Gene Literature Dashboard

An understanding of differential gene expression in highly metastatic osteosarcoma could provide gene targets for treatment of metastases. We compared gene expression profiles of high- (LM7) and low- (LM2) metastatic SaOS2-derived cell lines in an in vitro tissue culture model and examined several differentially regulated genes in vivo in a murine orthotopic xenograft model. We hypothesized an orthotopic inoculation of LM2 and LM7 cells would establish a primary lesion and the gene expression profile of cells grafted in this fashion would resemble the gene expression profile observed in an in vitro model. Thirty-five days after inoculation, animals were euthanized and both tibiae were harvested and rapidly frozen in liquid nitrogen. Human-specific GAPDH mRNA was present in two of four tibias inoculated with LM2 cells and three of four tibias inoculated with LM7 cells. Tibiae displaying the presence of human cells were assayed by semiquantitative reverse transcriptase polymerase chain reaction. We observed poor correspondence of in vitro to in vivo gene expression for either cell line. Accordingly, in vitro osteosarcoma gene expression data must be interpreted with caution until confirmed in vivo. Our orthotopic injection model allowed in vivo study of differential gene expression between these two cell lines but did not show radiographic evidence of an established primary lesion.

<h4>Background</h4>Few genetic factors predisposing to the sporadic form of amyotrophic lateral sclerosis (ALS) have been identified, but the pathology itself seems to be a true multifactorial disease in which complex interactions between environmental and genetic susceptibility factors take place. The purpose of this study was to approach genetic data with an innovative statistical method such as artificial neural networks to identify a possible genetic background predisposing to the disease. A DNA multiarray panel was applied to genotype more than 60 polymorphisms within 35 genes selected from pathways of lipid and homocysteine metabolism, regulation of blood pressure, coagulation, inflammation, cellular adhesion and matrix integrity, in 54 sporadic ALS patients and 208 controls. Advanced intelligent systems based on novel coupling of artificial neural networks and evolutionary algorithms have been applied. The results obtained have been compared with those derived from the use of standard neural networks and classical statistical analysis<h4>Results</h4>Advanced intelligent systems based on novel coupling of artificial neural networks and evolutionary algorithms have been applied. The results obtained have been compared with those derived from the use of standard neural networks and classical statistical analysis. An unexpected discovery of a strong genetic background in sporadic ALS using a DNA multiarray panel and analytical processing of the data with advanced artificial neural networks was found. The predictive accuracy obtained with Linear Discriminant Analysis and Standard Artificial Neural Networks ranged from 70% to 79% (average 75.31%) and from 69.1 to 86.2% (average 76.6%) respectively. The corresponding value obtained with Advanced Intelligent Systems reached an average of 96.0% (range 94.4 to 97.6%). This latter approach allowed the identification of seven genetic variants essential to differentiate cases from controls: apolipoprotein E arg158cys; hepatic lipase -480 C/T; endothelial nitric oxide synthase 690 C/T and glu298asp; vitamin K-dependent coagulation factor seven arg353glu, glycoprotein Ia/IIa 873 G/A and E-selectin ser128arg.<h4>Conclusion</h4>This study provides an alternative and reliable method to approach complex diseases. Indeed, the application of a novel artificial intelligence-based method offers a new insight into genetic markers of sporadic ALS pointing out the existence of a strong genetic background.

Alkylcarbamic acid biphenyl-3-yl esters are a class of fatty acid amide hydrolase (FAAH) inhibitors that comprises cyclohexylcarbamic acid 3'-carbamoylbiphenyl-3-yl ester (URB597), a compound with analgesic, anxiolytic-like and antidepressant-like properties in rat and mouse models. Here, we extended the structure-activity relationships (SARs) for this class of compounds by replacing the cyclohexyl ring of the parent compound cyclohexylcarbamic acid biphenyl-3-yl ester (URB524) (FAAH IC50 = 63 nM) with a selected set of substituents of different size, shape, flexibility, and lipophilicity. Docking experiments and linear interaction energy (LIE) calculations indicated that the N-terminal group of O-arylcarbamates fits within the lipophilic region of the substrate-binding site, mimicking the arachidonoyl chain of anandamide. Significant potency improvements were observed for the beta-naphthylmethyl derivative 4q (IC50 = 5.3 nM) and its 3'-carbamoylbiphenyl-3-yl ester 4z (URB880, IC50 = 0.63 nM), indicating that shape complementarity and hydrogen bonds are crucial to obtain highly potent inhibitors.

<h4>Background</h4>Genome-wide detection of single feature polymorphisms (SFP) in swine using transcriptome profiling of day 25 placental RNA by contrasting probe intensities from either Meishan or an occidental composite breed with Affymetrix porcine microarrays is presented. A linear mixed model analysis was used to identify significant breed-by-probe interactions.<h4>Results</h4>Gene specific linear mixed models were fit to each of the log2 transformed probe intensities on these arrays, using fixed effects for breed, probe, breed-by-probe interaction, and a random effect for array. After surveying the day 25 placental transcriptome, 857 probes with a q-value < or = 0.05 and |fold change| > or = 2 for the breed-by-probe interaction were identified as candidates containing SFP. To address the quality of the bioinformatics approach, universal pyrosequencing assays were designed from Affymetrix exemplar sequences to independently assess polymorphisms within a subset of probes for validation. Additionally probes were randomly selected for sequencing to determine an unbiased confirmation rate. In most cases, the 25-mer probe sequence printed on the microarray diverged from Meishan, not occidental crosses. This analysis was used to define a set of highly reliable predicted SFPs according to their probability scores.<h4>Conclusion</h4>By applying a SFP detection method to two mammalian breeds for the first time, we detected transition and transversion single nucleotide polymorphisms, as well as insertions/deletions which can be used to rapidly develop markers for genetic mapping and association analysis in species where high density genotyping platforms are otherwise unavailable.SNPs and INDELS discovered by this approach have been publicly deposited in NCBI's SNP repository dbSNP. This method is an attractive bioinformatics tool for uncovering breed-by-probe interactions, for rapidly identifying expressed SNPs, for investigating potential functional correlations between gene expression and breed polymorphisms, and is robust enough to be used on any Affymetrix gene expression platform.

<h4>Background</h4>Systematic search for genes whose gain-of-function by exogenous expression confers an advantage in cell-based selective screenings is a powerful method for unbiased functional exploration of the genome, and has the potential to disclose new targets for cancer therapy. A major limit of this approach resides in the labor-intensive cloning of resistant cells, identification of the integrated genes and validation of their ability to confer a selective advantage. Moreover, the selection has to be drastic and genes conferring a limited advantage are typically missed.<h4>Results</h4>We developed a new functional screening strategy based on transduction of mammalian cells of a given species with an expression library from another species, followed by one-shot quantitative tracing with DNA microarrays of all library-derived transcripts before and after selection. In this way, exogenous transcripts enriched after selection, and therefore likely to confer resistance, are readily detected. We transduced a retroviral cDNA expression library from mouse testis into human and canine cells, and optimized the use of commercial murine gene expression arrays for species-specific detection of library-derived transcripts. We then conducted a functional screening by growing library-transduced canine MDCK cells in suspension, to enrich for cDNAs conferring anchorage independence. Notably, these cells show partial resistance to loss of anchorage, and the selection can be of limited stringency, compromising approaches based on clonal selection or anyway requiring high stringency. Microarray analysis revealed reproducible enrichment after three weeks of growth on polyhema for seven genes, among which the Hras proto-oncogene and Sox5. When individually transduced into MDCK cells, Sox5 specifically promoted anchorage-independent growth, thereby confirming the validity and specificity of the approach.<h4>Conclusion</h4>The procedure described here brings substantial advantages to the field of expression cloning, being faster, more systematic and more sensitive. Indeed, this strategy allowed identification and validation of genes promoting anchorage-independent growth of epithelial cells under selection conditions not amenable to conventional expression cloning.

The myelodysplastic syndromes (MDS) are clonal stem cell disorders, characterized by ineffective and dysplastic hematopoiesis. The genetic and epigenetic pathways that determine disease stage and progression are largely unknown. In the current study we used gene expression microarray methodology to examine the gene expression differences between normal hematopoietic cells and hematopoietic cells from patients with MDS at different disease stages, using both unselected and CD34+ selected cells. Significant differences between normal and MDS hematopoietic cells were observed for several genes and pathways. Several genes promoting or opposing apoptosis were dysregulated in MDS cases, most notably MCL1 and EPOR. Progression from RA to RAEB(T) was associated with increased expression of several histone genes. In addition, the RAR-RXR pathway, critical for maintaining a balance between self-renewal and differentiation of hematopoietic stem cells, was found to be deregulated in hematopoietic cells from patients with advanced MDS compared to patients with refractory anemia. These findings provide new insights into the understanding of the pathophysiology and progression of MDS, and may guide to new targets for therapy. Taken together with previous published data, the present results also underscore the considerable complexity of the regulation of gene expression in MDS.

<h4>Background</h4>The transcription factor Pax6 is expressed by many cell types in the developing eye. Eyes do not form in homozygous loss-of-function mouse mutants (Pax6Sey/Sey) and are abnormally small in Pax6Sey/+ mutants. Eyes are also abnormally small in PAX77 mice expressing multiple copies of human PAX6 in addition to endogenous Pax6; protein sequences are identical in the two species. The developmental events that lead to microphthalmia in PAX77 mice are not well-characterised, so it is not clear whether over- and under-expression of Pax6/PAX6 cause microphthalmia through similar mechanisms. Here, we examined the consequences of over-expression for the eye and its axonal connections.<h4>Results</h4>Eyes form in PAX77+/+ embryos but subsequently degenerate. At E12.5, we found no abnormalities in ocular morphology, retinal cell cycle parameters and the incidence of retinal cell death. From E14.5 on, we observed malformations of the optic disc. From E16.5 into postnatal life there is progressively more severe retinal dysplasia and microphthalmia. Analyses of patterns of gene expression indicated that PAX77+/+ retinae produce a normal range of cell types, including retinal ganglion cells (RGCs). At E14.5 and E16.5, quantitative RT-PCR with probes for a range of molecules associated with retinal development showed only one significant change: a slight reduction in levels of mRNA encoding the secreted morphogen Shh at E16.5. At E16.5, tract-tracing with carbocyanine dyes in PAX77+/+ embryos revealed errors in intraretinal navigation by RGC axons, a decrease in the number of RGC axons reaching the thalamus and an increase in the proportion of ipsilateral projections among those RGC axons that do reach the thalamus. A survey of embryos with different Pax6/PAX6 gene dosage (Pax6Sey/+, Pax6+/+, PAX77+ and PAX77+/+) showed that (1) the total number of RGC axons projected by the retina and (2) the proportions that are sorted into the ipsilateral and contralateral optic tracts at the optic chiasm vary differently with gene dosage. Increasing dosage increases the proportion projecting ipsilaterally regardless of the size of the total projection.<h4>Conclusion</h4>Pax6 overexpression does not obviously impair the initial formation of the eye and its major cell-types but prevents normal development of the retina from about E14.5, leading eventually to severe retinal degeneration in postnatal life. This sequence is different to that underlying microphthalmia in Pax6+/- heterozygotes, which is due primarily to defects in the initial stages of lens formation. Before the onset of severe retinal dysplasia, Pax6 overexpression causes defects of retinal axons, preventing their normal growth and navigation through the optic chiasm.

<h4>Objectives</h4>Controversies concerning the pivotal trial and negative results from other Drotrecogin alfa activated (DAA) trials have raised questions about its efficacy in severe sepsis patients. Based on all available evidence, our study aimed to respond to: (1) What is the current probability that DAA is not better than the control? If the current probability is not small, e.g. greater than 0.05, then (2) How many patients will be needed for the DAA confirmatory trial?<h4>Design</h4>We applied the Bayesian methodology to all randomized trials which tested anti-thrombotic therapies for severe sepsis. Prior distributions were defined as severe skeptic (all anti-thrombotic trials or DAA trials); moderate skeptic (anti-thrombotic trials with high risk of death); mild skeptic (DAA trials only); enthusiastic (DAA trials with high risk of death).<h4>Results</h4>The total study sample includes 7,683 patients: DAA (N = 3,143); Anti-thrombin-III (N = 2,581); tissue factor pathway inhibitor (N = 1,959). Answer 1: All current probabilities that DAA is not better than the control (except for the enthusiastic analysis) range from 0.14 to 0.48, which strongly suggest the need for a confirmatory trial. Answer 2: The number of patients necessary for the DAA confirmatory trial ranges up to 8,350 for the severe skeptic; 730-810 for moderate skeptic; 550-685 for mild skeptic; and zero for enthusiastic analysis.<h4>Conclusions</h4>A confirmatory trial with approximately 600 patients with severe sepsis and high risk of death can provide a convincing answer for both the mild and moderate skeptic physicians concerning the efficacy of DAA in severe sepsis.

Cadherins and protocadherins are cell adhesion proteins that play an important role in neuronal migration, differentiation and synaptogenesis, properties that make them targets to consider in schizophrenia (SZ) and bipolar disorder (BD) pathogenesis. Consequently, allelic variation occurring in protocadherin and cadherin encoding genes that map to regions of the genome targeted in SZ and BD linkage studies are particularly strong candidates to consider. One such set of candidate genes is the 5q31-linked PCDH family, which consists of more than 50 exons encoding three related, though distinct family members--alpha, beta, and gamma--which can generate thousands of different protocadherin proteins through alternative promoter usage and cis-alternative splicing. In this study, we focused on a SNP, rs31745, which is located in a putative PCDHalpha enhancer mapped by ChIP-chip using antibodies to covalently modified histone H3. A striking increase in homozygotes for the minor allele at this locus was detected in patients with BD. Molecular analysis revealed that the SNP causes allele-specific changes in binding to a brain protein. The findings suggest that the 5q31-linked PCDH locus should be more thoroughly considered as a disease-susceptibility locus in psychiatric disorders.

<h4>Objectives</h4>Hemodialysis (HD) population commonly show high plasma ferritin levels with a poor diagnostic value. The objective of this study is to elucidate the meaning of HD hyperferritinemia through the analysis of its ferritin iron content (FIC).<h4>Design and methods</h4>FIC (iron atoms/ferritin molecule) was measured by atomic emission spectrometry. Ferritin and FIC values were correlated with iron storage and inflammation markers and the results of HD patients compared to those of septic and hemochromatosis patients.<h4>Results</h4>1) In the whole HD population, high ferritin levels were associated to low FIC values; 2) the correlation of ferritin with iron indices and inflammation markers in HD patients was intermediate in between that of septic and hemochromatosis patients; 3) the FIC level of HD patients was lower than that of the other two groups.<h4>Conclusions</h4>The high ferritin levels of HD patients are not synonymous with either inflammation or of high levels of iron storage. Their high levels and the low FIC values might be due to the presence inside the ferritin core of oligoelements other than iron.

In patients with malignant hematological disorders receiving immunosuppressive therapy, invasive pulmonary infections are serious complications that are associated with high morbidity and mortality. In immunocompromised hosts with impaired cellular immunity, two or more organisms may coexist leading to a wide range of clinical and radiological manifestations. Reported here is an old man who was diagnosed to have angioimmunoblastic T-cell lymphoma at King Faisal Specialist Hospital and Research Centre in Riyadh in December 2004. The lymphoma was treated with various immunosuppressive agents including alemtuzumab. In October 2006, the patient was admitted with severe bronchopneumonia caused by Nocardia asteroides and Aspergillus niger that was complicated by septic shock. The invasive pulmonary infections were successfully treated with trimethoprim-sulphamethoxazole, amikacin and liposomal amphotericin-B (amBisome).

Social context is increasingly recognized as essential for understanding complex human outcomes even among geneticists who focus on genetic influences. These outcomes typically involve multiple genes, multiple environmental factors, and the interactions between the two. In this paper, we propose a conceptual framework for gene-environment interaction and show how the interaction can be tested empirically using a sample of MZ twin, DZ twins, and full siblings. We test the hypothesis that the genetic contribution to adolescent drinking depends on the drinking behavior of their friends, using a sample of clusters of siblings and their friends from Add Health. Our analysis has yielded evidence supporting the gene-environment interaction hypothesis. High levels of alcohol use by one's best friend or among one's friends tend to bring about higher levels of genetic contribution to alcohol use. Lower levels of alcohol use by one's best friend or among one's friends tend to suppress the level of genetic contribution to alcohol use. Our findings suggest that friend behavior might be a particularly important environmental moderator of the expression of genetic disposition for adolescent drug use, smoking, dietary habits, and risky sexual behavior. Subsequent studies of these behaviors that use non-DNA twin samples or DNA measures of genetic variants should investigate peer influence as a significant environmental moderator.

Mutant huntingtin accumulates in the neuronal nuclei and processes, which suggests that its subcellular localization is critical for the pathology of Huntington's disease (HD). However, the contribution of cytoplasmic mutant huntingtin and its aggregates in neuronal processes (neuropil aggregates) has not been rigorously explored. We generated an intracellular antibody (intrabody) whose binding to a unique epitope of human huntingtin is enhanced by polyglutamine expansion. This intrabody decreases the cytotoxicity of mutant huntingtin and its distribution in neuronal processes. When expressed in the striatum of HD mice via adenoviral infection, the intrabody reduces neuropil aggregate formation and ameliorates neurological symptoms. Interaction of the intrabody with mutant huntingtin increases the ubiquitination of cytoplasmic huntingtin and its degradation. These findings suggest that the intrabody reduces the specific neurotoxicity of cytoplasmic mutant huntingtin and its associated neurological symptoms by preventing the accumulation of mutant huntingtin in neuronal processes and promoting its clearance in the cytoplasm.

