Gene Literature Dashboard

North Carolina macular dystrophy (NCMD) is an autosomal dominant macular disease, was mapped to 6q14-q16.2, the disease-causing gene has yet not been identified. It shares phenotypic similarity with age-related macular degeneration including drusen and choroidal neovascularization. We collected six families with NCMD including 75 members, and conducted clinical characterization and genetic mapping for these families. Forty-five patients were diagnosed as NCMD; all six NCMD families were mapped to MCDR1 locus using genetic linkage analysis. MCDR1 interval was refined to 3 cM (1.8mb) between D6S1716 to D6S1671 via fine mapping using microsatellite markers in these six families, all eleven annotated genes within the interval were analyzed by mutation screening in coding regions, no mutation was found, suggesting a potential novel gene or a new pathological mechanism causing NCMD. The refinement of MCDR1 locus will aid the disease-causing gene identification. Functional studies of NCMD genes should provide important insights into pathogenetic mechanisms of NCMD and age-related macular degeneration.

Despite the well established role of the frontal and posterior perisylvian cortices in many facets of human-cognitive specializations, including language, little is known about the developmental patterning of these regions in the human brain. We performed a genome-wide analysis of human cerebral patterning during midgestation, a critical epoch in cortical regionalization. A total of 345 genes were identified as differentially expressed between superior temporal gyrus (STG) and the remaining cerebral cortex. Gene ontology categories representing transcription factors were enriched in STG, whereas cell-adhesion and extracellular matrix molecules were enriched in the other cortical regions. Quantitative RT-PCR or in situ hybridization was performed to validate differential expression in a subset of 32 genes, most of which were confirmed. LIM domain-binding 1 (LDB1), which we show to be enriched in the STG, is a recently identified interactor of LIM domain only 4 (LMO4), a gene known to be involved in the asymmetric pattering of the perisylvian region in the developing human brain. Protocadherin 17 (PCDH17), a neuronal cell adhesion molecule, was highly enriched in focal regions of the human prefrontal cortex. Contactin associated protein-like 2 (CNTNAP2), in which mutations are known to cause autism, epilepsy, and language delay, showed a remarkable pattern of anterior-enriched cortical expression in human that was not observed in mouse or rat. These data highlight the importance of expression analysis of human brain and the utility of cross-species comparisons of gene expression. Genes identified here provide a foundation for understanding molecular aspects of human-cognitive specializations and the disorders that disrupt them.

Adult neural progenitor cells (aNPCs) exhibit limited migration in vivo with the exception of the rostral migratory stream and injury-induced movement. Surprisingly little is known regarding those signals regulating attraction or inhibition of the aNPC. These studies demonstrate that aNPCs respond principally to a repulsive cue expressed at the embryonic floor plate (FP) and also the injured adult CNS. Adult spinal cord progenitor cells (aSCPs) were seeded onto organotypic slice preparations of the intact embryonic or injured adult spinal cord. Cell migration assays combined with genetic and molecular perturbation of FP-derived migration cues or aSCP receptors establish netrin-1 (Ntn-1) but not Slit-2, Shh, or Ephrin-B3 as the primary FP-derived repellant. When slices were prepared from injured spinal cord, aSCP migration away from the injury core was Ntn-1-dependent. These studies establish Ntn-1 as a critical regulator of aSCP migration in the intact and injured CNS.

A study of the structure-activity relationships of 5'-O-[N-(salicyl)sulfamoyl]adenosine (6), a potent inhibitor of the bifunctional enzyme salicyl-AMP ligase (MbtA, encoded by the gene Rv2384) in Mycobacterium tuberculosis, is described, targeting the salicyl moiety. A systematic series of analogues was prepared exploring the importance of substitution at the C-2 position revealing that a hydroxy group is required for optimal activity. Examination of a series of substituted salicyl derivatives indicated that substitution at C-4 was tolerated. Consequently, a series of analogues at this position provided 4-fluoro derivative, which displayed an impressive MIC99 of 0.098 microM against whole-cell M. tuberculosis under iron-limiting conditions. Examination of other heterocyclic, cycloalkyl, alkyl, and aminoacyl replacements of the salicyl moiety demonstrated that these nonconservative modifications were poorly tolerated, a result consistent with the fairly strict substrate specificities of related non-ribosomal peptide synthetase adenylation enzymes.

<h4>Aims/hypothesis</h4>The slit diaphragm is an adhesion and signalling protein complex linking the interdigitating podocyte foot processes in the kidney glomerulus, and mutations in slit diaphragm-associated genes result in severe proteinuria. Here we report a genetic association analysis of four slit diaphragm genes, LRRC7, KIRREL, NPHS2 and ACTN4, in a Finnish diabetic nephropathy cohort.<h4>Materials and methods</h4>A total of 40 single nucleotide polymorphisms (SNPs) were genotyped in 1103 patients with type 1 diabetes. The patients were classified according to their renal status, and the genotype data were analysed in a cross-sectional case-control setting. To confirm positive associations, four SNPs were genotyped in 1,025 additional patients with type 1 diabetes.<h4>Results</h4>No associations with diabetic nephropathy were observed for any of the analysed SNPs. The SNPs were not associated with the time from the onset of diabetes to the diagnosis of nephropathy or with glomerular filtration rate or AER as quantitative variables. In a sex-specific sub-analysis, the variants rs979972 and rs749701 in the first intron of ACTN4 were nominally associated with diabetic nephropathy in females, with odds ratios of 1.81 (95% CI 1.18-2.79, p = 0.007) and 1.93 (95% CI 1.26-2.96, p = 0.003) respectively.<h4>Conclusions/interpretation</h4>Our study has not found any evidence that common variants in LRRC7, KIRREL, NPHS2 and ACTN4 contribute to susceptibility to diabetic nephropathy in Finnish patients with type 1 diabetes.

Huntington's disease is caused by a polyglutamine expansion in the huntingtin (htt) protein, and previous data indicate that over-activation of NMDA receptors (NMDARs) may be involved in the selective degeneration of cells expressing NR1/NR2B NMDARs. We used Kinetworkstrade mark multi-immunoblotting screens to examine expression of 76 protein kinases, 18 protein phosphatases, 25 heat shock/stress proteins, and 27 apoptosis proteins in human embryonic kidney 293 cells transfected with NR1/NR2B and htt containing 15 (htt-15Q; wild-type) or 138 (htt-138Q; mutant) glutamine repeats. Follow-up experiments revealed several proteins involved in the heat-shock response pathway to be up-regulated in the soluble fraction from cells expressing htt-138Q, including protein phosphatase 5 and cyclin-dependent kinase 5. Increased expression in the soluble fraction of htt-138Q-expressing cells was also noted for the stress- and calcium-activated protein-serine/threonine kinase casein kinase 2, a change which was confirmed in striatal tissue of yeast artificial chromosome transgenic mice expressing full-length mutant htt. Inhibition of casein kinase 2 activity in cultured striatal neurons from these mice significantly exacerbated NMDAR-mediated toxicity, as assessed by labeling of apoptotic nuclei. Our findings are consistent with up-regulation of components of the stress response pathway in the presence of polyglutamine-expanded htt and NR1/NR2B which may reflect an attempt at the cellular level to ameliorate the detrimental effects of mutant htt expression.

<h4>Background</h4>In developing neurons, somal migration and initiation of axon outgrowth often occur simultaneously and are regulated in part by similar classes of molecules. When neurons reach their final destinations, however, somal translocation and axon extension are uncoupled. Insights into the mechanisms underlying this process of disengagement came from our study of the behaviour of embryonic spinal motor neurons following ablation of boundary cap cells. These are neural crest derivatives that transiently reside at motor exit points, central nervous system (CNS):peripheral nervous system (PNS) interfaces where motor axons leave the CNS. In the absence of boundary cap cells, motor neuron cell bodies migrate along their axons into the periphery, suggesting that repellent signals from boundary cap cells regulate the selective gating of somal migration and axon outgrowth at the motor exit point. Here we used RNA interference in the chick embryo together with analysis of null mutant mice to identify possible boundary cap cell ligands, their receptors on motor neurons and cytoplasmic signalling molecules that control this process.<h4>Results</h4>We demonstrate that targeted knock down in motor neurons of Neuropilin-2 (Npn-2), a high affinity receptor for class 3 semaphorins, causes their somata to migrate to ectopic positions in ventral nerve roots. This finding was corroborated in Npn-2 null mice, in which we identified motor neuron cell bodies in ectopic positions in the PNS. Our RNA interference studies further revealed a role for Plexin-A2, but not Plexin-A1 or Plexin-A4. We show that chick and mouse boundary cap cells express Sema3B and 3G, secreted semaphorins, and Sema6A, a transmembrane semaphorin. However, no increased numbers of ectopic motor neurons are found in Sema3B null mouse embryos. In contrast, Sema6A null mice display an ectopic motor neuron phenotype. Finally, knockdown of MICAL3, a downstream semaphorin/Plexin-A signalling molecule, in chick motor neurons led to their ectopic positioning in the PNS.<h4>Conclusion</h4>We conclude that semaphorin-mediated repellent interactions between boundary cap cells and immature spinal motor neurons regulates somal positioning by countering the drag exerted on motor neuron cell bodies by their axons as they emerge from the CNS at motor exit points. Our data support a model in which BC cell semaphorins signal through Npn-2 and/or Plexin-A2 receptors on motor neurons via a cytoplasmic effector, MICAL3, to trigger cytoskeletal reorganisation. This leads to the disengagement of somal migration from axon extension and the confinement of motor neuron cell bodies to the spinal cord.

Hereditary hemochromatosis is an inherited disorder of increased iron absorption that can result in cirrhosis, diabetes, and other morbidities. We have investigated the mechanisms underlying supranormal glucose tolerance despite decreased insulin secretion in a mouse model of hemochromatosis with deletion of the hemochromatosis gene (Hfe(-/-)). Hfe(-/-) mice on 129Sv or C57BL/6J backgrounds have decreased glucose excursions after challenge compared with controls. In the C57BL/6J/ Hfe(-/-), for example, incremental area under the glucose curve is reduced 52% (p < 0.001) despite decreased serum insulin, and homeostasis model assessment insulin resistance is decreased 50% (p < 0.05). When studied by the euglycemic clamp technique 129Sv/Hfe(-/-) mice exhibit a 20% increase in glucose disposal (p < 0.05) at submaximal insulin but no increase at maximal insulin compared with wild types. [1,2-(13)C]D-glucose clearance from plasma is significantly increased in Hfe(-/-) mice (19%, p < 0.05), and lactate derived from glycolysis is elevated 5.1-fold in Hfe(-/-) mice (p < 0.0001). Basal but not insulin-stimulated glucose uptake is elevated in isolated soleus muscle from Hfe(-/-) mice (p < 0.03). Compared with controls Hfe(-/-) mice exhibit no differences in serum lipid, insulin, glucagon, or thyroid hormone levels; adiponectin levels are elevated 41% (p < 0.05), and the adiponectin message in adipocytes is increased 83% (p = 0.04). Insulin action measured by phosphorylation of Akt is not enhanced in muscle, but phosphorylation of AMP-dependent kinase is increased. We conclude that supranormal glucose tolerance in iron overload is characterized by increased glucose disposal that does not result from increased insulin action. Instead, the Hfe(-/-) mice demonstrate increased adiponectin levels and activation of AMP-dependent kinase.

<h4>Background</h4>Serum aminotransferase concentrations are reportedly strongly associated with insulin resistance, an established cardiovascular risk factor that is not routinely assessed in clinical practice. We therefore explored the possibility that serum aminotransferase concentrations are as closely related to large artery disease as insulin resistance in rheumatoid arthritis (RA).<h4>Methods</h4>Carotid artery plaque (ultrasonography), insulin resistance and liver enzymes (prior to methotrexate (MTX) were determined in 77 consecutive patients with RA (43 with and 34 without MTX).<h4>Results</h4>Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were associated with insulin resistance in univariate analysis (R = 0.54, p < 0.0001 and R = 0.36, p = 0.001, respectively) and after adjustment for age, gender and waist circumference (partial R = 0.43, p = 0.0001 and partial R = 0.37, p = 0.001, respectively). ALT and AST concentrations were higher in patients with plaque as compared to in those without plaque (ALT (u/l): 27 2223242526272829303132 versus 20 181920212223, p = 0.02; AST (u/l): 25 2122232425262728 versus 20 19202122, p = 0.02). The odds ratios [95% CI] for plaque were 1.92 [1.14-3.24] (p = 0.01), 1.93 [1.17-3.16] (p = 0.009) and 1.82 [1.13-2.93] (p = 0.01) for 1 SD increase in ALT (~10 u/l) and AST (approximately 6 u/l) concentrations and in logarithmically transformed homeostasis model assessment of insulin resistance (HOMA-IR) (~0.2 uU.mmol/ml.l), respectively. After adjustment for the potentially confounding characteristics of age, sex, hypertension and hypothyroidism in logistic regression models, ALT (p = 0.049) and AST concentrations (p = 0.027) remained associated with plaque whereas the HOMA-IR did not (p = 0.08). AST concentrations (p = 0.049) were associated with plaque independent of insulin resistance whereas the HOMA-IR (p = 0.1) was not associated with plaque independent of AST concentrations.<h4>Conclusion</h4>Within currently recommended reference ranges, serum aminotransferase concentrations may be strongly associated with insulin resistance and atherosclerosis in patients with RA. The measurement of aminotransferase concentrations could be a useful tool in cardiovascular risk stratification in patients with RA.

