, DG, and hypothalamus. Detailed examination of frontal cortical layer mRNA expression indicated TRPC4 mRNA is distributed throughout layers 2-6 of the prefrontal cortex (PFC), motor cortex (MCx), and somatosensory cortex (SCx). TRPC5 mRNA expression was concentrated specifically in the deep layers 5/6 and superficial layers 2/3 of the PFC and anterior cingulate. Patch-clamp recording indicated a strong metabotropic glutamate-activated cation current-mediated depolarization that was dependent on intracellular Ca(2+)and inhibited by protein kinase C in brain regions associated with dense TRPC4 or 5 expression and absent in regions lacking TRPC4 and 5 expression. Overall, the dense corticolimbic expression pattern suggests that these Gq/PLC coupled nonselective cation channels may be involved in learning, memory, and goal-directed behaviors.

The Huntington's disease (HD) CAG repeat, encoding a polymorphic glutamine tract in huntingtin, is inversely correlated with cellular energy level, with alleles over approximately 37 repeats leading to the loss of striatal neurons. This early HD neuronal specificity can be modeled by respiratory chain inhibitor 3-nitropropionic acid (3-NP) and, like 3-NP, mutant huntingtin has been proposed to directly influence the mitochondrion, via interaction or decreased PGC-1alpha expression. We have tested this hypothesis by comparing the gene expression changes due to mutant huntingtin accurately expressed in STHdh(Q111/Q111) cells with the changes produced by 3-NP treatment of wild-type striatal cells. In general, the HD mutation did not mimic 3-NP, although both produced a state of energy collapse that was mildly alleviated by the PGC-1alpha-coregulated nuclear respiratory factor 1 (Nrf-1). Moreover, unlike 3-NP, the HD CAG repeat did not significantly alter mitochondrial pathways in STHdh(Q111/Q111) cells, despite decreased Ppargc1a expression. Instead, the HD mutation enriched for processes linked to huntingtin normal function and Nf-kappaB signaling. Thus, rather than a direct impact on the mitochondrion, the polyglutamine tract may modulate some aspect of huntingtin's activity in extra-mitochondrial energy metabolism. Elucidation of this HD CAG-dependent pathway would spur efforts to achieve energy-based therapeutics in HD.

The present study investigated mRNA expression profiles of bovine oocytes and blastocysts by using a cross-species hybridization approach employing an array consisting of 15,529 human cDNAs as probe, thus enabling the identification of conserved genes during human and bovine preimplantation development. Our analysis revealed 419 genes that were expressed in both oocytes and blastocysts. The expression of 1,324 genes was detected exclusively in the blastocyst, in contrast to 164 in the oocyte including a significant number of novel genes. Genes indicative for transcriptional and translational control (ELAVL4, TACC3) were overexpressed in the oocyte, whereas cellular trafficking (SLC2A14, SLC1A3), proteasome (PSMA1, PSMB3), cell cycle (BUB3, CCNE1, GSPT1), and protein modification and turnover (TNK1, UBE3A) genes were found to be overexpressed in blastocysts. Transcripts implicated in chromatin remodeling were found in both oocytes (NASP, SMARCA2) and blastocysts (H2AFY, HDAC7A). The trophectodermal markers PSG2 and KRT18 were enriched 5- and 50-fold in the blastocyst. Pathway analysis revealed differential expression of genes involved in 107 distinct signaling and metabolic pathways. For example, phosphatidylinositol signaling and gluconeogenesis were prominent pathways identified in the blastocyst. Expression patterns in bovine and human blastocysts were to a large extent identical. This analysis compared the transcriptomes of bovine oocytes and blastocysts and provides a solid foundation for future studies on the first major differentiation events in blastocysts and identification of a set of markers indicative for regular mammalian development.

Genomewide association studies (GWAs) initially investigate hundreds of thousands of single-nucleotide polymorphisms (SNPs), and the most promising SNPs are further evaluated with additional subjects, for replication or a joint analysis. Deciding which SNPs merit follow-up is one of the most crucial aspects of these studies. We present here an approach for selecting the most-promising SNPs that incorporates into a hierarchical model both conventional results and other existing information about the SNPs. The model is developed for general use, its potential value is shown by application, and tools are provided for undertaking hierarchical modeling. By quantitatively harnessing all available information in GWAs, hierarchical modeling may more clearly distinguish true causal variants from noise.

Smad proteins are critical intracellular signaling mediators for the transforming growth factor beta (TGFbeta) superfamily. Here, we report that Erbin (for "ErbB2/Her2-interacting protein"), which contains leucine-rich repeats and a PDZ (PSD-95/DLG/ZO-1) domain, interacts specifically with Smad3 and, to a lesser extent, with Smad2 through a novel Smad-interacting domain (SID) adjacent to its PDZ domain. Increased expression of Erbin does not affect the level of TGFbeta-induced phosphorylation of Smad2/Smad3, but it physically sequesters Smad2/Smad3 from their association with Smad4 and hence negatively modulates TGFbeta-dependent transcriptional responses and cell growth inhibition. An isoform of Erbin encoded by an alternatively spliced transcript in human tissues lacks this SID and fails to inhibit TGFbeta responses. Consistently, knockdown of the endogenous Erbin gene with short hairpin RNA enhances TGFbeta-induced antiproliferative and transcriptional responses. In addition, Erbin suppresses activin/Smad2-dependent, but not BMP/Smad1-mediated, induction of endogenous gene expression in Xenopus embryos. Therefore, these results define Erbin as a novel negative modulator of Smad2/Smad3 functions and expand the physiological role of Erbin to the regulation of TGFbeta signaling.

<h4>Background</h4>The 3'-untranslated region (3'-UTR) of mRNA contains regulatory elements that are essential for the appropriate expression of many genes. These regulatory elements are involved in the control of nuclear transport, polyadenylation status, subcellular targetting as well as rates of translation and degradation of mRNA. Indeed, 3'-UTR mutations have been associated with disease, but frequently this region is not analyzed. To gain insights into congenital heart disease (CHD), we have been analyzing cardiac-specific transcription factor genes, including GATA4, which encodes a zinc finger transcription factor. Germline mutations in the coding region of GATA4 have been associated with septation defects of the human heart, but mutations are rather rare. Previously, we identified 19 somatically-derived zinc finger mutations in diseased tissues of malformed hearts. We now continued our search in the 609 bp 3'-UTR region of GATA4 to explore further molecular avenues leading to CHD.<h4>Methods</h4>By direct sequencing, we analyzed the 3'-UTR of GATA4 in DNA isolated from 68 formalin-fixed explanted hearts with complex cardiac malformations encompassing ventricular, atrial, and atrioventricular septal defects. We also analyzed blood samples of 12 patients with CHD and 100 unrelated healthy individuals.<h4>Results</h4>We identified germline and somatic mutations in the 3'-UTR of GATA4. In the malformed hearts, we found nine frequently occurring sequence alterations and six dbSNPs in the 3'-UTR region of GATA4. Seven of these mutations are predicted to affect RNA folding. We also found further five nonsynonymous mutations in exons 6 and 7 of GATA4. Except for the dbSNPs, analysis of tissue distal to the septation defect failed to detect sequence variations in the same donor, thus suggesting somatic origin and mosaicism of mutations. In a family, we observed c.+119A > T in the 3'-UTR associated with ASD type II.<h4>Conclusion</h4>Our results suggest that somatic GATA4 mutations in the 3'-UTR may provide an additional molecular rationale for CHD.

Previously, we showed that overexpression of ubiquilin reduces protein aggregates and toxicity of expanded polyglutamine proteins. Here, we investigated the mechanism of ubiquilin's protective effect. Immunofluorescence microscopy and immunoprecipitation studies indicated that ubiquilin colocalized and coimmunoprecipitated more with GFP-huntingtin-exon-1-fusion proteins containing a 74-polyglutamine tract than with GFP-huntingtin-fusion proteins containing a 28-polyglutamine tract or with GFP protein alone. Furthermore, overexpression of ubiquilin selectively enhanced the turnover of the expanded GFP-huntingtin-fusion protein. These results suggest that elevating ubiquilin levels could aid in the selective disposal of potentially toxic expanded polyglutamine proteins that are thought to cause several human diseases.

Netrins were first identified as neural guidance molecules, acting through receptors that are members of the DCC and UNC-5 family. All netrins share structural homology to the laminin N-terminal domains and the laminin epidermal growth factor-like domains of laminin short arms. Laminins use these domains to self-assemble into complex networks. Here we demonstrate that netrin-4 is a component of basement membranes and is integrated into the laminin polymer via interactions with the laminin gamma1 andgamma3 short arms. The binding is mediated through the laminin N-terminal domain of netrin-4. In contrast to netrin-4, other members of the netrin family do not bind to these laminin short arms. Moreover, a truncated form of netrin-4 completely inhibits laminin-111 self-assembly in vitro, and full-length netrin-4 can partially disrupt laminin self-interactions. When added to explant cultures, netrin-4 retards salivary gland branching morphogenesis.

APPL1 is an effector of the small GTPase Rab5. Together, they mediate a signal transduction pathway initiated by ligand binding to cell surface receptors. Interaction with Rab5 is confined to the amino (N)-terminal region of APPL1. We report the crystal structures of human APPL1 N-terminal BAR-PH domain motif. The BAR and PH domains, together with a novel linker helix, form an integrated, crescent-shaped, symmetrical dimer. This BAR-PH interaction is likely conserved in the class of BAR-PH containing proteins. Biochemical analyses indicate two independent Rab-binding sites located at the opposite ends of the dimer, where the PH domain directly interacts with Rab5 and Rab21. Besides structurally supporting the PH domain, the BAR domain also contributes to Rab binding through a small surface region in the vicinity of the PH domain. In stark contrast to the helix-dominated, Rab-binding domains previously reported, APPL1 PH domain employs beta-strands to interact with Rab5. On the Rab5 side, both switch regions are involved in the interaction. Thus we identified a new binding mode between PH domains and small GTPases.

Densin is a member of LAP (leucine-rich repeat and PDZ domain) protein family that localizes in kidney to slit diaphragms, which are essential components of the glomerular filtration barrier. We have previously shown that densin interacts with a crucial slit diaphragm protein, nephrin. Here, we searched for novel binding partners of densin by yeast-two hybrid assay and identified beta-catenin. The interaction was confirmed by reciprocal co-immunoprecipitation assay and the binding site in densin was determined by GST-pull down assays. The GST-tagged densin was also able to pull down P-cadherin together with beta-catenin from human kidney glomerular lysates. Furthermore, densin co-localized with beta-catenin and F-actin in cell-cell contacts in cultured mouse podocytes. During cell-cell contact disruption and reformation densin and beta-catenin were dislocated from and relocated back to plasma membrane in a similar fashion. These and our previous findings suggest that densin may associate with the cadherin-catenin and nephrin complex(es), and may be involved in the formation of the cell-cell contacts including the slit diaphragm.

Iron is an essential element of living cells and organisms as a component of numerous metabolic pathways. Hemoglobin and ferric-transferrin in vertebrate host blood are the two major iron sources for female mosquitoes. We used inductively coupled plasma mass spectrometry (ICP-MS) and radioisotope labeling to quantify the fate of iron supplied from hemoglobin or as transferrin in Aedes aegypti. At the end of the first gonotrophic cycle, approximately 87% of the ingested total meal heme iron was excreted, while 7% was distributed into the eggs and 6% was stored in different tissues. In contrast, approximately 8% of the iron provided as transferrin was excreted and of that absorbed, 77% was allocated to the eggs and 15% distributed in the tissues. Further analyses indicate that of the iron supplied in a blood meal, approximately 7% appears in the eggs and of this iron 98% is from hemoglobin and 2% from ferric-transferrin. Whereas, of iron from a blood meal retained in body of the female, approximately 97% is from heme and <1% is from transferrin. Evaluation of iron-binding proteins in hemolymph and egg following intake of (59)Fe-transferrin revealed that ferritin is iron loaded in these animals, and indicate that this protein plays a critical role in meal iron transport and iron storage in eggs in A. aegypti.