Significant levels of the 1, N(2)-gamma-hydroxypropano-dG adducts of the alpha,beta-unsaturated aldehydes acrolein, crotonaldehyde, and 4-hydroxy-2E-nonenal (HNE) have been identified in human DNA, arising from both exogenous and endogenous exposures. They yield interstrand DNA cross-links between guanines in the neighboring C.G and G.C base pairs located in 5'-CpG-3' sequences, as a result of opening of the 1,N(2)-gamma-hydroxypropano-dG adducts to form reactive aldehydes that are positioned within the minor groove of duplex DNA. Using a combination of chemical, spectroscopic, and computational methods, we have elucidated the chemistry of cross-link formation in duplex DNA. NMR spectroscopy revealed that, at equilibrium, the acrolein and crotonaldehyde cross-links consist primarily of interstrand carbinolamine linkages between the exocyclic amines of the two guanines located in the neighboring C.G and G.C base pairs located in 5'-CpG-3' sequences, that maintain the Watson-Crick hydrogen bonding of the cross-linked base pairs. The ability of crotonaldehyde and HNE to form interstrand cross-links depends upon their common relative stereochemistry at the C6 position of the 1,N(2)-gamma-hydroxypropano-dG adduct. The stereochemistry at this center modulates the orientation of the reactive aldehyde within the minor groove of the double-stranded DNA, either facilitating or hindering the cross-linking reactions; it also affects the stabilities of the resulting diastereoisomeric cross-links. The presence of these cross-links in vivo is anticipated to interfere with DNA replication and transcription, thereby contributing to the etiology of human disease. Reduced derivatives of these cross-links are useful tools for studying their biological processing.

During iron acquisition by the cell, complete homodimeric transferrin receptor 1 in an unknown state (R1) binds iron-loaded human serum apotransferrin in an unknown state (T) and allows its internalization in the cytoplasm. T also forms complexes with metals other than iron. Are these metals incorporated by the iron acquisition pathway and how can other proteins interact with R1? We report here a four-step mechanism for cobalt(III) transfer from CoNtaCO(3)(2-) to T and analyze the interaction of cobalt-loaded transferrin with R1. The first step in cobalt uptake by T is a fast transfer of Co(3+) and CO(3)(2-) from CoNtaCO(3)(2-) to the metal-binding site in the C-lobe of T: direct rate constant, k(1)=(1.1+/-0.1) x 10(6) M(-1) s(-1); reverse rate constant, k(-1)=(1.9+/-0.6) x 10(6) M(-1) s(-1); and equilibrium constant, K=1.7+/-0.7. This step is followed by a proton-assisted conformational change of the C-lobe: direct rate constant, k(2)=(3+/-0.3) x 10(6) M(-1) s(-1); reverse rate constant, k(-2)=(1.6+/-0.3) x 10(-2) s(-1); and equilibrium constant, K(2a)=5.3+/-1.5 nM. The two final steps are slow changes in the conformation of the protein (0.5 h and 72 h), which allow it to achieve its final thermodynamic state and also to acquire second cobalt. The cobalt-saturated transferrin in an unknown state (TCo(2)) interacts with R1 in two different steps. The first is an ultra-fast interaction of the C-lobe of TCo(2) with the helical domain of R1: direct rate constant, k(3)=(4.4+/-0.6)x10(10) M(-1) s(-1); reverse rate constant, k(-3)=(3.6+/-0.6) x 10(4) s(-1); and dissociation constant, K(1d)=0.82+/-0.25 muM. The second is a very slow interaction of the N-lobe of TCo(2) with the protease-like domain of R1. This increases the stability of the protein-protein adduct by 30-fold with an average overall dissociation constant K(d)=25+/-10 nM. The main trigger in the R1-mediated iron acquisition is the ultra-fast interaction of the metal-loaded C-lobe of T with R1. This step is much faster than endocytosis, which in turn is much faster than the interaction of the N-lobe of T with the protease-like domain. This can explain why other metal-loaded transferrins or a protein such as HFE-with a lower affinity for R1 than iron-saturated transferrin but with, however, similar or higher affinities for the helical domain than the C-lobe-competes with iron-saturated transferrin in an unknown state towards interaction with R1.

<h4>Background</h4>Many studies have shown that the abundance level of gene expression is heritable. Analogous to the traditional genetic study, most researchers treat the expression of one gene as a quantitative trait and map it to expression quantitative trait loci (eQTL). This is 1D-trait mapping. 1D-trait mapping ignores the trait-trait interaction completely, which is a major shortcoming.<h4>Results</h4>To overcome this limitation, we study the expression of a pair of genes and treat the variation in their co-expression pattern as a two dimensional quantitative trait. We develop a method to find gene pairs, whose co-expression patterns, including both signs and strengths, are mediated by genetic variations and map these 2D-traits to the corresponding genetic loci. We report several applications by combining 1D-trait mapping with 2D-trait mapping, including the contribution of genetic variations to the perturbations in the regulatory mechanisms of yeast metabolic pathways.<h4>Conclusion</h4>Our approach of 2D-trait mapping provides a novel and effective way to connect the genetic variation with higher order biological modules via gene expression profiles.

Aggresomes are juxtanuclear inclusion bodies that have been proposed to act as staging grounds for the disposal of protein aggregates via the autophagic route. To examine whether the composition of an aggresome influences its clearance by autophagy, we ectopically expressed a variety of aggregation-prone proteins in cultured cells to generate aggresomes that differ in their protein content. We found that whereas aggresomes generated in cells expressing mutant huntingtin or mutant tau, or co-expressing synphilin-1 and alpha-synuclein, are amenable to clearance by autophagy, those produced in AIMP2 (p38)- or mutant desmin-expressing cells are apparently resistant to autophagic clearance. Notably, AIMP2 (p38)- and desmin-positive inclusions fail to recruit key components of the autophagic/lysosomal system. However, by altering the composition of inclusions, 'autophagy-resistant' aggresomes could be rendered 'autophagy-susceptible'. Taken together, our results demonstrate that not all aggresomes are efficiently primed for autophagic clearance and highlight a certain degree of selectivity for the supposedly non-discriminative pathway.

Peroxiredoxins (Prdxs), a family of antioxidant and redox-signaling proteins, are plentiful within the heart; however, their cardiac functions are poorly understood. These studies were designed to characterize the complex changes in Prdxs induced by oxidant stress in rat myocardium. Hydrogen peroxide, a Prdx substrate, was used as the model oxidant pertinent to redox signaling during health and to injury at higher concentrations. Rat hearts were aerobically perfused with a broad concentration range of hydrogen peroxide by the Langendorff method, homogenized, and analyzed by immunoblotting. Heart extracts were also analyzed by size-exclusion chromatography under nondenaturing conditions. Hydrogen peroxide-induced changes in disulfide bond formation, nonreversible oxidation of cysteine (hyperoxidation), and subcellular localization were determined. Hydrogen peroxide induced an array of changes in the myocardium, including formation of disulfide bonds that were intermolecular for Prdx1, Prdx2, and Prdx3 but intramolecular within Prdx5. For Prdx1, Prdx2, and Prdx5, disulfide bond formation can be approximated to an EC(50) of 10-100, 1-10, and 100-1,000 microM peroxide, respectively. Hydrogen peroxide induced hyperoxidation, not just within monomeric Prdx (by SDS-PAGE), but also within Prdx disulfide dimers, and reflects a flexibility within the dimeric unit. Prdx oxidation was also associated with movement from the cytosolic to the membrane and myofilament-enriched fractions. In summary, Prdxs undergo a complex series of redox-dependent structural changes in the heart in response to oxidant challenge with its substrate hydrogen peroxide.

<h4>Rationale</h4>Exploring differences between mouse strains in drug effects in models of antidepressant-like activity may provide clues to the neurobiology of antidepressant responses.<h4>Objectives</h4>The objective of this study was to explore whether insensitivity to selective serotonin reuptake inhibitors (SSRIs) in NMRI mice in the tail suspension test can be related to 5-hydroxytryptamine (5-HT) function.<h4>Materials and methods</h4>We compared NMRI and C57Bl/6 mice, a SSRI-sensitive strain, in the tail suspension test following citalopram, paroxetine, or fluoxetine and determined 5-HT transporter (5-HTT) densities, 5-HT tissue and extracellular levels, 5-HT synthesis, tryptophan hydroxylase 2 (TPH2) genotypes and hypothermia induced by the 5-HT(1A) agonist 8-OH-DPAT. In NMRI mice, we tested if co-treatment with 5-HTP would increase 5-HT levels and confer SSRI sensitivity in the tail suspension test.<h4>Results</h4>C57Bl/6, but not NMRI, mice responded to SSRIs in the tail suspension test. 5-HTT densities in the frontal cortex and hippocampus were similar between the strains. NMRI mice had lower tissue 5-HT levels in these regions and decreased extracellular 5-HT in the frontal cortex at baseline and following citalopram. C57Bl/6 mice were more sensitive to 8-OH-DPAT-induced hypothermia. Both strains had the 1473C TPH2 genotype and similar 5-HT synthesis. In NMRI mice, 5-HTP co-treatment restored the tail suspension and extracellular 5-HT responses to SSRIs to levels equivalent to those seen in C57Bl/6 mice.<h4>Conclusion</h4>Low 5-HT function in NMRI mice may account for their insensitivity to SSRIs in the tail suspension test. As the tail suspension test is a predictor of clinical efficacy, the current data suggest that 5-HTP adjunct treatment may benefit SSRI treatment refractory patients.

<h4>Background</h4>The formation of new infectious human immunodeficiency type 1 virus (HIV-1) mainly relies on the homo-multimerization of the viral structural polyprotein Pr55Gag and on the recruitment of host factors. We have previously shown that the double-stranded RNA-binding protein Staufen 1 (Stau1), likely through an interaction between its third double-stranded RNA-binding domain (dsRBD3) and the nucleocapsid (NC) domain of Pr55Gag, participates in HIV-1 assembly by influencing Pr55Gag multimerization.<h4>Results</h4>We now report the fine mapping of Stau1/Pr55Gag association using co-immunoprecipitation and live cell bioluminescence resonance energy transfer (BRET) assays. On the one hand, our results show that the Stau1-Pr55Gag interaction requires the integrity of at least one of the two zinc fingers in the NC domain of Pr55Gag but not that of the NC N-terminal basic region. Disruption of both zinc fingers dramatically impeded Pr55Gag multimerization and virus particle release. In parallel, we tested several Stau1 deletion mutants for their capacity to influence Pr55Gag multimerization using the Pr55Gag/Pr55Gag BRET assay in live cells. Our results revealed that a molecular determinant of 12 amino acids at the N-terminal end of Stau1 is necessary to increase Pr55Gag multimerization and particle release. However, this region is not required for Stau1 interaction with the viral polyprotein Pr55Gag.<h4>Conclusion</h4>These data highlight that Stau1 is a modular protein and that Stau1 influences Pr55Gag multimerization via 1) an interaction between its dsRBD3 and Pr55Gag zinc fingers and 2) a regulatory domain within the N-terminus that could recruit host machineries that are critical for the completion of new HIV-1 capsids.

Axon migrations are guided by extracellular cues that induce asymmetric outgrowth activity in the growth cone. Several intracellular signaling proteins have been implicated in the guidance response. However, how these proteins interact to generate asymmetric outgrowth activity is unknown. Here, we present evidence that in C. elegans, the CED-10/Rac1 GTPase binds to and causes asymmetric localization of MIG-10/lamellipodin, a protein that regulates actin polymerization and has outgrowth-promoting activity in neurons. Genetic analysis indicates that mig-10 and ced-10 function together to orient axon outgrowth. The RAPH domain of MIG-10 binds to activated CED-10/Rac1, and ced-10 function is required for the asymmetric MIG-10 localization that occurs in response to the UNC-6/netrin guidance cue. We also show that asymmetric localization of MIG-10 in growth cones is associated with asymmetric concentrations of f-actin and microtubules. These results suggest that CED-10/Rac1 is asymmetrically activated in response to the UNC-6/netrin signal and thereby causes asymmetric recruitment of MIG-10/lamellipodin. We propose that the interaction between activated CED-10/Rac1 and MIG-10/lamellipodin triggers local cytoskeletal assembly and polarizes outgrowth activity in response to UNC-6/netrin.

As terminally differentiated vital cells, neurons may be specialized to fight viral infections without undergoing cellular self-destruction. The cellular lysosomal degradation pathway, autophagy, is emerging as one such mechanism of neuronal antiviral defence. Autophagy has diverse physiological functions, such as cellular adaptation to stress, routine organelle and protein turnover, and innate immunity against intracellular pathogens, including viruses. Most of the in vivo evidence for an antiviral role of autophagy is related to viruses that specifically target neurons, including the prototype alphavirus, Sindbis virus, and the alpha-herpesvirus, herpes simplex virus type 1 (HSV-1). In the case of HSV-1, viral evasion of autophagy is essential for lethal encephalitis. As basal autophagy is important in preventing neurodegeneration, and induced autophagy is important in promoting cellular survival during stress, viral antagonism of autophagy in neurons may lead to neuronal dysfunction and/or neuronal cell death. This review provides background information on the roles of autophagy in immunity and neuroprotection, and then discusses the relationships between autophagy and viral neurovirulence.

Transcriptional dysregulation has emerged as a central pathogenic mechanism in Huntington's disease (HD), which is associated with neuropathological changes predominantly in the striatum. Here we demonstrate that expression of Bcl11b (a.k.a. CTIP2), a transcription factor exhibiting highly-enriched localization in adult striatum, is significantly decreased in HD cells, mouse models and human subjects and that overexpression of Bcl11b attenuates toxic effects of mutant huntingtin in cultured striatal neurons. We show that Bcl11b directly activates the proximal promoter regions of striatal-enriched genes and can increase mRNA levels of striatal-expressing genes. We further demonstrate an interaction between Bcl11b and huntingtin protein in cultured cells and brain homogenates from HD R6/1 and YAC72 transgenic mice. We propose that sequestration and/or decreased expression of Bcl11b in HD is responsible, at least in part, for the dysregulation of striatal gene expression observed in HD and may contribute to the specificity of pathology observed in this disease.

One challenging question in neurogenesis concerns the identification of cues that trigger axonal growth and pathfinding to form stereotypic neuronal networks during the construction of a nervous system. Here, we show that in Drosophila, Engrailed (EN) and Gooseberry-Neuro (GsbN) act together as cofactors to build the posterior commissures (PCs), which shapes the ventral nerve cord. Indeed, we show that these two proteins are acting together in axon growth and midline crossing, and that this concerted action occurs at early development, in neuroblasts. More precisely, we identified that their expressions in NB 6-4 are necessary and sufficient to trigger the formation of the PCs, demonstrating that segmentation genes such as EN and GsbN play a crucial role in the determination of NB 6-4 in a way that will later influence growth and guidance of all the axons that form the PCs. We also demonstrate a more specific function of GsbN in differentiated neurons, leading to fasciculations between axons, which might be required to obtain PC mature axon bundles.

Specialized chromatin exists at centromeres and must be precisely transmitted during DNA replication. The mechanisms involved in the propagation of these structures remain elusive. Fission yeast centromeres are composed of two chromatin domains: the central CENP-A(Cnp1) kinetochore domain and flanking heterochromatin domains. Here we show that fission yeast Mcl1, a DNA polymerase alpha (Pol alpha) accessory protein, is critical for maintenance of centromeric chromatin. In a screen for mutants that alleviate both central domain and outer repeat silencing, we isolated several cos mutants, of which cos1 is allelic to mcl1. The mcl1-101 mutation causes reduced CENP-A(Cnp1) in the central domain and an aberrant increase in histone acetylation in both domains. These phenotypes are also observed in a mutant of swi7(+), which encodes a catalytic subunit of Pol alpha. Mcl1 forms S-phase-specific nuclear foci, which colocalize with those of PCNA and Pol alpha. These results suggest that Mcl1 and Pol alpha are required for propagation of centromere chromatin structures during DNA replication.

Archaeal class I CCA-adding enzymes use a ribonucleoprotein template to build and repair the universally conserved 3'-terminal CCA sequence of the acceptor stem of all tRNAs. A wealth of structural and biochemical data indicate that the Archaeoglobus fulgidus CCA-adding enzyme binds primarily to the tRNA acceptor stem through a long, highly conserved alpha-helix that lies nearly parallel to the acceptor stem and makes many contacts with its sugar-phosphate backbone. Although the geometry of this alpha-helix is nearly ideal in all available cocrystal structures, the helix contains a highly conserved, potentially helix-breaking proline or glycine near the N terminus. We performed a mutational analysis to dissect the role of this residue in CCA-addition activity. We found that the phylogenetically permissible P295G mutant and the phylogenetically absent P295T had little effect on CCA addition, whereas P295A and P295S progressively interfered with CCA addition (C74>C75>A76 addition). We also examined the effects of these mutations on tRNA binding and the kinetics of CCA addition, and performed a computational analysis using Rosetta Design to better understand the role of P295 in nucleotide transfer. Our data indicate that CCA-adding activity does not correlate with the stability of the pre-addition cocrystal structures visualized by X-ray crystallography. Rather, the data are consistent with a transient conformational change involving P295 of the tRNA-binding alpha-helix during or between one or more steps in CCA addition.

The effects of combined treatment with a glucocorticoid receptor (GR) antagonist, Org 34850, and a selective serotonin reuptake inhibitor (SSRI), fluoxetine, were investigated on pre- and postsynaptic aspects of 5-HT neurotransmission. Rats were treated for 14 days with Org 34850 (15 mg per kg per day subcutaneously), fluoxetine (10 mg per kg per day intraperitoneally), or a combination of both drugs. [(3)H]-citalopram binding (an index of 5-HT transporter (5-HTT) expression) was only slightly affected by Org 34850 alone: decreased in cortex and midbrain and increased in hippocampus. In contrast, chronic fluoxetine markedly decreased 5-HTT levels in all regions. Importantly, this decrease was significantly enhanced by combined Org 34850/fluoxetine treatment. There were no changes in the expression of 5-HTT mRNA, suggesting these effects were not due to changes in gene transcription. Expression of tryptophan hydroxylase mRNA and both 5-HT(1A) autoreceptor mRNA and protein (assessed using [(3)H]-8-OH-DPAT binding) were unchanged by any treatment. The expression of postsynaptic 5-HT(1A) receptor protein in the forebrain was unaltered by fluoxetine, Org 34850 or the combined Org 34850/fluoxetine treatment. This downregulation of 5-HTT by fluoxetine and its enhancement by Org 34850 can explain our recent observation that GR antagonists augment the SSRI-induced increase in extracellular 5-HT. In addition, these data suggest that the augmentation of forebrain 5-HT does not result in downregulation of forebrain 5-HT(1A) receptor expression. Given the importance of 5-HT(1A) receptor-mediated transmission in the forebrain to the antidepressant response, these data indicate that co-administration of GR antagonists may be effective in augmenting the antidepressant response to SSRI treatment.