To investigate how intracellular parasites manipulate their host cell environment at the molecular level, we undertook a quantitative proteomic study of cells following infection with the apicomplexan parasite Toxoplasma gondii. Using conventional two-dimensional electrophoresis, difference gel electrophoresis (DIGE), and mass spectrometry, we identified host proteins that were consistently modulated in expression following infection. We detected modification of protein expression in key metabolic pathways, including glycolysis, lipid and sterol metabolism, mitosis, apoptosis, and structural-protein expression, suggestive of global reprogramming of cell metabolism by the parasite. Many of the differentially expressed proteins had not been previously implicated in the response to the parasite, while others provide important corroborative protein evidence for previously proposed hypotheses of pathogen-cell interactions. Significantly, over one-third of all modulated proteins were mitochondrial, and this was further investigated by DIGE analysis of a mitochondrion-enriched preparation from infected cells. Comparison of our proteomic data with previous transcriptional studies suggested that a complex relationship exits between transcription and protein expression that may be partly explained by posttranslational modifications of proteins and revealed the importance of investigating protein changes when interpreting transcriptional data. To investigate this further, we used phosphatase treatment and DIGE to demonstrate changes in the phosphorylation states of several key proteins following infection. Overall, our findings indicate that the host cell proteome responds in a dramatic way to T. gondii invasion, in terms of both protein expression changes and protein modifications, and reveal a complex and intimate molecular relationship between host and parasite.

<h4>Background</h4>The homeobox genes are a large and diverse group of genes, many of which play important roles in the embryonic development of animals. Increasingly, homeobox genes are being compared between genomes in an attempt to understand the evolution of animal development. Despite their importance, the full diversity of human homeobox genes has not previously been described.<h4>Results</h4>We have identified all homeobox genes and pseudogenes in the euchromatic regions of the human genome, finding many unannotated, incorrectly annotated, unnamed, misnamed or misclassified genes and pseudogenes. We describe 300 human homeobox loci, which we divide into 235 probable functional genes and 65 probable pseudogenes. These totals include 3 genes with partial homeoboxes and 13 pseudogenes that lack homeoboxes but are clearly derived from homeobox genes. These figures exclude the repetitive DUX1 to DUX5 homeobox sequences of which we identified 35 probable pseudogenes, with many more expected in heterochromatic regions. Nomenclature is established for approximately 40 formerly unnamed loci, reflecting their evolutionary relationships to other loci in human and other species, and nomenclature revisions are proposed for around 30 other loci. We use a classification that recognizes 11 homeobox gene 'classes' subdivided into 102 homeobox gene 'families'.<h4>Conclusion</h4>We have conducted a comprehensive survey of homeobox genes and pseudogenes in the human genome, described many new loci, and revised the classification and nomenclature of homeobox genes. The classification scheme may be widely applicable to homeobox genes in other animal genomes and will facilitate comparative genomics of this important gene superclass.

<h4>Background & aims</h4>The influence of HFE gene mutations and liver iron overload on hepatocellular carcinoma (HCC) occurrence in patients with cirrhosis is subjected to controversial results. The aim of this work was to clarify this influence in a large cohort of prospectively followed-up cirrhotic patients classified according to the cause of their liver disease.<h4>Methods</h4>Three hundred one consecutive cirrhotic patients (162 alcoholics and 139 HCV-infected patients) were included at time of diagnosis of cirrhosis and followed-up. Liver iron overload on initial biopsy according to modified Deugnier's score and C282Y/H63D HFE gene mutations were assessed.<h4>Results</h4>In patients with alcoholic cirrhosis (mean iron score, 2.0 +/- 3.0; mean time of follow-up, 66.1 +/- 45.1 months), 40 (24.6%) developed HCC. Thirteen (8.02%) were heterozygotes for C282Y HFE gene mutation and had higher hepatic iron scores (3.6 +/- 3.8 vs 1.9 +/- 2.8, respectively, P = .05). In univariate analysis, liver iron overload as a continuous variable (HR, 1.23 [1.13-1.34], P < .001) or in binary coding with an optimal threshold of iron score >/=2.0 (HR, 4.1 [2.1-7.3], P < .0001) and C282Y mutation carriage (HR, 2.7 [1.2-6.3], P = .01) were risk factors for HCC. In multivariate analysis, liver iron and C282Y mutation carriage remained independent risk factors for HCC. In patients with HCV-related cirrhosis (C282Y mutation carriage, 17 [12.23%]; mean liver iron score, 0.9 +/- 1.9; mean time of follow-up, 85.5 +/- 42.1 months; HCC, 63 [45.32%] patients), C282Y mutation carriage and liver iron were not associated with HCC occurrence.<h4>Conclusions</h4>Liver iron overload and C282Y mutation are associated with a higher risk of HCC in patients with alcoholic but not HCV-related cirrhosis.

We carried out univariate and bivariate linkage analyses to identify genomic regions that may influence plasma levels of C-reactive protein (CRP) and fibrinogen and exert pleiotropic effects on both traits. Subjects included African American (AA, n=1310, mean age 62.7+/-9.4 years) and non-Hispanic white (NHW, n=796, mean age 58.4+/-9.8 years) belonging to hypertensive sibships. Plasma CRP was measured by an immunoturbidimetric assay and fibrinogen by the Clauss method. Genotyping was performed at 366 microsatellite marker loci spaced approximately 10 cM apart across the 22 autosomes. Estimation of heritability and linkage analyses was carried out using a variance components approach. Significant heritability was noted for CRP (0.38 in AA and 0.37 in NHW subjects) and fibrinogen (0.44 in AA and 0.28 in NHW subjects). Significant genetic correlation between CRP and fibrinogen was present in both AA (0.39) and NHW (0.40) subjects. In univariate linkage analysis, the maximum logarithm of odds (LOD) score for CRP was on chromosome 10q22 in NHW (LOD=1.69, 106.75 cM, P=0.0026) and for fibrinogen on chromosome 2 in AA (LOD=2.14, 55.5 cM, P=0.0009) subjects. Bivariate linkage analysis demonstrated suggestive evidence of linkage (defined as LOD score >or= 2.87) for both traits on chromosome 12 (LOD=3.44, 152.16 cM, P=0.0003) in AA and on chromosome 21 (LOD=3.03, 13.05 cM, P=0.0008) in NHW subjects. Plasma CRP and fibrinogen levels are heritable and genetically correlated. Linkage analyses identified several chromosomal regions that may harbour genes influencing CRP and fibrinogen levels and exert pleiotropic effects on both traits.

A transcriptional module (TM) is a collection of transcription factors (TF) that as a group, co-regulate multiple, functionally related genes. The task of identifying TMs poses an important biological challenge. Since TFs belong to evolutionarily and structurally related families, TF family members often bind to similar DNA motifs and can confound sequence-based approaches to TM identification. A previous approach to TM detection addresses this issue by pre-selecting a single representative from each TF family. One problem with this approach is that closely related transcription factors can still target sufficiently distinct genes in a biologically meaningful way, and thus, pre-selecting a single family representative may in principle miss certain TMs. Here we report a method-TREMOR (Transcriptional Regulatory Module Retriever). This method uses the Mahalanobis distance to assess the validity of a TM and automatically incorporates the inter-TF binding similarity without resorting to pre-selecting family representatives. The application of TREMOR on human muscle-specific, liver-specific and cell-cycle-related genes reveals TFs and TMs that were validated from literature and also reveals additional related genes.

Nucleosome-remodelling factors containing the ATPase ISWI, such as ACF, render DNA in chromatin accessible by promoting the sliding of histone octamers. Although the ATP-dependent repositioning of mononucleosomes is readily observable in vitro, it is unclear to which extent nucleosomes can be moved in physiological chromatin, where neighbouring nucleosomes, linker histones and the folding of the nucleosomal array restrict mobility. We assembled arrays consisting of 12 nucleosomes or 12 chromatosomes (nucleosomes plus linker histone) from defined components and subjected them to remodelling by ACF or the ATPase CHD1. Both factors increased the access to DNA in nucleosome arrays. ACF, but not CHD1, catalysed profound movements of nucleosomes throughout the array, suggesting different remodelling mechanisms. Linker histones inhibited remodelling by CHD1. Surprisingly, ACF catalysed significant repositioning of entire chromatosomes in chromatin containing saturating levels of linker histone H1. H1 inhibited the ATP-dependent generation of DNA accessibility by only about 50%. This first demonstration of catalysed chromatosome movements suggests that the bulk of interphase euchromatin may be rendered dynamic by dedicated nucleosome-remodelling factors.

Women and men differ in serotonin associated psychiatric conditions, such as depression, anxiety and suicide. Despite this, very few studies focus on sex differences in the serotonin system. Of the biomarkers in the serotonin system, serotonin(1A) (5-HT(1A)) receptor is implicated in depression, and anxiety and serotonin transporter (5-HTT) is a target for selective serotonin reuptake inhibitors, psychotropic drugs used in the treatment of these disorders. The objective of the present study was to study sex related differences in the 5-HT(1A) receptor and 5-HTT binding potentials (BP(ND)s) in healthy humans, in vivo. Positron emission tomography and selective radioligands [(11)C]WAY100635 and [(11)C]MADAM were used to evaluate binding potentials for 5-HT(1A) receptors (14 women and 14 men) and 5-HTT (8 women and 10 men). The binding potentials were estimated both on the level of anatomical regions and voxel wise, derived by the simplified reference tissue model and wavelet/Logan plot parametric image techniques respectively. Compared to men, women had significantly higher 5-HT(1A) receptor and lower 5-HTT binding potentials in a wide array of cortical and subcortical brain regions. In women, there was a positive correlation between 5-HT(1A) receptor and 5-HTT binding potentials for the region of hippocampus. Sex differences in 5-HT(1A) receptor and 5-HTT BP(ND) may reflect biological distinctions in the serotonin system contributing to sex differences in the prevalence of psychiatric disorders such as depression and anxiety. The result of the present study may help in understanding sex differences in drug treatment responses to drugs affecting the serotonin system.

The medical utility of proteins, e.g. therapeutic enzymes, is greatly restricted by their labile nature and inadequate delivery. Most therapeutic enzymes do not accumulate in their targets and are inactivated by proteases. Targeting of enzymes encapsulated into substrate-permeable polymer nano-carriers (PNC) impermeable for proteases might overcome these limitations. To test this hypothesis, we designed endothelial targeted PNC loaded with catalase, an H(2)O(2)-detoxifying enzyme, and tested if this approach protects against vascular oxidative stress, a pathological process implicated in ischemia-reperfusion and other disease conditions. Encapsulation of catalase (MW 247 kD), peroxidase (MW 42 kD) and xanthine oxidase (XO, MW 300 kD) into approximately 300 nm diameter PNC composed of co-polymers of polyethylene glycol and poly-lactic/poly-glycolic acid (PEG-PLGA) was in the range approximately 10% for all enzymes. PNC/catalase and PNC/peroxidase were protected from external proteolysis and exerted enzymatic activity on their PNC diffusible substrates, H(2)O(2) and ortho-phenylendiamine, whereas activity of encapsulated XO was negligible due to polymer impermeability to the substrate. PNC targeted to platelet-endothelial cell (EC) adhesion molecule-1 delivered active encapsulated catalase to ECs and protected the endothelium against oxidative stress in cell culture and animal studies. Vascular targeting of PNC-loaded detoxifying enzymes may find wide medical applications including management of oxidative stress and other toxicities.

<h4>Objective</h4>To clarify the pathogenesis of diabetes associated with mutations of the hemochromatosis (HFE) gene, 17 carriers, 9 normal glucose tolerant (NGT) and 8 diabetic, were evaluated in an interventional trial.<h4>Research design and methods</h4>At enrollment and after a 2-year bloodletting period, euglycemic-hyperinsulinemic clamp, oral glucose tolerance test (OGTT), liver histology (nonalcoholic fatty liver disease activity score [NAS]), and liver iron content (LIC) were assessed.<h4>Results</h4>NGT subjects had significantly higher baseline insulin sensitivity (P <or= 0.001), secretion, and insulinogenic index (calculated from the OGTT) (P <or= 0.0001 for both) and lower LIC (P = 0.004) and NAS (P = 0.02) than diabetic patients. Baseline LIC correlated negatively with insulin secretion (NGT r(0) = -0.676, P <or= 0.0001; diabetes r(0) = -0.589, P = 0.02) and insulin sensitivity (M value) (NGT r(0) = -0.597, P = 0.009; diabetes r(0) = -0.535, P = 0.03) and positively with NAS (diabetes r(0) = 0.649, P = 0.007) and triglycerides (NGT r(0) = 0.563, P = 0.015). At month 24, circulating iron was reduced by 179 +/- 26% in NGT and 284 +/- 54% in diabetic subjects. Insulin secretion (NGT 20 +/- 4%; diabetes 33 +/- 7%) and insulin sensitivity (NGT 25 +/- 5%; diabetes 18 +/- 3%) increased. LIC decreased in both groups (NGT 126 +/- 42%; diabetes 61 +/- 13%), and NAS ameliorated (NGT 65.1 +/- 6.5 vs. 38.1 +/- 6.83; P <or= 0.0001; diabetes 2.1 +/- 10.7 vs. 69.9 +/- 10; P <or= 0.0001).<h4>Conclusions</h4>Iron depletion ameliorates insulin secretion and sensitivity in NGT and diabetic carriers of HFE gene mutations. This amelioration occurs in parallel with decreased LIC and improved NAS. These results justify glucose tolerance testing and prophylactic iron depletion in asymptomatic carriers as well.

Members of group E and group D of the Sox gene family function as important transcriptional regulators of glial development in the central nervous system. Here, we have examined Sox gene expression in 60 human primary gliomas. Transcripts from each of the six group E and group D genes were expressed in gliomas of various types and malignancy grades, but with significant differences. SOX5, SOX9 and SOX10 were generally expressed at levels similar to or below those in adult brain tissue. In contrast, many oligodendrogliomas exhibited upregulation of SOX6, SOX8 and SOX13. Furthermore, loss of heterozygosity on chromosomal arms 1p and 19q was associated with significantly higher SOX8 mRNA levels. Low-grade astrocytomas, but not glioblastomas, also showed elevated SOX8 transcript levels. Taken together, the expression pattern of Sox genes in gliomas is heterogeneous and overall compatible with the less differentiated state of glioma cells as compared with their normal adult counterparts. Despite their restricted expression in astrocytes and oligodendrocytes during normal development, none of the Sox genes was selectively expressed in tumours of the oligodendroglial or astrocytic lineage. This is compatible with an origin of gliomas from neuroepithelial stem or precursor cells.