A series of amphiphilic cationic graft polymers (PEC) were synthesized by coupling poly(epsilon-caprolactone) of differing molecular weights (MW) to low MW branched polyethylenimine via an amide group. IR, (1)H-NMR and GPC were employed to characterize the graft copolymers. The self-assembly characteristics of these copolymers in an aqueous solution were studied by fluorescence techniques. The critical micelle concentration (CMC) varied from 0.044 to 0.032g/L when the MW of poly(epsilon-caprolactone) increased from 1,800 to 5,500. The micelles formed electrostatic complexes with a reporter gene (pCMV-Luc) after an anticancer drug, Doxorubicin (DOX), was loaded by dialysis method. Gel retardation studies proved that micelles with or without DOX were able to complex with DNA completely at an equivalent N/P ratio of around 2.0, indicating that drug loading did not interfere in the interaction between the PEI shell and DNA. Particle size slightly decreased at higher N/P ratios of polyplexes, but increased with drug encapsulation. It was also noted that DNA/micelle complexes were significantly less toxic to HepG2 cells than blank PEC micelles, and improved gene transfection efficiency (about 3 orders of magnitude greater than PEI 25K alone at most) whether DOX was present in the system or not. These results suggest that this group of cationic graft polymers may be a potential candidate for the development of a drug delivery system that can examine the synergistic effects of combined drug and gene therapy.

A 30-year-old Japanese woman with acquired severe aplastic anemia (SAA), diagnosed 20 years ago, was referred to our institution for allogeneic stem cell transplantation (SCT). As an unusual case of long-standing SAA, the patient was complicated with moderate heart failure due to secondary hemochromatosis. After successful SCT using a non-myeloablative conditioning regimen, she needed no transfusion. Five years after SCT, echocardiography showed a dramatic improvement of her cardiac function. This case indicates that the cardiac function in secondary hemochromatosis could be reversed once iron overload from multitransfusions is stopped.

<h4>Background</h4>MeCP2, methyl-CpG-binding protein 2, binds to methylated cytosines at CpG dinucleotides, as well as to unmethylated DNA, and affects chromatin condensation. MECP2 mutations in females lead to Rett syndrome, a neurological disorder characterized by developmental stagnation and regression, loss of purposeful hand movements and speech, stereotypic hand movements, deceleration of brain growth, autonomic dysfunction and seizures. Most mutations occur de novo during spermatogenesis. Located at Xq28, MECP2 is subject to X inactivation, and affected females are mosaic. Rare hemizygous males suffer from a severe congenital encephalopathy.<h4>Methods</h4>To identify the pathways mis-regulated by MeCP2 deficiency, microarray-based global gene expression studies were carried out in cerebellum of Mecp2 mutant mice. We compared transcript levels in mutant/wildtype male sibs of two different MeCP2-deficient mouse models at 2, 4 and 8 weeks of age. Increased transcript levels were evaluated by real-time quantitative RT-PCR. Chromatin immunoprecipitation assays were used to document in vivo MeCP2 binding to promoter regions of candidate target genes.<h4>Results</h4>Of several hundred genes with altered expression levels in the mutants, twice as many were increased than decreased, and only 27 were differentially expressed at more than one time point. The number of misregulated genes was 30% lower in mice with the exon 3 deletion (Mecp2tm1.1Jae) than in mice with the larger deletion (Mecp2tm1.1Bird). Between the mutants, few genes overlapped at each time point. Real-time quantitative RT-PCR assays validated increased transcript levels for four genes: Irak1, interleukin-1 receptor-associated kinase 1; Fxyd1, phospholemman, associated with Na, K-ATPase;Reln, encoding an extracellular signaling molecule essential for neuronal lamination and synaptic plasticity; and Gtl2/Meg3, an imprinted maternally expressed non-translated RNA that serves as a host gene for C/D box snoRNAs and microRNAs. Chromatin immunoprecipitation assays documented in vivo MeCP2 binding to promoter regions of Fxyd1, Reln, and Gtl2.<h4>Conclusion</h4>Transcriptional profiling of cerebellum failed to detect significant global changes in Mecp2-mutant mice. Increased transcript levels of Irak1, Fxyd1, Reln, and Gtl2 may contribute to the neuronal dysfunction in MeCP2-deficient mice and individuals with Rett syndrome. Our data provide testable hypotheses for future studies of the regulatory or signaling pathways that these genes act on.

Down syndrome (DS) is characterized by extensive phenotypic variability, with most traits occurring in only a fraction of affected individuals. Substantial gene-expression variation is present among unaffected individuals, and this variation has a strong genetic component. Since DS is caused by genomic-dosage imbalance, we hypothesize that gene-expression variation of human chromosome 21 (HSA21) genes in individuals with DS has an impact on the phenotypic variability among affected individuals. We studied gene-expression variation in 14 lymphoblastoid and 17 fibroblast cell lines from individuals with DS and an equal number of controls. Gene expression was assayed using quantitative real-time polymerase chain reaction on 100 and 106 HSA21 genes and 23 and 26 non-HSA21 genes in lymphoblastoid and fibroblast cell lines, respectively. Surprisingly, only 39% and 62% of HSA21 genes in lymphoblastoid and fibroblast cells, respectively, showed a statistically significant difference between DS and normal samples, although the average up-regulation of HSA21 genes was close to the expected 1.5-fold in both cell types. Gene-expression variation in DS and normal samples was evaluated using the Kolmogorov-Smirnov test. According to the degree of overlap in expression levels, we classified all genes into 3 groups: (A) nonoverlapping, (B) partially overlapping, and (C) extensively overlapping expression distributions between normal and DS samples. We hypothesize that, in each cell type, group A genes are the most dosage sensitive and are most likely involved in the constant DS traits, group B genes might be involved in variable DS traits, and group C genes are not dosage sensitive and are least likely to participate in DS pathological phenotypes. This study provides the first extensive data set on HSA21 gene-expression variation in DS and underscores its role in modulating the outcome of gene-dosage imbalance.

Effect of heat treatment of Ag-Cu-Pd dispersant particles on the structure, mechanical properties and mercury vapor release rate of an Ag-Cu-Sn/Ag-Cu-Pd-based dental amalgam has been investigated. Experimental results indicate that crystallinity of dispersant Ag-Cu-Pd alloy increases with increasing HTT, with most notable increase occurring between 100 and 200 degrees C. Increasing HTT of Ag-Cu-Pd alloy does not change much of the mercury/alloy ratio for amalgamation, but largely reduces working/setting time of the amalgam. The Ag-Cu-Pd particles in 7 d-aged amalgam are comprised primarily of an outer Sn/Cu/Pd-rich zone and an inner Ag/Cu/Pd-rich zone with eutectic-type morphology and chemical distribution. The annealing-enhanced Pd segregation effect is most significantly observed in the amalgam derived from 300 degrees C-annealed Ag-Cu-Pd dispersant. This amalgam also has the highest compressive strength, highest DTS, and lowest creep rate. Higher annealing temperature causes mechanical property of the amalgam to deteriorate. The initial mercury vapor release rates of amalgams derived from 100, 200 and 300 degrees C-annealed Ag-Cu-Pd dispersant are significantly lower than that derived from 400 degrees C-annealed dispersant.

Chx10/Vsx2 and Vsx1 are the only Paired-like CVC (Prd-L:CVC) homeobox genes in the mouse genome. Both are expressed in the retina and have important but distinct roles in retinal development. Mutations in Chx10/Vsx2 cause reduced retinal progenitor cell (RPC) proliferation and an absence of bipolar cells, while mutations in Vsx1 impair differentiation of cone bipolar cells. Given their structural similarities and importance in retinal development, we sought to determine if a regulatory interaction exists between these genes and whether inactivation of both genes blocks initiation of retinal development. We found that Chx10/Vsx2 binds to a specific sequence in the Vsx1 5'-intergenic region and represses the activity of a luciferase reporter under the control of the Vsx1 promoter. This is consistent with our observation that there is an inverse relationship between the levels of Chx10/Vsx2 and Vsx1 immunostaining within the bipolar cell class. Furthermore, Vsx1 mRNA is upregulated in the RPCs of Chx10/Vsx2 deficient mice and zebrafish embryos injected with a chx10/vsx2 morpholino. In mice deficient for both Chx10/Vsx2 and Vsx1 and zebrafish embryos co-injected with chx10/Vsx2 and vsx1 morpholinos, the changes in embryonic retinal development and marker expression are similar in magnitude to embryos with Chx10/Vsx2 loss of function only. From these studies, we propose that Vsx1 is a direct target of Chx10/Vsx2-mediated transcriptional repression. Although Vsx1 mRNA is upregulated in Chx10/Vsx2 deficient RPCs, Vsx1 does not genetically compensate for loss of Chx10/Vsx2, demonstrating that Prd-L:CVC genes, although important, are not absolutely required to initiate retinal development.

In an effort to identify proteins involved in the disease process of acute liver failure (ALF), we investigated changes in the plasma proteome associated with d-galactosamine/lipopolysaccharide (GalN/LPS) treatment of BALB/c mice. The plasma samples from mice with ALF and control were screened for potential differences using two-dimensional electrophoresis followed by liquid chromatography-electrospray ionization-tandem mass spectrometry or matrix associated laser desorption ionization-time-of-flight mass spectrometry. The expression levels of candidate protein named phosphatidylethanolamine binding protein (PEBP) in plasma and liver, brain tissues were confirmed by western blot and RT-PCR analyses. Results were confirmed in plasma samples of human beings. Seven proteins existed in plasma of GalN/LPS-treatment animals only but not in controls. They included PEBP, regucalcin, Cu/Zn superoxide dismutase, glyoxalase 1, malate dehydrogenase, proteasome subunit alpha type 1, and HPMS haptoglobin precursor. Two proteins, proteasome subunit alpha type 5 and apolipoprotein A-I precursor, were up-regulated by GalN/LPS, and one protein, HPMS haptoglobin precursor, was down-regulated by this treatment. Western blot analysis confirmed the results that PEBP protein levels increased significantly in plasma and liver tissues only in ALF mice, but not in surviving mice treated with GalN/LPS. Further analysis revealed that GalN/LPS also induced up-regulation of PEBP mRNA levels in liver tissues. Importantly, plasma obtained from ALF patients, but not from healthy volunteers or from hepatitis patients, also contained detectable levels of PEBP. The present study show that PEBP may be a potential plasma biomarker for ALF diagnosis and participate in the pathphysiological process of ALF.

During embryonic development post-mitotic neurons of the precerebellar neuroepithelium, migrate from the rhombic lip to the ventral part of the neural tube to form the precerebellar nuclei of the pons and medulla oblongata. In this study, we show that neural basic helix-loop-helix transcription factors NSCL-1 and -2 are expressed in all cells of the anterior extramural migration stream (aes), which forms the precerebellar nuclei. The combined inactivation of NSCL-1 and -2 led to a complete absence of the pontine nuclei and a strong reduction in the reticulotegmental nuclei. We demonstrate that NSCL-1/2 were required for a sustained expression of the netrin receptor and cell guidance molecule Dcc in the aes. Unc5H3, a second netrin receptor, which serves as a stop signal for migratory cells was up-regulated in NSCL-1/2 deficient cells, which ceased migration and accumulated ectopically. Furthermore, we observed a massive increase of apoptosis in cells of the aes in the absence of NSCL-1/2, which together with the arrest of migration might explain the virtually complete loss of aes-derived structures in NSCL-1/2 mutant mice. We conclude that NSCL-1/2 maintain migration and survival of cells in the aes.

<h4>Aim</h4>To determine the prevalence of selected HFE polymorphisms (C282Y, H63D and S65C) among patients with chronic viral hepatitis B and C and to investigate their role in the progression of liver disease.<h4>Methods</h4>A total of 207 subjects with chronic B or C viral hepatitis and 243 healthy controls were enrolled in the case-control study. Cases were further classified into three groups according to the clinical stage of liver disease: (A) virus carriers; (B) compensated liver disease; and (C) decompensated liver disease. HFE polymorphisms were detected by polymerase chain reaction-based methodology. Fisher's exact test, chi(2) and Kruskal-Wallis tests were used to test for differences in variables studied between groups. Haplotypes were inferred in silico and their distribution compared by permutation test. Modified survival (time-to-event) analysis was used to test for the differences in the progression to the decompensated liver disease in carriers of C282Y wild-type versus mutated genotypes.<h4>Results</h4>The frequency of HFE genotypes, alleles and haplotypes differed neither between HBV nor HCV patients versus controls. In HCV subjects: (i) the frequency of the 282Y allele was significantly higher in the (C) group compared to (B) group (12.5 vs 2.2%, respectively, P = 0.002, Fisher's exact test); and (ii) carriers of the 282Y mutation exhibited significantly faster progression to decompensated liver disease than wild-type carriers (P = 0.044, log-rank test).<h4>Conclusion</h4>Carriage of the minor HFE C282Y polymorphism is associated with decompensated liver disease and its earlier onset in the subjects with chronic viral hepatitis C in the Czech population.