LAP (leucine-rich repeats (LRR) and PSD-95/Dlg/ZO-1 (PDZ)) family proteins, including Scribble, LET-413, ERBIN, Densin-180 and Lano, are involved in the regulation of cell polarity. The LRR domains of LAP proteins were reported to mediate their basolateral membrane localization and to be essential for their function. To further dissect the mechanism of the plasma membrane localization of ERBIN, we introduced various mutants of ERBIN into cultured cells and observed the intracellular localization. When an LRR domain mutant lacking amino acid residues 1-32 at the amino (N) terminal region was over-expressed in cells, the mutant did not localize at the plasma membrane, but localized in the cytoplasm. We found that cysteines 14 and 16 at the N-terminal region of ERBIN are in vivo palmitoylated. Over-expressed mutants in which cysteine 14 and/or cysteine 16 were changed to serines did not localize at the plasma membrane, indicating that the palmitoylation of ERBIN is necessary for its plasma membrane localization. The over-expressed 1-196 amino acids fragment of ERBIN, which lacked the latter half of LRR, was palmitoylated but did not localize at the plasma membrane. These results suggest that both palmitoylation and LRR are required for the plasma membrane localization of ERBIN.

Conjugation to SUMO is a reversible post-translational modification that regulates several transcription factors involved in cell proliferation, differentiation, and disease. The p53 tumor suppressor can be modified by SUMO-1 in mammalian cells, but the functional consequences of this modification are unclear. Here, we demonstrate that the Drosophila homolog of human p53 can be efficiently sumoylated in insect cells. We identify two lysine residues involved in SUMO attachment, one at the C terminus, between the DNA binding and oligomerization domains, and one at the N terminus of the protein. We find that sumoylation helps recruit Drosophila p53 to nuclear dot-like structures that can be marked by human PML and the Drosophila homologue of Daxx. We demonstrate that mutation of both sumoylation sites dramatically reduces the transcriptional activity of p53 and its ability to induce apoptosis in transgenic flies, providing in vivo evidence that sumoylation is critical for Drosophila p53 function.

<h4>Background/aims</h4>Liver transplantation usually cures hepatocellular carcinoma when the Milan selection criteria are applied, whereas there is substantial risk of posttransplant recurrence with tumors beyond these criteria. This study uses molecular data to identify a subgroup of patients who, despite having hepatocellular carcinoma beyond Milan criteria, have favorable outcomes.<h4>Methods</h4>Allelic imbalance of 18 microsatellites was analyzed in 70 consecutive patients (35 within Milan, 35 beyond Milan criteria) transplanted for hepatocellular carcinoma of whom 24 had recurrence and 46 survived at least 5 years recurrence-free. Fractional allelic imbalance (the fraction of significant microsatellites that demonstrated allelic imbalance) and relevant clinical/pathological variables were tested for correlation with time to recurrence.<h4>Results</h4>Allelic imbalance in 9/18 microsatellites correlated with recurrence. Fractional allelic imbalance > 0.27 and macrovascular invasion were independent predictors of recurrence in patients with tumors beyond Milan criteria; the probability of recurrence at 5 years was 85% with fractional allelic imbalance > or = 0.27 vs. 10% when < 0.27 (p=0.0002). An algorithm including Milan criteria and fractional allelic imbalance status is 89% accurate in predicting tumor recurrence after transplantation.<h4>Conclusion</h4>Analysis of allelic imbalance of 9 microsatellites identifies a subgroup of patients who, despite having hepatocellular carcinoma beyond Milan criteria, have a low risk of posttransplant recurrence.

<h4>Background/aims</h4>Iron overload can cause liver toxicity and increase the risk of liver failure or hepatocellular carcinoma in humans. Curcumin (diferuloylmethane), a component of the food spice turmeric, has antioxidant, iron binding and hepatoprotective properties. The aim of this study was to quantify its effects on iron overload and the resulting downstream toxic effects in cultured T51B rat liver epithelial cells.<h4>Methods</h4>T51B cells were loaded with ferric ammonium citrate (FAC) with or without the iron delivery agent 8-hydroxyquinoline. Cytotoxicity was measured by methylthiazolyldiphenyl-tetrazolium bromide assay. Iron uptake and iron bioavailability were documented by chemical assay, quench of calcein fluorescence and ferritin induction. Reactive oxygen species (ROS) were measured by a fluorescence assay using 2',7'-dichlorodihydrofluorescein diacetate. Oxidative stress signalling to jnk, c-jun and p38 was measured by a Western blot with phospho-specific antibodies.<h4>Results</h4>Curcumin bound iron, but did not block iron uptake or bioavailability in T51B cells given FAC. However, it reduced cytotoxicity, blocked the generation of ROS and eliminated signalling to cellular stress pathways caused by iron. Inhibition was observed over a wide range of FAC concentrations (50-500 microM), with an apparent IC(50) in all cases between 5 and 10 microM curcumin. In contrast, desferoxamine blocked both iron uptake and toxic effects of iron at concentrations that depended on the FAC concentration. The effects of curcumin also differed from those of alpha-tocopherol, which did not bind iron and was less effective at blocking iron-stimulated ROS generation.<h4>Conclusions</h4>Curcumin reduced iron-dependent oxidative stress and iron toxicity in T51B cells without blocking iron uptake.

An African American male of West Indies descent was diagnosed to have elevated transferrin saturation, hyperferritinemia, severe iron deposition in hepatocytes, and hepatic cirrhosis at age 4. He was treated with serial phlebotomy to maintain a normal serum ferritin concentration thereafter. We evaluated him at age 23 and confirmed that he had normal serum ferritin levels, severe iron deposition in hepatocytes, hepatic cirrhosis, and portal hypertension. He did not have endocrinopathy, cardiomyopathy, or arthropathy. He was homozygous for the novel hemojuvelin (HJV) premature stop-codon mutation R54X (exon 3; c.160A-->T). He did not have either HFE C282Y, H63D, or S65C, or deleterious coding region mutations of SLC40A1, TFR2, or HAMP. His erythrocyte measures and hemoglobin electrophoresis were consistent with alpha-thalassemia trait. We conclude that homozygosity for HJV R54X accounts for his severe, early age-of-onset hemochromatosis; his phenotype was probably modified by serial phlebotomy therapy.

Non-human primates are valuable for modelling human disorders and for developing therapeutic strategies; however, little work has been reported in establishing transgenic non-human primate models of human diseases. Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder characterized by motor impairment, cognitive deterioration and psychiatric disturbances followed by death within 10-15 years of the onset of the symptoms. HD is caused by the expansion of cytosine-adenine-guanine (CAG, translated into glutamine) trinucleotide repeats in the first exon of the human huntingtin (HTT) gene. Mutant HTT with expanded polyglutamine (polyQ) is widely expressed in the brain and peripheral tissues, but causes selective neurodegeneration that is most prominent in the striatum and cortex of the brain. Although rodent models of HD have been developed, these models do not satisfactorily parallel the brain changes and behavioural features observed in HD patients. Because of the close physiological, neurological and genetic similarities between humans and higher primates, monkeys can serve as very useful models for understanding human physiology and diseases. Here we report our progress in developing a transgenic model of HD in a rhesus macaque that expresses polyglutamine-expanded HTT. Hallmark features of HD, including nuclear inclusions and neuropil aggregates, were observed in the brains of the HD transgenic monkeys. Additionally, the transgenic monkeys showed important clinical features of HD, including dystonia and chorea. A transgenic HD monkey model may open the way to understanding the underlying biology of HD better, and to the development of potential therapies. Moreover, our data suggest that it will be feasible to generate valuable non-human primate models of HD and possibly other human genetic diseases.

Cidofovir (HPMPC, 1), a broad-spectrum antiviral agent, is currently used to treat AIDS-related human cytomegalovirus (HCMV) retinitis and has recognized therapeutic potential for orthopox virus infections, but is limited by its low oral bioavailability. Cyclic cidofovir (2) displays decreased nephrotoxicity compared to 1, while also exhibiting potent antiviral activity. Here we describe in detail the synthesis and evaluation as prodrugs of four cHPMPC dipeptide conjugates in which the free POH of 2 is esterified by the Ser side chain alcohol group of an X-L-Ser(OMe) dipeptide: 3 (X=L-Ala), 4 (X=L-Val), 5 (X=L-Leu), and 6 (X=L-Phe). Perfusion studies in the rat establish that the mesenteric permeability to 4 is more than 20-fold greater than to 1, and the bioavailability of 4 is increased 6-fold relative to 1 in an in vivo murine model. In gastrointestinal and liver homogenates, the cHPMPC prodrugs are rapidly hydrolyzed to 2. Prodrugs 3, 4, and 5 are nontoxic at 100 microM in HFF and KB cells and in cell-based plaque reduction assays had IC 50 values of 0.1-0.5 microM for HCMV and 10 microM for two orthopox viruses (vaccinia and cowpox). The enhanced transport properties of 3-6, conferred by incorporation of a biologically benign dipeptide moiety, and the facile cleavage of the Ser-O-P linkage suggest that these prodrugs represent a promising new approach to enhancing the bioavailability of 2.

The phosphorylated glycoprotein osteopontin (OPN) is involved in the regulation of biomineralization under normal and pathological conditions. Its actions include inhibiting apatite crystal growth and promoting the formation and function of mineral resorbing cells, including osteoclasts (OCL). The purpose of this study was to develop stable apatitic mineral surfaces and determine their influence on OCL formation and mineral resorption from bone marrow macrophages derived from OPN wild-type (OPN+/+) and OPN deficient (OPN-/-) mice. We demonstrated that these mineral coatings were stable and supported bone marrow-derived macrophage differentiation to OCL under our culture conditions. Macrophages harvested from OPN-/- mice had a greater capacity to form OCL than macrophages from OPN+/+ mice when allowed to differentiate on tissue culture plastic. In contrast, when allowed to differentiate on a mineral surface, no difference in OCL formation was observed. Interestingly, OPN+/+ OCL were more efficient at mineral dissolution than OPN-/- OCL, and this difference was observed regardless of differentiating surface. Our results suggest that mineralized substrates as well as ability to synthesize OPN both control OCL function in our model system. The exact nature of these effects may be dependent on variables related to mineral substrate presentation.

<h4>Background</h4>T-cell activation is an essential step of the immune response and relies on the tightly controlled orchestration of hundreds of genes/proteins, yet the cellular and molecular events underlying this complex process are not fully understood, especially at the genome-scale. Significantly, a comparative genome-scale transcriptional analysis of two T-cell subsets (CD4+ and CD8+) against each other and against the naturally mixed population (CD3+ cells) remains unexplored.<h4>Results</h4>Comparison of the microarray-based gene expression patterns between CD3+ T cells, and the CD4+ and CD8+ subsets revealed largely conserved, but not identical, transcriptional patterns. We employed a Gene-Ontology-driven transcriptional analysis coupled with protein abundance assays in order to identify novel T-cell activation genes and cell-type-specific genes associated with the immune response. We identified potential genes involved in the communication between the two subsets (including IL23A, NR4A2, CD83, PSMB2, -8, MIF, IFI16, TNFAIP1, POU2AF1, and OTUB1) and would-be effector-function-specific genes (XCL2, SLAMF7, TNFSF4, -5, -9, CSF3, CD48 and CD244). Chemokines induced during T-cell activation, but not previously identified in T cells, include CCL20, CXCL9, -10, -11 (in all three populations), and XCL2 (preferentially in CD8+ T cells). Increased expression of other unexpected cytokines (GPI, OSM and MIF) suggests their involvement in T-cell activation with their functions yet to be examined. Differential expression of many receptors, not previously reported in the context of T-cell activation, including CCR5, CCR7, IL1R2, IL1RAP, IL6R, TNFRSF25 and TNFRSF1A, suggests their role in this immune process. Several receptors involved in TCR activation (CD3D, CD3G, TRAT1, ITGAL, ITGB1, ITGB2, CD8A and B (CD8+ T-cell specific) along with LCK, ZAP70 and TYROBP were synchronously downregulated. Members of cell-surface receptors (HLA-Ds and KLRs), none previously identified in the context of T-cell activation, were also downregulated.<h4>Conclusion</h4>This comparative genome-scale, transcriptional analysis of T-cell activation in the CD4+ and CD8+ subsets and the mixed CD3+ populations made possible the identification of many immune-response genes not previously identified in the context of T-cell activation. Significantly, it made possible to identify the temporal patterns of many previously known T-cell activation genes, and also identify genes implicated in effector functions of and communication between CD4+ and CD8+ T cells.

Fear and reward learning can occur through direct experience or observation. Both channels can enhance survival or create maladaptive behavior. We used fMRI to isolate neural mechanisms of observational fear and reward learning and investigate whether neural response varied according to individual differences in neuroticism and extraversion. Participants learned object-emotion associations by observing a woman respond with fearful (or neutral) and happy (or neutral) facial expressions to novel objects. The amygdala-hippocampal complex was active when learning the object-fear association, and the hippocampus was active when learning the object-happy association. After learning, objects were presented alone; amygdala activity was greater for the fear (vs. neutral) and happy (vs. neutral) associated object. Importantly, greater amygdala-hippocampal activity during fear (vs. neutral) learning predicted better recognition of learned objects on a subsequent memory test. Furthermore, personality modulated neural mechanisms of learning. Neuroticism positively correlated with neural activity in the amygdala and hippocampus during fear (vs. neutral) learning. Low extraversion/high introversion was related to faster behavioral predictions of the fearful and neutral expressions during fear learning. In addition, low extraversion/high introversion was related to greater amygdala activity during happy (vs. neutral) learning, happy (vs. neutral) object recognition, and faster reaction times for predicting happy and neutral expressions during reward learning. These findings suggest that neuroticism is associated with an increased sensitivity in the neural mechanism for fear learning which leads to enhanced encoding of fear associations, and that low extraversion/high introversion is related to enhanced conditionability for both fear and reward learning.

<h4>Objective</h4>The molecular basis for the antithrombin (AT) deficiency and dilated cardiomyopathy (DCM) combined in a Japanese patient was investigated.<h4>Methods</h4>We analyzed candidate genes -SERPINC1 for AT deficiency, and TNNT2 and LMNA for DCM. In addition, we examined the characteristics of recombinant mutant AT and evaluated the LMNA mutation associated with DCM by molecular modeling.<h4>Results</h4>Genome sequencing of SERPINC1 revealed a C-to-A transversion in exon 6 that resulted in a p.Pro439Thr mutation of AT, which was previously reported as a pleiotropic effect type II AT deficiency (AT Budapest5). However, expression experiments with recombinant 439Thr-AT showed normal heparin affinity, slightly reduced secretion, and low specific activity, which suggested that this mutation exhibits an intermediate feature of type I and type II AT deficiencies. In a survey of gene abnormalities causing DCM, we found no causative gene defect in TNNT2; however, we identified a G-to-C transversion in LMNA that resulted in a novel p.Asp357His mutation in lamin A/C. This acidic-to-basic residue substitution might have impaired the head-to-tail association of two lamin dimers leading to DCM. Further, we identified both SERPINC1 and LMNA mutations in the patient's daughter and son, both of whom had AT deficiency. These data suggested that a p.Pro439Thr mutation in SERPINC1 and a p.Asp357His mutation in LMNA might have cosegregated in this family, associated with AT deficiency and DCM, respectively.<h4>Conclusions</h4>We identified missense mutations in SERPINC1 and LMNA genes to be associated with AT deficiency and DCM, respectively, which might have cosegregated in the family of the patient.

We previously reported that cardiotonic steroids stimulate collagen synthesis by cardiac fibroblasts in a process that involves signaling through the Na-K-ATPase pathway (Elkareh et al. Hypertension 49: 215-224, 2007). In this study, we examined the effect of cardiotonic steroids on dermal fibroblasts collagen synthesis and on wound healing. Increased collagen expression by human dermal fibroblasts was noted in response to the cardiotonic steroid marinobufagenin in a dose- and time-dependent fashion. An eightfold increase in collagen synthesis was noted when cells were exposed to 10 nM marinobufagenin for 24 h (P < 0.01). Similar increases in proline incorporation were seen following treatment with digoxin, ouabain, and marinobufagenin (10 nM x 24 h, all results P < 0.01 vs. control). The coadministration of the Src inhibitor PP2 or N-acetylcysteine completely prevented collagen stimulation by marinobufagenin. Next, we examined the effect of digoxin, ouabain, and marinobufagenin on the rate of wound closure in an in vitro model where human dermal fibroblasts cultures were wounded with a pipette tip and monitored by digital microscopy. Finally, we administered digoxin in an in vivo wound healing model. Olive oil was chosen as the digoxin carrier because of a favorable partition coefficient observed for labeled digoxin with saline. This application significantly accelerated in vivo wound healing in rats wounded with an 8-mm biopsy cut. Increased collagen accumulation was noted 9 days after wounding (both P < 0.01). The data suggest that cardiotonic steroids induce increases in collagen synthesis by dermal fibroblasts, as could potentially be exploited to accelerate wound healing.

Type I collagen, the predominant protein of vertebrates, polymerizes with type III and V collagens and non-collagenous molecules into large cable-like fibrils, yet how the fibril interacts with cells and other binding partners remains poorly understood. To help reveal insights into the collagen structure-function relationship, a data base was assembled including hundreds of type I collagen ligand binding sites and mutations on a two-dimensional model of the fibril. Visual examination of the distribution of functional sites, and statistical analysis of mutation distributions on the fibril suggest it is organized into two domains. The "cell interaction domain" is proposed to regulate dynamic aspects of collagen biology, including integrin-mediated cell interactions and fibril remodeling. The "matrix interaction domain" may assume a structural role, mediating collagen cross-linking, proteoglycan interactions, and tissue mineralization. Molecular modeling was used to superimpose the positions of functional sites and mutations from the two-dimensional fibril map onto a three-dimensional x-ray diffraction structure of the collagen microfibril in situ, indicating the existence of domains in the native fibril. Sequence searches revealed that major fibril domain elements are conserved in type I collagens through evolution and in the type II/XI collagen fibril predominant in cartilage. Moreover, the fibril domain model provides potential insights into the genotype-phenotype relationship for several classes of human connective tissue diseases, mechanisms of integrin clustering by fibrils, the polarity of fibril assembly, heterotypic fibril function, and connective tissue pathology in diabetes and aging.