We have previously demonstrated that over-expression of spike protein (S) of severe acute respiratory syndrome coronavirus (SARS-CoV) or its C-terminal subunit (S2) is sufficient to induce apoptosis in vitro. To further investigate the possible roles of S2 in SARS-CoV-induced apoptosis and pathogenesis of SARS, we characterized the host expression profiles induced upon S2 over-expression in Vero E6 cells by oligonucleotide microarray analysis. Possible activation of mitochondrial apoptotic pathway in S2 expressing cells was suggested, as evidenced by the up-regulation of cytochrome c and down-regulation of the Bcl-2 family anti-apoptotic members. Inhibition of Bcl-2-related anti-apoptotic pathway was further supported by the diminution of S2-induced apoptosis in Vero E6 cells over-expressing Bcl-xL. In addition, modulation of CCN E2 and CDKN 1A implied the possible control of cell cycle arrest at G1/S phase. This study is expected to extend our understanding on the pathogenesis of SARS at a molecular level.

No abstract available.

The short (s) variant of the serotonin transporter (5-HTT) gene linked functional polymorphic region (5-HTTLPR) is associated with depression. Stressful life events, gender, and race have been shown to moderate this association. We examined the relationship between 5-HTTLPR genotype and symptoms of depression in two samples. Study 1 = 288 participants from a study of caregiver stress; and Study 2 = 142 participants from a study examining psychosocial stressors, genetics, and health. Main effects of 5-HTTLPR on symptoms of depression were examined, along with moderation by stress (caregiving status or low childhood socioeconomic status (SES), gender, and race. The 5-HTTLPR x stress group x gender interaction was significant in both samples (P < 0.003, and P < 0.008, respectively). For females, the s allele, combined with caregiving stress (Study 1) or low childhood SES (Study 2), was associated with higher depression scores as compared to participants in the non-stressor group and those with the long (l) allele; whereas, in males, the l allele, combined with a stressor, was associated with higher depression scores as compared to those in the non-stressor group and those with the s allele. Findings from two independent samples suggest that the association of 5-HTTLPR with depression varies according to gender and stressful life events.

Mutations in the transmembrane glycoproteins transferrin receptor 2 (TfR2) and HFE are associated with hereditary hemochromatosis. Interactions between HFE and transferrin receptor 1 (TfR1) have been mapped to the alpha1- and alpha2-helices in HFE and to the helical domain of TfR1. Recently, TfR2 was also reported to interact with HFE in transfected mammalian cells. To test whether similar HFE residues are important for both TfR1 and TfR2 binding, a mutant form of HFE (W81AHFE) that has an approximately 5,000-fold lower affinity for TfR1 than HFE was employed. As expected, W81AHFE does not interact with TfR1. However, we found that the same mutation in HFE does not affect the TfR2/HFE interaction. This finding indicates that the TfR2/HFE and TfR1/HFE interactions are distinct. We further observed that, unlike TfR1/HFE, Tf does not compete with HFE for binding to TfR2 and that binding is independent of pH (pH 6-7.5). TfR2-TfR1 and HFE-HLA-B7 chimeras were generated to map the domains of the TfR2/HFE interaction. TfR1 and HLA-B7 were chosen because of their similar overall structures with TfR2 and HFE, respectively. We mapped the interacting domains to the putative stalk and protease-like domains of TfR2 located between residues 104 and 250 and to the alpha3 domain of HFE, both of which differ from the TfR1/HFE interacting domains. Furthermore, we found that HFE increases TfR2 levels in hepatic cells independent of holo-Tf.

Topoisomerase II (topo II) is a major component of mitotic chromosomes, and its unique decatenating activity has been implicated in many aspects of chromosome dynamics, of which chromosome segregation is the most seriously affected by loss of topo II activity in living cells. There is considerable evidence that topo II plays a role at the centromere including: the centromere-specific accumulation of topo II protein; cytogenetic/molecular mapping of the catalytic activity of topo II to active centromeres; the influence of sumoylated topo II on sister centromere cohesion; and its involvement in the activation of a Mad2-dependent spindle checkpoint. By using a human cell line with a conditional-lethal mutation in the gene encoding DNA topoisomerase IIalpha, we find that depletion of topo IIalpha, while leading to a disorganised metaphase plate, does not have any overt effect on general assembly of kinetochores. Fluorescence in situ hybridisation suggested that centromeres segregate normally, most segregation errors being chromatin bridges involving longer chromosome arms. Strikingly, a linear human X centromere-based minichromosome also displayed a significantly increased rate of missegregation. This sensitivity to depletion of topo IIalpha might be linked to structural alterations within the centromere domain, as indicated by a significant shortening of the distance across metaphase sister centromeres and the abnormal persistence of PICH-coated connections between segregating chromatids.

We study the striatal susceptibility to NMDA receptor (NMDAR)-mediated injury of two Huntington's disease (HD) transgenic mice: R6/1 and R6/1:BDNF(+/-). We found that R6/1:BDNF(+/-) mice--which express reduced levels of BDNF--were more resistant than R6/1 mice to intrastriatal injection of quinolinate. This increased resistance is related to a differential reduction in expression of NMDAR scaffolding proteins, MAGUKs (PSD-95, PSD-93, SAP-102 and SAP-97) but not to altered levels or synaptic location of NMDAR. A robust reorganization of postsynaptic density (PSD) was detected in HD transgenic mice, shown by a switch of PSD-93 by PSD-95 in PSD. Furthermore, NMDAR signaling pathways were affected by different BDNF levels in HD mice; we found a reduction of synaptic alpha CaMKII (but not of nNOS) in R6/1:BDNF(+/-) compared to R6/1 mice. The specific regulation of MAGUKs and alpha CaMKII in striatal neurons may reflect a protective mechanism against expression of mutant huntingtin exon-1.

The morphological events forming the body's musculature are sensitive to genetic and environmental perturbations with high incidence of congenital myopathies, muscular dystrophies and degenerations. Pattern formation generates branching series of states in the genetic regulatory network. Different states of the network specify pre-myogenic progenitor cells in the head and trunk. These progenitors reveal their myogenic nature by the subsequent onset of expression of the master switch gene MyoD and/or Myf5. Once initiated, the myogenic progression that ultimately forms mature muscle appears to be quite similar in head and trunk skeletal muscle. Several genes that are essential in specifying pre-myogenic progenitors in the trunk are known. Pax3, Lbx1, and a number of other homeobox transcription factors are essential in specifying pre-myogenic progenitors in the dermomyotome, from which the epaxial and hypaxial myoblasts, which express myogenic regulatory factors (MRFs), emerge. The proteins involved in specifying pre-myogenic progenitors in the head are just beginning to be discovered and appear to be distinct from those in the trunk. The homeobox gene Pitx2, the T-box gene Tbx1, and the bHLH genes Tcf21 and Msc encode transcription factors that play roles in specifying progenitor cells that will give rise to branchiomeric muscles of the head. Pitx2 is expressed well before the onset of myogenic progression in the first branchial arch (BA) mesodermal core and is essential for the formation of first BA derived muscle groups. Anterior-posterior patterning events that occur during gastrulation appear to initiate the Pitx2 expression domain in the cephalic and BA mesoderm. Pitx2 therefore contributes to the establishment of network states, or kernels, that specify pre-myogenic progenitors for extraocular and mastication muscles. A detailed understanding of the molecular mechanisms that regulate head muscle specification and formation provides the foundation for understanding congenital myopathies. Current technology and mouse model systems help to elucidate the molecular basis on etiology and repair of muscular degenerative diseases.

<h4>Background</h4>A polymorphism in the serotonin transporter (5-HTT) gene modulates the association between adverse early experiences and risk for major depression in adulthood. Although human imaging studies have begun to elucidate the neural circuits involved in the 5-HTT x environment risk factor, a molecular understanding of this phenomenon is lacking. Such an understanding might help to identify novel targets for the diagnosis and therapy of mood disorders. To address this need, we developed a gene-environment screening paradigm in the mouse.<h4>Methods</h4>We established a mouse model in which a heterozygous null mutation in 5-HTT moderates the effects of poor maternal care on adult anxiety and depression-related behavior. Biochemical analysis of brains from these animals was performed to identify molecular substrates of the gene, environment, and gene x environment effects.<h4>Results</h4>Mice experiencing low maternal care showed deficient gamma-aminobutyric acid-A receptor binding in the amygdala and 5-HTT heterozygous null mice showed decreased serotonin turnover in hippocampus and striatum. Strikingly, levels of brain-derived neurotrophic factor (BDNF) messenger RNA in hippocampus were elevated exclusively in 5-HTT heterozygous null mice experiencing poor maternal care, suggesting that developmental programming of hippocampal circuits might underlie the 5-HTT x environment risk factor.<h4>Conclusions</h4>These findings demonstrate that serotonin plays a similar role in modifying the long-term behavioral effects of rearing environment in diverse mammalian species and identifies BDNF as a molecular substrate of this risk factor.

<h4>Background</h4>We evaluated the magnitude of the reported associations between amygdala activation and the serotonin transporter gene linked polymorphic region (5-HTTLPR) and the likely effect size of this relationship.<h4>Methods</h4>We used meta-analytic techniques to combine data from existing published and unpublished studies. We also tested for possible publication bias and explored possible moderating influences on any association, such as sample ancestry.<h4>Results</h4>Our results provide support for the association of the 5-HTTLPR polymorphism and amygdala activation and suggest that this locus may account for up to 10% of phenotypic variance. Although we did not observe evidence for potential publication bias in our main analysis, this was due in part to efforts to obtain unpublished data pertinent to this meta-analysis, and when three unpublished data sets were excluded we did observe evidence of such bias. We also observed evidence that the first published study may provide an overestimate of the true effect size, which is consistent with findings from genetic association studies of other phenotypes.<h4>Conclusions</h4>Although our analysis provides support for the association of the 5-HTTLPR polymorphism and amygdala activation, it also suggests that most studies to date are nevertheless lacking in statistical power. Increasing the sample sizes of future imaging genetics studies will allow a more accurate characterization of any true effect size and afford adequate power to examine the impact of multiple polymorphisms that likely work in concert to affect gene function and, in turn, bias neural processes mediating dispositional traits such as temperament and personality.

A 23-year-old man with an elevated blood hemoglobin was found to have hemoglobin (Hb) Johnstown, a high oxygen-affinity hemoglobin and an elevated red cell hemoglobin content. The hemoglobin mutation was present in all family members who consented to molecular investigation. No elevation of the erythropoietin level was found in the carriers. A high ferritin level was observed in one family member. When carriers of the hemoglobin mutation were analyzed for mutations in the HFE-genes (C282Y, H63D and S65C), only the individual with the high ferritin level was a compound heterozygous for the H63D/S65C genotype. This genotype normally does not confer the hemochromatosis phenotype but may contribute to iron overload when present in an individual with increased hemoglobin synthesis. The original report of this hemoglobin variant was in the United States. Additional descriptions followed from Spain and Argentina. The family in this report is the first described in Central Europe carrying the beta109 (Val-->Leu) mutation.

Diminished expression of the metastasis suppressor protein RKIP was previously reported in a number of cancers. The underlying mechanism remains unknown. Here, we show that the expression of RKIP negatively correlates with that of Snail zinc-transcriptional repressor, a key modulator of normal and neoplastic epithelial-mesenchymal transition (EMT) program. With a combination of loss-of-function and gain-of-function approaches, we showed that Snail repressed the expression of RKIP in metastatic prostate cancer cell lines. The effect of Snail on RKIP was on the level of transcriptional initiation and mediated by a proximal E-box on the RKIP promoter. Our results therefore suggest that RKIP is a novel component of the Snail transcriptional regulatory network important for the progression and metastasis of cancer.

To identify genomic abnormalities characteristic of pancreatic ductal adenocarcinoma (PDAC) in vivo, a panel of 27 microdissected PDAC specimens were analysed using high-density microarrays representing approximately 116 000 single nucleotide polymorphism (SNP) loci. We detected frequent gains of 1q, 2, 3, 5, 7p, 8q, 11, 14q and 17q (> or =78% of cases), and losses of 1p, 3p, 6, 9p, 13q, 14q, 17p and 18q (> or =44%). Although the results were comparable with those from array CGH, regions of those genetic changes were defined more accurately by SNP arrays. Integrating the Ensembl public data, we have generated 'gene' copy number indices that facilitate the search for novel candidates involved in pancreatic carcinogenesis. Copy numbers in a subset of the genes were validated using quantitative real-time PCR. The SKAP2/SCAP2 gene (7p15.2), which belongs to the src family kinases, was most frequently (63%) amplified in our sample set and its recurrent overexpression (67%) was confirmed by reverse transcription-PCR. Furthermore, fluorescence in situ hybridization and in situ RNA hybridization analyses for this gene have demonstrated a significant correlation between DNA copy number and mRNA expression level in an independent sample set (P<0.001). These findings indicate that the dysregulation of SKAP2/SCAP2, which is mostly caused by its increased gene copy number, is likely to be associated with the development of PDAC.

We have recently published the precise definition of an aminoterminal membrane association domain in huntingtin, capable of targeting to the endoplasmic reticulum and late endosomes as well as autophagic vesicles. In response to ER stress induced by several pathways, huntingtin releases from membranes and rapidly translocates into the nucleus. Huntingtin is then capable of nuclear export and re-association with the ER in the absence of stress. This release is inhibited when huntingtin contains the polyglutamine expansion seen in Huntington's disease. As a result, mutant huntingtin expressing cells have a perturbed ER and an increase in autophagic vesicles. Here, we discuss the potential function of the huntingtin protein as an ER sentinel, potentially regulating autophagy in response to ER stress. We compare these recent findings to the well characterized mammalian target of rapamycin, mTor, a protein described over a decade ago as related to huntingtin structurally by leucine-rich, repetitive HEAT sequences. Since then, the described functional similarities between Huntingtin and mTor are striking, and this new information about huntingtin's direct association with autophagic vesicles indicates that this structural similarity may extend to functional similarities having an impact upon ER functionality and autophagy.