Skilled movements, such as reaching and grasping, have classically been considered as originating in the primate lineage. For this reason, the use of rodents to investigate the genetic and molecular machinery of reaching and grasping has been limited in research. A few studies in rodents have now shown that these movements are not exclusive to primates. Here we present a new test, the Mouse Reaching and Grasping (MoRaG) performance scale, intended to help researchers in the characterization of these motor behaviors in the mouse. Within the MoRaG test battery we identified early phenotypes for the characterization of motor neurone (Tg[SOD1-G93A](dl)1Gur mice) and neurodegenerative (TgN(HD82Gln)81Dbo transgenic mice) disease models in addition to specific motor deficits associated with aging (C3H/HeH inbred strain). We conclude that the MoRaG test can be used to further investigate complex neuromuscular, neurological, neurodegenerative and behavioral disorders. Moreover, our study supports the validity of the mouse as a model for reaching and grasping studies.

HMG-CoA-reductase inhibitors (statins) influence lipid metabolism and have pleiotropic effects. Several statins reduce various forms of pulmonary hypertension (PH) in animal models. The relationship between atorvastatin and expression of serotonin transporter protein (5-HTT) remains unknown. This study focused on the effects of atorvastatin on the course of monocrotaline (MCT)-induced PH and its relation to 5-HTT expression. Male Sprague-Dawley rats were challenged with MCT with or without subsequent daily oral treatment with 0.1, 1, and 10 mg/kg of atorvastatin for 28 days. Over the 4-wk course, the progression of PH was followed by transthoracic echocardiography [pulmonary artery pressure was assessed by pulmonary artery flow acceleration time (PAAT), an estimate reciprocal to pulmonary artery pressure], and, at the end of the 4-wk course, invasive right ventricular pressure, right ventricular weight, quantitative morphology, and 5-HTT expression were measured. MCT caused significant PH as early as 7 days after injection. Atorvastatin treatment increased PAAT and reduced right ventricular pressure, right ventricular hypertrophy, and vascular remodeling over the 4-wk course. MCT challenge was associated with increased pulmonary vascular 5-HTT expression, and atorvastatin treatment reduced the 5-HTT expression. MCT-induced PH over the course of 4 wk can be easily followed by transthoracic echocardiography, and atorvastatin is effective in reducing the PH. Atorvastatin's effects are associated with a decrease of 5-HTT expression.

Gene expression profiling is a powerful method by which alterations in gene expression can be interrogated in a single experiment. The disease familial adenomatous polyposis (FAP) is associated with germline mutations in the APC gene, which result in aberrant beta-catenin control. The molecular mechanisms underlying colorectal cancer development in FAP are being characterised but limited information is available about other symptoms that occur in this disorder. Although extremely rare in the general population, desmoid tumours in approximately 10% of FAP patients. The aim of this study was to determine the similarities and differences in gene expression profiles in adenomas and compare them to those observed in desmoid tumours. Illumina whole genome gene expression BeadChips were used to measure gene expression in FAP adenomas and desmoid tumours. Similarities between gene expression profiles and mechanisms important in regulating formation of FAP adenomas and desmoid tumours were identified. This study furthers our understanding of the mechanisms underlying FAP and desmoid tumour formation.

An abnormal diastolic function of left ventricle represents the main pathophysiological mechanism responsible for different clinical states such as restrictive cardiomyopathy, infiltrative myocardial disease and, specially, diastolic heart failure (also called heart failure with preserved systolic function), which is present in a large number of patients with a clinical picture of pulmonary congestion.Although the invasive approach, through cardiac catheterization allowing the direct measurement of left ventricular filling pressure, myocardial relaxation and compliance, is considered the gold standard for the identification of diastolic dysfunction, several noninvasive methods have been proposed for the study of left ventricular diastolic function.Doppler echocardiography represents an excellent noninvasive technique to fully characterize the diastolic function in health and disease.

The therapeutic benefits of fungal beta-glucans have been demonstrated as immuno-stimulating agents. In this study, we aimed to explore the mechanisms used by yeast beta-glucan-rich particles to activate murine resident macrophages for cytokine secretion. We demonstrated that resident macrophages were effectively activated by whole yeast beta-glucan particles (WGPs), such as with the upregulation of co-stimulatory molecules and the secretion of cytokines. The binding ability of WGPs and the levels of cytokine secretion in resident macrophages were significantly inhibited by soluble yeast beta-glucan but not by blockade of zymosan glucan receptor dectin-1. In addition, WGP-stimulated cytokine secretion was partially dependent on the MyD-88 pathway but was not significantly affected in CR3-deficient (CR3(-/-)) mice. Furthermore, we showed that Syk kinase was recruited upon WGP stimulation and was required for the production of cytokines. Taken together, these observations suggest that beta-glucan recognition is necessary but not sufficient to induce inflammatory response on resident macrophages. In addition, beta-glucan particles may use differential mechanisms for cytokine secretion in resident macrophages that may modulate both innate and adaptive immunity.

The molecular mechanisms controlling differentiation of mesenchymal precursor cells into chondrocytes (chondrogenesis) are not completely understood. We have recently shown that the small GTPase RhoA inhibits this process. Here we demonstrate that a different Rho GTPase family member, Rac1, promotes chondrogenesis. Pharmacological inhibition of Rac1 expression in micromass culture resulted in reduced mRNA levels of the chondrogenic markers collagen II and aggrecan, and decreased accumulation of glycosaminoglycans. Expression of the essential chondrogenic transcription factors Sox9, Sox5, and Sox6 was also reduced upon inhibition of Rac1 signaling. In contrast, overexpression of Rac1 in the chondrogenic ATDC5 cell line increased mRNA transcripts of Sox9, 5, and 6, collagen II, and aggrecan. Inhibition of Rac1 resulted in a reduction in the number, size, and organization of cellular condensations and decreased expression of N-cadherin. Overexpression of Rac1 resulted in an increase in N-cadherin expression levels. Furthermore, genetic ablation of Rac1 in primary micromass cultures resulted in reduced expression of chondrogenic markers. Additionally, we provide evidence that Cdc42 also promotes chondrogenesis. Overexpression of Cdc42 in ATDC5 cells resulted in increased expression of Sox5, Sox9, and collagen II but not Sox6, aggrecan, or N-cadherin. Therefore, we demonstrate that Rac1 and Cdc42 are positive regulators of chondrogenesis, but act at least in part through different cellular and molecular mechanisms.

Enterovirus 71 (EV71) is a causative agent of hand, foot, and mouth disease and is also sometimes associated with serious neurological disorders. In this study, we characterized the antigenicity and tissue specificity of an attenuated strain of EV71 [EV71(S1-3')], which belongs to genotype A, in a monkey infection model. Three cynomolgus monkeys were inoculated with EV71(S1-3'), followed by lethal challenge with the parental virulent strain EV71(BrCr-TR) via an intravenous route on day 45 postinoculation of EV71(S1-3'). Monkeys inoculated with EV71(S1-3') showed a mild neurological symptom (tremor) but survived lethal challenge by virulent EV71(BrCr-TR) without exacerbation of the symptom. The immunized monkey sera showed a broad spectrum of neutralizing activity against different genotypes of EV71, including genotypes A, B1, B4, C2, and C4. For the strains examined, the sera showed the highest neutralization activity against the homotype (genotype A) and the lowest neutralization activity against genotype C2. The order of decreasing neutralization activity of sera was as follows: A > B1 > C4 > B4 > C2. To examine the tissue specificity of EV71(S1-3'), two monkeys were intravenously inoculated with EV71(S1-3'), followed by examination of virus distribution in the central nervous system (CNS) and extraneural tissues. In the CNS, EV71(S1-3') was isolated only from the spinal cord. These results indicate that EV71(S1-3') acts as an effective antigen, although this attenuated strain was still neurotropic when inoculated via the intravenous route.

Huntington's disease (HD) is one of nine neurodegenerative disorders caused by expansion of CAG repeats encoding polyglutamine in their respective, otherwise apparently unrelated proteins. Despite these proteins having widespread and overlapping expression patterns in the brain, a specific and unique subset of neurons exhibits particular vulnerability in each disease. It has been hypothesized that perturbation of normal protein function contributes to the specificity of neuronal vulnerability; however, the normal biological functions of many of these proteins including the HD gene product, Huntingtin (Htt), are unclear. To explore the roles of Htt, we have used antisense morpholino oligonucleotides to observe the effects of Htt deficiency in early zebrafish development. Knockdown of Htt expression resulted in a variety of developmental defects. Most notably, Htt-deficient zebrafish had hypochromic blood due to decreased hemoglobin production, despite the presence of iron within blood cells. Furthermore, transferrin receptor 1 transcripts were increased, suggesting cellular iron starvation. Provision of iron to the cytoplasm in a bio-available form restored hemoglobin production in Htt-deficient embryos. Since erythroid cells acquire iron via receptor-mediated endocytosis of transferrin, these results suggest a role for Htt in making endocytosed iron accessible for cellular utilization. Iron is required for oxidative energy production, and defects in iron homeostasis and energy metabolism are features of HD pathogenesis that are most pronounced in the major region of neurodegeneration. It is therefore plausible that perturbation of Htt's normal role in the iron pathway (by polyglutamine tract expansion) contributes to HD pathology, and particularly to its neuronal specificity.

<h4>Background</h4>Ovarian folliculogenesis in mammals is a complex process involving interactions between germ and somatic cells. Carefully orchestrated expression of transcription factors, cell adhesion molecules and growth factors are required for success. We have identified a germ-cell specific, basic helix-loop-helix transcription factor, FIGLA (Factor In the GermLine, Alpha) and demonstrated its involvement in two independent developmental processes: formation of the primordial follicle and coordinate expression of zona pellucida genes.<h4>Results</h4>Taking advantage of Figla null mouse lines, we have used a combined approach of microarray and Serial Analysis of Gene Expression (SAGE) to identify potential downstream target genes. Using high stringent cutoffs, we find that FIGLA functions as a key regulatory molecule in coordinating expression of the NALP family of genes, genes of known oocyte-specific expression and a set of functionally un-annotated genes. FIGLA also inhibits expression of male germ cell specific genes that might otherwise disrupt normal oogenesis.<h4>Conclusion</h4>These data implicate FIGLA as a central regulator of oocyte-specific genes that play roles in folliculogenesis, fertilization and early development.

<h4>Background</h4>Tryptophan hydroxylase 2 (TPH2) is the rate-limiting enzyme in brain serotonin (5-HT) biosynthesis. Although dysfunction of 5-HT neurotransmission has been implicated in a variety of neuropsychiatric conditions, the human TPH2 promoter has not been characterized in vitro.<h4>Methods</h4>The functional relevance of TPH2 promoter polymorphisms was determined with luciferase assays in primary serotonergic neurons from rat raphe nuclei and in human small cell lung carcinoma cells (SHP-77 cells). We also investigated transcription factor binding to the variant promoter sequence with electrophoretic mobility shift assay (EMSA).<h4>Results</h4>The polymorphism rs11178997 of the human TPH2 promoter significantly reduced TPH2 transcriptional activity by 22% and 7% in primary serotonergic neurons and in SHP-77 cells, respectively. In contrast, no significant differences in promoter activity were observed for the G- and T-alleles of rs4570625. The EMSA revealed reduced binding of the transcription factor POU3F2 (also known as Brn-2, N-Oct-3) to the A-allele of the polymorphism rs11178997. Overexpression of POU3F2 resulted in a robust activation of the TPH2 promoter (2.7-fold).<h4>Conclusions</h4>Our data suggest that the human TPH2 promoter polymorphism rs11178997 impacts on gene expression, which might have implications for the development and function of the serotonergic system in the brain.

The bulge region of the hair follicle serves as a repository for epithelial stem cells that can regenerate the follicle in each hair growth cycle and contribute to epidermis regeneration upon injury. Here we describe a population of multipotential stem cells in the hair follicle bulge region; these cells can be identified by fluorescence in transgenic nestin-GFP mice. The morphological features of these cells suggest that they maintain close associations with each other and with the surrounding niche. Upon explantation, these cells can give rise to neurosphere-like structures in vitro. When these cells are permitted to differentiate, they produce several cell types, including cells with neuronal, astrocytic, oligodendrocytic, smooth muscle, adipocytic, and other phenotypes. Furthermore, upon implantation into the developing nervous system of chick, these cells generate neuronal cells in vivo. We used transcriptional profiling to assess the relationship between these cells and embryonic and postnatal neural stem cells and to compare them with other stem cell populations of the bulge. Our results show that nestin-expressing cells in the bulge region of the hair follicle have stem cell-like properties, are multipotent, and can effectively generate cells of neural lineage in vitro and in vivo.