<h4>Background</h4>alpha-Methylacyl-CoA racemase (AMACR), an immunomarker for prostatic adenocarcinoma, has been shown to be expressed in a variety of other neoplasms. This study aims to evaluate immunohistochemical expression of the AMACR in neoplastic and nonneoplastic liver lesions, and assess its value in the diagnosis of hepatocellular carcinoma (HCC).<h4>Methods</h4>Formalin-fixed paraffin-embedded tissue sections of 51 HCC (14 well, 22 moderately and 15 poorly differentiated), 9 hepatocellular adenoma (HCA), 48 cirrhotic nodules (CN) and 16 normal liver tissues (NLT) were immunostained for AMACR.<h4>Results</h4>Expression of AMACR is significantly enhanced in HCC tissue compared with non-HCC tissue. High expression of AMACR was found in 82% of HCC including 86% of well-differentiated HCC. In contrast, only 11% of HCA, 13% of CN and 6% of NLT showed high expression for AMACR. Clinicopathological evaluation showed a significant correlation between AMACR expression and venous invasion and capsular invasion by HCC.<h4>Conclusion</h4>Our results suggest that AMACR staining may serve as a useful marker for the differential diagnosis of well-differentiated HCC from HCA. Increased AMACR expression and its association with tumor venous invasion suggest that AMACR may play a role in HCC development and progression.

Remarkably, although cardiac disease accounts for the largest proportion of adult mortality and morbidity in the industrialized world, the genetic programs controlling early cardiogenesis are largely incompletely understood. To better understand this process, we set out to identify genes whose expression is enriched within early cardiac fated populations, obtaining the transcriptional signatures of mouse embryonic stem cells (mESCs) at defined intervals during their differentiation along a cardiac path. We compared the RNA profiles of cardiac precursors cells (CPCs) with time-matched non-CPCs and undifferentiated mESCs, using a transgenic mESC line harboring an Nkx2-5 cardiac-specific regulatory sequence driving green fluorescent protein (GFP) to facilitate selection of CPCs. We identify 176 transcripts that are significantly elevated in their abundance within CPCs compared with other assayed populations, predicting that they will likely play a role in cardiogenesis. Of note, approximately 24% (43/176) of the cardiogenic candidate transcripts have known roles in cardiac function or development. Importantly, we evaluated the biological relevance of a significant subset 31/133 (23%) of the remaining candidate genes by in situ hybridization at multiple time points during development (embryonic day, E7.5-9.5) and report that all were expressed in key cardiac structures during cardiogenesis. Furthermore 9/31, of which many were previously uncharacterized, were detected as early as the formation of the cardiac crescent. These data demonstrate the potential power of integrating genomic approaches with mESC differentiation to illuminate developmental processes, and provides a valuable resource that may be mined to further elucidate the genetic programs underlying cardiogenesis.

<h4>Objective</h4>To quantify the relative risk reductions achieved with different regimens to lower blood pressure in younger and older adults.<h4>Design</h4>Meta-analyses and meta-regression analyses used to compare the effects on the primary outcome between two age groups (<65 v > or =65 years). Evidence for an interaction between age and the effects of treatment sought by fitting age as a continuous variable and estimating overall effects across trials.<h4>Main outcome measures</h4><h4>Primary outcome</h4>total major cardiovascular events.<h4>Results</h4>31 trials, with 190 606 participants, were included. The meta-analyses showed no clear difference between age groups in the effects of lowering blood pressure or any difference between the effects of the drug classes on major cardiovascular events (all P> or =0.24). Neither was there any significant interaction between age and treatment when age was fitted as a continuous variable (all P>0.09). The meta-regressions also showed no difference in effects between the two age groups for the outcome of major cardiovascular events (<65 v > or =65; P=0.38).<h4>Conclusions</h4>Reduction of blood pressure produces benefits in younger (<65 years) and older (> or =65 years) adults, with no strong evidence that protection against major vascular events afforded by different drug classes varies substantially with age.

We engineered mutants into residues of SMC2 to dissect the role of ATPase function in the condensin complex. These residues are predicted to be involved in ATP binding or hydrolysis and in the Q-loop, which is thought to act as a mediator of conformational changes induced by substrate binding. All the engineered ATPase mutations resulted in lethality when introduced into SMC2 null cells. We found that ATP binding, but not hydrolysis, is essential to allow stable condensin association with chromosomes. How SMC proteins bind and interact with DNA is still a major question. Cohesin may form a ring structure that topologically encircles DNA. We examined whether condensin behaves in an analogous way to its cohesin counterpart, and we have generated a cleavable form of biologically active condensin with PreScission protease sites engineered into the SMC2 protein. This has allowed us to demonstrate that topological integrity of the SMC2-SMC4 heterodimer is not necessary for the stability of the condensin complex in vitro or for its stable association with mitotic chromosomes. Thus, despite their similar molecular organization, condensin and cohesin exhibit fundamental differences in their structure and function.

Acidic fibroblast growth factor (aFGF; also known as FGF-1) is a potent neurotrophic factor that affects neuronal survival in the injured spinal cord. However, the pathological changes that occur with spinal cord injury (SCI) and the attribution to aFGF of a neuroprotective effect during SCI are still elusive. In this study, we demonstrated that rat SCI, when treated with aFGF, showed significant functional recovery as indicated by the Basso, Beattie, and Bresnahan locomotor rating scale and the combined behavior score (p < 0.01-0.001). Furthermore proteomics and bioinformatics approaches were adapted to investigate changes in the global protein profile of the damaged spinal cord tissue when experimental rats were treated either with or without aFGF at 24 h after injury. We found that 51 protein spots, resolvable by two-dimensional PAGE, had significant differential expression. Using hierarchical clustering analysis, these proteins were categorized into five major expression patterns. Noticeably proteins involved in the process of secondary injury, such as astrocyte activation (glial fibrillary acidic protein), inflammation (S100B), and scar formation (keratan sulfate proteoglycan lumican), which lead to the blocking of injured spinal cord regeneration, were down-regulated in the contusive spinal cord after treatment with aFGF. We propose that aFGF might initiate a series of biological processes to prevent or attenuate secondary injury and that this, in turn, leads to an improvement in functional recovery. Moreover the quantitative expression level of these proteins was verified by quantitative real time PCR. Furthermore we identified various potential neuroprotective protein factors that are induced by aFGF and may be involved in the spinal cord repair processes of SCI rats. Thus, our results could have a remarkable impact on clinical developments in the area of spinal cord injury therapy.

There is an increased interest in the development of bioactive polymeric dental composites and related materials that have potential for mineralized tissue regeneration and preservation. This study explores how the substitution of ethyl alpha-hydroxymethylacryate (EHMA) for 2-hydroxyethyl methacrylate (HEMA) in photo-activated 2,2-bis[p-(2'-hydroxy-3'-methacryloxypropoxy)phenyl]propane (Bis-GMA) and Bis-GMA/tri(ethylene glycol) dimethacrylate (TEGDMA) resins affected selected physicochemical properties of the polymers and their amorphous calcium phosphate (ACP) composites. Rate of polymerization and the degree of conversion (DC) of polymers {EHMA (E), HEMA (H), Bis-GMA/EHMA (BE), Bis-GMA/HEMA (BH), Bis-GMA/TEGDMA/EHMA (BTE) and Bis-GMA/TEGDMA/HEMA (BTH)} were assessed by photo-differential scanning calorimetry and Fourier-Transform Infrared (FTIR) spectroscopy. ACP/BTE and ACP/BTH composites were evaluated for DC, biaxial flexure strength (BFS), water sorption (WS) and mineral ion release. Mid-FTIR and near-IR measurements revealed the following order of decreasing DC: [E, H polymers (97.0%)] > [BE copolymer (89.9%)] > [BH copolymer (86.2%)] > [BTE, BTH copolymers (85.5%)] > [ACP/BTH composite (82.6%)] > [ACP/BTE composite (79.3%)]. Compared to HEMA, EHMA did not adversely affect the BFS of its copolymers and/or ACP composites. Lower WS of BTE copolymers and composites (28% and 14%, respectively, compared to the BTH copolymers and composites) only marginal reduced the ion release from ACP/BTE composites compared to ACP/BTH composites. More hydrophobic ACP composites with acceptable ion-releasing properties were developed by substituting the less hydrophilic EHMA for HEMA.

The aim of this study was to create a gene expression profile to better define the phenotype of human adipose-derived stromal cells (HADSCs) during in vitro chondrogenesis, osteogenesis and adipogenesis. A novel aspect of this work was the analysis of the same subset of genes during HADSC differentiation into all three lineages. Chondrogenic induction resulted in increased mRNA expression of Sox transcription factors, COL2A1,COL10A1, Runx2, and Osterix. This is the first report demonstrating significant upregulation in expression of osteogenesis-related transcription factors Runx2 and Osterix by TGF-beta3 induction of HADSCs during in vitro chondrogenesis. These findings suggest that the commonly-used chondrogenic induction reagents promote differentiation suggestive of hypertrophic chondrocytes and osteoblasts. We conclude that alternative strategies are required to induce efficient articular chondrocyte differentiation in order for HADSCs to be of clinical use in cartilage tissue engineering.

Lasting anxiogenic effects of predator stress in rodents may model aspects of post-traumatic stress disorder (PTSD). There is a link between genetic variation in the serotonin (5-HT) transporter (SERT) and anxiety in humans, prompting the generation of SERT knockout mice. This review brings together studies of SERT knockout male mice, normal female mice, and different 5-HT receptors in predator stress effects on anxiety. These studies provide for a link between vulnerability to the anxiogenic effects of predator stress and abnormalities of 5-HT transmission induced by a life long reduction in 5-HT reuptake in male mice, which creates a vulnerability like that seen in normal female mice. Data reviewed suggest abnormalities in 5-HT transmission contribute to vulnerability to lasting anxiogenic effects of species relevant stressors. To the extent to which predator stress effects model aspects of PTSD, and in the light of relevant human literature, these considerations implicate abnormalities of 5-HT transmission in vulnerability to PTSD per se, and as a potential contributor to enhanced female vulnerability to PTSD.

Huntington's disease is due to the mutation of the IT15 gene coding for Huntingtin protein (Htt). This mutation leads to the expression of an abnormal repeat of polyglutamines in the N-terminal region of Htt. The pathophysiology of Huntington's disease remains to be elucidated. Various mechanisms have been proposed to account for neuronal dysfunction and death, and include both a gain of toxic function of the mutated Htt and a loss of function of the wild type Htt. Among these mechanisms, transcriptional dysregulation, mitochondrial dysfunction, increased excitotoxicity, as well as alteration of neuritic transport and synaptic transmission have been proposed. Interestingly, there is a prominent vulnerability of the striatal neurons, despite the ubiquitous expression of Htt. This may be due to the particular morphological and functional characteristics of these neurons, or to their location within the cerebral networks and the inputs they receive. As multiple mechanisms are involved in neuronal death, the use of drugs association should be considered in future neuroprotective therapeutic trials.

The tumor suppressor p53 is a sequence-specific transcription factor, which regulates the expression of target genes involved in different stress responses. To understand p53's essential transcriptional functions, unbiased analysis of its DNA-binding repertoire is pivotal. In a genome-wide tiling ChIP-on-chip approach, we have identified and characterized 1546 binding sites of p53 upon Actinomycin D treatment. Among those binding sites were known as well as novel p53 target sites, which included regulatory regions of potentially novel transcripts. Using this collection of genome-wide binding sites, a new high-confidence algorithm was developed, p53scan, to identify the p53 consensus-binding motif. Strikingly, this motif was present in the majority of all bound sequences with 83% of all binding sites containing the motif. In the surrounding sequences of the binding sites, several motifs for potential regulatory cobinders were identified. Finally, we show that the majority of the genome-wide p53 target sites can also be bound by overexpressed p63 and p73 in vivo, suggesting that they can possibly play an important role at p53 binding sites. This emphasizes the possible interplay of p53 and its family members in the context of target gene binding. Our study greatly expands the known, experimentally validated p53 binding site repertoire and serves as a valuable knowledgebase for future research.

<h4>Objectives</h4>Neonatal hemochromatosis is the result of severe fetal liver injury that seems to result from maternal-fetal alloimmunity. Women who have had an infant affected with neonatal hemochromatosis are at high risk in subsequent pregnancies for having another affected infant. This study was designed to determine whether therapy directed at limiting the severity of gestational alloimmunity can reduce the occurrence of severe neonatal hemochromatosis in infants of women at risk. A secondary objective was to use a prospectively collected data set to examine questions of vital interest about neonatal hemochromatosis.<h4>Methods</h4>Women with a history of pregnancy ending in documented neonatal hemochromatosis were treated with intravenous immunoglobulin at 1 g/kg of body weight weekly from week 18 until the end of gestation. Extensive data were prospectively collected regarding the gestational histories of the subjects. The outcomes of treated pregnancies were compared with those of previous affected pregnancies, which were used as historical controls.<h4>Results</h4>Forty-eight women were enrolled to be treated during 53 pregnancies. The gestational histories of these women demonstrated the high risk of occurrence of neonatal hemochromatosis: 92% of pregnancies at risk resulted in intrauterine fetal demise, neonatal death, or liver failure necessitating transplant. In contrast, with gestational therapy, the 53 at-risk gestations resulted in 3 failures and 52 infants who survived intact with medical therapy alone. When compared on a per-woman or per-infant basis, the outcome of gestation at risk for neonatal hemochromatosis was improved by gestational therapy.<h4>Conclusions</h4>Neonatal hemochromatosis seems to be the result of a gestational alloimmune disease, and occurrence of severe neonatal hemochromatosis in at-risk pregnancies can be significantly reduced by treatment with high-dose intravenous immunoglobulin during gestation.

<h4>Background</h4>Loss of control of mucosal crypt cell proliferation resulting in a hyperproliferative field change occurs early in the adenoma-carcinoma sequence. Ki-67, the current gold-standard marker of cellular proliferation, is a cell cycle protein that may lack sensitivity in demonstrating altered mucosal crypt cell dynamics. Minichromosome maintenance protein 2 (MCM2) has a specific role in DNA replication and has been proposed as a new marker for screening for colorectal cancer.<h4>Aim</h4>To compare the expression of Ki-67 with that of MCM2 in colorectal mucosa associated with colorectal cancer.<h4>Methods</h4>Ki-67 and MCM2 immunostaining was performed on serial sections taken from formalin-fixed, paraffin-embedded specimens. Labelling indices were calculated by counting the proportion of positively stained nuclei in representative areas of adenocarcinoma, and in equivalent superficial, middle and basal crypt compartments of mucosa sampled 1 cm from tumour (Ca1) and 10 cm from tumour (Ca10).<h4>Results</h4>Specimens were obtained from 43 patients (27 adenocarcinoma, 16 no-cancer controls). Most nuclei in specimens of adenocarcinoma stained positively for MCM2 and Ki-67. In Ca1 and Ca10 samples, significantly greater staining of MCM2 than Ki-67 was seen in all crypt compartments. Receiver operator characteristic curve analysis suggested that proliferation changes (assessed by either MCM2 or Ki-67 staining) in Ca10, but not in Ca1, mucosa significantly predicted origin from a carcinoma-associated colon.<h4>Conclusions</h4>MCM2 was more sensitive than Ki-67 in identifying colorectal mucosal proliferation. Increased proliferation (assessed by either MCM2 or Ki-67 staining) in mucosa at 10 cm, but not at 1 cm, from carcinoma significantly predicted origin from a carcinoma-associated colon.

<h4>Aim</h4>To investigate polymorphisms in the serotonin transporter (5-HTT) gene in cases of sudden infant death syndrome (SIDS) and controls, and further to elucidate a possible relationship between 5-HTT genotypes and external risk factors for SIDS.<h4>Method</h4>The subjects investigated consist of 163 SIDS cases and 243 controls. Polymorphisms in both the promoter and intron 2 of the 5-HTT gene were investigated, and the genotypes were determined using polymerase chain reaction (PCR) and gel electrophoresis.<h4>Results</h4>In the promoter, there was a tendency for the L allele and L/L genotype to be found more often in the SIDS cases than in the controls (p=0.05 and p=0.07, respectively). Regarding the intron 2 polymorphism, there were no differences between the groups, and the SIDS cases were not found to have a higher frequency of either the L/L-12/12 genotype or the L-12 haplotype than the controls. When investigating possible correlations between genotype and risk factors for SIDS, there was a tendency towards different distribution of the promoter genotypes in cases found dead prone compared to cases found dead in other sleeping positions (p=0.06).<h4>Conclusion</h4>Polymorphisms in the promoter of the 5-HTT gene may be of importance with regard to SIDS.

Netrins, a family of secreted molecules, have critical functions in axon guidance and cell migration during neuronal development. In addition to its role as a chemotropic molecule, netrin-1 also acts as a survival factor. Both UNC5 (that is, UNC5A, UNC5B, UNC5C or UNC5D) and DCC are transmembrane receptors for netrin-1 (Refs 8, 9). In the absence of netrin-1, DCC and UNC5 act as dependence receptors and trigger apoptosis. However, how netrin-1 suppresses the apoptotic activity of the receptors remains elusive. Here we show that netrin-1 induces interaction of UNC5B with the brain-specific GTPase PIKE-L. This interaction triggers the activation of PtdIns-3-OH kinase signalling, prevents UNC5B's pro-apoptotic activity and enhances neuronal survival. Moreover, this process relies strongly on Fyn because PIKE-L is tyrosine phosphorylated in response to netrin-1, and the netrin-1-mediated interaction of UNC5B with PIKE-L is inhibited in Fyn-null mice. Thus, PIKE-L acts as a downstream survival effector for netrin-1 through UNC5B in the nervous system.

Mature striatal medium size spiny neurons express the dopamine and cAMP-regulated phosphoprotein, 32 kDa (DARPP-32), but little is known about the mechanisms regulating its levels, or the specification of fully differentiated neuronal subtypes. Cell extrinsic molecules that increase DARPP-32 mRNA and/or protein levels include retinoic acid (RA), brain-derived neurotrophic factor, and estrogen (E(2)). We now demonstrate that RA regulates DARPP-32 mRNA and protein in primary striatal neuronal cultures. Furthermore, DARPP-32 induction by RA in vitro requires phosphatidylinositide 3-kinase, but is independent of tropomyosin-related kinase B, cyclin-dependent kinase 5, and protein kinase B. Using pharmacologic inhibitors of various isoforms of protein kinase C (PKC), we also demonstrate that DARPP-32 induction by RA in vitro is dependent on PKC zeta (PKCzeta). Thus, the signal transduction pathways mediated by RA are very different than those mediating DARPP-32 induction by brain-derived neurotrophic factor. These data support the presence of multiple signal transduction pathways mediating expression of DARPP-32 in vitro, including a novel, important pathway via which phosphatidylinositide 3-kinase regulates the contribution of PKCzeta.