The mitochondrial DNA (mtDNA) may play an essential role in the pathogenesis of the respiratory chain complex activities in neurodegenerative disorders such as Huntington's disease (HD). Research studies were conducted to determine the possible levels of mitochondrial defect (deletion) in HD patients and consideration of interaction between the expanded Huntingtin gene as a nuclear gene and mitochondria as a cytoplasmic organelle. To determine mtDNA damage, we investigated deletions based in four areas of mitochondrial DNA, in a group of 60 Iranian patients clinically diagnosed with HD and 70 healthy controls. A total of 41 patients out of 60 had CAG expansion (group A). About 19 patients did not show expansion but had the clinical symptoms of HD (group B). MtDNA deletions were classified into four groups according to size; 9 kb, 7.5 kb, 7 kb, and 5 kb. We found one of the four-mtDNA deletions in at least 90% of samples. Multiple deletions have also been observed in 63% of HD patients. None of the normal control (group C) showed mtDNA deletions. The sizes or locations of the deletions did not show a clear correlation with expanded CAG repeat and age in our samples. The study presented evidence that HD patients had higher frequencies of mtDNA deletions in lymphocytes in comparison to the controls. It is thus proposed that CAG repeats instability and mutant Htt are causative factor in mtDNA damage.

Aberrant protein aggregation and misfolding are key pathological features of many neurodegenerative disorders, including Huntington's and Parkinson's diseases. Compounds that offer protection from toxicity associated with aggregation-prone neurodegenerative proteins may have applications for the treatment of a multitude of disorders. A high-throughput bioassay system with parallel electrospray ionization mass spectrometry screening has been designed for critical evaluation of milligram quantities of natural product extracts, including dietary substances, for compounds of pharmacological relevance to the treatment of human neurodegenerative diseases. Using Saccharomyces cerevisiae strains engineered to express mutant human huntingtin and alpha-synuclein, we are able to identify extracts and compounds that protect cells from toxicity associated with these proteins. Applying this screening paradigm, we determined that a bioactive green tea extract contains an assemblage of catechins that were individually characterized for their respective protective effects against huntingtin and alpha-synuclein toxicity.

<h4>Background</h4>Hereditary hemochromatosis (HH) encompasses genetic disorders of iron overload characterized by deficient expression or function of the iron-regulatory hormone hepcidin. Mutations in 5 genes have been linked to this disease: HFE, TFR2 (encoding transferrin receptor 2), HAMP (encoding hepcidin), SLC40A1 (encoding ferroportin) and HJV (encoding hemojuvelin). Hepcidin inhibits iron export from cells into plasma. Hemojuvelin, an upstream regulator of hepcidin expression, is expressed in mice mainly in the heart and skeletal muscle. It has been suggested that soluble hemojuvelin shed by the muscle might reach the liver to influence hepcidin expression. Heart muscle is one of the target tissues affected by iron overload, with resultant cardiomyopathy in some HH patients. Therefore, we investigated the effect of iron overload on gene expression in skeletal muscle and heart using Illuminatrade mark arrays containing over 47,000 probes. The most apparent changes in gene expression were confirmed using real-time RT-PCR.<h4>Results</h4>Genes with up-regulated expression after iron overload in both skeletal and heart muscle included angiopoietin-like 4, pyruvate dehydrogenase kinase 4 and calgranulin A and B. The expression of transferrin receptor, heat shock protein 1B and DnaJ homolog B1 were down-regulated by iron in both muscle types. Two potential hepcidin regulatory genes, hemojuvelin and neogenin, showed no clear change in expression after iron overload.<h4>Conclusion</h4>Microarray analysis revealed iron-induced changes in the expression of several genes involved in the regulation of glucose and lipid metabolism, transcription and cellular stress responses. These may represent novel connections between iron overload and pathological manifestations of HH such as cardiomyopathy and diabetes.

<h4>Background</h4>New genetic forms of hereditary hemochromatosis (HH) or hereditary hyperferritinemia (HF) have been identified over the last few years, and abnormalities of various genes may interact in a single patient. This study aimed to develop a rapid automated method for sequencing the main genes involved.<h4>Methods</h4>We used a standard 96-well microplate with a single PCR condition in an adaptation of the SCAIP (single-condition amplification with internal primer) method to sequence the HFE (hemochromatosis), HAMP (hepcidin antimicrobial peptide), HFE2/HJV [hemochromatosis type 2 (juvenile)], SLC40A1 (ferroportin), and TFR2 (transferrin receptor 2) genes, and the 5' untranslated region of the FTL (ferritin, light polypeptide) gene. To further simplify the method, we adjusted PCR conditions to avoid the use of an internal primer and applied this single-condition amplification method to 38 selected, unrelated patients. We tailored the genetic investigation according to the clinical picture, with the patients falling into 2 groups. Group 1 consisted of patients with hyperferritinemia and high transferrin saturation (TS) (classic adult and juvenile HH forms, groups 1A and 1B, respectively), and group 2 consisted of patients with hyperferritinemia and low, typical, or slightly increased TS, with or without iron overload (groups 2A and 2B, respectively).<h4>Results</h4>With this strategy we identified single-gene and multigene abnormalities, including 6 previously undescribed abnormalities in HFE (c.794dupA), HFE2 (c.-89-4dupT), and SLC40A1 (c.262A>G, c.533G>A, c.1468G>A, and c.-59_-45del).<h4>Conclusion</h4>This method is a simple approach for investigating hereditary iron overload or HF and allows rapid evaluation of patients.

Expansion of polymorphic glutamine (Q) numbers present at the protein Huntingtin (Htt) beyond 36Q results in its misfolding and aggregation, and the aggregates recruit several other proteins. Here we show that HYPK, initially identified as an Htt-interacting partner by yeast two-hybrid assay, physically interacts with N-terminal Htt in Neuro2A cells and alters the numbers and distribution of aggregates formed by N-terminal Htt with 40Q. HYPK also alters the kinetics of mutated N-terminal Htt-mediated aggregate formation. Fluorescence recovery after photobleaching studies reveal that over-expression of HYPK results in the appearance of Htt poly Q aggregates, which upon bleaching recovers approximately 80% of initial fluorescence intensity within 6 min. Fluorescence loss in photobleaching studies indicate loss off fluorescence intensity of the aggregates with time in presence of HYPK. Over-expression of this protein reduces poly Q-mediated caspase-2, caspase-3 and caspase-8 activations, whereas gamma ray-induced activations of these enzymes are not affected. In vitro and in vivo studies demonstrate that HYPK possesses a novel chaperone-like activity. We conclude that HYPK, without having any sequence similarity with known chaperones, plays an effective role in protecting neuronal cells against apoptosis induced by mutated N-terminal Htt by modulating the aggregate formation.

Huntington's disease (HD) is an autosomal dominant, late onset, neurodegenerative disease characterized by motor deficits and dementia that is caused by expansion of a CAG repeat in the HD gene. Clinical manifestations result from selective neuronal degeneration of predominantly GABAergic striatal medium-sized spiny neurons (MSNs). A growing number of studies demonstrate that personality, mood and cognitive disturbances are some of the earliest signs of HD and may reflect synaptic dysfunction prior to neuronal loss. Previous studies in striatal MSNs demonstrated early alterations in NMDA-type glutamate receptor currents in several HD mouse models, as well as evidence for presynaptic dysfunction prior to disease manifestations in the R6/2 HD fragment mouse model. We have compared corticostriatal synaptic function in full-length, human HD gene-carrying YAC transgenic mice expressing a non-pathogenic CAG repeat (YAC18; control) with three increasingly severe variants of pathogenic HD gene-expressing mice (YAC72 and two different lines of YAC128), at ages that precede any detectable disease phenotype. We report presynaptic dysfunction and a propensity towards synaptic depression in YAC72 and YAC128 compared to YAC18 mice, and, in the most severe model, we also observed altered AMPA receptor function. When normalized to evoked AMPAR currents, postsynaptic NMDAR currents are augmented in all three pathogenic HD YAC variants. These findings demonstrate multiple perturbations to corticostriatal synaptic function in HD mice, furthering our understanding of the early effects of the HD mutation that may contribute to cognitive dysfunction, mood disorders and later development of more serious dysfunction. Furthermore, this study provides a set of neurophysiological sequelae against which to test and compare other mouse models and potential therapies in HD.

In Caenorhabditis elegans, sex is determined by the opposing actions of X-signal elements (XSEs) and autosomal signal elements (ASEs), which communicate the ratio of X chromosomes to sets of autosomes (X:A signal). This study delves more deeply into the mechanism by which XSEs transmit X chromosome dose. We determined the relative contributions of individual XSEs to the X:A signal and showed the order of XSE strength to be sex-1 > sex-2 > fox-1 > ceh-39 >/= region 1 XSE. sex-1 exerts a more potent influence on sex determination and dosage compensation than any other XSE by functioning in two separate capacities in the pathway: sex-1 acts upstream as an XSE to repress xol-1 and downstream as an activator of hermaphrodite development and dosage compensation. Furthermore, the process of dosage compensation affects expression of the very XSEs that control it; XSEs become fully dosage compensated once sex is determined. The X:A signal is then equivalent between XO and XX animals, causing sexual differentiation to be controlled by genes downstream of xol-1 in the sex-determination pathway. Prior to the onset of dosage compensation, the difference in XSE expression between XX and XO embryos appears to be greater than twofold, making X chromosome counting a robust process.

<h4>Objective</h4>High mobility group box 1 (HMGB1) is an important factor in the development of sepsis. Previous work suggests that antithrombin III (ATIII) inhibits inflammation, but the mechanism of action is still poorly understood.<h4>Design and setting</h4>Prospective controlled animal study in a university laboratory.<h4>Materials</h4>Rats were randomly divided into a lipopolysaccharide (LPS)-induced sepsis control group and an ATIII-treated experimental group. Animals in the experimental group received a bolus of 250 units/kg of ATIII injected into the tail vein.<h4>Measurements and results</h4>Animals receiving high-dose ATIII (250 units/kg) had significantly improved lung histopathology and survival compared to the control rats. We measured serum and lung levels of various cytokines and HMGB1 at regular intervals from 0 to 12 h after the induction of sepsis and demonstrated lower HMGB1 levels over time in ATIII-treated animals. In an in vitro experiment, we stimulated the mouse macrophage cell line RAW 264.7 with LPS in the presence or absence of ATIII. Subsequent measurement of HMGB1 concentrations in the supernatant and cell signaling molecules in cell lysates revealed an ATIII dose-dependent decrease in HMGB1 release. Furthermore, inhibition of IkB and p42 phosphorylation was observed with the administration of ATIII, suggestive of downstream signaling pathways.<h4>Conclusions</h4>High-dose ATIII decreases lung pathology and reduces mortality in a rat sepsis model. This finding may be mediated by the inhibition of HMGB1.

Huntington disease (HD) is a neurodegenerative disorder due to an excessive number of CAG repeats in the IT15 gene on chromosome 4. Studies of cognitive function in asymptomatic gene carriers have yielded contradictory results. This study compared cognitive performance in 44 subjects with the HD mutation (group of carriers) who had no clinical signs of HD and 39 at-risk individuals without HD mutation (group of non-carriers). Neuropsychological evaluation focused on global cognitive efficiency, psychomotor speed, attentional, executive and memory functions. Significant differences, with lower performances in the group of gene carriers, were detected for some measures of psychomotor speed, attention and executive functioning (all P < 0.01). More differences between groups were observed for memory measures, in particular on the California Verbal Memory Test. Complementing these observations, cognitive scores were correlated with age in the group of gene carriers, but not in the group of non-carriers. This suggests that the cognitive changes precede the appearance of the motor and psychiatric symptoms in HD and that tests proved to be sensitive to early HD deficiencies are better suited than global cognitive efficiency scales to observe them.

Previously we demonstrated the ability of ethyl glycinato substituted polyphosphazenes to neutralize the acidic degradation products and control the degradation rate of poly(lactic acid-glycolic acid) (PLAGA) by blending. In this study, blends of high strength poly[(50% ethyl alanato) (50% p-phenyl phenoxy) phosphazene] (PNEA(50)PhPh(50)) and 85:15 PLAGA were prepared using a mutual solvent approach. Three different solvents, methylene chloride (MC), chloroform (CF) and tetrahydrofuran (THF) were studied to investigate solvent effects on blend miscibility. Three different blends were then fabricated at various weight ratios namely 25:75 (BLEND25), 50:50 (BLEND50), and 75:25 (BLEND75) using THF as the mutual solvent. The miscibility of the blends was evaluated by scanning electron microscopy (SEM), differential scanning calorimetry (DSC), and Fourier transform infrared spectroscopy (FTIR). Among these, BLEND25 was miscible while BLEND50 and BLEND75 were partially miscible. Furthermore, BLEND25 formed apatite layers on its surface as evidenced in a biomimetic study performed. These novel blends showed cell adhesion and proliferation comparable to PLAGA. However, the PNEA(50)PhPh(50) component in the blends was able to increase the phenotypic expression and mineralized matrix synthesis of the primary rat osteoblasts (PRO) in vitro. Blends of high strength PNEA(50)PhPh(50) and 85:15 PLAGA are promising biomaterials for a variety of musculoskeletal applications.

Toxicity in amyloid diseases is intimately linked to the nature of aggregates, with early oligomeric species believed to be more cytotoxic than later fibrillar aggregates. Yet mechanistic understanding of how aggregating species evolve with time is currently lacking. We have explored the aggregation process of a chimera composed of a globular protein (cellular retinoic acid-binding protein, CRABP) and huntingtin exon 1 with polyglutamine tracts either above (Q53) or below (Q20) the pathological threshold using Escherichia coli cells as a model intracellular environment. Previously we showed that fusion of the huntingtin exon 1 sequence with >40Q led to structural perturbation and decreased stability of CRABP (Ignatova, Z., and Gierasch, L. M. (2006) J. Biol. Chem. 281, 12959-12967). Here we report that the Q53 chimera aggregates in cells via a multistep process: early stage aggregates are spherical and detergent-soluble, characteristics of prefibrillar aggregates, and appear to be dominated structurally by CRABP, in that they can promote aggregation of a CRABP variant but not oligoglutamine aggregation, and the CRABP domain is relatively sequestered based on its protection from proteolysis. Late stage aggregates appear to be dominated by polyGln; they are fibrillar, detergent-resistant, capable of seeding aggregation of oligoglutamine but not the CRABP variant, and show relative protection of the polyglutamine-exon1 domain from proteolysis. These results point to an evolution of the dominant sequences in intracellular aggregates and may provide molecular insight into origins of toxic prefibrillar aggregates.