Slit, which mediates its function by binding to the Roundabout (Robo) receptor, has been shown to regulate neuronal, dendritic, and leukocyte migration. However, the molecular mechanism by which the Slit/Robo complex inhibits the migration of cells is not well defined. Here, we showed that Slit-2 can inhibit the CXCL12-induced chemotaxis and transendothelial migration of T cells and monocytes. We observed that CXCR4 associates with Robo-1 and that Slit-2 treatment enhances this association with the Robo-1 receptor. Robo-1 is a single-pass transmembrane receptor whose intracellular region contains four conserved motifs designated as CC0, CC1, CC2, and CC3. Structural and functional analyses of Robo receptors revealed that interaction of the CC3 motif with the CXCR4 receptor may regulate the CXCL12-induced chemotaxis of T cells. We further characterized Slit-2-mediated inhibition of the CXCL12/CXCR4 chemotactic pathway and found that Slit-2 can block the CXCL12-induced activation of the Src and Lck kinases but not Lyn kinase. Although Slit-2 did not inhibit the CXCL12-induced activation of MAPKs, it did inhibit the Akt phosphorylation and Rac activation induced by this chemokine. Altogether, our studies indicate a novel mechanism by which the Slit/Robo complex may inhibit the CXCR4/CXCL12-mediated chemotaxis of T cells.

<h4>Background</h4>The lipogenic enzyme fatty acid synthase (FAS) is up-regulated in a wide variety of cancers, and is considered a potential metabolic oncogene by virtue of its ability to enhance tumor cell survival. Inhibition of tumor FAS causes both cell cycle arrest and apoptosis, indicating FAS is a promising target for cancer treatment.<h4>Results</h4>Here, we used gene expression profiling to conduct a global study of the cellular processes affected by siRNA mediated knockdown of FAS in MDA-MB-435 mammary carcinoma cells. The study identified 169 up-regulated genes (> or = 1.5 fold) and 110 down-regulated genes (< or = 0.67 fold) in response to knockdown of FAS. These genes regulate several aspects of tumor function, including metabolism, cell survival/proliferation, DNA replication/transcription, and protein degradation. Quantitative pathway analysis using Gene Set Enrichment Analysis software further revealed that the most pronounced effect of FAS knockdown was down-regulation in pathways that regulate lipid metabolism, glycolysis, the TCA cycle and oxidative phosphorylation. These changes were coupled with up-regulation in genes involved in cell cycle arrest and death receptor mediated apoptotic pathways.<h4>Conclusion</h4>Together these findings reveal a wide network of pathways that are influenced in response to FAS knockdown and provide new insight into the role of this enzyme in tumor cell survival and proliferation.

Increased levels of the repressor element 1/neuron restrictive silencer element (RE1/NRSE) silencing activity promoter, and a consequent reduction in the transcription of many RE1/NRSE-bearing neuronal genes, including brain-derived neurotrophic factor (BDNF), have been demonstrated in Huntington disease (HD) and represent one possible effector of its selective neuronal vulnerability. Restoring the expression levels of neuronal genes in diseased neurons therefore seems to be an attractive therapeutic approach. To this end, we have developed a cell-based reporter assay for monitoring RE1/NRSE silencing activity and validated it by genetically inactivating the RE1/NRSE or pharmacologically stimulating global transcription. In a pilot compound screen, we identified three closely related structural analogues that up-regulate reporter expression at low nanomolar concentrations, and follow-up studies have shown that they efficaciously increase endogenous BDNF levels in HD cells. Moreover, one of the compounds increases the viability of HD cells. Our findings suggest a new avenue for the development of drugs for HD and other neurodegenerative disorders based on the pharmacological up-regulation of the production of the neuronal survival factor BDNF and of other RE1/NRSE-regulated neuronal genes.

Yersinia enterocolitica is a common cause of food-borne gastrointestinal disease leading to self-limiting diarrhea and mesenteric lymphadenitis. Occasionally, focal abscess formation in the livers and spleens of certain predisposed patients (those with iron overload states such as hemochromatosis) is observed. In the mouse oral infection model, yersiniae produce a similar disease involving the replication of yersiniae in the small intestine, the invasion of Peyer's patches, and dissemination to the liver and spleen. In these tissues and organs, yersiniae are known to replicate predominantly extracellularly and to form microcolonies. By infecting mice orally with a mixture of equal amounts of green- and red-fluorescing yersiniae (yersiniae expressing green or red fluorescent protein), we were able to show for the first time that yersiniae produce exclusively monoclonal microcolonies in Peyer's patches, the liver, and the spleen, indicating that a single bacterium is sufficient to induce microcolony and microabscess formation in vivo. Furthermore, we present evidence for the clonal invasion of Peyer's patches from the small intestine. The finding that only very few yersiniae are required to establish microcolonies in Peyer's patches is due to both Yersinia-specific and host-specific factors. We demonstrate that yersiniae growing in the small intestinal lumen show strongly reduced levels of invasin, the most important factor for the early invasion of Peyer's patches. Furthermore, we show that the host severely restricts sequential microcolony formation in previously infected Peyer's patches.

DNA microarrays currently provide measurements of sufficiently high quality to allow a wide variety of sound inferences about gene regulation and the coordination of cellular processes to be drawn. Nonetheless, a desire for greater precision in the measurements continues to drive the microarray research community to seek higher measurement quality through improvements in array fabrication and sample labeling and hybridization. We prepared oligonucleotide microarrays by printing 65-mer on aldehyde functional group-derivatized slides as described in a previous study. We could improve the reliability of data by removing enzymatic bias during probe labeling and hybridizing under a more stringent condition. This optimized method was used to profile gene expression patterns for nine different mouse tissues and organs, and multidimensional scaling (MDS) analysis of data showed both strong similarity between like samples and a clear, highly reproducible separation between different tissue samples. Three other microarrays were fabricated on commercial substrates and hybridized following the manufacturer's instructions. The data were then compared with in-house microarray data and reverse transcription-polymerase chain reaction (RT-PCR) data. The microarray printed on the custom aldehyde slide was superior to microarrays printed on commercially available substrate slides in terms of signal intensities, background, and hybridization characteristics. The data from the custom substrate microarray generally showed good agreement in quantitative changes up to 100-fold changes of transcript abundance with RT-PCR data. However, more accurate comparisons will be made as more genomic sequence information is gathered in the public data domain.

<h4>Background</h4>It has been estimated that more than 1 million workers in the United States are exposed to cobalt. Occupational exposure to 59 Co occurs mainly via inhalation and leads to various lung diseases. Cobalt is classified by the IARC as a possible human carcinogen (group 2B). Although there is evidence for in vivo and in vitro toxicity, the mechanisms of cobalt-induced lung toxicity are not fully known. The purpose of this work was to identify potential signatures of acute cobalt exposure using a toxicogenomic approach. Data analysis focused on some cellular processes and protein targets that are thought to be relevant for carcinogenesis, transport and biomarker research.<h4>Results</h4>A time course transcriptome analysis was performed on A549 human pulmonary cells, leading to the identification of 85 genes which are repressed or induced in response to soluble 59 Co. A group of 29 of these genes, representing the main biological functions, was assessed by quantitative RT-PCR. The expression profiles of six of them were then tested by quantitative RT-PCR in a time-dependent manner and three modulations were confirmed by Western blotting. The 85 modulated genes include potential cobalt carriers (FBXL2, ZNT1, SLC12A5), tumor suppressors or transcription factors (MAZ, DLG1, MYC, AXL) and genes linked to the stress response (UBC, HSPCB, BNIP3L). We also identified nine genes coding for secreted proteins as candidates for biomarker research. Of those, TIMP2 was found to be down-regulated and this modulation was confirmed, in a dose-dependent manner, at protein level in the supernatant of exposed cells.<h4>Conclusion</h4>Most of these genes have never been described as related to cobalt stress and provide original hypotheses for further study of the effects of this metal ion on human lung epithelial cells. A putative biomarker of cobalt toxicity was identified.

Maintain stemness, commit to a specific lineage, differentiate, proliferate, or die. These are essential decisions that every cell is constantly challenged to make in multi-cellular organisms to ensure proper development, adult maintenance, and adaptability. SRY-related high-mobility-group box (Sox) transcription factors have emerged in the animal kingdom to help cells effect such decisions. They are encoded by 20 genes in humans and mice. They share a highly conserved high-mobility-group box domain that was originally identified in SRY, the sex-determining gene on the Y chromosome, and that has derived from a canonical high-mobility-group domain characteristic of chromatin-associated proteins. The high-mobility-group box domain binds DNA in the minor groove and increases its DNA binding affinity and specificity by interacting with many types of transcription factors. It also bends DNA and may thereby confer on Sox proteins a unique and critical role in the assembly of transcriptional enhanceosomes. Sox proteins fall into eight groups. Most feature a transactivation or transrepression domain and thereby also act as typical transcription factors. Each gene has distinct expression pattern and molecular properties, often redundant with those in the same group and overlapping with those in other groups. As a whole the Sox family controls cell fate and differentiation in a multitude of processes, such as male differentiation, stemness, neurogenesis, and skeletogenesis. We review their specific molecular properties and in vivo roles, stress recent advances in the field, and suggest directions for future investigations.

<h4>Background</h4>To assess the interaction between the expression of netrin-1 or of its receptors to the prognosis of ductal adenocarcinoma of the pancreas.<h4>Methods</h4>In 82 patients with resectable pancreatic adenocarcinoma who underwent curative operation, the expression patterns of netrin-1, deleted in colorectal carcinomas (DCC), UNC5H3, and neogenin were determined by immunohistochemical staining. Kaplan-Meier analysis was performed to assess the prognostic relevance of the examined expression patterns.<h4>Results</h4>Median follow-up was 15 +/- 19.9 months (range, 4-108 months). Patients suffering from tumors with no or little expression of netrin-1 (n = 67) had a median recurrence-free survival of 10 months (95% CI, 7-13 months), while a middle to strong expression (n = 15) was associated with a significantly worse median recurrence-free survival of only four months (95% CI, three to five months, p = 0.0165). Overall and recurrence-free survival showed no significant differences between the different expression patterns of DCC, UNC5H3 or neogenin. Netrin-1 expression had significant impact (p = 0.001) on overall survival of patients suffering from poorly differentiated tumors. Stratification according to the nodal status revealed significant influence (p = 0.007) of UNC5H3 expression on the overall survival of patients with pN1 status.<h4>Conclusion</h4>Expression of netrin-1 has significant impact on time to tumor relapse in adenocarcinoma of the pancreas. Netrin-1 expression is associated with worse outcome in poorly differentiated pancreatic adenocarcinomas. Risk-stratification according to the UNC5H3 receptor expression pattern shows that node positive patients (pN1) with no to little UNC5H3 expression carry a significantly worse prognosis than those with middle to strong UNC5H3 expression.

Huntingon's disease is a progressive neurodegenerative disease arising from expansion of a polyglutamine (polyQ) tract in the protein huntingtin (Htt) resulting in aggregation of mutant Htt into nuclear and/or cytosolic inclusions in neurons. Mutant Htt affects multiple processes including protein degradation, transcription, signal transduction, fast axonal transport and endocytosis [reviewed in Ross, C.A. and Poirier, M.A. (2005) Opinion: what is the role of protein aggregation in neurodegeneration? Nat. Rev. Mol. Cell. Biol., 6, 891-898]. Here, we report that the endocytic and signal transduction scaffold intersectin (ITSN) increased aggregate formation by mutant Htt through activation of the c-Jun-NH(2)-terminal kinase (JNK)-MAPK pathway. Conversely, silencing ITSN or inhibiting JNK attenuated aggregate formation. Using a Drosophila model for polyQ repeat disease, we observed that ITSN enhanced polyQ-mediated neurotoxicity. A reciprocal relationship was observed between ITSN and Htt. While ITSN enhanced Htt aggregation and toxicity, Htt, in turn, inhibited the cooperativity between ITSN and the epidermal growth factor receptor signal transduction pathway. Finally, we observed that ITSN overexpression enhanced aggregation of polyQ-expanded androgen receptor (AR) as well as wild-type versions of both Htt and AR suggesting a broader involvement of ITSN in neurodegenerative diseases through destabilization of polyQ-containing proteins.