Conditions causing high iron levels, such as hemochromatosis, are proposed risk factors for esophageal adenocarcinoma. Although this hypothesis is supported by animal models, no human data currently exist. We conducted a case-control study of persons with a new Barrett's esophagus diagnosis (cases), persons with gastroesophageal reflux disease (GERD) (without Barrett's esophagus), and population controls. Subjects completed detailed examinations and assays for hemochromatosis mutations and serum iron stores. We evaluated 317 cases, 306 GERD patients, and 308 population controls. There was no significant association between Barrett's esophagus and any hemochromatosis gene defect (odds ratio [OR] = 1.32, 95% confidence interval [CI]: 0.95-1.84), a moderate or severe mutation (OR = 1.54, 95% CI: 0.94-2.52), or a severe mutation (C282Y homozygote or C282Y/H63D heterozygote; OR = 0.77, 95% CI: 0.24-2.48) compared with the population controls. As expected, gene defects were associated with increased iron stores. We can conclude from our findings that Barrett's esophagus was not associated with hemochromatosis gene defects, although we cannot exclude small effects.

The high mobility group (HMG) proteins, including HMGA, HMGB and HMGN, are abundant and ubiquitous nuclear proteins that bind to DNA, nucleosome and other multi-protein complexes in a dynamic and reversible fashion to regulate DNA processing in the context of chromatin. All HMG proteins, like histone proteins, are subjected to extensive post-translational modifications (PTMs), such as lysine acetylation, arginine/lysine methylation and serine/threonine phosphorylation, to modulate their interactions with DNA and other proteins. There is a growing appreciation for the complex relationship between the PTMs of HMG proteins and their diverse biological activities. Here, we reviewed the identified covalent modifications of HMG proteins, and highlighted how these PTMs affect the functions of HMG proteins in a variety of cellular processes.

The directional migration of cells within multicellular organisms is governed by gradients of both chemical attractants and repellents in diverse processes, including leukocyte trafficking and neuronal pathfinding in vivo. These complex extracellular environments direct the orchestrated bidirectional trafficking of leukocytes between the vasculature and tissues. Substantial progress has been made in dissecting the molecular mechanisms involved in orchestrating the directed movement of leukocytes into host tissues; however, less is known about the reverse migration of leukocytes from the tissues to the vasculature. In this article, we discuss the functional interplay between chemoattraction and chemorepulsion in the bidirectional movement of cells in complex in vivo environments, and we describe how these mechanisms influence both normal physiology and human disease.

The phosphate, uracil, and ribose moieties of uracil nucleotides were varied structurally for evaluation of agonist activity at the human P2Y(2), P2Y(4), and P2Y(6) receptors. The 2-thio modification, found previously to enhance P2Y(2) receptor potency, could be combined with other favorable modifications to produce novel molecules that exhibit high potencies and receptor selectivities. Phosphonomethylene bridges introduced for stability in analogues of UDP, UTP, and uracil dinucleotides markedly reduced potency. Truncation of dinucleotide agonists of the P2Y(2) receptor, in the form of Up(4)-sugars, indicated that a terminal uracil ring is not essential for moderate potency at this receptor and that specific SAR patterns are observed at this distal end of the molecule. Key compounds reported in this study include 9, alpha,beta-methylene-UDP, a P2Y(6) receptor agonist; 30, Up(4)-phenyl ester and 34, Up(4)-[1]glucose, selective P2Y(2) receptor agonists; dihalomethylene phosphonate analogues 16 and 41, selective P2Y(2) receptor agonists; 43, the 2-thio analogue of INS37217 (P(1)-(uridine-5')-P(4)-(2'-deoxycytidine-5')tetraphosphate), a potent and selective P2Y(2) receptor agonist.

As an alternative to cell specific cancer targeting strategies (which are often afflicted with the heterogeneity of cancer cells as with most biological systems), a novel polymeric micelle constitute of two block copolymers of poly(L-lactic acid)-b-poly(ethylene glycol)-b-poly(L-histidine)-TAT (transactivator of transcription) and poly(L-histidine)-b-poly(ethylene glycol) was developed. The micelle formed via the dialysis method was approximately 95 nm in diameter and contained 15 wt.% of doxorubicin (DOX) by weight. The micelle surface hides TAT during circulation, which has the strong capability to translocate the micelle into cells, and exposes TAT at a slightly acidic tumor extracellular pH to facilitate the internalization process. The micelle core was engineered for disintegration in early endosomal pH of tumor cells, quickly releasing DOX. The ionization process of the block copolymers and ionized polymers assisted in disrupting the endosomal membrane. This processes permitted high DOX concentrations in the cytosol and its target site of the nucleus, thus increasing DOX potency in various wild and multidrug resistant (MDR) cell lines (3.8-8.8 times lower IC50 than free DOX, depending on cell line). When tested with the xenografted tumors of human ovarian tumor drug-resistant A2780/AD, human breast tumor drug-sensitive MCF-7, human lung tumor A549 and human epidermoid tumor KB in a nude mice model, all tumors significantly regressed in size by three bolus injections at a dose of DOX 10 mg equivalent/kg body per injection of DOX-loaded micelle at three day interval, while minimum weight loss was observed. This approach may replace the need for cell-specific antibodies or targeting ligands, thereby providing a general strategy for solid tumor targeting.

The phenylethylene diamines are a class of sigma receptor ligands with excellent selectivity over other biological systems and with anti-cocaine actions that involve antagonism of sigma1 receptors. In order to increase the potency of the aromatic methoxyl substituted analogues, trifluoromethoxyl groups were introduced to prevent metabolic demethylation. The para-substituted trifluoromethoxyl substituted analogues were shown to have increased sigma receptor affinity and represent the most potent anti-cocaine phenylethylene diamines yet described.

Axonal receptors for class 3 semaphorins (Sema3s) are heterocomplexes of neuropilins (Nrps) and Plexin-As signalling coreceptors. In the developing cerebral cortex, the Ig superfamily cell adhesion molecule L1 associates with Nrp1. Intriguingly, the genetic removal of L1 blocks axon responses of cortical neurons to Sema3A in vitro despite the expression of Plexin-As in the cortex, suggesting either that L1 substitutes for Plexin-As or that L1 and Plexin-A are both required and mediate distinct roles. We report that association of Nrp1 with L1 but not Plexin-As mediates the recruitment and activation of a Sema3A-induced focal adhesion kinase-mitogen-activated protein kinase cascade. This signalling downstream of L1 is needed for the disassembly of adherent points formed in growth cones and subsequently their collapse response to Sema3A. Plexin-As and L1 are coexpressed and present in common complexes in cortical neurons and both dominant-negative forms of Plexin-A and L1 impair their response to Sema3A. Consistently, Nrp1-expressing cortical projections are defective in mice lacking Plexin-A3, Plexin-A4 or L1. This reveals that specific signalling activities downstream of L1 and Plexin-As cooperate for mediating the axon guidance effects of Sema3A.

Atherosclerotic lesions contain T lymphocytes, which orchestrate adaptive immunity and regulate many innate immune pathways. This study examined the influence of Th1 and Th2 helper cell subsets on atherogenesis in two ApoE(-/-) mouse strains, C57BL/6 and BALB/c, which display opposite T-cell subset polarizations. ApoE(-/-) BL/6 mice showed predominant Th1-like immune responses on polyclonal stimulation of splenic CD4(+) T cells and had IgG2a antibodies to oxidized low-density lipoprotein (a disease-related antigen) whereas ApoE(-/-) BALB/c mice displayed predominant Th2 responses by CD4(+) T cells and an IgG1 isotype response toward oxidized low-density lipoprotein. ApoE(-/-) BL/6 and BALB/c mice consumed a high-cholesterol diet for 10, 16, and 24 weeks with equivalent cholesterolemic responses. The Th1-slanted BL/6 mice developed significantly more atherosclerosis in the aortic root and abdominal aorta at all time points compared with BALB/c mice, supporting a proatherogenic role for Th1 response. Progression of atherosclerosis was associated with increased levels of interleukin (IL)-6 in mouse serum and CD4(+) T-cell culture supernatants and increased levels of the acute-phase protein, serum amyloid A (SAA). Both IL-6 and SAA levels rose significantly in ApoE(-/-) BL/6 mice compared with BALB/c mice. The circulating cytokine milieu (IL-6) and acute phase reactants such as SAA may reflect alterations in the Th1/Th2 balance. The results presented here illustrate how genetically determined modifiers of both immune and inflammatory responses can modulate atherogenesis independently of lipid levels.

Increasing evidence exists that iron overload, a common finding in chronic hepatitis C virus (HCV) infection, plays an important role in the pathophysiology of this disease. The mechanisms by which iron excess induces liver damage along with the benefit of iron depletion via phlebotomy on biochemical and histological outcomes in patients with chronic HCV infection have been discussed in this review. Finally, we focus on the effect of iron reduction on the rate of response to interferon antiviral therapy.

<h4>Summary</h4>In recent years, transcriptomics and proteomics have provided us with a great deal of information about the expression profiles of various cell types and how these change under different conditions. Stem cell research is one area where this has had a major impact by providing an insight into events at the molecular level that control stem cell growth and differentiation. This includes mesenchymal stem cell (MSC) biology where knowledge about the mechanisms governing differentiation is vital for the development of future therapeutic strategies. Although there is still much to learn, we are starting to build up a picture of the main events in these differentiation processes. This review will discuss control of MSC differentiation at the transcriptional level. Not all the factors which have been shown to play a role in lineage-specific mesenchymal differentiation can be covered here. Instead, we will focus specifically on the key factors that contribute to the regulation of osteogenesis, adipogenesis, and chondrogenesis.

Netrin-1 is a critical molecule for axonal pathfinding during embryo development, and because of its structural homology to the endothelial mitogens, it may share its effects on vascular network formation. Using an adeno-associated viral netrin-1 vector (AAV-NT-1) gene transfer, we demonstrated that netrin-1 was able to stimulate the proliferation and migration of human cerebral endothelial cells (HCECs) and human aortic smooth muscle cells (HASMCs) compared with the control (P<0.05), and could also promote HCEC tube formation on matrigel (P<0.05) in vitro. Moreover, netrin-1 hyperstimulation could promote focal neovascularization (P<0.05) in the adult brain in vivo. Unlike VEGF-induced microvessel increase, netrin-1-induced newly formed vessels showed an artery-like phenotype, with an intact endothelial cell monolayer surrounded by multiple cell layers, including smooth muscle cells and an astrocyte-connected outer layer. Our findings suggest that netrin-1 plays an important role in promoting blood vessel formation in the adult rodent central nervous system, and could have broad implication in cerebrovascular development and remodeling.

<h4>Background</h4>The signaling pathways that may modulate the pathogenesis of diseases induced by expanded polyglutamine proteins are not well understood.<h4>Methodologies/principal findings</h4>Herein we demonstrate that expanded polyglutamine protein cytotoxicity is mediated primarily through activation of p38MAPK and that the atypical PKC iota (PKCiota) enzyme antagonizes polyglutamine-induced cell death through induction of the ERK signaling pathway. We show that pharmacological blockade of p38MAPK rescues cells from polyglutamine-induced cell death whereas inhibition of ERK recapitulates the sensitivity observed in cells depleted of PKCiota by RNA interference. We provide evidence that two unrelated proteins with expanded polyglutamine repeats induce p38MAPK in cultured cells, and demonstrate induction of p38MAPK in an in vivo model of neurodegeneration (spinocerebellar ataxia 1, or SCA-1).<h4>Conclusions/significance</h4>Taken together, our data implicate activated p38MAPK in disease progression and suggest that its inhibition may represent a rational strategy for therapeutic intervention in the polyglutamine disorders.

By producing ATP and regulating intracellular calcium levels, mitochondria are vital for the function and survival of neurons. Oxidative stress and damage to mitochondrial DNA during the aging process can impair mitochondrial energy metabolism and ion homeostasis in neurons, thereby rendering them vulnerable to degeneration. Mitochondrial abnormalities have been documented in all of the major neurodegenerative disorders-Alzheimer's, Parkinson's and Huntington's diseases, and amyotrophic lateral sclerosis. Mitochondrial DNA damage and dysfunction may be downstream of primary disease processes such as accumulation of pathogenic proteins. However, recent experimental evidence demonstrates that mitochondrial DNA damage responses play important roles in aging and in the pathogenesis of neurodegenerative diseases. Therapeutic interventions that target mitochondrial regulatory systems have been shown effective in cell culture and animal models, but their efficacy in humans remains to be established.

Depression and fatigue are frequent side effects of interferon-alpha (IFN-alpha) treatment, and there is compelling evidence that the inflammatory response system (including interleukin-6, IL-6) and the serotonergic system is important in the pathophysiology of such symptoms. Functional polymorphisms in the promoter region of the IL-6 gene (rs1800795) and serotonin transporter gene (5-HTTLPR) have been identified as regulating these systems. The present study aimed to determine if these polymorphisms were associated with the development of depression and fatigue during IFN-alpha and ribavirin treatment. Ninety-eight Caucasian patients receiving pegylated IFN-alpha and ribavirin treatment for chronic hepatitis C virus at King's College Hospital, London, and Emory University Hospital, Atlanta, participated in this prospective cohort study. Symptoms of depression and fatigue were measured before treatment and at weeks 4, 8, 12 and 24 during treatment. The 'low IL-6' synthesizing genotype (CC) was associated with significantly fewer symptoms of depression (effect size = 0.7 at week 24; F = 9.4, d.f. = 436, P = 0.002). The 'high transcription' serotonin transporter (5-HTT) genotype (LL) was also associated with significantly fewer symptoms of depression, but with a much smaller effect (effect size = 0.2 at week 24; F = 4.5, d.f. = 436, P = 0.03). Neither polymorphisms were associated with symptoms of fatigue (IL-6: F = 1.2, d.f. = 430, P = 0.2; 5-HTT: F = 0.5, d.f. = 430, P = 0.5). The smaller effects of the 5-HTT polymorphism on depression may be explained by an interaction between the genes (F = 5.0, d.f. = 434, P = 0.02): the 'protective' effect of the 5-HTTLPR polymorphism was evident only in the presence of the 'low IL-6' genotype (F = 5.4, d.f. = 64, P = 0.02), not in the presence of the 'high IL-6' genotype (F = 2.2, d.f. = 369, P = 0.1). The association between the IL-6 polymorphism and reduced risk of depressive symptoms confirms the role of the inflammatory response system in the pathophysiology of IFN-alpha-induced depression; in contrast, the effect of the 5-HTT gene was small and perhaps dependent on the status of the inflammatory response.

An intricate interplay between DNA methylation and polycomb-mediated gene silencing has been highlighted recently. Here we provided evidence that Nervous System Polycomb 1 (NSPc1), a BMI1 homologous polycomb protein, plays important roles in promoting H2A ubiquitination and cooperates with DNA methylation in HOX gene silencing. We showed that NSPc1 stimulates H2A ubiquitination in vivo and in vitro through direct interaction with both RING2 and H2A. RT-PCR analysis revealed that loss of NSPc1, EZH2 or DNA methyltransferase 1 (Dnmt1), or inhibition of DNA methylation in HeLa cells de-represses the expression of HOXA7. Chromatin immunoprecipitation (ChIP) assays demonstrated that NSPc1, EZH2 and Dnmt1 bind to the promoter of HOXA7, which is frequently hypermethylated in tumors. Knockdown of NSPc1 results in significant reduction of H2A ubiquitination and DNA demethylation as well as Dnmt1 dissociation in the HOXA7 promoter. Meanwhile Dnmt1 deficiency affects NSPc1 recruitment and H2A ubiquitination, whereas on both cases EZH2-mediated H3K27 trimethylation remains unaffected. When EZH2 was depleted, however, NSPc1 and Dnmt1 enrichment was abolished concomitant with local reduction of H3K27 trimethylation, H2A ubiquitination and DNA methylation. Taken together, our findings indicated that NSPc1-mediated H2A ubiquitination and DNA methylation, both being directed by EZH2, are interdependent in long-term target gene silencing within cancer cells.

This work represents a maiden effort to systematically screen the transcriptome of the healing wound-edge tissue temporally using high-density GeneChips. Changes during the acute inflammatory phase of murine excisional wounds were characterized histologically. Sets of genes that significantly changed in expression during healing could be segregated into the following five sets: up-early (6-24 h; cytokine-cytokine receptor interaction pathway), up-intermediary (12-96 h; leukocyte-endothelial interaction pathway), up-late (48-96 h; cell-cycle pathway), down-early (6-12 h; purine metabolism) and down-intermediary (12-96 h; oxidative phosphorylation pathway). Results from microarray and real-time PCR analyses were consistent. Results listing all genes that were significantly changed at any specific time point were further mined for cell-type (neutrophils, macrophages, endothelial, fibroblasts, and pluripotent stem cells) specificity. Candidate genes were also clustered on the basis of their functional annotation, linking them to inflammation, angiogenesis, reactive oxygen species (ROS), or extracellular matrix (ECM) categories. Rapid induction of genes encoding NADPH oxidase subunits and downregulation of catalase in response to wounding is consistent with the fact that low levels of endogenous H2O2 is required for wound healing. Angiogenic genes, previously not connected to cutaneous wound healing, that were induced in the healing wound-edge included adiponectin, epiregulin, angiomotin, Nogo, and VEGF-B. This study provides a digested database that may serve as a valuable reference tool to develop novel hypotheses aiming to elucidate the biology of cutaneous wound healing comprehensively.