Dissociation is defined as the disruption of the usually integrated functions of consciousness, such as memory, identity, and perceptions of the environment. Causes include various psychological, neurological and neurobiological mechanisms, none of which have been consistently supported. To our knowledge, the role of gene-environment interactions in dissociative experiences in obsessive-compulsive disorder (OCD) has not previously been investigated. Eighty-three Caucasian patients (29 male, 54 female) with a principal diagnosis of OCD were included. The Dissociative Experiences Scale was used to assess dissociation. The role of childhood trauma (assessed with the Childhood Trauma Questionnaire), and a functional 44-bp insertion/deletion polymorphism in the promoter region of the serotonin transporter, or 5-HTT, in mediating dissociation, was investigated using multiple regression analysis and path analysis using the partial least squares model. Both analyses indicated that an interaction between physical neglect and the S/S genotype of the 5-HTT gene significantly predicted dissociation in patients with OCD. Dissociation may be a predictor of poorer treatment outcome in patients with OCD; therefore, a better understanding of the mechanisms that underlie this phenomenon may be useful. Here, two different but related statistical techniques (multiple regression and partial least squares), confirmed that physical neglect and the 5-HTT genotype jointly play a role in predicting dissociation in OCD.

A variety of novel heterocyclic compounds were synthesized and evaluated for MMP inhibition. Broad spectrum inhibition of MMPs 1, 2, 9, and 12 was found with pyridinone-based compounds while N-heterocyclic triazoles and tetrazoles were largely ineffective. A highly selective tetrazole inhibitor for MMP-2 was discovered.

Huntington's disease (HD) is a neurodegenerative disorder caused by expansion of a CAG repeat in the huntingtin (Htt) gene. HD is autosomal dominant and, in theory, amenable to therapeutic RNA silencing. We introduced cholesterol-conjugated small interfering RNA duplexes (cc-siRNA) targeting human Htt mRNA (siRNA-Htt) into mouse striata that also received adeno-associated virus containing either expanded (100 CAG) or wild-type (18 CAG) Htt cDNA encoding huntingtin (Htt) 1-400. Adeno-associated virus delivery to striatum and overlying cortex of the mutant Htt gene, but not the wild type, produced neuropathology and motor deficits. Treatment with cc-siRNA-Htt in mice with mutant Htt prolonged survival of striatal neurons, reduced neuropil aggregates, diminished inclusion size, and lowered the frequency of clasping and footslips on balance beam. cc-siRNA-Htt was designed to target human wild-type and mutant Htt and decreased levels of both in the striatum. Our findings indicate that a single administration into the adult striatum of an siRNA targeting Htt can silence mutant Htt, attenuate neuronal pathology, and delay the abnormal behavioral phenotype observed in a rapid-onset, viral transgenic mouse model of HD.

A 55-year-old Japanese woman was hospitalized because liver function tests showed an abnormality. Transaminases and biliary enzymes were markedly elevated with hyperferritinemia. Her imaging tests revealed no significant abnormality. She had been taking various non-prescription supplements for over approximately 6 months. After the supplements were discontinued her liver function gradually improved. This clinical course was suggestive of supplement-induced hepatitis. She had no history of taking supplements containing iron, so it was interesting that she had hyperferritinemia. We examined C282Y and H63D, which are important mutations in theiron-metabolizing gene, HFE. She was found to be heterozygous for the H63D mutation. The interaction between hyperferritinemia and supplements is unknown, but it can be speculated that some interaction between iron overload and supplements may have underlain the pathogenesis of her liver injury.

The pathology of Alzheimer's disease (AD) begins years prior to clinical diagnosis. The development of antecedent biomarkers that indicate the presence of AD pathology and predict risk for decline in both cognitively normal and mildly impaired individuals will be useful as effective therapies are developed. While cerebrospinal fluid (CSF) markers such as amyloid-β (Aβ) 42 and tau are useful, additional biomarkers are needed. To identify new markers, we utilized 2-D difference gel electrophoresis (2-D DIGE) of individual CSF samples from subjects with very mild AD versus controls after depletion of high-abundant proteins. Protein spots displaying differential abundance between the two groups were identified with MS. A number of candidate biomarkers were identified in 18 gel features. Selected candidates were quantified in a larger clinical set using ELISA. The mean levels of α1-antichymotrypsin (ACT), antithrombin III (ATIII), and zinc-α2-glycoprotein (ZAG) were significantly higher in the mild AD group, and the mean level of carnosinase 1 (CNDP1) was decreased. When these biomarkers are optimally combined, there is a strong trend toward greater specificity and sensitivity based on clinical diagnosis than when used individually. Our findings provide novel biomarker candidates for very mild and mild AD that can be further assessed as antecedent markers and predictors of clinical progression.

<h4>Background</h4>The retinal vasculature is a capillary network of blood vessels that nourishes the inner retina of most mammals. Developmental abnormalities or microvascular complications in the retinal vasculature result in severe human eye diseases that lead to blindness. To exploit the advantages of zebrafish for genetic, developmental and pharmacological studies of retinal vasculature, we characterised the intraocular vasculature in zebrafish.<h4>Results</h4>We show a detailed morphological and developmental analysis of the retinal blood supply in zebrafish. Similar to the transient hyaloid vasculature in mammalian embryos, vessels are first found attached to the zebrafish lens at 2.5 days post fertilisation. These vessels progressively lose contact with the lens and by 30 days post fertilisation adhere to the inner limiting membrane of the juvenile retina. Ultrastructure analysis shows these vessels to exhibit distinctive hallmarks of mammalian retinal vasculature. For example, smooth muscle actin-expressing pericytes are ensheathed by the basal lamina of the blood vessel, and vesicle vacuolar organelles (VVO), subcellular mediators of vessel-retinal nourishment, are present. Finally, we identify 9 genes with cell membrane, extracellular matrix and unknown identity that are necessary for zebrafish hyaloid and retinal vasculature development.<h4>Conclusion</h4>Zebrafish have a retinal blood supply with a characteristic developmental and adult morphology. Abnormalities of these intraocular vessels are easily observed, enabling application of genetic and chemical approaches in zebrafish to identify molecular regulators of hyaloid and retinal vasculature in development and disease.

Mitotic chromatin condensation is essential for cell division in eukaryotes. Posttranslational modification of the N-terminal tail of histone proteins, particularly by phosphorylation by mitotic histone kinases, may facilitate this process. In mammals, aurora B is believed to be the mitotic histone H3 Ser10 kinase; however, it is not sufficient to phosphorylate H3 Ser10 with aurora B alone. We show that histone H3 is phosphorylated by vaccinia-related kinase 1 (VRK1). Direct phosphorylation of Thr3 and Ser10 in H3 by VRK1 both in vitro and in vivo was observed. Loss of VRK1 activity was associated with a marked decrease in H3 phosphorylation during mitosis. Phosphorylation of Ser10 by VRK1 is similar to that by aurora B. Moreover, expression and chromatin localization of VRK1 depended on the cell cycle phase. Overexpression of VRK1 resulted in a dramatic condensation of nuclei. Our findings collectively support a role of VRK1 as a novel mitotic histone H3 kinase in mammals.

The differential susceptibility of inbred mouse strains DBA/2J (susceptible) and C57BL/6J (resistant) to pulmonary tuberculosis following aerosol infection is under complex genetic control. In this report, transcriptional profiling with RNAs from Mycobacterium tuberculosis-infected lungs was used to investigate the physiological response, cell type, and biochemical pathways underlying differential susceptibility to infection. Statistical analysis of cDNA-based microarrays revealed that 1,097 transcripts showed statistically significant changes in abundance (changes of > or = 1.5-fold) in at least one of four experimental group comparisons (C57BL/6J [day 0] versus DBA/2J [day 0] mice, C57BL/6J [day 90] versus DBA/2J [day 90] mice, C57BL/6J [day 90] versus C57BL/6J [day 0] mice, or DBA/2J [day 90] versus DBA/2J [day 0] mice). A group of genes showing very high degrees of significance (changes of > or = 2.0-fold) displayed enrichment for transcripts associated with tissue remodeling and the fibrotic response. The differential expression of fibrotic response genes (Sparc, Col1a1, Col1a2, Col4a1, and Col4a2) in the infected lungs of the two mouse strains was validated by another microarray platform (Affymetrix oligonucleotide chips) and by reverse transcription-PCR. Furthermore, the differential expression of additional genes known to be associated with fibrosis (Mmp2, Timp1, and Arg1) was also validated by these approaches. Overall, these results identify the differential fibrotic response as a pathological basis for the high susceptibility of DBA/2J mice to pulmonary tuberculosis.

<h4>Aims/hypothesis</h4>Glucose-stimulated insulin secretion is dependent on the electrical activity of beta cells; hence, genes encoding beta cell ion channels are potential candidate genes for type 2 diabetes. The gene encoding the voltage-dependent Ca(2+) channel Ca(V)2.3 (CACNA1E), telomeric to a region that has shown suggestive linkage to type 2 diabetes (1q21-q25), has been ascribed a role for second-phase insulin secretion.<h4>Methods</h4>Based upon the genotyping of 52 haplotype tagging single nucleotide polymorphisms (SNPs) in a type 2 diabetes case-control sample (n = 1,467), we selected five SNPs that were nominally associated with type 2 diabetes and genotyped them in the following groups (1) a new case-control sample of 6,570 individuals from Sweden; (2) 2,293 individuals from the Botnia prospective cohort; and (3) 935 individuals with insulin secretion data from an IVGTT.<h4>Results</h4>The rs679931 TT genotype was associated with (1) an increased risk of type 2 diabetes in the Botnia case-control sample [odds ratio (OR) 1.4, 95% CI 1.0-2.0, p = 0.06] and in the replication sample (OR 1.2, 95% CI 1.0-1.5, p = 0.01 one-tailed), with a combined OR of 1.3 (95% CI 1.1-1.5, p = 0.004 two-tailed); (2) reduced insulin secretion [insulinogenic index at 30 min p = 0.02, disposition index (D (I)) p = 0.03] in control participants during an OGTT; (3) reduced second-phase insulin secretion at 30 min (p = 0.04) and 60 min (p = 0.02) during an IVGTT; and (4) reduced D (I) over time in the Botnia prospective cohort (p = 0.05).<h4>Conclusions/interpretation</h4>We conclude that genetic variation in the CACNA1E gene contributes to an increased risk of the development of type 2 diabetes by reducing insulin secretion.

Intestinal disorders such as inflammatory bowel disease (IBD) result in chronic illness requiring lifelong therapy. Our aim was to evaluate the efficacy of recombinant adeno-associated virus (AAV) vector-mediated gene delivery to intestinal epithelial cells in vitro and in vivo. Human colon epithelial cell lines and colon biopsies were transduced using AAV pseudotypes 2/1, 2/2, and 2/5 encoding green fluorescence protein (GFP). Mice were administered the same vectors through oral, enema, intraperitoneal (IP) injection and superior mesenteric artery (SMA) injection routes. Tropism and efficiency were determined by microscopy, flow cytometry, immunohistochemistry and PCR. Caco2 cells were more permissive to AAV transduction. Human colon epithelial cells in organ culture were more effectively transduced by AAV2/2. SMA injection provided the most effective means of vector gene transfer to small intestine and colonic epithelial cells in vivo. Transgene detection 80 days post AAV treatment suggests transduction of crypt progenitor cells. This study shows the feasibility of AAV-mediated intestinal gene delivery, applicable for the investigation of IBD pathogenesis and novel therapeutic options, but also revealed the need for further studies to identify more efficient pseudotypes.

The neurofibromatosis 2 (NF2) tumor suppressor protein merlin is commonly mutated in human benign brain tumors. The gene altered in NF2 was located on human chromosome 22q12 in 1993 and the encoded protein named merlin and schwannomin. Merlin has homology to ERM family proteins, ezrin, radixin, and moesin, within the protein 4.1 superfamily. In efforts to determine merlin function several groups have discovered 34 merlin interacting proteins, including ezrin, radixin, moesin, CD44, layilin, paxillin, actin, N-WASP, betaII-spectrin, microtubules, TRBP, eIF3c, PIKE, NHERF, MAP, RalGDS, RhoGDI, EG1/magicin, HEI10, HRS, syntenin, caspr/paranodin, DCC, NGB, CRM1/exportin, SCHIP1, MYPT-1-PP1delta, RIbeta, PKA, PAK (three types), calpain and Drosophila expanded. Many of the proteins that interact with the merlin N-terminal domain also bind ezrin, while other merlin interacting proteins do not bind other members of the ERM family. Merlin also interacts with itself. This review describes these proteins, their possible roles in NF2, and the resultant hypothesized merlin functions. Review of all of the merlin interacting proteins and functional consequences of losses of these interactions reveals multiple merlin actions in PI3-kinase, MAP kinase and small GTPase signaling pathways that might be targeted to inhibit the proliferation of NF2 tumors.