The apolipoprotein E4 (APOE4) gene is a well-known risk factor for Alzheimer's disease (AD) and other neurological disorders. Post-menopausal women with AD who express at least one APOE4 gene have more severe neuropathology and worsened cognitive scores than their non-expressing counterparts. Since 17beta-estradiol down-regulates inflammation as part of its neuroprotective role, we examined the effect of 17beta-estradiol on the response of microglia to immune activation as a function of APOE genotype. Our data show that the anti-inflammatory activity of 17beta-estradiol is significantly reduced in APOE4 targeted replacement mice compared to APOE3 mice. A significant interaction between APOE genotype and the response to 17beta-estradiol was observed for NO and cytokine production by immune activated microglia. The genotype specific effect was not restricted to brain macrophages since peritoneal macrophages from APOE4 ovariectomized mice also demonstrated a significant difference in 17beta-estradiol responsiveness. ERbeta protein levels in APOE4 microglia were higher than APOE3 microglia, suggesting a difference in post-translational protein regulation in the presence of the APOE4 gene. Overall, our data indicate that the APOE genotype may be a critical component in assessing the effectiveness of 17beta-estradiol's action and may impact the neuroprotective role of 17beta-estradiol and of hormone replacement therapy on brain function when the APOE4 gene is expressed.

Phenotypic expression of the homozygous C282Y/C282Y mutation of the HFE gene has been classified in five stages, and appropriate management recommended for each stage. Phlebotomy is indicated for stages>or=2, that is, with elevated transferrin saturation and serum ferritin levels >300 microg/L in men and >200 microg/L in women. Maximal volume per phlebotomy is 7 mL/kg and should not exceed 550 mL. The main goal of this iron-depletion therapy is to reach and maintain serum ferritin levels<or=50 microg/L. Phlebotomies performed at home by nurses should be encouraged, under strict conditions. Because genetic counseling and family screening should follow individual diagnosis, the physician must recommend that the patient inform siblings, adult children, and parents. Screening should include simultaneous genetic testing and serum iron markers.

A yeast artificial chromosome (YAC) mouse model of Huntington's disease (YAC128) develops motor abnormalities, age-dependent striatal atrophy and neuronal loss. Alteration of neurotransmitter receptors, particularly glutamate and dopamine receptors, is a pathological hallmark of Huntington's disease. We therefore analyzed neurotransmitter receptors in symptomatic YAC128 Huntington's disease mice. We found significant increases in N-methyl-d-aspartate, AMPA and metabotropic glutamate receptor binding, which were not due to increases in receptor subunit mRNA expression levels. Subcellular fractionation analysis revealed increased levels of glutamate receptor subunits in synaptic membrane fractions from YAC128 mice. We found no changes in dopamine, GABA or adenosine receptor binding, nor did we see alterations in dopamine D1, D2 or adenosine A2a receptor mRNA levels. The receptor abnormalities in YAC128 transgenic mice thus appear limited to glutamate receptors. We also found a significant decrease in preproenkephalin mRNA in the striatum of YAC128 mice, which contrasts with the lack of change in levels of mRNA encoding neurotransmitter receptors. Taken together, the abnormal and selective increases in glutamate receptor subunit expression and binding are not due to increases in receptor subunit expression and may exert detrimental effects. The decrease in preproenkephalin mRNA suggests a selective transcriptional deficit, as opposed to neuronal loss, and could additionally contribute to the abnormal motor symptoms in YAC128 mice.

Cytoplasmic dynein is a multisubunit microtubule motor complex that, together with its activator, dynactin, drives vesicular cargo toward the minus ends of microtubules. Huntingtin (Htt) is a vesicle-associated protein found in both neuronal and nonneuronal cells that is thought to be involved in vesicular transport. In this study, we demonstrate through yeast two-hybrid and affinity chromatography assays that Htt and dynein intermediate chain interact directly; endogenous Htt and dynein co-immunoprecipitate from mouse brain cytosol. Htt RNAi in HeLa cells results in Golgi disruption, similar to the effects of compromising dynein/dynactin function. In vitro studies reveal that Htt and dynein are both present on vesicles purified from mouse brain. Antibodies to Htt inhibited vesicular transport along microtubules, suggesting that Htt facilitates dynein-mediated vesicle motility. In vivo inhibition of dynein function results in a significant redistribution of Htt to the cell periphery, suggesting that dynein transports Htt-associated vesicles toward the cell center. Together these findings indicate that Htt binds to dynein and acts in a complex along with dynactin and Htt-associated protein-1 to facilitate vesicular transport.

<h4>Background and objectives</h4>Adhesion molecules are implicated in the progression of colorectal cancer (CRC). Despite the evidence of association between their expression and patients' prognosis, the data have not been examined simultaneously in a same study; thus, the relative clinical value remained largely unknown. The aim of this study was to identify the adhesion factors that display the most significant prognostic value for CRC patients to guide clinical decision-making regarding appropriate treatment.<h4>Patients and methods</h4>We examined by immunohistochemistry, the expression of E-cadherin and its associated catenins, alpha(alpha)-catenin and beta(beta)-catenin, DCC, and CD44 and its partner, MT1-MMP in a series of 140 CRC tissues at intermediate Stage II and Stage III to determine their prognostic significance.<h4>Results</h4>Clinicopathological survey indicated an inverse relationship between E-cadherin expression and tumor differentiation, and an association between CD44 expression and venous invasion. Univariate and multivariate analyses showed that loss of expression of E-cadherin and CD44 significantly correlated to poor survival, especially in Stage II. Combination studies indicated that loss of E-cadherin and loss of CD44 had the worst impact on patient prognosis, particularly in colon cancer.<h4>Conclusion</h4>Immunohistochemical staining of E-cadherin and CD44 may help to identify a subgroup of high-risk patients with Stage II CRC, especially in colon cancer, who may need intensive follow-up and appropriate therapeutic strategy.

Huntington's disease (HD) is a progressive, autosomal dominant neurodegenerative disease caused by an abnormally expanded CAG repeat in the HD gene. Ubiquitylated aggregates containing mutant huntingtin protein in neurons are hallmarks of HD. Misfolded mutant huntingtin monomers, oligomers, or aggregates may be a result of, and cause, ubiquitin- proteasome dysfunction. To investigate the ubiquitin-proteasome system we designed a series of firefly luciferase reporters targeted selectively to different points along this pathway. These reporters were used to monitor ubiquitin-proteasome system function in a striatal cell culture model of HD. Ubiquitylation processes were not reduced in mutant huntingtin cells but recognition or degradation of ubiquitylated substrates was decreased. We also found mutant huntingtin expressing cells had slight but significant decreases in chymotrypsin-like and caspase-like activities, and an unexpected increase in trypsin-like activity of the proteasome core. General proteasome core inhibitors, as well as selective caspase-like activity inhibitors, were less effective in mutant cells. Finally, treatment with 3-nitropropionic acid, a succinate dehydrogenase inhibitor, had opposite effects on the ubiquitin-proteasome system with activation in wild-type and decreased activity in mutant huntingtin expressing cells. The results of these experiments show clearly selective disruption of the ubiquitin-proteasome system in this cell culture model of HD. The high throughput tools that we have designed and optimized will also be useful in identifying compounds that alter ubiquitin-proteasome system function and to investigate other neurodegenerative diseases such Alzheimer's disease and Parkinson's disease.

NH is a rare disorder of iron storage in newborns resulting in rapid liver failure. Outcomes are dismal with 20-30% survival. We report our experience in eight children with NH. Assessment of liver function included admission PT and serum levels of FV and FVII. Medical treatment (antioxidant cocktail) was started in all patients, with chelation therapy in six. Of these six, three survived with medical treatment alone. The other three underwent liver transplant. One died 158 days after transplant to sepsis: two are well more than five yr after transplant. The two neonates who did not receive chelation therapy, died to multi-organ failure and sepsis. In summary, five children (62.5%) survived long-term. In the three transplanted, one- and five-yr-survival was 66%. Older children with compromised synthetic liver function (FVII levels < or = 15%) required liver replacement for survival. Early referral to a tertiary care center is essential to increase survival of these children with a rare and otherwise fatal disease. Single center experience of children with NH is here presented. Potentials for survival improvement with of medical and surgical treatment are examined.

<h4>Purpose</h4>To determine the effects of traditionally prepared kava beverages on the liver function tests of regular kava beverage consumers in a population of Tongan and non-Tongan residents of Hawaii (Oahu).<h4>Methods</h4>The liver function tests of 31 healthy adult kava drinkers were compared against a control group of 31 healthy adult non-kava drinkers. Subjects were recruited from the general population, a kava bar, and Tongan kava drinking circles. The liver function profile included AST, ALT, ALP, GGT, and bilirubin (total and direct). Other tests included total protein, albumin, and screens for viral hepatitis and hemochromatosis when indicated.<h4>Results</h4>Chronic kava beverage consumption was associated with elevation of GGT in 65% of the kava drinkers versus 26% in the controls (P = .005). ALP was elevated in 23% of kava drinkers versus 3% in the controls (P = .053).<h4>Conclusion</h4>Heavy kava beverage consumption was associated with significantly elevated GGT levels.

Hereditary deposition of iron (primary haemochromatosis) or copper (Wilson's disease) are autosomal recessive metabolic disease characterized by progressive liver pathology and subsequent involvement of various other organs. The prevalence of primary haemochromatosis is approximately 0.5%, about 200 times higher than the prevalence of Wilson's disease. The two diseases are characterized by homozygous occurrences of mutations in the HFE gene on chromosome 6 (primary haemochromatosis) and the ATP7B gene on chromosome 13 (Wilson's disease). Unlike most other inherited conditions, these diseases can be successfully treated, emphasizing the importance of early diagnosis. Serum ferritin values, transferrin saturation and genetic analysis are used when diagnosing haemochromatosis. The diagnostics of Wilson's disease depends on the use of urinary copper values, serum ceruloplasmin and liver biopsy. If untreated, both of these genetic diseases result in rapidly progressing multiorgan damage and early death. The key treatment for haemochromatosis is phlebotomy, for Wilson's disease chelation or Zn treatment. Although the present treatments considerably improve the prognosis of patients, they may be inadequate in patients diagnosed so late that extensive body deposits of metal have been developed. The main research needs in this field are to further clarify molecular mechanisms of disease progression and to develop new chelators that are more effective and less toxic than those presently available.

The Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) family of proteins is required for filopodia formation in growth cones and plays a crucial role in guidance cue-induced remodeling of the actin cytoskeleton. In vivo studies with pharmacological inhibitors of actin polymerization have previously provided evidence for the view that filopodia are needed for growth cone navigation in the developing visual pathway. Here we have re-examined this issue using an alternative strategy to generate growth cones without filopodia in vivo by artificially targeting Xena/XVASP (Xenopus homologs of Ena/VASP) proteins to mitochondria in retinal ganglion cells (RGCs). We used the specific binding of the EVH1 domain of the Ena/VASP family of proteins with the ligand motif FP4 to sequester the protein at the mitochondria surface. RGCs with reduced function of Xena/XVASP proteins extended fewer axons out of the eye and possessed dynamic lamellipodial growth cones missing filopodia that advanced slowly in the optic tract. Surprisingly, despite lacking filopodia, the axons navigated along the optic pathway without obvious guidance errors, indicating that the Xena/XVASP family of proteins and filopodial protrusions are non-essential for pathfinding in retinal axons. However, depletion of Xena/XVASP proteins severely impaired the ability of growth cones to form branches within the optic tectum, suggesting that this protein family, and probably filopodia, plays a key role in establishing terminal arborizations.

We examined mortality and recurrence of hepatocellular carcinoma (HCC) among 106 transplant candidates with cirrhosis and HCC who had a potential living donor evaluated between January 1998 and February 2003 at the nine centers participating in the Adult-to-Adult Living Donor Liver Transplantation Cohort Study (A2ALL). Cox regression models were fitted to compare time from donor evaluation and time from transplant to death or HCC recurrence between 58 living donor liver transplant (LDLT) and 34 deceased donor liver transplant (DDLT) recipients. Mean age and calculated Model for End-Stage Liver Disease (MELD) scores at transplant were similar between LDLT and DDLT recipients (age: 55 vs. 52 years, p = 0.21; MELD: 13 vs. 15, p = 0.08). Relative to DDLT recipients, LDLT recipients had a shorter time from listing to transplant (mean 160 vs. 469 days, p < 0.0001) and a higher rate of HCC recurrence within 3 years than DDLT recipients (29% vs. 0%, p = 0.002), but there was no difference in mortality or the combined outcome of mortality or recurrence. LDLT recipients had lower relative mortality risk than patients who did not undergo LDLT after the center had more experience (p = 0.03). Enthusiasm for LDLT as HCC treatment is dampened by higher HCC recurrence compared to DDLT.