We present here a genome-wide map of abnormalities found in diagnostic samples from 45 adults and adolescents with acute lymphoblastic leukemia (ALL). A 500K SNP array analysis uncovered frequent genetic abnormalities, with cryptic deletions constituting half of the detected changes, implying that microdeletions are a characteristic feature of this malignancy. Importantly, the pattern of deletions resembled that recently reported in pediatric ALL, suggesting that adult, adolescent, and childhood cases may be more similar on the genetic level than previously thought. Thus, 70% of the cases displayed deletion of one or more of the CDKN2A, PAX5, IKZF1, ETV6, RB1, and EBF1 genes. Furthermore, several genes not previously implicated in the pathogenesis of ALL were identified as possible recurrent targets of deletion. In total, the SNP array analysis identified 367 genetic abnormalities not corresponding to known copy number polymorphisms, with all but two cases (96%) displaying at least one cryptic change. The resolution level of this SNP array study is the highest used to date to investigate a malignant hematologic disorder. Our findings provide insights into the leukemogenic process and may be clinically important in adult and adolescent ALL. Most importantly, we report that microdeletions of key genes appear to be a common, characteristic feature of ALL that is shared among different clinical, morphological, and cytogenetic subgroups.

In Drosophila, dosage compensation augments X chromosome-linked transcription in males relative to females. This process is achieved by the Dosage Compensation Complex (DCC), which associates specifically with the male X chromosome. We previously found that the morphology of this chromosome is sensitive to the amounts of the heterochromatin-associated protein SU(VAR)3-7. In this study, we examine the impact of change in levels of SU(VAR)3-7 on dosage compensation. We first demonstrate that the DCC makes the X chromosome a preferential target for heterochromatic markers. In addition, reduced or increased amounts of SU(VAR)3-7 result in redistribution of the DCC proteins MSL1 and MSL2, and of Histone 4 acetylation of lysine 16, indicating that a wild-type dose of SU(VAR)3-7 is required for X-restricted DCC targeting. SU(VAR)3-7 is also involved in the dosage compensated expression of the X-linked white gene. Finally, we show that absence of maternally provided SU(VAR)3-7 renders dosage compensation toxic in males, and that global amounts of heterochromatin affect viability of ectopic MSL2-expressing females. Taken together, these results bring to light a link between heterochromatin and dosage compensation.

Homeobox (HOX) A10 is essential for fertility as demonstrated in transgenic mice, specifically affecting implantation and decidualization. Its role in human decidualization, however, remains unknown. In this study, we used gene silencing followed by microarray analysis to decipher the role of HOXA10 during decidualization of human endometrial stromal cells (HESCs). HOXA10 was knocked down using siRNA oligonucleotide transfection and cells were treated with estradiol, medroxyprogesterone acetate and dibutyryl cAMP (H + cAMP) to induce decidualization. Genes significantly regulated were identified using the Affymetrix microarray chip. With this method, 2361 transcripts were significantly altered by 1.5-fold or higher (P < 0.05) with H + cAMP treatment only. Of these genes, 258 were significantly up-regulated by HOXA10 knockdown and 236 transcripts were significantly down-regulated by more than 1.5-fold, totaling 494 genes that were regulated by HOXA10 during decidualization. Data analysis using the Ingenuity System revealed that many of the genes regulated by HOXA10 knockdown during H + cAMP treatment were associated with cell cycle. Real-time PCR was used to confirm that HOXA10 knockdown decreased expression of the cell cycle genes CDC2 and CCNB2. In addition, a higher percentage of cells were arrested in the G2/M phase. Next, we observed that cell proliferation as measured by BrdU incorporation was decreased upon HOXA10 knockdown and H + cAMP treatment. Apoptosis, on the other hand, as measured by Annexin V staining was not influenced by siHOXA10 in decidualizing cells. Together, these data demonstrate that during decidualization of HESC, HOXA10 is actively involved in promoting cell proliferation through the regulation of hundreds of genes.

NASP has been described as a histone H1 chaperone in mammals. However, the molecular mechanisms involved have not yet been characterized. Here, we show that this protein is not only present in mammals but is widely distributed throughout eukaryotes both in its somatic and testicular forms. The secondary structure of the human somatic version consists mainly of clusters of alpha-helices and exists as a homodimer in solution. The protein binds nonspecifically to core histone H2A-H2B dimers and H3-H4 tetramers but only forms specific complexes with histone H1. The formation of the NASP-H1 complexes is mediated by the N- and C-terminal domains of histone H1 and does not involve the winged helix domain that is characteristic of linker histones. In vitro chromatin reconstitution experiments show that this protein facilitates the incorporation of linker histones onto nucleosome arrays and hence is a bona fide linker histone chaperone.

Peroxiredoxins (PRDXs) are a superfamily of thiol-dependent peroxidases found in all phyla. PRDXs are mechanistically divided into three subfamilies, namely typical 2-Cys, atypical 2-Cys, and 1-Cys PRDXs. To reduce peroxides, the N-terminal peroxidatic Cys of PRDXs is first oxidized into sulfenic acid. This intermediate is reduced by forming a disulfide bond either with a resolving Cys of another monomeric entity (typical 2-Cys) or of the same molecule (atypical 2-Cys). In 1-Cys PRDXs, the resolving Cys is missing and the sulfenic acid of the peroxidatic Cys is reduced by a heterologous thiol-containing reductant. In search of a homolog of human 1-Cys PRDX6 in Arenicola marina, an annelid worm living in intertidal sediments, we have cloned and characterized a PRDX exhibiting high sequence homology with its mammalian counterpart. However, A. marina PRDX6 possesses five Cys among which two Cys function as peroxidatic and resolving Cys of typical 2-Cys PRDXs. Thus, A. marina PRDX6 belongs to a transient group exhibiting sequence homologies with mammalian 1-Cys PRDX6 but must be mechanistically classified into typical 2-Cys PRDXs. Moreover, PRDX6 is highly expressed in tissues directly exposed to the external environment, suggesting that this PRDX may be of particular importance for protection against exogenous oxidative attacks.

The epithelium-specific ETS (ESE)-1 transcription factor is induced in chondrocytes by interleukin-1beta (IL-1beta). We reported previously that early activation of EGR-1 by IL-1beta results in suppression of the proximal COL2A1 promoter activity by displacement of Sp1 from GC boxes. Here we report that ESE-1 is a potent transcriptional suppressor of COL2A1 promoter activity in chondrocytes and accounts for the sustained, NF-kappaB-dependent inhibition by IL-1beta. Of the ETS factors tested, this response was specific to ESE-1, since ESE-3, which was also induced by IL-1beta, suppressed COL2A1 promoter activity only weakly. In contrast, overexpression of ETS-1 increased COL2A1 promoter activity and blocked the inhibition by IL-1beta. These responses to ESE-1 and ETS-1 were confirmed using siRNA-ESE1 and siRNA-ETS1. In transient cotransfections, the inhibitory responses to ESE-1 and IL-1beta colocalized in the -577/-132 bp promoter region, ESE-1 bound specifically to tandem ETS sites at -403/-381 bp, and IL-1-induced binding of ESE-1 to the COL2A1 promoter was confirmed in vivo by ChIP. Our results indicate that ESE-1 serves a potent repressor function by interacting with at least two sites in the COL2A1 promoter. However, the endogenous response may depend upon the balance of other ETS factors such as ETS-1, and other IL-1-induced factors, including EGR-1 at any given time. Intracellular ESE-1 staining in chondrocytes in cartilage from patients with osteoarthritis (OA), but not in normal cartilage, further suggests a fundamental role for ESE-1 in cartilage degeneration and suppression of repair.

We have previously shown that a fraction of newly expressed GRP78 is translocated to the cell surface in association with the co-chaperone MTJ-1. Proteinase and methylamine-activated alpha(2)M (alpha(2)M*) bind to cell surface-associated GRP78 activating phosphoinositide-specific phospholipase C coupled to a pertussis toxin-insensitive heterotrimeric G protein, generating IP(3)/calcium signaling. We have now studied the association of pertussis toxin-insensitive Galphaq11, with GRP78/MTJ-1 complexes in the plasma membranes of alpha(2)M*-stimulated macrophages. When GRP78 was immunoprecipitated from plasma membranes of macrophages stimulated with alpha(2)M*, Galphaq11, and MTJ-1 were co-precipitated. Likewise Galphaq11 and GRP78 co-immunoprecipitated with MTJ-1 while GRP78 and MTJ-1 co-immunoprecipitated with Galphaq11. Silencing GRP78 expression with GRP78 dsRNA or MTJ-1 with MTJ-1 dsRNA greatly reduced the levels of Galphaq11 co-precipitated with GRP78 or MTJ-1. In conclusion, we show here that plasma membrane-associated GRP78 is coupled to pertussis toxin-insensitive Galphaq11 and forms a ternary signaling complex with MTJ-1.

We previously reported identifying SOX6 as a glioma antigen by serological screening using a testis cDNA library. Its preferential expression and frequent IgG responses in glioma patients indicate that SOX6 may be a useful target for immunotherapy. To examine whether cytotoxic T-lymphocyte (CTL) responses specific for SOX6 to destroy glioma can be generated in vivo, we treated glioma-bearing mice by vaccination with a plasmid DNA encoding murine full-length SOX6 protein. Following SOX6-DNA vaccination, CTLs specific for SOX6-expressing glioma cells were induced, while normal autologous-cells that had restrictedly expressed SOX6 during embryogenesis were not destroyed. Furthermore, DNA vaccination with SOX6 exerted protective and therapeutic antitumor responses in the glioma-bearing mice. This antitumor activity was abrogated by the depletion of CD4 positive T cells and/or CD8 positive T cells. These results suggest that the SOX6 protein has multiple CTL and helper epitopes to induce antitumor activity and the effectiveness of SOX6-DNA vaccine for the prevention and treatment of glioma.

Gamma-Secretase is a promiscuous protease that cleaves bitopic membrane proteins within the lipid bilayer. Elucidating both the mechanistic basis of gamma-secretase proteolysis and the precise factors regulating substrate identification is important because modulation of this biochemical degradative process can have important consequences in a physiological and pathophysiological context. Here, we briefly review such information for all major classes of intramembranously cleaving proteases (I-CLiPs), with an emphasis on gamma-secretase, an I-CLiP closely linked to the etiology of Alzheimer's disease. A large body of emerging data allows us to survey the substrates of gamma-secretase to ascertain the conformational features that predispose a peptide to cleavage by this enigmatic protease. Because substrate specificity in vivo is closely linked to the relative subcellular compartmentalization of gamma-secretase and its substrates, we also survey the voluminous body of literature concerning the traffic of gamma-secretase and its most prominent substrate, the amyloid precursor protein.

Recent evidence suggests that additional risk loci for RA are present in the major histocompatibility complex (MHC), independent of the class II HLA-DRB1 locus. We have now tested a total of 1,769 SNPs across 7.5Mb of the MHC located from 6p22.2 (26.03 Mb) to 6p21.32 (33.59 Mb) derived from the Illumina 550K Beadchip (Illumina, San Diego, CA, USA). For an initial analysis in the whole dataset (869 RA CCP + cases, 1,193 controls), the strongest association signal was observed in markers near the HLA-DRB1 locus, with additional evidence for association extending out into the Class I HLA region. To avoid confounding that may arise due to linkage disequilibrium with DRB1 alleles, we analyzed a subset of the data by matching cases and controls by DRB1 genotype (both alleles matched 1:1), yielding a set of 372 cases with 372 controls. This analysis revealed the presence of at least two regions of association with RA in the Class I region, independent of DRB1 genotype. SNP alleles found on the conserved A1-B8-DR3 (8.1) haplotype show the strongest evidence of positive association (P ~ 0.00005) clustered in the region around the HLA-C locus. In addition, we identified risk alleles that are not present on the 8.1 haplotype, with maximal association signals (P ~ 0.001-0.0027) located near the ZNF311 locus. This latter association is enriched in DRB1*0404 individuals. Finally, several additional association signals were found in the extreme centromeric portion of the MHC, in regions containing the DOB1, TAP2, DPB1, and COL11A2 genes. These data emphasize that further analysis of the MHC is likely to reveal genetic risk factors for rheumatoid arthritis that are independent of the DRB1 shared epitope alleles.

Pancreatic cancer (PC) cell lines provide a useful starting point for the discovery and functional analysis of genes driving the genesis and progression of this lethal cancer. To increase our understanding of the gene copy number changes in pancreatic carcinomas and to identify key amplification and deletion targets, we applied genome-wide array-based comparative genomic hybridization using in-house array (MCG Cancer Array-800) to 24 PC cell lines. Overall, the analyses revealed high genomic complexity, with several copy number changes detected in each line. Homozygous deletions (log(2)ratio < -2) of eight genes (clones) were seen in 14 of the 24 cell lines, whereas high-level amplifications (log(2)ratio > 2) of 10 genes (clones) were detected in seven lines. Among them, we focused on high-level amplification at 7q22.1, because target genes for this alteration remain unknown. Through precise mapping of the altered region by fluorescence in situ hybridization, determination of the expression status of genes located within those regions, and functional analysis using knockdown of the gene expression or the ectopic overexpression approach in PC cell lines, as well as immunohistochemical analyses of candidates in primary tumors of PC, we successfully identified SMURF1 as having the greatest potential as a 7q21.3-22.1 amplification target. SMURF1 may work as a growth-promoting gene in PC through overexpression and might be a good candidate as a therapeutic target. Our results suggest that array-based comparative genomic hybridization analysis combined with further genetic and functional examinations is a useful approach for identifying novel tumor-associated genes involved in the pathogenesis of this lethal disease.

<h4>Purpose of review</h4>The peptide hormone hepcidin regulates iron metabolism in response to erythropoietic demand, iron stores and inflammation. Major advances have been made in understanding the regulation of hepcidin production, and consequently the availability of iron for erythropoiesis.<h4>Recent findings</h4>It is becoming clear that the bone morphogenetic protein (BMP) pathway plays a major role in setting the baseline hepcidin level and, with the assistance of BMP2/4 and hemochromatosis-related proteins hemojuvelin, HFE and transferrin receptor 2, also regulates hepcidin expression in response to iron. Regulation of hepcidin in anemias has now been linked to increased erythropoietic activity and is likely mediated by factor(s) secreted by erythroid precursors. GDF-15 was identified as a candidate for one of the erythroid factors suppressing hepcidin. Tissue hypoxia may also directly contribute to hepcidin suppression in anemias. Regulation of hepcidin by inflammation may include multiple cytokines and the Toll-like receptors pathways. Although it has not yet been shown that increased hepcidin is indispensible for the development of anemia of inflammation, transgenic overexpression of hepcidin was sufficient to replicate its key features.<h4>Summary</h4>Regulation of hepcidin and iron availability for erythropoiesis has revealed unexpected pathways and much complexity. The renaissance of the study of iron regulation continues to reward researchers with interesting biology and potential therapeutic targets.

<h4>Unlabelled</h4>HFE-related hereditary hemochromatosis results in hepatic iron overload. Hepatocytes acquire transferrin-bound iron via transferrin receptor (Tfr) 1 and Tfr1-independent pathways (possibly Tfr2-mediated). In this study, the role of Hfe in the regulation of hepatic transferrin-bound iron uptake by these pathways was investigated using Hfe knockout mice. Iron and transferrin uptake by hepatocytes from Hfe knockout, non-iron-loaded and iron-loaded wild-type mice were measured after incubation with 50 nM (125)I-Tf-(59)Fe (Tfr1 pathway) and 5 microM (125)I-Tf-(59)Fe (Tfr1-independent or putative Tfr2 pathway). Tfr1 and Tfr2 messenger RNA (mRNA) and protein expression were measured by real-time polymerase chain reaction and western blotting, respectively. Tfr1-mediated iron and transferrin uptake by Hfe knockout hepatocytes were increased by 40% to 70% compared with iron-loaded wild-type hepatocytes with similar iron levels and Tfr1 expression. Iron and transferrin uptake by the Tfr1-independent pathway was approximately 100-fold greater than by the Tfr1 pathway and was not affected by the absence of Hfe. Diferric transferrin increased hepatocyte Tfr2 protein expression, resulting in a small increase in transferrin but not iron uptake by the Tfr1-independent pathway.<h4>Conclusion</h4>Tfr1-mediated iron uptake is regulated by Hfe in hepatocytes. The Tfr1-independent pathway exhibited a much greater capacity for iron uptake than the Tfr1 pathway but it was not regulated by Hfe. Diferric transferrin up-regulated hepatocyte Tfr2 protein expression but not iron uptake, suggesting that Tfr2 may have a limited role in the Tfr1-independent pathway.

<h4>Background</h4>Essential hypertension is a complex disorder that results from the interaction of a number of susceptibility genes and environmental factors. The TNFRSF4 (tumor necrosis factor receptor superfamily, member 4) gene was one of the genes that showed altered renal expression in long-term salt loading in mice. Moreover, association of the TNFRSF4 and TNFSF4 (tumor necrosis factor (ligand) superfamily, member 4) genes with myocardial infarction was recently reported. Since essential hypertension is a well-known risk factor for myocardial infarction, we hypothesized that TNFRSF4 could be a susceptibility gene for essential hypertension.<h4>Methods</h4>We performed a case-control study of TNFRSF4 in two independent population.<h4>Results</h4>Extensive investigation of single nucleotide polymorphisms of the entire gene suggested that it resided in one linkage disequilibrium block, and four single nucleotide polymorphisms in the 5' flanking region sufficiently represented major haplotypes. In the combined population, the frequency of the most frequent haplotype, C-C-A-A, was significantly lower (P = 8.07 x 10(-5)) and that of the second most frequent haplotype, C-T-G-A, was significantly higher (P = 6.07 x 10(-4)) in hypertensive subjects than in control subjects. This difference was observed only in female patients. The C-T-G-A haplotype showed a lower promoter activity than other haplotypes, suggesting a relationship with disease susceptibility.<h4>Conclusion</h4>Our results suggest that TNFRSF4 is a female-specific susceptible gene for essential hypertension.