Several studies suggest that the histocompatibility complex (HLA) class I region harbours genes modulating multiple sclerosis (MS) susceptibility independently from the effect of class II alleles. A candidate gene in this region is MOG, encoding the myelin oligodendrocyte glycoprotein. A significant association with the missense variation V142L (rs2857766) was previously reported in a small sample of 50 Italian MS patients. We confirmed this result in two independent Italian sample sets consisting of 878 MS patients and 890 matched controls (P=6.6 x 10(-4)) and 246 trio families (P=1.5 x 10(-3)). The comparison of genotype frequencies suggested a dominant-protective effect of L142. In the combined sample sets L142 conferred an odds ratio (OR)=0.70 (95% confidence interval (CI): 0.60-0.82) that remained similar after accounting for HLA-DRB1(*)15 carrier status. The association with MOG V142L was still significant after conditioning for all DRB1 alleles (P=0.035). Eleven additional single nucleotide polymorphisms in the MOG gene (namely -1077T/C, -910T/C, -875A/G, -93T/C, S5S, Indel L22, V145I, +814C/T, +900A/G, +1024A/T, +1059C/T), two microsatellites in the MOG 5' flanking (MOGCA) and 3' untranslated (MOGTAAA) regions and four microsatellites in the HLA-class I region, from HLA-B to HFE, (namely MIB, D6S265, D6S1683 and D6S2239) were tested by transmission disequilibrium test in 199 trio families. None of these polymorphisms or of their haplotypic combinations showed a significant transmission distortion, in the absence of V142L. In conclusion, MOG V142L, or an untested variant in tight-linkage disequilibrium with it, is an independent MS susceptibility-modulating factor in the HLA class I region.

N-Hydroxysuccinimide (NHS) esters are widely used as leaving groups to activate covalent coupling of amine-containing biomolecules onto surfaces in academic and commercial surface immobilizations. Their intrinsic hydrolytic instability is well-known and remains a concern for maintaining stable, reactive surface chemistry, especially for reliable longer term storage. In this work, we use X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry (TOF-SIMS) to investigate surface hydrolysis in NHS-bearing organic thin films. Principal component analysis (PCA) of both positive and negative ion TOF-SIMS data was used to correlate changes in the well-defined NHS ester oligo(ethylene glycol) (NHS-OEG) self-assembled monolayers to their surface treatment. From PCA results, multivariate peak intensity ratios were developed for monitoring NHS reactivity, thin-film thickness, and oxidation of the monolayers during surface hydrolysis. Aging in ambient air for up to 7 days resulted in hydrolysis of some fraction of bound NHS groups, oxidation of some resident thiol groups, and deposition of adventitious hydrocarbon contaminants onto the monolayers. Overnight film immersion under water produced complete hydrolysis and removal of the NHS chemistry, as well as removal of some of the thiolated OEG chains. NHS regeneration of the hydrolyzed surfaces was assessed using the same multivariable peak intensity ratio as well as surface coupling with amine-terminated molecules. Both aqueous and organic NHS regeneration methods produced surfaces with bound NHS concentrations approximately 50% of the bound NHS concentration on freshly prepared NHS-OEG monolayers. Precise methods for quantifying NHS chemistry on surfaces are useful for quality control processes required in surface technologies that rely on reliable and reproducible reactive ester coupling. These applications include microarray, microfluidic, immunoassay, bioreactor, tissue engineer-ing, and biomedical device fabrication.

<h4>Rationale</h4>Not all family members with BMPR2 mutations develop pulmonary arterial hypertension (PAH), implying that additional modifier genes or proteins are necessary for full expression of the disease.<h4>Objectives</h4>To determine whether protein expression is altered in patients with familial PAH (FPAH) compared with obligate carriers and nondiseased control subjects.<h4>Methods</h4>Protein extracts from transformed blood lymphocytes from four patients with FPAH, three obligate carriers, and three married-in control subjects from one family with a known BMPR2 mutation (exon 3 T354G) were labeled with either Cy3 or Cy5. Cy3/5 pairs were separated by standard two-dimensional differential gel electrophoresis using a Cy2-labeled internal standard of all patient samples. Log volume ratios were analyzed using a linear mixed-effects model. Proteins were identified by matrix-assisted laser desorption ionization, time-of-flight mass spectrometry (MALDI-TOF MS) and tandem TOF/TOF MS/MS.<h4>Measurements and main results</h4>Hierarchical clustering, heat-map, and principal components analysis revealed marked changes in protein expression in patients with FPAH when compared with obligate carriers. Significant changes were apparent in expression of 16 proteins (P < 0.05) when affected patients were compared with obligates: nine showed a significant increase and seven showed a significant reduction.<h4>Conclusions</h4>A series of novel proteins with altered expression were found that could distinguish affected patients from obligate carriers and married-in controls in a single family with a BMPR2 mutation. These differences provide new information highlighting proteins that may be involved in the mechanism(s) that differentiates those individuals with a BMPR2 mutation who develop FPAH from those who do not.

While initially thought to be essentially a developmental characteristic, observed in artificial in vitro models, local protein synthesis in growth cones has been described in the adult, and more interestingly, during nerve regeneration. This emerging field is under intense investigation, revealing new functions of localized protein synthesis that include axon guidance, growth cone adaptation and sensitivity modulation at intermediate targets or axon regeneration. Here, we will review these functions and provide a short survey of the current knowledge on mechanisms of mRNA transport and regulation of localized protein synthesis. In addition, we will consider what lessons can be learned from localized protein synthesis in dendrites, and what developments can be expected next in the field. This latter question relates to the crucial point of which technical strategy to adopt for an ideal and pertinent analysis of the phenomenon.

Netrin-1 is a guidance cue molecule fundamental to the organization of neuronal connectivity during development. Netrin-1 and its receptors, deleted in colorectal cancer (DCC) and UNC-5 homologues (UNC-5), continue to be expressed in the adult brain, although neither their function nor the kinds of events that activate their expression are known. Two lines of evidence suggest a role for netrin-1 in amphetamine-induced dopamine plasticity in the adult. First, DCC is highly expressed by adult dopamine neurons. Second, adult mice with reduced DCC levels do not develop amphetamine-induced behavioral sensitization. To explore the role of netrin-1 in amphetamine-induced plasticity, we examined the effects of sensitizing treatment regimens of amphetamine on DCC and/or UNC-5 protein expression in the adult rat. These treatments produced striking and enduring increases in DCC and UNC-5 expression in the cell body, but not terminal regions, of the mesocorticolimbic dopamine system. Notably, neuroadaptations in the cell body region of mesocorticolimbic dopamine neurons underlie the development of sensitization to the effects of amphetamine. Furthermore, these localized amphetamine-induced changes were prevented by co-treatment with an N-methyl-d-aspartate receptor antagonist, a treatment known to block the development of amphetamine-induced sensitization of behavioral activation, dopamine release and motivated behavior. Using immunohistochemistry, we showed that both DCC and UNC-5 receptors are highly expressed by adult mesocorticolimbic dopamine neurons. These results provide the first evidence that repeated exposure to a stimulant drug such as amphetamine affects netrin-1 receptor expression in the adult brain. Taken together, our findings suggest that changes in netrin-1 receptor expression may play a role in the lasting effects of exposure to amphetamine and other stimulant drugs.

Activation of the nuclear factor (NF)-kappaB signaling pathway may be associated with the development of cardiac hypertrophy and its transition to heart failure (HF). The transgenic Myo-Tg mouse develops hypertrophy and HF as a result of overexpression of myotrophin in the heart associated with an elevated level of NF-kappaB activity. Using this mouse model and an NF-kappaB-targeted gene array, we first determined the components of NF-kappaB signaling cascade and the NF-kappaB-linked genes that are expressed during the progression to cardiac hypertrophy and HF. Second, we explored the effects of inhibition of NF-kappaB signaling events by using a gene knockdown approach: RNA interference through delivery of a short hairpin RNA against NF-kappaB p65 using a lentiviral vector (L-sh-p65). When the short hairpin RNA was delivered directly into the hearts of 10-week-old Myo-Tg mice, there was a significant regression of cardiac hypertrophy, associated with a significant reduction in NF-kappaB activation and atrial natriuretic factor expression. Our data suggest, for the first time, that inhibition of NF-kappaB using direct gene delivery of sh-p65 RNA results in regression of cardiac hypertrophy. These data validate NF-kappaB as a therapeutic target to prevent hypertrophy/HF.

Huntington's disease (HD) is a progressive neurodegenerative disease caused by a glutamine expansion within huntingtin protein. The exact pathological mechanisms determining disease onset and progression remain unclear. However, aggregates of insoluble mutant huntingtin (mhtt), a hallmark of HD, are readily detected within neurons in HD brain. Although aggregated polyglutamines may not be inherently toxic, they constitute a biomarker for mutant huntingtin useful for developing therapeutics. We previously reported that the small molecule, C2-8, inhibits polyglutamine aggregation in cell culture and brain slices and rescues degeneration of photoreceptors in a Drosophila model of HD. In this study, we assessed the therapeutic potential of C2-8 in the R6/2 mouse model of HD, which has been used to provide proof-of-concept data in considering whether to advance therapies to human HD. We show that, at nontoxic doses, C2-8 penetrates the blood-brain barrier and is present in brain at a high concentration. C2-8-treated mice showed improved motor performance and reduced neuronal atrophy and had smaller huntingtin aggregates. There have been no prior drug-like, non-toxic, brain-penetrable aggregation inhibitors to arise from cell-based high-throughput screens for reducing huntingtin aggregation that is efficacious in preclinical in vivo models. C2-8 provides an essential tool to help elucidate mechanisms of neurodegeneration in HD and a therapeutic lead for further optimization and development.

The Neisseria gonorrhoeae type IV secretion system secretes chromosomal DNA that acts in natural transformation. To examine the mechanism of DNA processing for secretion, we made mutations in the putative relaxase gene traI and used nucleases to characterize the secreted DNA. The nuclease experiments demonstrated that the secreted DNA is single-stranded and blocked at the 5' end. Mutation of traI identified Tyr93 as required for DNA secretion, while substitution of Tyr201 resulted in intermediate levels of DNA secretion. TraI exhibits features of relaxases, but also has features that are absent in previously characterized relaxases, including an HD phosphohydrolase domain and an N-terminal hydrophobic region. The HD domain residue Asp120 was required for wild-type levels of DNA secretion. Subcellular localization studies demonstrated that the TraI N-terminal region promotes membrane interaction. We propose that Tyr93 initiates DNA processing and Tyr201 is required for termination or acts in DNA binding. Disruption of an inverted-repeat sequence eliminated DNA secretion, suggesting that this sequence may serve as the origin of transfer for chromosomal DNA secretion. The TraI domain architecture, although not previously described, is present in 53 uncharacterized proteins, suggesting that the mechanism of TraI function is a widespread process for DNA donation.

Oxidative lesions account for much of the spontaneously occurring DNA damage in normal cells and, left unrepaired, can be mutagenic or cytotoxic. We have investigated the capacity of purified human enzymes to initiate the base excision repair (BER) of oxidative lesions in model nucleosomes. In a construct where the minor groove of a thymine glycol lesion faced outward from the histone octamer, the human DNA glycosylase NTH1 (hNTH1) processed the lesion with nearly the same efficiency as in naked DNA. The hNTH1 reaction did not generate free DNA, indicating that the first step in BER occurred without irreversibly disrupting nucleosomes. Instead, lesion processing entailed the formation of nucleosome-hNTH1 ternary complexes that could be visualized in a gel mobility shift assay. These complexes contained both processed and unprocessed DNA. hNTH1 processing of lesions whose minor groove faced toward the histone octamer was poor at low hNTH1 concentrations but increased substantially as hNTH1 concentrations increased to nearly physiological levels. Additionally, an inward-facing lesion near the nucleosome edge was more efficiently processed than one closer to the nucleosome dyad. These observations suggest that access to sterically occluded lesions entails the partial, reversible unwrapping of DNA from the histone octamer, allowing hNTH1 to capture its DNA substrate when it is in an unwound state.

A highly convergent and efficient synthesis of a new sialyl Lewis(x) (sLe(x)) mimic, which was predicted by computational studies to fulfil the spacial requirements for a selectin antagonist, has been developed. With a beta(2,3)-amino acid residue l-galactose (bioisostere of the l-fucose moiety present in the natural sLe(x)) and succinate are linked, leading to a mimic of sLe(x) that contains all the required pharmacophores, namely the 3- and 4-hydroxy group of l-fucose, the 4- and 6-hydroxy group of d-galactose and the carboxylic acid of N-acetylneuraminic acid. The key step of the synthesis involves a tandem reaction consisting of a N-deprotection and a suitable O-->N intramolecular acyl migration reaction which is promoted by cerium ammonium nitrate (CAN). Finally, the new sialyl Lewis(x) mimic was biologically evaluated in a competitive binding assay.

No abstract available.

Reactive oxygen species and nitric oxide (NO) are capable of both mediating redox-sensitive signal transduction and eliciting cell injury. The interplay between these messengers is quite complex, and intersection of their signaling pathways as well as regulation of their fluxes requires tight control. In this regard, peroxiredoxins (Prxs), a recently identified family of six thiol peroxidases, are central because they reduce H2O2, organic peroxides, and peroxynitrite. Here we provide evidence that endogenously produced NO participates in protection of murine primary macrophages against oxidative and nitrosative stress by inducing Prx I and VI expression at mRNA and protein levels. We also show that NO prevented the sulfinylation-dependent inactivation of 2-Cys Prxs, a reversible overoxidation that controls H2O2 signaling. In addition, studies using macrophages from sulfiredoxin (Srx)-deficient mice indicated that regeneration of 2-Cys Prxs to the active form was dependent on Srx. Last, we show that NO increased Srx expression and hastened Srx-dependent recovery of 2-Cys Prxs. We therefore propose that modulation by NO of Prx expression and redox state, as well as up-regulation of Srx expression, constitutes a novel pathway that contributes to antioxidant response and control of H2O2-mediated signal transduction in mammals.