Child maltreatment is associated with a huge burden of suffering, yet there are serious gaps in knowledge about its epidemiology and approaches to intervention. This article describes the development of a proposed national research framework in child maltreatment, as requested by the Department of Justice, Canada, based on (1) a review of the literature, (2) consultation with experts, and (3) application of evaluation criteria for considering research priorities. The article identifies gaps in knowledge about child maltreatment in Canada and proposes a research agenda to make evidence-based policy decisions more likely. Although this work was driven by gaps in Canada's knowledge about child maltreatment, the international scope of the review and consultation process could make the findings useful to broader research and policy audiences.

Ki67 immunohistochemistry is a widely used marker of the tumor proliferative fraction. Apart from the nuclear staining of dividing cells, MIB-1 monoclonal antibody was also found to stain the cell membrane of some tumor types. Indeed, such membrane reactivity was proposed as a diagnostic feature of hyalinizing trabecular tumor (HTT) of the thyroid. To verify the diagnostic role of Ki67 membrane pattern, 6 HTTs, 8 pulmonary sclerosing hemangiomas (SH), and 6 other human tumors with MIB-1 cell membrane immunoreactivity were stained by immunoperoxidase with 5 different anti-Ki67 antibodies in different experimental conditions. We show here that the cell membrane reactivity reported in HTT is produced only by MIB-1 and not by other antibodies to Ki67 (including commercially available mouse and rabbit monoclonal antibodies). In addition, this peculiar pattern is obtained only if the reaction is performed at room temperature, because automated immunostainers which operate at 37 degrees C do not produce any MIB-1 membrane localization. The same findings were obtained in the other 6 tumors. Conversely, sclerosing hemangioma of the lung did not produce any MIB-1 cell membrane reactivity in our hands. A cross-reactivity of the MIB-1 monoclonal antibody with an epitope expressed at the cell membrane level (rather than an artifact) seems the most likely explanation for this finding, because the immunoreactivity is generally intense and uniform in the membrane positive tumors. We conclude that when Ki67 immunohistochemistry is used for diagnostic purposes in a suspected HTT, only MIB-1 clone at room temperature should be employed.

Much information has been reported on the genetic and genomic alterations in colorectal cancer (CRC) in literature; however, nonrandom chromosomal alterations in Chinese CRC patients have only one report in Hong Kong. To further identify genomic alteration in primary sporadic colorectal carcinomas (SCRC) in Chinese patients and understand the molecular mechanisms in CRC development, progress, and metastasis, we used comparative genomic hybridization to screen for losses and/or gains of DNA copies along chromosomes in 24 SCRC tissues from 24 patients. Comparative genomic hybridization was applied to investigate the genomic imbalance in 24 cases of primary SCRC and compared the differences between tumors in different loci and between tumors with and without metastasis. The common chromosomal alterations in the SCRC included gains of chromosomes 1q, 2q, 4q, 7q, 8q, 11q, 13q, 20q and also losses of chromosomes 9p, 16q, 17p, 18q. Among them, gains of 1q, 7q, 20q and losses of 17p, 18q were related with lymph node metastasis of SCRC (P<0.05). The gains of 4q, 7q, 20q and losses of 9p, 18q were related with the sites (P<0.05), colon and rectum, respectively; gain of 20q and loss of 9p were commonly found in the colon cancer; gain of 4q, 7q and loss of 18q were easily seen in the rectal cancer. There are multiple regions of chromosomes with copy-number changes in SCRC. The tumor suppressor genes and oncogenes on these regions may be involved in the development and progress of SCRC. The chromosome 1q, 2q, 4q, 7q, 8q, 11q, 13q, 20q regions may have oncogenes such as epidermal growth factor, MET, platelet-derived growth factor receptor A, and 9p, 16q, 17p, 18q regions may have tumor suppressor genes such as p53,DCC, IGFR1 associated with occurrence of SCRC. The chromosome 1q, 7q, 20q, 17p, 18q regions may have genes related with metastasis of SCRC. The development mechanisms of colon cancer and rectal cancer may not be completely similar. Additionally, gain of chromosome 1q was verified by the second technique-Real-time reverse transcription PCR.

Classic hereditary hemochromatosis is an autosomal recessive disorder characterized by iron overload and sequence variants in the HFE gene. The HFE gene is located at 6p21.3 and contains 2 common single nucleotide polymorphisms (SNPs) C282Y and H63D, which are routinely tested for in the molecular diagnostics laboratory. In this study, we used DNA samples from 59 patients in which clinicians wanted to confirm or rule-out hereditary hemochromatosis that had been previously tested for the HFE SNPs using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay and the ABI 7700 real time PCR assay with a MGB Eclipse ASR Probe system. The new assay used TAQman SNP Genotyping Assays, which were performed on the ABI 7500 FAST real time PCR platform. Allelic discrimination was determined during a postamplification plate read. Of the 59 samples genotyped, 7 were homozygous for C282Y, 6 were heterozygous for C282Y, 9 were homozygous for H63D, 10 were heterozygous for H63D, 6 were compound heterozygotes, and 20 were wild type. With the exception of one sample that was indeterminate by the TAQman SNP Genotyping Assay, all others showed 100% concordance between the 3 assays. The one indeterminate sample was heterozygous for C282Y by the PCR-RFLP and ABI 7700 real time PCR assays, but there was an insufficient quantity of DNA to perform the TAQman SNP Genotyping Assay. Our study suggests that the ABI 7500 FAST TAQman SNP Genotyping Assay is comparable with the PCR-RFLP and ABI 7700 real time PCR methods in detecting and characterizing these 2 HFE SNPs. Improved software and thermocycling capabilities have resulted in a very robust TAQman assay with the advantage of a much improved turn-around-time and throughput.

<h4>Background</h4>The embryonic developmental process in avian species is quite different from that in mammals. The first cleavage begins 4 h after fertilization, but the first differentiation does not occur until laying of the egg (Eyal-Giladi and Kochav (EK) stage X). After 12 to 13 h of incubation (Hamburger and Hamilton (HH) stage 3), the three germ layers form and germ cell segregation in the early chick embryo are completed. Thus, to identify genes associated with early embryonic development, we compared transcript expression patterns between undifferentiated (stage X) and differentiated (HH stage 3) embryos.<h4>Results</h4>Microarray analysis primarily showed 40 genes indicating the significant changes in expression levels between stage X and HH stage 3, and 80% of the genes (32/40) were differentially expressed with more than a twofold change. Among those, 72% (23/32) were relatively up-regulated at stage X compared to HH stage 3, while 28% (9/32) were relatively up-regulated at HH stage 3 compared to stage X. Verification and gene expression profiling of these GeneChip expression data were performed using quantitative RT-PCR for 32 genes at developmental four points; stage X (0 h), HH stage 3 (12 h), HH stage 6 (24 h), and HH stage 9 (30 h). Additionally, we further analyzed four genes with less than twofold expression increase at HH stage 3. As a result, we identified a set of stage-specific genes during the early chick embryo development; 21 genes were relatively up-regulated in the stage X embryo and 12 genes were relatively up-regulated in the HH stage 3 embryo based on both results of microarray and quantitative RT-PCR.<h4>Conclusion</h4>We identified a set of genes with stage-specific expression from microarray Genechip and quantitative RT-PCR. Discovering stage-specific genes will aid in uncovering the molecular mechanisms involved the formation of the three germ layers and germ cell segregation in the early chick embryos.

The ion channel genome is still being defined despite numerous publications on the subject. The ion channel transcriptome is even more difficult to assess. Using high-throughput computational tools, we surveyed all available inner ear cDNA libraries to identify genes coding for ion channels. We mapped over 100,000 expressed sequence tags (ESTs) derived from human cochlea, mouse organ of Corti, mouse and zebrafish inner ear, and rat vestibular end organs to Homo sapiens, Mus musculus, Danio rerio, and Rattus norvegicus genomes. A survey of EST data alone reveals that at least a third of the ion channel genome is expressed in the inner ear, with highest expression occurring in hair cell-enriched mouse organ of Corti and rat vestibule. Our data and comparisons with other experimental techniques that measure gene expression show that every method has its limitations and does not per se provide a complete coverage of the inner ear ion channelome. In addition, the data show that most genes produce alternative transcripts with the same spectrum across multiple organisms, no ion channel gene variants are unique to the inner ear, and many splice variants have yet to be annotated. Our high-throughput approach offers a qualitative computational and experimental analysis of ion channel genes in inner ear cDNA collections. A lack of data and incomplete gene annotations prevent both rigorous statistical analyses and comparisons of entire ion channelomes derived from different tissues and organisms.

The carboxyl terminus of the Hsc70-interacting protein (CHIP) is an Hsp70 co-chaperone as well as an E3 ubiquitin ligase that protects cells from proteotoxic stress. The abilities of CHIP to interact with Hsp70 and function as a ubiquitin ligase place CHIP at a pivotal position in the protein quality control system, where its entrance into Hsp70-substrate complexes partitions nonnative proteins toward degradation. However, the manner by which Hsp70 substrates are selected for ubiquitination by CHIP is not well understood. We discovered that CHIP possesses an intrinsic chaperone activity that enables it to selectively recognize and bind nonnative proteins. Interestingly, the chaperone function of CHIP is temperature-sensitive and is dramatically enhanced by heat stress. The ability of CHIP to recognize nonnative protein structure may aid in selection of slow folding or misfolded polypeptides for ubiquitination.

The t(2;11)(q31;p15) chromosomal translocation results in a fusion between the NUP98 and HOXD13 genes and has been observed in patients with myelodysplastic syndrome (MDS) or acute myelogenous leukemia. We previously showed that expression of the NUP98-HOXD13 (NHD13) fusion gene in transgenic mice results in an invariably fatal MDS; approximately one third of mice die due to complications of severe pancytopenia, and about two thirds progress to a fatal acute leukemia. In the present study, we used retroviral insertional mutagenesis to identify genes that might collaborate with NHD13 as the MDS transformed to an acute leukemia. Newborn NHD13 transgenic mice and littermate controls were infected with the MOL4070LTR retrovirus. The onset of leukemia was accelerated, suggesting a synergistic effect between the NHD13 transgene and the genes neighboring retroviral insertion events. We identified numerous common insertion sites located near protein-coding genes and confirmed dysregulation of a subset of these by expression analyses. Among these genes were Meis1, a known collaborator of HOX and NUP98-HOX fusion genes, and Mn1, a transcriptional coactivator involved in human leukemia through fusion with the TEL gene. Other putative collaborators included Gata2, Erg, and Epor. Of note, we identified a common insertion site that was >100 kb from the nearest coding gene, but within 20 kb of the miR29a/miR29b1 microRNA locus. Both of these miRNA were up-regulated, demonstrating that retroviral insertional mutagenesis can target miRNA loci as well as protein-coding loci. Our data provide new insights into NHD13-mediated leukemogenesis as well as retroviral insertional mutagenesis mechanisms.

Proline-, glutamic acid-, leucine-rich protein 1 (PELP1), a novel nuclear receptor coactivator, and its expression is deregulated in hormone-dependent cancers, including those of the breast, endometrium, and ovary. PELP1 interacts with estrogen receptor and modulates its genomic and nongenomic functions. In this study, we examined whether PELP1 functions as an oncogene. The overexpression of PELP1 in fibroblasts and epithelial model cells resulted in cellular transformation. PELP1 also enhanced the transformation potential of c-Src kinase in focus formation assays, and PELP1 overexpression potentiated estradiol-mediated cell migratory potential and anchorage-independent growth. Using PELP1-small interfering RNA, we provided evidence that endogenous PELP1 plays an essential role in E2-mediated anchorage-independent growth, cell migration, and cytoskeletal changes. When compared with control vector transfectants, breast cancer cells stably overexpressing PELP1 showed a rapid tumor growth in xenograft studies. Immunohistochemical analysis of PELP1 expression using a tumor progression array of 252 breast carcinomas and normal breast tissue specimens revealed that PELP1 expression is deregulated to a greater degree in higher grade node-positive invasive tumors than in normal breast tissue or ductal carcinoma in situ. Our data suggest that PELP1 is a potential oncogene, that its expression is deregulated during cancer progression, and that PELP1 may play a role in oncogenesis.