<h4>Background</h4>HFE-related genetic haemochromatosis (GH) is the commonest inherited genetic disorder in Caucasian populations with approximately one in 180 of individuals in the west of Scotland homozygous for the common C282Y mutation. The clinical diagnosis of GH, however, remains relatively uncommon - suggesting either under diagnosis or low clinical penetrance.<h4>Aim</h4>We aimed to assess the biochemical and clinical penetrance of GH in first-degree relatives of patients with known GH, who subsequently themselves screened positive for the common GH mutations.<h4>Methods</h4>Individuals were identified from two large teaching hospitals in North Glasgow from July 1997 to July 2005 diagnosed with GH after predictive genetic testing after a relative was found to have GH. Details of patient history, biochemistry and known comorbidity at diagnosis and results of related further investigations were collected.<h4>Results</h4>Sixty-three individuals were identified, 31 (49%) of whom were males. Fifty-five individuals (87%) were C282Y homozygous and the remaining eight were compound heterozygotes for C282Y and H63D. All 31 male patients were found to have evidence of iron overload as opposed to 63% of females. Elevated liver enzyme levels were encountered in 15 patients (24%). All except one had evidence of iron overload. Four individuals underwent a liver biopsy, two of whom had hepatic fibrosis. Four patients were found to be diabetic. A full clinical history was obtained from 54 of 63 individuals, 38 (70%) of whom were entirely asymptomatic. Thirteen individuals complained of joint pains and a further nine complained of fatigue.<h4>Conclusion</h4>This study suggests that although biochemical penetrance of GH is high, the clinical penetrance is low.

RNA-binding proteins (RBP) influence RNA editing, localization, stability and translation and may contribute to oocyte developmental competence by regulating the stability and turnover of oogenetic mRNAs. The expression of Staufen 1 and 2 and ELAVL1, ELAVL2 RNA-binding proteins during cow early development was characterized. Cumulus-oocyte complexes were collected from slaughterhouse ovaries, matured, inseminated and subjected to embryo culture in vitro. Oocyte or preimplantation embryo pools were processed for RT-PCR and whole-mount immunofluorescence analysis of mRNA expression and protein distribution. STAU1 and STAU2 and ELAVL1 mRNAs and proteins were detected throughout cow preimplantation development from the germinal vesicle (GV) oocyte to the blastocyst stage. ELAVL2 mRNAs were detectable from the GV to the morula stage, whereas ELAVL2 protein was in all stages examined and localized to both cytoplasm and nuclei. The findings provide a foundation for investigating the role of RBPs during mammalian oocyte maturation and early embryogenesis.

<h4>Purpose of review</h4>Studies are reviewed from the past year concerning the histopathology of liver and biliary diseases and their pathogenesis.<h4>Recent findings</h4>Several cases of acute hepatitis E showed portal and periportal hepatitis, with polarization of neutrophils to the interface region and lymphocytes more centrally in the portal tracts. Transfection of hepatitis C virus into cultured fetal hepatocytes resulted in sustained growth of 50-90 nm diameter viral particles. The ductular reaction in nonalcoholic steatohepatitis appears to evolve with fibrosis in response to hepatocyte replicative senescence. Hepatocellular release of hepcidin is critical for iron homeostasis in a manner analogous to pancreatic insulin for glucose homeostasis; this 'endocrine' focus was elaborated in an overview of hemochromatosis. Specific microscopic features of liver-cell adenomas combined with genetic analysis for hepatocyte nuclear factor 1alpha and beta-catenin mutations allows differentiation into four variants. Steroid-sensitive biliary strictures resembling primary sclerosing cholangitis but with increased serum immunoglobulin G4 and infiltrating immunoglobulin G4-positive plasma cells ('immunoglobulin associated cholangitis') are part of a spectrum of disorders including autoimmune pancreatitis and inflammatory pseudotumor.<h4>Summary</h4>Pathologic features of viral hepatitis C and E, immunohistochemistry for the ductular reaction and malignant liver tumors and several systemic disorders are among recent important pathology studies.

Imaging genetics has been a highly effective and increasingly applied strategy for identifying the impact of genetic polymorphisms on individual differences in neural circuitry supporting complex behaviors. The application of imaging genetics towards further elucidating neural circuitry associated with the pathophysiology of psychiatric illness is of particular interest given its potential to guide the development and improvement of current therapeutic methods. The identification of genetic variants that contribute to or predict the disruption of specific neural pathways associated with psychopathology may also serve as useful markers of risk demarcating individuals with elevated susceptibility for psychiatric illness and affording early or even preemptive treatment strategies. In the continued development of this technique, recent multimodal neuroimaging strategies and studies examining the effects of multiple genes in concert within large subject populations have shown promise in the development of a more complete understanding of the interrelationships between genes, brain function, behavior and associated risk for psychopathology.

This study demonstrates, for the first time, that murine regulatory T (Treg) cells in the tumor microenvironment display both enhanced proliferation and reduced functionality. This enhanced proliferation, combined with decreased apoptosis, leads to an intratumoral accumulation of Treg cells with a unique phenotype: CD4(+)CD25(+)FoxP3(+)GITR(high)CD27(low)CD62L(-). The loss of functionality is associated with down-regulation of the TCR signaling complex, including IL-2-inducible T cell kinase. It is also demonstrated that tumor-infiltrating Treg cells have impaired TCR-mediated signaling and calcium influx. Based on these findings, this study supports the hypothesis that 1) tumor-infiltrating Treg cells lose functionality due to their diminished ability to become effectively activated and 2) intratumoral accumulation of Treg cells may compensate for the impaired functionality, thus maintaining immune tolerance to the tumor.

The antibiotic valanimycin is a naturally occurring azoxy compound produced by Streptomyces viridifaciens MG456-hF10. Precursor incorporation experiments showed that valanimycin is derived from l-valine and l-serine via the intermediacy of isobutylamine and isobutylhydroxylamine. Enzymatic and genetic investigations led to the cloning and sequencing of the valanimycin biosynthetic gene cluster, which was found to contain 14 genes. A novel feature of the valanimycin biosynthetic gene cluster is the presence of a gene (vlmL) that encodes a class II seryl-tRNA synthetase. Previous studies suggested that the role of this enzyme is to provide seryl-tRNA for the valanimycin biosynthetic pathway. Here, we report the results of investigations to elucidate the role of seryl-tRNA in valanimycin biosynthesis. A combination of enzymatic and chemical studies has revealed that the VlmA protein encoded by the valanimycin biosynthetic gene cluster catalyzes the transfer of the seryl residue from seryl-tRNA to the hydroxyl group of isobutylhydroxylamine to produce the ester O-seryl-isobutylhydroxylamine. These findings provide an example of the involvement of an aminoacyl-tRNA in an antibiotic biosynthetic pathway.

Hepcidin, a liver-derived protein that restricts enteric iron absorption, is the key regulator of body iron content. Several proteins induce expression of the hepcidin-encoding gene Hamp in response to infection or high levels of iron. However, mechanism(s) of Hamp suppression during iron depletion are poorly understood. We describe mask: a recessive, chemically induced mutant mouse phenotype, characterized by progressive loss of body (but not facial) hair and microcytic anemia. The mask phenotype results from reduced absorption of dietary iron caused by high levels of hepcidin and is due to a splicing defect in the transmembrane serine protease 6 gene Tmprss6. Overexpression of normal TMPRSS6 protein suppresses activation of the Hamp promoter, and the TMPRSS6 cytoplasmic domain mediates Hamp suppression via proximal promoter element(s). TMPRSS6 is an essential component of a pathway that detects iron deficiency and blocks Hamp transcription, permitting enhanced dietary iron absorption.

<h4>Objective</h4>To investigate whether citrus fruit, noncitrus fruit, and other dietary factors act as environmental modifiers of iron status in the absence or presence of hemochromatotic HFE gene mutations.<h4>Participants and methods</h4>Iron studies, HFE genotypic analyses, and dietary data from a survey conducted from March 21, 1994, through December 15, 1995, were analyzed for a group of 2232 residents (1105 men, 1127 women) aged 20 to 79 years recruited from the community electoral roll of Busselton in Western Australia. Data were analyzed by linear regression analysis and analysis of covariance.<h4>Results</h4>Higher levels of fresh fruit intake (excluding citrus fruits and citrus juices) had a significant protective effect (P=.002) against high body iron status as gauged by ferritin levels in men, irrespective of HFE genotype. Consumption of 2 or more pieces of fruit per day on average reduced mean serum ferritin levels by 20% compared with average consumption of less than 1 piece of fruit per day. This effect was not observed in women. Consumption of citrus fruits and citrus juices had no significant effects in either sex. No protective effects were observed for tea consumption or any other dietary factors studied. Red meat and alcohol consumption correlated with high body iron stores (P<.05), consistent with previous studies, but did not interact with fruit with regard to effects on serum ferritin (P>.05).<h4>Conclusion</h4>Noncitrus fruits are environmental modifiers of iron status independent of HFE genotype. This could have important implications for the provision of evidence-based dietary advice to patients with other iron-storage disorders.

Stem cell differentiation involves changes in transcription, but little is known about translational control during differentiation. We comprehensively profiled gene expression during differentiation of murine embryonic stem cells (ESCs) into embryoid bodies by integrating transcriptome analysis with global assessment of ribosome loading. While protein synthesis was parsimonious during self-renewal, differentiation induced an anabolic switch, with global increases in transcript abundance, polysome content, protein synthesis, and protein content. Furthermore, 78% of transcripts showed increased ribosome loading, thereby enhancing translational efficiency. Transcripts under exclusive translational control included the transcription factor ATF5, the tumor suppressor DCC, and the beta-catenin agonist Wnt1. We show that a hierarchy of translational regulators, including mTOR, 4EBP1, and the RNA-binding proteins DAZL and GRSF1, control global and selective protein synthesis during ESC differentiation. Parsimonious translation in pluripotent state and hierarchical translational regulation during differentiation may be important quality controls for self-renewal and choice of fate in ESCs.

In an attempt to further elucidate the pathomechanisms in oral squamous cell carcinoma (OSCC), gene expression profiling was performed using a whole-transcriptome chip that contains 35,035 gene-specific 70 mere oligonucleotides (Human OligoSet 4.0; Operon, Cologne, Germany) to a set of 35 primary OSCCs. Altogether, 7390 genes were found differentially expressed between OSCC tumor samples and oral mucosa. To characterize the major biologic processes in this tumor collection, MAPPFinder, a component of GenMAPP version 2.1, was applied to this data set to generate a statistically ranked list of molecular signaling pathways. Among others, cancer-related pathways, such as mitogen-activated protein (MAP) kinase signaling (z score = 4.6, P < .001), transforming growth factor-beta signaling (z score = 3.0, P = .015), and signaling pathways involved in apoptosis (z score = 2.1, P = .037), were found deregulated in the OSCC collection analyzed. Focusing on the MAP kinase signaling pathway, subsequent tissue microarray analyses by immunohistochemistry revealed an increase in protein expression of MAP kinase-related proteins ERK1 in 22.8% (48 of 209) and ERK5 in 27.4% (76 of 277), respectively. An association of high ERK5 but not of high ERK1 expression with advanced tumor stage and the presence of lymph node metastases was found (P = .008 and P = .016, respectively). Our analysis demonstrates the reliability of the combined approach of gene expression profiling, signaling pathway analyses, and tissue microarray analysis to detect novel distinct molecular aberrations in OSCC.

Vertebrate hearts depend on highly specialized cardiomyocytes that form the cardiac conduction system (CCS) to coordinate chamber contraction and drive blood efficiently and unidirectionally throughout the organism. Defects in this specialized wiring system can lead to syncope and sudden cardiac death. Thus, a greater understanding of cardiac conduction development may help to prevent these devastating clinical outcomes. Utilizing a cardiac-specific fluorescent calcium indicator zebrafish transgenic line, Tg(cmlc2:gCaMP)(s878), that allows for in vivo optical mapping analysis in intact animals, we identified and analyzed four distinct stages of cardiac conduction development that correspond to cellular and anatomical changes of the developing heart. Additionally, we observed that epigenetic factors, such as hemodynamic flow and contraction, regulate the fast conduction network of this specialized electrical system. To identify novel regulators of the CCS, we designed and performed a new, physiology-based, forward genetic screen and identified for the first time, to our knowledge, 17 conduction-specific mutations. Positional cloning of hobgoblin(s634) revealed that tcf2, a homeobox transcription factor gene involved in mature onset diabetes of the young and familial glomerulocystic kidney disease, also regulates conduction between the atrium and the ventricle. The combination of the Tg(cmlc2:gCaMP)(s878) line/in vivo optical mapping technique and characterization of cardiac conduction mutants provides a novel multidisciplinary approach to further understand the molecular determinants of the vertebrate CCS.

Recent studies have demonstrated that the topography of thalamocortical (TC) axon projections is initiated before they reach the cortex, in the ventral telencephalon (VTel). However, at this point, the molecular mechanisms patterning the topography of TC projections in the VTel remains poorly understood. Here, we show that a long-range, high-rostral to low-caudal gradient of Netrin-1 in the VTel is required in vivo for the topographic sorting of TC axons to distinct cortical domains. We demonstrate that Netrin-1 is a chemoattractant for rostral thalamic axons but functions as a chemorepulsive cue for caudal thalamic axons. In accordance with this model, DCC is expressed in a high-rostromedial to low-caudolateral gradient in the dorsal thalamus (DTh), whereas three Unc5 receptors (Unc5A-C) show graded expression in the reverse orientation. Finally, we show that DCC is required for the attraction of rostromedial thalamic axons to the Netrin-1-rich, anterior part of the VTel, whereas DCC and Unc5A/C receptors are required for the repulsion of caudolateral TC axons from the same Netrin-1-rich region of the VTel. Our results demonstrate that a long-range gradient of Netrin-1 acts as a counteracting force from ephrin-A5 to control the topography of TC projections before they enter the cortex.

<h4>Purpose</h4>Raf Kinase Inhibitory Protein (RKIP) plays a pivotal role in cancer by regulating apoptosis induced by chemotherapeutic agents, or immune-mediated stimuli and is a metastasis suppressor protein. The signal transducer and activator of transcription 3 (STAT3) is a transcription factor that is frequently activated in gastric adenocarcinomas, thereby promoting tumor growth. We examined the expression patterns of RKIP and STAT3 with regard to human gastric cancer, predicting that elevated RKIP status may favor clinical outcome.<h4>Experimental design</h4>Tissue microarrays were created from samples from 143 patients with gastric adenocarcinomas. The microarrays were immunohistochemically stained for RKIP and STAT3, and the intensity and extent of the staining was semiquantitatively scored.<h4>Results</h4>In intestinal-type gastric adenocarcinomas, RKIP and STAT3, expression were inversely associated. Cytoplasmic RKIP expression directly correlated with patient survival. Nuclear STAT3 expression inversely correlated with survival. In the diffuse tumor type, no significant correlation was found between RKIP and patient outcome. In the intestinal-type gastric adenocarcinoma, multivariate analysis adjusted for treatment types revealed RKIP and tumor stage to be significant independent predictors of survival. In the diffuse tumor type, stage was the only significant predictor of survival.<h4>Conclusion</h4>These results indicate the predictive and protective role of cytoplasmic RKIP expression in gastric adenocarcinoma of the intestinal subtype. In contrast, nuclear STAT3 expression is associated with poor patient prognosis in the intestinal subtype. Significantly, we show an inverse association between RKIP and STAT3 and a positive correlation between RKIP and patient survival.

The activation of the tumor suppressor p53 facilitates the cellular response to genotoxic stress; however, the p53 response can only be executed if its interaction with its inhibitor Mdm2 is abolished. There have been conflicting reports on the question of whether p53 posttranslational modifications, such as phosphorylation or acetylation, are essential or only play a subtle, fine-tuning role in the p53 response. Thus, it remains unclear whether p53 modification is absolutely required for its activation. We have now identified all major acetylation sites of p53. Although unacetylated p53 retains its ability to induce the p53-Mdm2 feedback loop, loss of acetylation completely abolishes p53-dependent growth arrest and apoptosis. Notably, acetylation of p53 abrogates Mdm2-mediated repression by blocking the recruitment of Mdm2 to p53-responsive promoters, which leads to p53 activation independent of its phosphorylation status. Our study identifies p53 acetylation as an indispensable event that destabilizes the p53-Mdm2 interaction and enables the p53-mediated stress response.

PELP1 (proline-rich, glutamic acid-rich, and leucine-rich protein-1) is a potential proto-oncogene that functions as a coregulator of estrogen receptor (ER), and its expression is deregulated during breast cancer progression. Emerging evidence suggests growth factor signaling crosstalk with ER as one possible mechanism by which breast tumors acquire resistance to therapy. In this study, we examined mechanisms by which growth factors modulate PELP1 functions, leading to activation of ER. Using in vivo labeling assays, we have found that growth factors promote phosphorylation of PELP1. Utilizing a panel of substrate-specific phosphorylated antibodies, we discovered that growth factor stimulation promotes phosphorylation of PELP1 that is recognized by a protein kinase A (PKA) substrate-specific antibody. Accordingly, growth factor-mediated PELP1 phosphorylation was effectively blocked by PKA-specific inhibitor H89. Utilizing purified PKA enzyme and in vitro kinase assays, we obtained evidence of direct PELP1 phosphorylation by PKA. Using deletion and mutational analysis, we identified PELP1 domains that are phosphorylated by PKA. Interestingly, site-directed mutagenesis of the putative PKA site in PELP1 compromised growth factor-induced activation and subnuclear localization of PELP1 and also affected PELP1-mediated transactivation function. Utilizing MCF-7 cells expressing a PELP1 mutant that cannot be phosphorylated by PKA, we provide mechanistic insights by which growth factor signaling regulates ER transactivation in a PELP1-dependent manner. Collectively, these findings suggest that growth factor signals promote phosphorylation of ER coactivator PELP1 via PKA pathway, and such modification may have functional implications in breast tumors with deregulated growth factor signaling.

Condensin is the core activity responsible for chromosome condensation in mitosis. In the yeast S. cerevisiae, condensin binding is enriched at the regions where DNA replication terminates. Therefore, we investigated whether DNA replication completion determines the condensin-binding proficiency of chromatin. In order to fulfill putative mitotic requirements for condensin activity we analyzed chromosome condensation and condensin binding to unreplicated chromosomes in mitosis. For this purpose we used pGAL:CDC6 cdc15-ts cells that are known to enter mitosis without DNA replication if CDC6 transcription is repressed prior to S-phase. Both the condensation of nucleolar chromatin and proper condensin targeting to rDNA sites failed when unreplicated chromosomes were driven in mitosis. We propose that the DNA replication results in structural and/or biochemical changes to replicated chromatin, which are required for two-phase condensin binding and proper chromosome condensation.