Little is known about the tyrosine kinase c-Src's function in the systemic circulation, in particular its role in arterial responses to hormonal stimuli. In human aortic and venous endothelial cells, c-Src is indispensable for 17beta-estradiol (E2)-stimulated phosphatidylinositol 3-kinase/Akt/endothelial NO synthase (eNOS) pathway activation, a possible mechanism in E2-mediated vascular protection. Here we show that c-Src supports basal and E2-stimulated NO production and is required for E2-induced vasorelaxation in murine aortas. Only membrane c-Src is structurally and functionally involved in E2-induced eNOS activation. Independent of c-Src kinase activity, c-Src is associated with an N-terminally truncated estrogen receptor alpha variant (ER46) and eNOS in the plasma membrane through its "open" (substrate-accessible) conformation. In the presence of E2, c-Src kinase is activated by membrane ER46 and in turn phosphorylates ER46 for subsequent ER46 and c-Src membrane recruitment, the assembly of an eNOS-centered membrane macrocomplex, and membrane-initiated eNOS activation. Overall, these results provide insights into a critical role for the tyrosine kinase c-Src in estrogen-stimulated arterial responses, and in membrane-initiated rapid signal transduction, for which obligate complex assembly and localization require the c-Src substrate-accessible structure.

Exploring the development of nonviral nucleic acid delivery vectors with progressive, specific, and novel designs in molecular architecture is a fundamental way to investigate how aspects of chemical and physical structure impact the transfection process. In this study, macromolecules comprised of a four-arm star poly(ethylene glycol) and termini modified with one of five different heparin binding peptides have been investigated for their ability to bind, compact, and deliver DNA to mammalian cells in vitro. These new delivery vectors combine a PEG-derived stabilizing moiety with peptides that exhibit unique cell-surface binding ability in a molecular architecture that permits multivalent presentation of the cationic peptides. Five peptide sequences of varying heparin binding affinity were studied; each was found to sufficiently bind heparin for biological application. Additionally, the macromolecules were able to bind and compact DNA into particles of proper size for endocytosis. In biological studies, the PEG-star peptides displayed a range of toxicity and transfection efficiency dependent on the peptide identity. The vectors equipped with peptides of highest heparin binding affinity were found to bind DNA tightly, increase levels of cellular internalization, and display the most promising transfection qualities. Our results suggest heparin binding peptides with specific sequences hold more potential than nonspecific cationic polymers to optimize transfection efficiency while maintaining cell viability. Furthermore, the built-in multivalency of these macromolecules may allow simultaneous binding of both DNA at the core of the polyplex and heparan sulfate on the surface of the cell. This scheme may facilitate a bridging transport mechanism, tethering DNA to the surface of the cell and subsequently ushering therapeutic nucleic acids into the cell. This multivalent star shape is therefore a promising architectural feature that may be exploited in the design of future polycationic gene delivery vectors.

HFE gene mutations are associated with over 80% of cases of hereditary hemochromatosis (HH), an iron-overload disease in which the liver is the most frequently affected organ. Research on HFE has traditionally focused on its interaction with the transferrin receptor. More recent studies have suggested a more complex function for this nonclassical MHC-I protein. The aim of this study was to examine how HFE and its two most common mutations affect the expression of selected genes in a hepatocyte-like cell line. Gene expression was analyzed in HepG2 cells overexpressing wild-type and mutant HFE. The effect of HFE in iron import and oxidative stress levels was assessed. Unfolded protein response (UPR)-activated gene expression was analyzed in peripheral blood mononuclear cells from characterized HH patients. C282Y HFE down-regulated hepcidin and enhanced calreticulin mRNA expression. Calreticulin levels correlated with intracellular iron increase and were associated with protection from oxidative stress. In C282Y(+/+) patients calreticulin levels correlated with the expression of the UPR marker BiP and showed a negative association with the number of hereditary hemochromatosis clinical manifestations. The data show that expression of C282Y HFE triggers a stress-protective response in HepG2 cells and suggest a role for calreticulin as a modifier of the clinical expression of HH.

<h4>Purpose</h4>Develop a Cremophor and solvent free formulation of paclitaxel using amphiphilic block co-polymer micelles of poly(ethylene glycol)-b-poly(epsilon-caprolactone) (PEG-b-PCL) and characterize their release, solubility, cytotoxicity, tolerability, and disposition.<h4>Methods</h4>Hydrophobic prodrugs of paclitaxel were synthesized via DCC/DMAP or anhydride chemistry to overcome the poor loading (<1% w/w) of paclitaxel in micelles of PEG-b-PCL. Micelles were prepared by a co-solvent extraction technique. A micellar formulation of paclitaxel prodrug (PAX7'C(6)) was dosed intravenously to rats (10 mg/kg) and compared to Taxol (paclitaxel in CrEL:EtOH) and PAX7'C(6) in CrEL:EtOH as controls at the same dose. Pharmacokinetic parameters and tissue distribution were assessed.<h4>Results</h4>Paclitaxel prodrugs had solubilities >5 mg/ml in PEG-b-PCL micelles. Resulting PEG-b-PCL micelles contained 17-22% w/w prodrug and were less than 50 nm in diameter. PEG-b-PCL micelles released paclitaxel prodrugs over several days, t(1/2)>3 d. Only the 7'derivative of paclitaxel with the shortest acylchain 7'hexonoate (PAX7'C(6)) maintained cytotoxic activity similar to unmodified paclitaxel. PAX7'C(6) micelles demonstrated an increase in area under the curve, half-life, and mean residence time while total clearance and volume of distribution decreased.<h4>Conclusions</h4>Paclitaxel prodrugs in PEG-b-PCL micelle nanocarriers augment the disposition and increase tolerability making further studies on tumor efficacy warranted.

During early stages of limb development, the vasculature is subjected to extensive remodeling that leaves the prechondrogenic condensation avascular and, as we demonstrate hereafter, hypoxic. Numerous studies on a variety of cell types have reported that hypoxia has an inhibitory effect on cell differentiation. In order to investigate the mechanism that supports chondrocyte differentiation under hypoxic conditions, we inactivated the transcription factor hypoxia-inducible factor 1alpha (HIF1alpha) in mouse limb bud mesenchyme. Developmental analysis of Hif1alpha-depleted limbs revealed abnormal cartilage and joint formation in the autopod, suggesting that HIF1alpha is part of a mechanism that regulates the differentiation of hypoxic prechondrogenic cells. Dramatically reduced cartilage formation in Hif1alpha-depleted micromass culture cells under hypoxia provided further support for the regulatory role of HIF1alpha in chondrogenesis. Reduced expression of Sox9, a key regulator of chondrocyte differentiation, followed by reduction of Sox6, collagen type II and aggrecan in Hif1alpha-depleted limbs raised the possibility that HIF1alpha regulation of Sox9 is necessary under hypoxic conditions for differentiation of prechondrogenic cells to chondrocytes. To study this possibility, we targeted Hif1alpha expression in micromass cultures. Under hypoxic conditions, Sox9 expression was increased twofold relative to its expression in normoxic condition; this increment was lost in the Hif1alpha-depleted cells. Chromatin immunoprecipitation demonstrated direct binding of HIF1alpha to the Sox9 promoter, thus supporting direct regulation of HIF1alpha on Sox9 expression. This work establishes for the first time HIF1alpha as a key component in the genetic program that regulates chondrogenesis by regulating Sox9 expression in hypoxic prechondrogenic cells.

<h4>Background</h4>The altered status of iron metabolism is reported in hereditary haemochromatosis and in non-alcoholic liver fatty disease. We investigated the relation between the H63D HFE mutation gene and non-alcoholic steatohepatitis (NASH).<h4>Methods</h4>We studied as outpatients, 272 Italian persons with NASH and compared them with 430 healthy subjects. Genetic screening for haemochromatosis, haematochemical tests, liver ultrasound examination and liver biopsies were carried out.<h4>Results</h4>The prevalence of heterozygosity for the H63D mutation in NASH patients was not significantly greater than controls. In assessing the C282Y HFE gene mutation alone, the percentage of heterozygosis for C282Y was not different in subjects with NASH compared with controls. As regards a mutation C282Y/H63D there was no significant difference between the two groups. The mean fibrosis score was not significantly different between subjects of group A, with and without HFE mutations (1 +/- 8 and 1 +/- 9, respectively); we did not find a significant correlation between hepatic iron concentration and histological score between subjects.<h4>Conclusion</h4>We have not found a significantly increased prevalence of the mutation H63D in the HFE gene in our patients with NASH. In these patients there was no more severe hepatic histological score when compared with NASH subjects without HFE mutations.

Elevated mean corpuscular volume (MCV) is common in persons with hemochromatosis associated with HFE C282Y homozygosity. We evaluated data from the subset of non-Hispanic white participants in the Hemochromatosis and Iron Overload Screening Study to determine if elevated MCV in C282Y homozygotes is related to this genotype or to serum iron measures. Regression analysis was used to model MCV and Hb from transferrin saturation (TfSat), serum ferritin (SF), mean corpuscular hemoglobin concentration, red blood cell count, age, HFE genotype, Field Center, and presence of liver-related abnormalities in C282Y homozygotes and control subjects without HFE mutations (wt/wt genotype). Mean MCV was higher in C282Y homozygotes than in HFE wt/wt controls (94.4 vs. 89.7 fL in women; 95.3 vs. 91.2 fL in men; P < 0.0001 for both). These differences were largely associated with increased mean TfSat and SF in C282Y homozygotes. Adjusted mean MCV was 92.0 fL (95% confidence interval, 91.1, 92.9) in female C282Y homozygotes and 90.9 fL (90.3, 91.5) in controls. Among women with SF in the reference range 20-200 microg/L, adjusted mean MCV was 92.9 fL, (91.7, 94.2) in C282Y homozygotes, 1.8 fL higher than in controls (P = 0.013). The adjusted mean MCV of male C282Y homozygotes and controls was similar (P = 0.30). Adjusted mean Hb was 0.2 g/dL higher in women with C282Y/C282Y than in controls. Greater mean MCV in C282Y homozygosity reflects increased mean TfSat and mean SF in men and women; an additional effect of genotype on MCV and Hb was detected in women.

The HMG-box domain of approximately 75 amino acid residues was originally identified as the domain that mediates the DNA-binding of chromatin-associated high-mobility group (HMG) proteins of the HMGB type. In the last few years, HMG-box domains have been found in various DNA-binding proteins including transcription factors and subunits of chromatin-remodeling complexes. HMG-box domains mediate either non-sequence-specific (e.g., HMGB-type proteins) or sequence-specific (e.g., transcription factors) DNA binding. Both types of HMG-box domains bind non-B-type DNA structures (bent, kinked and unwound) with high affinity. In addition, HMG-box domains are involved in a variety of protein-protein interactions. Here, we have examined the human and plant genomes for genes encoding HMG-box domains. Compared to plants, human cells contain a larger variety of HMG-box proteins. Whereas in humans transcription factors are the most divergent group of HMG-box proteins, in plants the chromosomal HMGB-type proteins are most variable.

Serial analysis of gene expression (SAGE) provides quantitative and comprehensive expression profiling in a given cell population. In our efforts to define gene expression alterations in Barrett's-related adenocarcinomas (BA), we produced eight SAGE libraries and obtained a total of 457,894 expressed tags with 32,035 (6.9%) accounting for singleton tags. The tumor samples produced an average of 71,804 tags per library, whereas normal samples produced an average of 42,669 tags per library. Our libraries contained 67,200 unique tags representing 16,040 known gene symbols. Five hundred and sixty-eight unique tags were differentially expressed between BAs and normal tissue samples (at least twofold; P<or=0.05), 395 of these matched to known genes. Interestingly, the distribution of altered genes was not uniform across the human genome. Overexpressed genes tended to cluster in well-defined hot spots located in certain chromosomes. For example, chromosome 19 had 26 overexpressed genes, of which 18 mapped to 19q13. Using the gene ontology approach for functional classification of genes, we identified several groups that are relevant to carcinogenesis. We validated the SAGE results of five representative genes (ANPEP, ECGF1, PP1201, EIF5A1, and GKN1) using quantitative real-time reverse-transcription PCR on 31 BA samples and 26 normal samples. In addition, we performed an immunohistochemistry analysis for ANPEP, which demonstrated overexpression of ANPEP in 6/7 (86%) Barrett's dysplasias and 35/65 (54%) BAs. ANPEP is a secreted protein that may have diagnostic and/or prognostic significance for Barrett's progression. The use of genomic approaches in this study provided useful information about the molecular pathobiology of BAs.

During development, multiple guidance cues direct the formation of appropriate synaptic connections. Factors that guide developing axons are known for various pathways throughout the mammalian brain; however, signals necessary to establish auditory connections are largely unknown. In the auditory brainstem the neurons whose axons traverse the midline in the ventral acoustic stria (VAS) are primarily located in the ventral cochlear nucleus (VCN) and project bilaterally to the superior olivary complex (SOC). The circumferential trajectory taken by developing VCN axons is similar to that of growing axons of spinal commissural neurons. Therefore, we reasoned that netrin-DCC and slit-robo signaling systems function in the guidance of VCN axons. VCN neurons express the transcription factor, mafB, as early as embryonic day (E) 13.5, thereby identifying the embryonic VCN for these studies. VCN axons extend toward the midline as early as E13, with many axons crossing by E14.5. During this time, netrin-1 and slit-1 RNAs are expressed at the brainstem midline. Additionally, neurons within the VCN express RNA for DCC, robo-1, and robo-2, and axons in the VAS are immunoreactive for DCC. VCN axons do not reach the midline of the brainstem in mice mutant for either the netrin-1 or DCC gene. VCN axons extend in pups lacking netrin-1, but most DCC-mutant samples lack VCN axonal outgrowth. Stereological cell estimates indicate only a modest reduction of VCN neurons in DCC-mutant mice. Taken together, these data show that a functional netrin-DCC signaling system is required for establishing proper VCN axonal projections in the auditory brainstem.