There is increasing evidence that genome-wide association (GWA) studies represent a powerful approach to the identification of genes involved in common human diseases. We describe a joint GWA study (using the Affymetrix GeneChip 500K Mapping Array Set) undertaken in the British population, which has examined approximately 2,000 individuals for each of 7 major diseases and a shared set of approximately 3,000 controls. Case-control comparisons identified 24 independent association signals at P < 5 x 10(-7): 1 in bipolar disorder, 1 in coronary artery disease, 9 in Crohn's disease, 3 in rheumatoid arthritis, 7 in type 1 diabetes and 3 in type 2 diabetes. On the basis of prior findings and replication studies thus-far completed, almost all of these signals reflect genuine susceptibility effects. We observed association at many previously identified loci, and found compelling evidence that some loci confer risk for more than one of the diseases studied. Across all diseases, we identified a large number of further signals (including 58 loci with single-point P values between 10(-5) and 5 x 10(-7)) likely to yield additional susceptibility loci. The importance of appropriately large samples was confirmed by the modest effect sizes observed at most loci identified. This study thus represents a thorough validation of the GWA approach. It has also demonstrated that careful use of a shared control group represents a safe and effective approach to GWA analyses of multiple disease phenotypes; has generated a genome-wide genotype database for future studies of common diseases in the British population; and shown that, provided individuals with non-European ancestry are excluded, the extent of population stratification in the British population is generally modest. Our findings offer new avenues for exploring the pathophysiology of these important disorders. We anticipate that our data, results and software, which will be widely available to other investigators, will provide a powerful resource for human genetics research.

A pharmacogenetic study suggests the 5-HTT LPR polymorphism predicts response to alosetron, and another study describes a possible association of the GNbeta3 C825T polymorphism with IBS in patients with dyspepsia. We performed a case-control association study to determine whether these polymorphisms are associated with irritable bowel syndrome (IBS). The study aim was to compare allele and genotype frequencies between cases and controls for the 5-HTT LPR and the GNbeta3 C825T polymorphism. Cases were 50 GI outpatients; controls were 53 General Medicine outpatients matched to cases for age, gender and race at a major medical centre. Participants completed a questionnaire and donated blood. DNA was genotyped using polymerase chain reaction based assays. Eighty-two per cent of cases met Rome II criteria for IBS: 12% constipation-, 46% diarrhoea-, and 42% mixed-IBS. Genotype and allele frequencies for both polymorphisms did not differ between cases and controls. However, the allele frequency of the short (S) allele of the 5-HTT LPR polymorphism was greater in those with mixed-IBS compared with controls (68%vs 45%, P < 0.05). This study suggests that the 5-HTT LPR polymorphism may be associated with mixed-IBS, but not IBS overall. No association was observed for the GNbeta3 C825T polymorphism with IBS overall or subtypes.

<h4>Background</h4>Screening of target populations for hemochromatosis (HFE) gene allele status has been recommended. Alcoholic liver disease may be associated with iron overload and there is evidence of excessive alcohol consumption among patients with hereditary hemochromatosis. This study determined the HFE gene allele status in alcohol-dependent patients and explored the associations between iron status, liver enzymes, and HFE status.<h4>Methods</h4>A total of 151 consecutive patients admitted for alcohol detoxification were tested for HFE mutations, iron status, and liver function tests. The prevalence data were compared with those from a New Zealand population. manova was used to compare liver function tests and iron status for subjects with different HFE mutations.<h4>Results</h4>Three compound heterozygotes, one homozygote for C282Y, and one homozygote for H63D were found among the 151 patients. For the remaining 146 patients, there was no difference in the distribution of heterozygote status by allele, compared to the general New Zealand population. No HFE mutation: general population 64.4%, alcohol-dependent patients 64.4%; H63D: general population 23.6%, alcohol-dependent patients 28.1%; C282Y: general population 11.9%, alcohol-dependent patients 7.5% (P = 0.20). There was no relationship between liver function tests or iron status and HFE mutation status among the study group.<h4>Conclusions</h4>No evidence has been found in the present that HFE allele status prevalence is different from the general population or associated with different liver function or iron status among alcohol-dependent patients. The cause of altered iron status among alcohol-dependent patients does not appear to be related to HFE status.

While major inroads have been made in identifying the genetic causes of rare Mendelian disorders, little progress has been made in the discovery of common gene variations that predispose to complex diseases. The single gene variants that have been shown to associate reproducibly with complex diseases typically have small effect sizes or attributable risks. However, the joint actions of common gene variants within pathways may play a major role in predisposing to complex diseases (the paradigm of complex genetics). The goal of this study was to determine whether polymorphism in a candidate pathway (axon guidance) predisposed to a complex disease (Parkinson disease [PD]). We mined a whole-genome association dataset and identified single nucleotide polymorphisms (SNPs) that were within axon-guidance pathway genes. We then constructed models of axon-guidance pathway SNPs that predicted three outcomes: PD susceptibility (odds ratio = 90.8, p = 4.64 x 10(-38)), survival free of PD (hazards ratio = 19.0, p = 5.43 x 10(-48)), and PD age at onset (R(2) = 0.68, p = 1.68 x 10(-51)). By contrast, models constructed from thousands of random selections of genomic SNPs predicted the three PD outcomes poorly. Mining of a second whole-genome association dataset and mining of an expression profiling dataset also supported a role for many axon-guidance pathway genes in PD. These findings could have important implications regarding the pathogenesis of PD. This genomic pathway approach may also offer insights into other complex diseases such as Alzheimer disease, diabetes mellitus, nicotine and alcohol dependence, and several cancers.

To screen factors related to spermatogonial stem cell (SSC) proliferation, and to investigate the mechanism of infertility caused by cryptorchidism, ten-day-old Kunming (KM) mice were used and experimental cryptorchidism was conducted. On the 35th day after cryptorchid operation, the left testes were fixed in Bouin's fluid and used for histological analysis. The testes of 45-day-old mice were subjected to the same histological analysis, and it was found that they contained germ cells at every stage of development, from SSCs to sperm, indicating that the animals were fully sexually mature at this age. While in experimental cryptorchid mice, the spermatogenesis was arrested at the stage of spermatocytes, and only spermatogonia and primary spermatocytes were present in cryptorchid testes. The proportion of spermatogonia to other types of germ cells was much higher than that in sexually mature mice. On the other hand, the right testes were used for proteomic analysis. The total protein in testes was extracted on the 35th day after cryptorchid operation. The differentially expressed proteins in cryptorchid mice and sexually mature mice were screened and compared by the proteomic techniques. Through the separation of two-dimensional gel electrophoresis (2-DE), 20 differential protein spots were found, and 9 of them were digested and identified by the matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrum. In cryptorchid mice, 6 out of 9 proteins were down-regulated, and 3 were up-regulated. Among these proteins, 4 proteins were identified, and they were Stathmin, phosphatidylethanolamine-binding protein1 (PEBP1), HES-related basic helix-loop-helix protein (HERP), and one unnamed protein (we temporarily named it Px). More Stathmin, PEBP1 and Px were expressed in sexually mature mice than in experimental cryptorchid mice. But HERP1 was the other way round. In the present study, we have screened 4 proteins related to cryptorchidism. It is helpful to study the mechanism of SSC proliferation and infertility caused by cryptorchidism.

Neuromodulation changes the cellular and synaptic properties of neurons, thereby enabling individual neuronal circuits to generate multiple activity patterns. However, distinct modulatory inputs could conceivably also cona different motor circuits to generate similar activity patterns. Using the isolated stomatogastric ganglion (STG) of the crab Cancer borealis, we showed previously that pyrokinin (PK) peptides activate the gastric mill (chewing) rhythm without the participation of the projection neuron modulatory commissural neuron 1 (MCN1). MCN1, which does not contain the PK peptide, also activates the gastric mill rhythm and, at these times, is a gastric mill central pattern generator (CPG) neuron. Here, we show that the gastric mill rhythms elicited by PK superfusion and MCN1 stimulation in the isolated STG are comparable, in contrast to the distinct gastric mill rhythms elicited by other input pathways. We also identified several cellular and synaptic mechanisms underlying the PK- and MCN1-elicited gastric mill rhythms that are distinct, including additional differences in their core CPG neurons. For example, the presence of the inhibitory synapse from the pyloric pacemaker neuron anterior burster onto the gastric mill CPG was necessary only for generation of the PK-elicited gastric mill rhythm. Similarly, the dorsal gastric motor neuron regulated only the PK rhythm, apparently because of PK-mediated enhancement of its synaptic actions. Thus, we demonstrate that different modulatory inputs can elicit comparable, as well as distinct activity patterns from the same neuronal ensemble. Moreover, these comparable rhythms can result from distinct CPGs using overlapping, but distinct sets of cellular and synaptic mechanisms.

Short-term synaptic plasticity influences how presynaptic spike patterns control the firing of postsynaptic targets. Here we investigated whether specific mechanisms of short-term plasticity are regulated in a target-dependent manner by comparing synapses made by cerebellar granule cell parallel fibers onto Golgi cells (PF-->GC synapse) and Purkinje cells (PF-->PC synapse). Both synapses exhibited similar facilitation, suggesting that any differential short-term plasticity does not reflect differences in the initial release probability. PF-->PC synapses were highly sensitive to stimulus bursts, which could result in either depression of subsequent responses, mediated by endocannabinoid-dependent retrograde signaling, or enhancement of responses through posttetanic potentiation (PTP). In contrast, stimulus bursts had remarkably little effect on the strength of PF-->GC synapses. Unlike PCs, GCs were unable to regulate their PF synapses by releasing endocannabinoids. Moreover, PTP was reduced at the PF-->GC synapse compared to the PF-->PC synapse. Thus, the target-dependence of PF synapses arises from the differential expression of both retrograde signaling and PTP.

<h4>Backgrounds/aims</h4>There are controversies on the role of iron overload in the mechanism of liver injury in nonalcoholic fatty liver disease (NAFLD). The aim of this study was to evaluate the prevalence of peripheral iron overload, and to study the presence of HFE mutations (C282Y, H63D, S65C) in a cohort of Korean NAFLD patients.<h4>Methods</h4>255 patients with NAFLD were included. The patients had been diagnosed as having NAFLD by the criteria of elevated aminotransferase levels, compatible ultrasonographic findings and exclusion of other etiologies. Blood samples were tested for chemistry, iron profile, and mutational analysis for HFE gene (C282Y, H63D, S65C).<h4>Results</h4>Of the 255 NAFLD patients, the prevalence of peripheral iron overload was 19.2% according to the cutoff level of transferrin saturation (TS) > 45%, and 3.9% of NAFLD patients were having hyperferritinemia over 400 ng/mL. Hyperferritinemia was significantly associated with elevated serum levels of fasting glucose, AST and TS. We found the presence of H63D mutation, either heterozygote or homozygote, among the NAFLD patients with peripheral iron overload.<h4>Conclusions</h4>The prevalence of peripheral iron overload in the Korean NAFLD patients was not rare, and the presence of H63D mutation among NALFD patients was identified. Further studies on the significance of iron overload or HFE mutation in the pathogenesis of NAFLD are needed.

<h4>Aim</h4>To identify the frequency of iron overload and study the three mutations in the HFE gene (C282Y, H63D, and S65C) in patients with chronic liver disorders (CLD) and controls.<h4>Methods</h4>To identify patients with iron overload (transferrin saturation > 45% in females and > 50% in males and serum ferritin > 1000 ng/mL) we evaluated 236 patients with CLD, including 59 with non-alcoholic steatohepatitis (NASH), 22 with alcoholic liver disease (ALD), 19 of cirrhosis due to viruses (HBV, HCV), and 136 with cryptogenic cirrhosis. Mutations of the HFE gene were analyzed by PCR-RE. hundred controls were screened for iron status and the mutations.<h4>Results</h4>Seventeen patients with CLD showed evidence of iron overload. Fifteen cases of iron overload had cryptogenic cirrhosis and two had ALD. None of the controls showed iron overload. We did not find any individual with 282Y or 65C either in the cases or in the controls. The prevalence of H63D heterozygosity was 12% in normal individuals, 14.8% in 236 patients (16.9% in NASH, 13.6% in ALD, 26.3% in viral and 12.5% in cryptogenic cirrhosis) and the overall prevalence was 13.98%. Only two of the 17 patients with primary iron overload were heterozygous for H63D. One patient with NASH and one normal individual who were homozygous for H63D showed no iron overload.<h4>Conclusion</h4>Primary iron overload in Indians is non-HFE type, which is different from that in Europeans and further molecular studies are required to determine the defect in various iron regulatory genes.