Dysregulation of iron homeostasis is implicated in Alzheimer's disease (AD). In this pilot study, common variants of the apolipoprotein E (APOE) and HFE genes resulting in the iron overload disorder of hereditary hemochromatosis (C282Y, H63D and S65C) were evaluated as factors in sporadic AD in an Ontario sample in which folic acid fortification has been mandatory since 1998. Laboratory studies also were done to search for genetic effects on blood markers of iron status, red cell folates and serum B12. Participants included 58 healthy volunteers (25 males, 33 females) and 54 patients with probable AD (20 males, 34 females). Statistical analyses were interpreted at the 95% confidence level. Contingency table and odds ratio analyses supported the hypothesis that in females of the given age range, E4 significantly predisposed to AD in the presence but not absence of H63D. In males, E4 significantly predisposed to AD in the absence of H63D, and H63D in the absence of E4 appeared protective against AD. Among E4+ AD patients, H63D was associated with significant lowering of red cell folate concentration, possibly as the result of excessive oxidative stress. However, folate levels in the lowest population quartile did not affect the risk of AD. A model is presented to explain the experimental findings.

The liver is one of the major target organs affected in sepsis that are usually accompanied with free radical formation. The use of minus charge for the prevention and cure of various radical related diseases is gaining wide importance in the medicinal field. Here, we investigate whether minus charge stimulation (MCS) inhibits nitric oxide (NO) production induced by lipopolysaccharide (LPS) in the mice liver. The survival rate was compared in LPS-treated group with MCS group. The liver NO radical was measured using electron spin resonance technique. Serum alanine transaminase (ALT) was estimated for liver injury. MCS significantly improved the survival rate of LPS-treated mice and inhibited increase of ALT in serum levels. MCS also reduced NO radical production significantly in the LPS-treated mice liver tissue. In conclusion, our results indicate that MCS prevents LPS-induced liver injury, which may be through the inhibition of liver NO radical production.

<h4>Background and purpose</h4>The aim of this study was to perform DNA analysis in patients with clinical diagnosis of Huntington's disease (HD) after molecular exclusion of HD and further molecular examinations for other neurodegenerative diseases such as Huntington's disease-like 2 (HDL-2; gene JPH3), dentatorubral pallidoluysian atrophy (DRPLA; gene ATN1) and spinocerebellar ataxia type 17 (SCA17; gene TBP).<h4>Material and methods</h4>The material comprised 224 DNA samples isolated from peripheral blood from patients suspected of HD and 100 DNA samples from unaffected controls. The control group was used to determine the normal range of the number of CAG/CTG repeats in genes JPH3, ATN1 and TBP in the Polish population. Molecular analysis was carried out by PCR reaction, embracing microsatellite repeats in genes JPH3, ATN1 and TBP with specific, fluorescently labelled primers. PCR products were separated in polyacrylamide gels. The normal ranges of the number of repeats established for the control group in genes JPH3, ATN1 and TBP were 7-19, 9-27 and 29-45, respectively.<h4>Results</h4>Molecular analysis of DNA from 224 individuals suspected of HD (117 women and 107 men) revealed one case of dynamic mutation - 55 CAG repeats - in the TBP locus (SCA17). No cases of DRPLA or HDL-2 were detected. The range of CAG/CTG repeats for the JPH3 gene in the patient group was 11-19, with the most common alleles containing 14 and 16 repeats. For the ATN1 gene in patients the range of 8-27 repeats was established and the most frequent allele with 16 triplets was present.<h4>Conclusions</h4>The study on 244 patients referred with the clinical diagnosis of HD and without mutation of the IT15 gene revealed one case of SCA17 but did not disclose the presence of two other diseases with a similar clinical manifestation: DRPLA and HDL2.

Several pharmacogenetics studies have analyzed the influence of specific genetic polymorphisms on the toxicity of antiretroviral treatment. The present review describes some of the adverse effects of antiretroviral drugs in which a genetic predisposition may be involved: efavirenz-induced neurological toxicity, generally associated with the 516G>T polymorphism of liver enzyme cytochrome P450 2B6 (CYP2B6); hypersensitivity reactions to nevirapine, associated with specific alleles of major histocompatibility complex, mainly the HLA-DRB1*0101 allele, which, in combination with a high CD4 lymphocyte count, has been associated with systemic reactions and hepatitis in Caucasians, and the HLA-Cw8 allele, which is associated with hypersensitivity reactions in persons from the Italian island of Sardinia and from Japan; nevirapine-induced hepatotoxicity associated with the C>T polymorphism in position 3435T of the ABCB1 (MDR-1) gene codifying for glycoprotein P (lower risk); hyperbilirubinemia in patients exposed to atazanavir or indinavir carrying the UGT1A1*28 polymorphism; peripheral neuropathy with nucleoside analogues associated with haplogroup T of the mitochondrial genome (higher risk) and with the HFE C282Y genotype of the hemochromatosis gene (lower risk); the mutation in codon 964 (R964C) of the POLG gene that codifies the mitochondrial polymerase DNA gamma described in a Thai patient with lactic acidosis; the ABCC2 gene haplotypes associated with tenofovir-induced proximal tubulopathy, and the risk of pancreatitis in persons with mutations in the CFTR and SPINK-1 genes.

<h4>Background/aims</h4>The diagnosis of hereditary hemochromatosis (HH) is based on qualitative measurement of tissue iron concentration and genetic tests. The aim of this study was to evaluate the correlation between the presence of iron deposits in the liver and the HFE gene mutations in patients with chronic liver diseases (CLD).<h4>Methodology</h4>The 182 patients, age range 18-71 years, were hospitalized in Gdansk because of CLD. The C282Y, H63D and S65C HFE mutations were screened by PCR-RFLP analysis. Liver function tests, serological examinations for viral hepatitis, serum iron and ferritin concentration and semiquantitative assessment of liver iron were done in all subjects. Patients were divided into Group A without iron deposits in the liver, and Group B with deposits. The most frequent etiology of CLD was chronic hepatitis C.<h4>Results</h4>Biochemical parameters indicating iron storage and ALT activity were significantly higher in Group B. Either typical for diagnosis HH homozygotes C282Y/C282Y and combined heterozygotes C282Y/H63D or carriers of other HFE gene mutations were found significantly more frequently in Group B.<h4>Conclusions</h4>The finding of iron deposits in routinely obtained liver specimen correlates with occurrence of the different HFE gene mutations.

A new sensitive, selective and simple immune resonance scattering spectral assay was proposed for the determination of trace amounts of Antithrombin-III (AT-III). It was based on the immune reaction of AT-III with the goat-anti-human AT-III antibody and the resonance scattering effect of the immunocomplex particles in pH 7.2 Tris-HCl buffer solution and in the presence of polyethelene glycol (PEG)6000. The results showed that the resonance scattering signal of AT-III and goat-anti-human AT-III antibody was very weak. However, AT-III was combined with goat-anti-human AT-III antibody specifically, and aggregated to form immunocomplex particles, which enhanced the resonance scattering intensity greatly and produced three resonance scattering peaks at 368 nm, 491 nm and 536 nm respectively. The strongest resonance scattering peak was at 491 nm. In the present paper, the influences of pH, goat-anti-human AT-III antibody and PEG-6000 concentration, incubation temperature, reactive time, and foreign substances were investigated. The result showed that the resonance scattering intensity at 491 nm (I(RS)) is linear to the AT-III concentration in the range of 62.5 to 850 ng x mL(-1), under the optimum conditions of 0.30 mL goat-anti-human AT-III antibody, 30 mg x mL(-1) polyethelene glycol-6000, being incubated at 37 degrees C for 15 min, the voltage at 400 V, and the excitation and emission slit width both at 5.0 nm. Its regress equation is deltaI(RS) 0.062 5c + 1.36, and a relative coefficient of 0.996, with a detection limit of 29.4 ng x mL(-1). The results of co-existing substance tolerance test showed that 1.40 x 10(5) ng x mL(-1) glycine, 9.0 x 10(3) ng x mL(-1) L-glutamic acid, 5.0 x 10(5) ng x mL(-1) glucose, 5.0 x 10(4) ng x mL(-1) urea, 1.5 x 10(4) ng x mL(-1) IgG, 3.0 x 10(4) ng x mL(-1) human serum albumin, and 1.5 x 10(4) ng x mL(-1) bovine serum albumin did not interfere with the resonance scattering determination of 2.50 x 10(2) ng x mL(-1) AT-III, when the relative error was within +/- 10%. Four polyethelene glycols, i. e. polyethelene glycol-4000, polyethelene glycol-6000, polyethelene glycol-10000 and polyethelene glycol-20000, on the immune resonance scattering spectral system were examined in details. The results show that polyethelene glycol-6000 has low blank and high deltaI(RS) value, and was chosen for use. The new resonance scattering spectral method features high sensitivity, good selectivity, simplicity and rapidity, and was applied to the quantitative analysis of AT-III in plasma and serum samples with satisfactory results. Its recovery is in the range of 90.2%-108.9%.

The review summarizes the results of the state-of-the-art studies of hemochromatosis (HC): iron-regulatory genes (HFE, HJV, HAMP, TFR2, SLC40A1) have been discovered; the HC types caused by mutations in these genes (types 1, 2, 3, and 4 in the OMIM register) have been identified; the inflammation anemia (IA) mediator - the polypeptide hepatic hormone hepcidin that is an important constituent of the natural immunity system - has been found. This gives an idea of hypersiderosis and dissiderosis as types of iron microelementosis. Types 1, 2, and 3 HC in which iron absorption and its total reserves are increased in the body serve as examples of hypersiderosis. Dissiderosis is characterized by the redistribution of iron between the functional and spare funds inherent in type 4 HC and IA. By taking into account their findings, the authors briefly discuss the leading clinical and morphological manifestations of HC and IA, difficulties in differential diagnosis, and treatment ways and prospects.

C282Y homozygosity is necessary (but insufficient in isolation) for the onset of hemochromatosis, as indicated by its low biochemical penetrance (75 % in men and 50 % in women) and clinical penetrance (25 % in men and 0 % in women). Factors modulating iron load may be acquired (diet, alcohol, metabolic syndrome, drugs, etc.) or, more importantly, genetic (digenism, polymorphism of genes involved in the regulation of hepcidin synthesis). Factors modulating iron-related organ damage include alcohol consumption, the metabolic syndrome, and the TGF-beta1 (hepatic fibrosis) and superoxide dismutase genes (cardiomyopathy). Further studies of these modifiers are needed to improve the management of C282Y homozygotes, at both the individual and the population levels.

Synthesis of a new class of phosphatidylcholine analogues derived from glyceric acid is reported for spectroscopic studies of phospholipases and conformation of phospholipid side-chains in biological membranes, using fluorescence resonance energy transfer (FRET) techniques.

The myocardium is mainly composed of long-lived postmitotic cells with, if there is any at all, a very low rate of replacement through the division and differentiation of stem cells. As a consequence, cardiac myocytes gradually undergo pronounced age-related alterations which, furthermore, occur at a rate that inversely correlates with the longevity of species. Basically, these alterations represent the accumulation of structures that have been damaged by oxidation and that are useless and often harmful. These structures (so-called 'waste' materials), include defective mitochondria, aberrant cytosolic proteins, often in aggregated form, and lipofuscin, which is an intralysosomal undegradable polymeric substance. The accumulation of 'waste' reflects the insufficient capacity for autophagy of the lysosomal compartment, as well as the less than perfect functioning of proteasomes, calpains and other cellular digestive systems. Senescent mitochondria are usually enlarged, show reduced potential over their inner membrane, are deficient in ATP production, and often produce increased amounts of reactive oxygen species. The turnover of damaged cellular structures is hindered by an increased lipofuscin loading of the lysosomal compartment. This particularly restricts the autophagic turnover of enlarged, defective mitochondria, by diverting the flow of lysosomal hydrolases from autophagic vacuoles to lipofuscin-loaded lysosomes where the enzymes are lost, since lipofuscin is not degradable by lysosomal hydrolases. As a consequence, aged lipofuscin-rich cardiac myocytes become overloaded with damaged mitochondria, leading to increased oxidative stress, apoptotic cell death, and the gradual development of heart failure. Defective lysosomal function also underlies myocardial degeneration in various lysosomal storage diseases, while other forms of cardiomyopathies develop due to mitochondrial DNA mutations, resulting in an accumulation of abnormal mitochondria that are not properly eliminated by autophagy. The degradation of iron-saturated ferritin in lysosomes mediates myocardial injury in hemochromatosis, an acquired or hereditary disease associated with iron overload. Lysosomes then become sensitized to oxidative stress by the overload of low mass, redox-active iron that accumulates when iron-saturated ferritin is degraded following autophagy. Lysosomal destabilization is of importance in the induction and/or execution of programmed cell death (either classical apoptotic or autophagic), which is a common manifestation of myocardial aging and a variety of cardiac pathologies.

No abstract available.

<h4>Introduction</h4> HIV infection is a global health issue. The typical pattern of HIV primary infection is characterized by high levels of virus in blood followed by a progressive loss of CD4+T cells, elevation of CD8+T cells and progressive impairment of T cell functions. <h4>Objective:</h4> To analyze the lymphocyte subsets changes in HIV positive patients. <h4>Materials and method:</h4> A retrospective study was conducted on HIV infected patients in HUSM during the period of 2005 till 2007. The patients’ records and results were reviewed and analysed in term of percentage and absolute count to determine the lymphocyte changes which will indirectly reflect the degree of severity of the disease. <h4>Results:</h4> In HIV infected subjects, the alteration of lymphocytes subsets from 2005 to 2007 mainly occur in CD4+ T cell counts (< 200cells/mm3 (70.8%, 84.6%, 81.1%), CD8+ T cells (<500cells/mm3 (85.4%, 94.8%, 86.5%). The absolute lymphocyte counts also drops down in all CD3, CD19/20, and CD16/56. <h4>Discussion and conclusion:</h4> Majority of HIV infected subjects have CD4+ T cells count less than 200 cells/mm3, low CD8+T cells with ratio of less than 1.0. This may suggest most of the HIV infected subjects in HUSM fall in the late stage of the disease which later might end up with lots of major complications and early death if no prompt treatment action taken. <h4>Objective</h4> To localize the motor neuron somata of the sternocleidomastoid muscle in rat. <h4>Subjects and method:</h4> Ten Sprague-Dawley rats were used. Under general anaesthesia, the right sternocleidomastoid (SCM) was injected 0.05 ml of 30% horseradish peroxidase. After 48 hours, the animals were perfused with normal saline, then with 1.25% glutaraldehyde and 1% paraformaldehyde in O.1 M phosphate buffer (pH 7.4), and finally, with 10% sucrose in same buffer. After perfusion, the medulla oblongata and 1st to 6th cervical segments (C-1 to C-6) of spinal cord were placed in above sucrose buffer solution for 24 hours, and their serial transverse sections cut in a cryostat. The sections were treated according to tetramethyl benzidine-HRP method of Mesulam (1978). <h4>Results:</h4> The HRP labeled neuron somata were located in the caudal 0.9 to 1.2 mm of medulla oblongata, whole lengths of C-1 and C-2 and rostral three-fourths of C-3. In the medulla oblongata, they were located ventrolateral to the pyramidal fibres that pass dorsolaterally after their decussation. In C-1, they were located in dorsomedial and central columns, in C-2, they were found in dorsomedial, central, and ventrolateral columns and in rostral three-fourths of C-3, they were located in the ventrolateral column. <h4>Discussion and conclusion:</h4> The motor neuron somata of the sternocleidomastoid muscle in rat are located in the caudal part of the medulla oblongata, the whole lengths of 1st and 2nd cervical segments and the rostral three-fourths of the 3rd cervical segment of the spinal cord. <h4>Introduction</h4> 4-hydroxy-3-methoxycinnamic acid which is better known as ferulic acid is a cinnamic acid derivatives existing ubiquitously in the plant kingdom. It is easily absorbed and metabolized in the human body (Ou et.al, 2004). It has become an effective component of Chinese medicine herbs such as Angelica sinensis and Cimicifuga heracleifolia (Fang, 1998, Sakai et.al, 1999). Its good effect on human has brought ferulic acid to the many physiological functions, including anti-oxidant, anti-microbial, anti-inflammatory, anti-thrombosis, and anti-cancer activities (Ou et.al, 2007). It is also used for treatment in sperm viability (Zhang et.al, 1996) and protects against coronary disease and lower cholesterol in liver (Ou et.al, 2004). <h4>Objective:</h4> To study on the synthesis of three ferulic acid derivatives which is known as methyl ferulate, allyl ferulate and epoxy ferulate. These derivatives are believed to have better medicinal properties than ferulic acid. <h4>Materials and method:</h4> The first step of this work is the protection of the carboxylic group of ferulic acid by the methyl group through the esterification process. The mixture of ferulic acid, dichloromethane (DCM), dimethylaminopyridine (DMAP), methanol, dichlorocarbamide (DCC), and glycerol was stirred for 3 hours using the magnetic stirrer under the constant temperature of 0° C. The product formed was filtered and washed using DCM. This product was then reacted separately with allyl and epoxyde at its hydroxyl group. In this step, the mixture of the first product with tetrahydofuran (THF), sodium hydroxide (KOH) and tetrabuthylammonium idodide (TBAI) was stirred for 6 hours under the room temperature. All the products were purified by column chromatography and characterized by FTIR and 1HNMR spectroscopy. <h4>Results:</h4> 88.70% yield of the first product was formed, while 48.89% and 1.53% yield of the second and third product were formed. The FTIR spectroscopy of the three products shows all the suspected peaks in the range of 4000cm−1 to 400cm−1. Some of the important peaks such as OH peak and C=O peak which is slightly sharp can be seen clearly through the spectroscopy. 1 HNMR spectroscopy proved that the three products are pured after column chromatography since all the peaks of the protons appeared, suit the protons in the structure. In other words, the experimental result is similar to the theoretical result of the work. <h4>Discussion and conclusion:</h4> Three ferulic acid derivatives has been synthesized and after characterization through FTIR and HNMR, it was conformed that the products are methyl ferulate (( E)-2-metoksi-4-(3-metoksibuta-1,3-dienil)fenol), allyl ferulate (3-aliloksi-4-hidroksifenil-3-metoksiferulat ) and epoxy ferulate (4-hidroksi-3-(oksiran-2-iloksifenil)-3-metoksi ferulat). It is hope that these derivatives will be able to help the researchers in studying its beneficial properties especially in medical field.