<h4>Aim</h4>To explore the prognostic variables in rectal cancer patients undergoing curative total mesorectal excision and the effect of postoperative chemotherapy in advanced rectal cancer.<h4>Methods</h4>A total of 259 consecutive rectal cancer patients treated with curative total mesorectal excision between 1999 and 2004 were collected. p53, p21, PCNA, and CD44v6 were examined using immunohistochemistry (IHC). The correlation between clinicopathological or molecular variables and clinical outcomes, including local recurrence, metastasis, disease-free survival and overall survival, was analyzed.<h4>Results</h4>The median follow-up was 44 mo. Five-year survival rates and 5-year disease free survival rates were 75.43% and 70.32%, respectively. Multi-analysis revealed TNM staging, preoperative CEA, and CD44v6 level were independent risk factors predicting overall survival or disease free survival. The hazard ratio of peroperative CEA was 2.65 (95% CI 1.4-5) and 3.10 (95% CI 1.37-6.54) for disease free survival and overall survival, respectively. The hazard ratio of CD44v6 was 1.93 (95% CI 1.04-3.61) and 2.21 (95% CI 1.01-4.88) for disease free survival and overall survival, respectively. TNM staging was the only risk factor predicting local recurrence. Postoperative chemotherapy without radiotherapy did not improve patients' outcome.<h4>Conclusion</h4>TNM staging, preoperative CEA and CD44v6 were independent prognostic factors for rectal cancer patients with total mesorectal excision. Postoperative chemotherapy may be only used together with radiotherapy for rectal cancer patients.

Huntingtin is a protein that is mutated in Huntington's disease (HD), a dominant inherited neurodegenerative disorder. We previously proposed that, in addition to the gained toxic activity of the mutant protein, selective molecular dysfunctions in HD may represent the consequences of the loss of wild-type protein activity. We first reported that wild-type huntingtin positively affects the transcription of the brain-derived neurotrophic factor (BDNF) gene, a cortically derived survival factor for the striatal neurons that are mainly affected in the disease. Mutation in huntingtin decreases BDNF gene transcription. One mechanism involves the activation of repressor element 1/neuron-restrictive silencer element (RE1/NRSE) located within the BDNF promoter. We now show that increased binding of the RE1 silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) repressor occurs at multiple genomic RE1/NRSE loci in HD cells, in animal models, and in postmortem brains, resulting in a decrease of RE1/NRSE-mediated gene transcription. The same molecular phenotype is produced in cells and brain tissue depleted of endogenous huntingtin, thereby directly validating the loss-of-function hypothesis of HD. Through a ChIP (chromatin immunoprecipitation)-on-chip approach, we examined occupancy of multiple REST/NRSF target genes in the postmortem HD brain, providing the first example of the application of this technology to neurodegenerative diseases. Finally, we show that attenuation of REST/NRSF binding restores BDNF levels, suggesting that relief of REST/NRSF mediated repression can restore aberrant neuronal gene transcription in HD.

The aim of this study was to ascertain the folate receptor (FR) targetability by an in vitro study and to acquire FR-targeted images in vivo models by using synthetic folate conjugates. PEG-folate was synthesized and labeled with (99m)Tc and fluorescein isothiocynate (FITC). Cell uptake studies were carried out in KB cells (FR-positive) and A549 cells (FR-negative) using FITC- and the (99m)Tc-labeled conjugates. The radiolabeled conjugate was intravenously injected to KB tumor xenografted mice. After it was injected, gamma images were recorded at 30 min, 1, 2, 3 and 4 hr. Cell uptake studies showed a difference between the KB cells and the A549 cells by flow cytometry analysis and gamma counting. On in vivo images, the tumor-to-normal muscle ratio was greater than 4. It ascertained that the PEG-folate conjugate specifically binds to the FR expressed on tumor cells in vitro. Moreover, it was possible to acquire the FR-targeted gamma images using PEG-folate conjugates in tumor models.

Neuropathic pain is a difficult state to treat, characterized by alterations in sensory processing that can include allodynia (touch-evoked pain). Evidence exists for nerve damage-induced plasticity in both transmission and modulatory systems, including changes in voltage-dependent calcium channel (VDCC) expression and function; however, the role of Ca(v)2.3 calcium channels has not clearly been defined. Here, the effects of SNX-482, a selective Ca(v)2.3 antagonist, on sensory transmission at the spinal cord level have been investigated in the rat. The spinal nerve ligation (SNL) model of chronic neuropathic pain [Kim & Chung, (1992)Pain, 50, 355-363] was used to induce mechanical allodynia, as tested on the ipsilateral hindpaw. In vivo electrophysiological measurements of dorsal horn neuronal responses to innocuous and noxious electrical and natural stimuli were made after SNL and compared to sham-operated animals. Spinal SNX-482 (0.5-4 microg/50 microL) exerted dose-related inhibitions of noxious C-fibre- and Adelta-fibre-mediated neuronal responses in conditions of neuropathy, but not in sham-operated animals. Measures of spinal cord hyperexcitability and nociception were most susceptible to SNX-482. In contrast, non-noxious Abeta-mediated responses were not affected by SNX-482. Moreover, responses to innocuous mechanical and also thermal stimuli were more sensitive to SNX-482 in SNL than control animals. This study is the first to demonstrate an antinociceptive role for SNX-482-sensitive channels in dorsal horn neurons during neuropathy. These data are consistent with plasticity in Ca(V)2.3 calcium channel expression and suggest a potential selective target to reduce nociceptive transmission during conditions of nerve damage.

Transthoracic intra-aortic balloon pump (IABP) insertion has been a relatively rare and uncommon procedure. However, it is an established beneficial option in patients with severe peripheral vascular disease (PVD) accompanied with bi-lateral femoral arterial occlusion. There are several viable alternatives to trans-aortic IABP insertion, including trans-axillary or in abdominal aorta (requiring a laparotomy). Cardiac surgery has the advantage of an open sternum, facilitating effortless direct intra-aortic balloon (IAB) insertion into the aorta. The IAB can be inserted either through a 9-mm graft or directly into the ascending aorta. During cardiac surgery, direct insertion into the ascending aorta with the balloon tip lying distally in the abdominal aorta is facilitated with an open sternum. The base of the balloon lies approximately 2 cm below the left subclavian and can be confirmed through a trans-esophageal echocardiogram (TEE). Elimination of a graft insertion saves the team from time-consuming maneuvers and additional hemorrhagic complications. In our experience, postoperative vasoplegic syndrome coupled with myocardial edema contributed to patent instability and was treated with vasopressin and transthoracic IAB insertion. The CS 100 (Datascope Corp., Mahwah, NJ) console allowed the ability to time the balloon accurately. This case report details our experience with one such patient and establishes trans-aortic counter-pulsation as a safe and viable option in patients with severe PVD, where percutaneous insertion is precluded or has failed.

<h4>Aim</h4>To identify clinically significant molecular markers of endometrial cancer.<h4>Materials and methods</h4>Cancer and normal endometrial tissue samples from 20 patients of the Gynecology Clinic of Odessa State Medical University (Odessa, Ukraine) with confirmed endometrial cancer were compared for SSR and ISSR polymorphisms. Identified polymorphic fragments from anonymous genome regions situated between microsatellite repeats underwent direct DNA sequencing; analysis of their homology to sequences from human genome database has been performed.<h4>Results</h4>No significant variability for the microsatellite loci adjacent to the E2F1, BAX, TCF7L2, C-MYC, WNT1, FES, DCC, P27, THRA, APC, CYP19 and P53 genes was detected. Search for new molecular markers of endometrial cancer within anonymous DNA sequences located between microsatellite repeats revealed 100 bp and 174 bp polymorphic fragments. These fragments were detected correspondingly in 60% and 35% of patients. 100 bp fragment appeared to be homologous to a region within the NFKB gene, 174 bp fragment - to a sequence within the DDR1 gene.<h4>Conclusions</h4>NFKB1 and DDR1 genes may be regarded as potential markers for some types of endometrial cancer. This is a first report about possible association of these genes with endometrial cancer.

Although serotonin (5-hydroxytryptamine, 5-HT) has been found to be a potent vasoconstrictor, a pivotal role of 5-HT in the control of appetite and mood control by the modulation of neuronal synapse has also been proposed. Selective 5-HT reuptake inhibitors (SSRIs) are frequently used to suppress appetite and treat depressive disorder, and the target protein of SSRIs is the 5-HT transporter (5-HTT) in the neuronal synapse. However, SSRIs may increase the free 5-HT concentration in circulating blood because platelets and vascular smooth muscles express functional 5-HTT. In addition, enhanced vasoactive action of 5-HT and alterations in 5-HT receptor subtypes have been reported in some types of hypertension. Therefore, we can infer that the use of drugs such as SSRIs in some hypertensive patients is potentially risky. Altered functional expression of ion channels in vascular smooth muscle is suggested to be a mechanism for the enhanced vasoconstriction by vasoactive agonists, including 5-HT. In this brief review, we compared the electrophysiological properties of mesenteric artery myocytes and their modulation by 5-HT between sham-operated control and deoxycorticosterone acetate (DOCA)-salt hypertensive rats.

<h4>Background</h4>Hereditary hemochromatosis (HH) is a disorder of iron storage that results in iron overload. It's prevalence in Puerto Rico (PR) is unknown and the condition frequently undiagnosed.<h4>Purpose</h4>Determine the prevalence of HH in the PR Veteran population.<h4>Methods</h4>Patients attending the San Juan VA Medical Center Laboratory for routine tests were invited to participate. Basic demographic data, symptoms related questionnaire, fasting samples for unbound iron binding capacity (UIBC) and serum iron were obtained. Transferrin saturation (TS) > or =45% was considered as iron overload. Patients with elevated TS had a second test done to confirm iron overload. Genetic testing was performed to patients with sustained elevated TS.<h4>Results</h4>Data from 521 of the 559 recruited patients was available for analysis. 59 patients had TS > or =45%. Iron overload was confirmed in eleven patients. Two patients were lost to follow up; one had secondary causes for iron overload. Eight patients underwent genetic testing. Genetic mutations associated with hemochromatosis were found in four patients.<h4>Conclusion</h4>The estimated prevalence of confirmed iron overload in our population is 2%, similar to one in the United States, while the prevalence of genetic mutations associated to hemochromatosis is 0.76%, which is slightly higher. Both genetic mutations (C282Y, H63D) are equally seen in the evaluated population which is different from reports elsewhere. Physician awareness of the prevalence of HH in PR may result in increased screening and early identification of the condition.

Premature ejaculation (PE) is the most common male sexual disorder, estimated to affect up to 30% of men. Over the past one or two decades, clinical investigators have participated in an increasing number of studies that are helping in our understanding of PE, which will undoubtedly facilitate future treatments. Apart from a number of behavioral approaches, the treatment of PE consists of primarily off-label use of oral selective serotonin reuptake inhibitors (SSRIs) via either on-demand or daily delivery. However, various undesirable side-effects of these medications have led researchers to search for and develop new therapeutic approaches for PE. Dapoxetine is a short-acting SSRI developed specifically for the treatment of PE. Early trials with dapoxetine have documented successful outcomes without serious short- or long-term side-effects. This review addresses the definition, classification, diagnosis, physiology, and neurobiopathology of PE, and evaluates therapeutic strategies with novel treatments for PE.

The current study examined the hypothesis that amygdala activation serves as a neural precondition for negative affective experience. Participants' affective experience was measured by asking them to report on their momentary experiences several times a day over the course of a month using an electronic experience-sampling procedure. One year later, participants viewed backwardly masked depictions of fear while functional magnetic resonance imaging was used to measure their amygdala and fusiform gyrus activation. Negative affect, as measured during the experience-sampling procedure 1-year prior, was positively correlated with amygdala activation in response to these brief presentations of fear depictions. Furthermore, descriptive analyses indicated that fusiform gyrus activation and negative affective experience in the scanner were associated for participants reporting increased nervousness during the imaging procedure. The results are consistent with the interpretation that the amygdala contributes to negative affective experience by increasing perceptual sensitivity for negative stimuli.

The nucleation processes involved in calcium phosphate formation in tooth enamel are not well understood but are believed to involve proteins in the extracellular matrix. The ability of one enamel protein, amelogenin, to promote the nucleation and growth of calcium phosphate was studied in an in vitro system involving metastable supersaturated solutions. It was found that recombinant amelogenin (rM179 and rp(H)M180) promoted the nucleation of calcium phosphate compared to solutions without protein. The amount of calcium phosphate increased with increasing supersaturation of the solutions and increasing protein concentrations up to 6.5 μg/mL. At higher protein concentrations, the amount of calcium phosphate decreased. The kinetics of nucleation was studied in situ and in real time using a quartz crystal microbalance (QCM) and showed that the protein reduced the induction time for nucleation compared to solutions without protein. This work shows a nucleation role for amelogenin in vitro which may be promoted by the association of amelogenin into nanosphere templates, exposing charged functionality at the surface.

No abstract available.