<h4>Background</h4>Altered serotonergic function is implicated in the aetiology and pathogenesis of a host of psychiatric disorders, and structural variations/polymorphisms in genes encoding the serotonin transporter and various serotonin receptor subtypes are attractive candidates to investigate the biological component underlying these disorders. Specific phenotypic subtypes, that perhaps represent homogeneous forms of the disorder, may increase the power to detect genes in complex diseases.<h4>Objective</h4>We investigated regulatory and functional polymorphic DNA markers of serotonergic candidate genes using a case-control approach in puerperal psychosis and bipolar affective disorder probands.<h4>Methods</h4>We genotyped 320 female participants (104 puerperal psychosis probands, 102 bipolar disorder participants and 114 controls) at the serotonin transporter SERT (5-HTT) 5-HTTVNTR and 5-HTTLPR locus; serotonin receptor 2A (5-HT2A)-T102C and His452Tyr loci, the serotonin receptor 2C (5-HT2C)-Cys23Ser locus, and seven unrelated Alu polymorphic markers.<h4>Results</h4>We observed an association of the puerperal psychosis phenotype with the allele 10 of 5-HTTVNTR of SERT (P=0.004) and a modest association with the genotypic frequencies of the 5-HTTLPR (P=0.036). A nominal P value of 0.006 was observed with the S-10 haplotype in the PP group as compared with bipolar affective disorder probands. Significant association was observed with bipolar affective disorder phenotype with Tyr allele of the 5-HT2A His452Tyr gene polymorphism (P=0.00043) even after a conservative multiple test correction. No association was observed, however, with the 5-HT2A T102C locus, and the distribution of the other seven Alu markers did not differ between the groups.<h4>Conclusion</h4>The association between polymorphisms in serotonergic genes (SERT and 5-HT2A, 5-HT2C) suggests that these genetic factors can modulate vulnerability to puerperal psychosis in female bipolar participants.

Monocyte chemoattractant protein-1 (MCP-1) is a major lymphocyte and inflammatory chemokine associated with persistent inflammatory states. Several abnormalities in the immune status of patients with hereditary hemochromatosis (HH) have been reported, suggesting an imbalance in their immune function. This may include persistent production of, or exposure to, inflammatory cytokines contributing to the pathogenesis of this disorder. The aim of this study was to assess MCP-1 levels in patients with HH and correlate these results with HFE status and iron indexes. One hundred and thirty-nine subjects diagnosed with HH (C282Y homozygotes = 87, C282Y/H63D = 26 heterozygotes, H63D homozygotes = 26), 27 healthy control subjects with no HFE mutation (N/N), and 18 normal subjects heterozygous for the H63D mutation served as age- and sex-matched controls. Ferritin and transferrin saturation and the presence of HFE mutation status were correlated with MCP-1 levels. Full white blood cell count analysis was also performed. We found a strongly significant decrease in MCP-1 protein levels in the C282Y homozygotes compared with the H63D homozygotes (P = 0.0009) and C282Y/H63D heterozygotes (P = 0.002). Similarly, MCP-1 protein levels in the C282Y homozygotes were decreased compared with the healthy controls (P = 0.00076). Furthermore, MCP-1 serum levels were elevated in H63D patients compared with the healthy controls (P = 0.0008). This study suggests for the first time that a differential expression of MCP-1 protein in patients with HH is associated with the specific HFE genetic component for iron overload. Therefore, these findings offer a possible explanation in the variable clinical spectrum of pathogenesis in patients with HH through abnormalities of an imbalance in the immune states of patients with HH.

Significant associations between human leucocyte antigen (HLA)-A and -B alleles and CD8+ T-lymphocyte numbers have been reported in the literature in both healthy populations and in HFE-haemochromatosis patients. In order to address whether HLA alleles themselves or alleles at linked genes are responsible for these associations, several genetic markers at the MHC class I region were typed on a population of 147 apparently healthy unrelated subjects phenotypically characterized for their CD8+ and CD4+ T-lymphocyte numbers. By using a machine learning approach, a set of rules was generated that predict the number of CD8+ T-lymphocyte numbers on the basis of the information of the D6S105 microsatellite alleles only. We demonstrate that the previously reported associations with HLA-A and -B alleles are due to the presence of common long (up to 4 megabases long) haplotypes that increased in frequency recently due to positive selection and that encompass a region where a putative gene contributing to the setting of CD8+ T lymphocytes is located, in the neighbourhood of microsatellite locus D6S105, in the 6p21.3 region.

Patients with alcoholic liver disease frequently exhibit increased body iron stores, as reflected by elevated serum iron indices (transferrin saturation, ferritin) and hepatic iron concentration. Even mild to moderate alcohol consumption has been shown to increase the prevalence of iron overload. Moreover, increased hepatic iron content is associated with greater mortality from alcoholic cirrhosis, suggesting a pathogenic role for iron in alcoholic liver disease. Alcohol increases the severity of disease in patients with genetic hemochromatosis, an iron overload disorder common in the Caucasian population. Both iron and alcohol individually cause oxidative stress and lipid peroxidation, which culminates in liver injury. Despite these observations, the underlying mechanisms of iron accumulation and the source of the excess iron observed in alcoholic liver disease remain unclear. Over the last decade, several novel iron-regulatory proteins have been identified and these have greatly enhanced our understanding of iron metabolism. For example, hepcidin, a circulatory antimicrobial peptide synthesized by the hepatocytes of the liver is now known to play a central role in the regulation of iron homeostasis. This review attempts to describe the interaction of alcohol and iron-regulatory molecules. Understanding these molecular mechanisms is of considerable clinical importance because both alcoholic liver disease and genetic hemochromatosis are common diseases, in which alcohol and iron appear to act synergistically to cause liver injury.

<h4>Background information</h4>FLRG (follistatin-related gene) is a secreted glycoprotein which is very similar to follistatin. As observed for follistatin, FLRG is involved in the regulation of various biological processes through its binding to members of the TGFbeta (transforming growth factor beta) superfamily, activin, BMPs (bone morphogenetic proteins) and myostatin. Unlike follistatin, FLRG has been found to be both secreted and localized within the nucleus of many FLRG-producing cells, suggesting the existence of specific intracellular functions of the protein.<h4>Results</h4>In order to analyse the function of the nuclear form of FLRG, we performed a yeast two-hybrid screen, in which we identified AF10 [ALL1 (acute lymphoblastic leukaemia) fused gene from chromosome 10], a translocation partner of the MLL (mixed-lineage leukaemia) oncogene in human leukaemia, as a FLRG-interacting protein. This interaction was confirmed by far-Western-blot analysis and co-immunoprecipitation with transfected COS-7 cells. The N-terminal region of AF10, including the PHD (plant homeodomain), is sufficient to mediate this interaction, and has been shown to be involved in AF10 homo-oligomerization. By immunoprecipitation experiments, we showed that FLRG enhances the homo-oligomerization of AF10. Functional studies demonstrated that FLRG enhances the transactivation properties of the AF10 protein fused to Gal4 DNA-binding domains in transient transfection assays.<h4>Conclusions</h4>Our present study provides novel insights into the function of the nuclear form of the FLRG protein, which is revealed as a novel regulator of transcription. The nuclear isoform of FLRG lacks an intrinsic transactivation domain, but enhances AF10-mediated transcription, probably through promoting the homo-oligomerization of AF10, thus facilitating the recruitment of co-activators.

Plasminogen activator inhibitor-1 (PAI-1) is a physiological inhibitor of urokinase (uPA), a serine protease known to promote cell migration and invasion. Intuitively, increased levels of PAI-1 should be beneficial in downregulating uPA activity, particularly in cancer. By contrast, in vivo, increased levels of PAI-1 are associated with a poor prognosis in breast cancer. This phenomenon is termed the "PAI-1 paradox". Many factors are responsible for the upregulation of PAI-1 in the tumor microenvironment. We hypothesize that there is a breast cancer predisposition to a more aggressive stage when PAI-1 is upregulated as a consequence of Metabolic Syndrome (MetS). MetS exerts a detrimental effect on the breast tumor microenvironment that supports cancer invasion. People with MetS have an increased risk of coronary heart disease, stroke, peripheral vascular disease and hyperinsulinemia. Recently, MetS has also been identified as a risk factor for breast cancer. We hypothesize the existence of the "PAI-1 cycle". Sustained by MetS, adipocytokines alter PAI-1 expression to promote angiogenesis, tumor-cell migration and procoagulant microparticle formation from endothelial cells, which generates thrombin and further propagates PAI-1 synthesis. All of these factors culminate in a chemotherapy-resistant breast tumor microenvironment. The PAI-1 cycle may partly explain the PAI-1 paradox. In this hypothesis paper, we will discuss further how MetS upregulates PAI-1 and how an increased level of PAI-1 can be linked to a poor prognosis.

<h4>Aim</h4>To study the value of biochemical and ultraso-nographic parameters in prediction of presence and size of esophageal varices.<h4>Methods</h4>The study includes selected cirrhotic patients who underwent a complete biochemical workup, upper digestive endoscopic and ultrasonographic examinations. Albumin/right liver lobe diameter and platelet count/spleen diameter ratios were calculated. The correlation between calculated ratio and the presence and degree of esophageal varices was evaluated.<h4>Results</h4>Ninety-four subjects (62 males, 32 females), with a mean age of 52.32 +/- 13.60 years, were studied. Child-Pugh class A accounted for 42.6%, class B 37.2%, whereas class C 20.2%. Esophageal varices (OE) were not demonstrated by upper digestive endoscopy in 24.5%, while OE grade I was found in 22.3% patients, grade II in 33.0%, grade III in 16.0%, and grade IV in 4.3%. The mean value of right liver lobe diameter/albumin ratio was 5.51 +/- 1.82 (range from 2.76 to 11.44), while the mean platelet count/spleen diameter ratio was 1017.75 +/- 729.36 (range from 117.39 to 3362.50), respectively. Statistically significant correlation was proved by Spearman's test between OE grade and calculated ratios. The P values were 0.481 and -0.686, respectively.<h4>Conclusion</h4>The right liver lobe diameter/albumin and platelet count/spleen diameter ratios are non-invasive parameters providing accurate information pertinent to determination of presence of esophageal varices, and their grading in patients with liver cirrhosis.

This review acknowledges the recent and dramatic advancement in the field of hemochromatosis and highlights the surprising analogies with a prototypic endocrine disease, diabetes. The term hemochromatosis should refer to a unique clinicopathologic subset of iron-overload syndromes that currently includes the disorder related to the C282Y homozygote mutation of the hemochromatosis protein HFE (by far the most common form of hemochromatosis) and the rare disorders more recently attributed to the loss of transferrin receptor 2, HAMP (hepcidin antimicrobial peptide), or hemojuvelin or to certain ferroportin mutations. The defining characteristic of this subset is failure to prevent unneeded iron from entering the circulatory pool as a result of genetic changes compromising the synthesis or activity of hepcidin, the iron hormone. Like diabetes, hemochromatosis results from the complex, nonlinear interaction between genetic and acquired factors. Depending on the underlying mutation, the coinheritance of modifier genes, the presence of nongenetic hepcidin inhibitors, and other host-related factors, the clinical manifestation may vary from simple biochemical abnormalities to severe multiorgan disease. The recognition of the endocrine nature of hemochromatosis suggests intriguing possibilities for new and more effective approaches to diagnosis and treatment.

TRPM2 is a calcium-permeable nonselective cation channel that is opened by the binding of ADP-ribose (ADPR) to a C-terminal nudix domain. Channel activity is further regulated by several cytosolic factors, including cyclic ADPR (cADPR), nicotinamide adenine dinucleotide phosphate (NAADP), Ca(2+) and calmodulin (CaM), and adenosine monophosphate (AMP). In addition, intracellular ions typically used in patch-clamp experiments such as Cs(+) or Na(+) can alter ADPR sensitivity and voltage dependence, complicating the evaluation of the roles of the various modulators in a physiological context. We investigated the roles of extra- and intracellular Ca(2+) as well as CaM as modulators of ADPR-induced TRPM2 currents under more physiological conditions, using K(+)-based internal saline in patch-clamp experiments performed on human TRPM2 expressed in HEK293 cells. Our results show that in the absence of Ca(2+), both internally and externally, ADPR alone cannot induce cation currents. In the absence of extracellular Ca(2+), a minimum of 30 nM internal Ca(2+) is required to cause partial TRPM2 activation with ADPR. However, 200 microM external Ca(2+) is as efficient as 1 mM Ca(2+) in TRPM2 activation, indicating an external Ca(2+) binding site important for proper channel function. Ca(2+) facilitates ADPR gating with a half-maximal effective concentration of 50 nM and this is independent of extracellular Ca(2+). Furthermore, TRPM2 currents inactivate if intracellular Ca(2+) levels fall below 100 nM irrespective of extracellular Ca(2+). The facilitatory effect of intracellular Ca(2+) is not mimicked by Mg(2+), Ba(2+), or Zn(2+). Only Sr(2+) facilitates TRPM2 as effectively as Ca(2+), but this is due to Sr(2+)-induced Ca(2+) release from internal stores rather than a direct effect of Sr(2+) itself. Together, these data demonstrate that cytosolic Ca(2+) regulates TRPM2 channel activation. Its facilitatory action likely occurs via CaM, since the addition of 100 microM CaM to the patch pipette significantly enhances ADPR-induced TRPM2 currents at fixed [Ca(2+)](i) and this can be counteracted by calmidazolium. We conclude that ADPR is responsible for TRPM2 gating and Ca(2+) facilitates activation via calmodulin.

<h4>Objective</h4>The objective of the study was to examine the effect of selective fetoscopic laser photocoagulation (SFLP) vs serial amnioreduction (AR) on perinatal mortality in severe twin-twin transfusion syndrome (TTTS).<h4>Study design</h4>This was a 5 year multicenter, prospective, randomized controlled trial. The primary outcome variable was 30 day postnatal survival of donors and recipients.<h4>Results</h4>There was no statistically significant difference in 30-day postnatal survival between SFLP or AR treatment for donors at 55% (11 of 20) vs 55% (11 of 20) (P = 1.0, odds ratio [OR] 1, 95% confidence interval [CI] 0.242 to 4.14) or recipients at 30% (6 of 20) vs 45% (9 of 20) (P = .51, OR 1.88, 95% CI 0.44 to 8.64). There was no difference in 30 day survival of 1 or both twins on a per-pregnancy basis between AR at 75% (15 of 20) and SFLP at 65% (13 of 20) (P = .73, OR 1.62, 95% CI 0.34 to 8.09). Overall survival (newborns divided by the number of fetuses treated) was not statistically significant for AR at 60% (24 of 40) vs SFLP 45% (18 of 40) (P = .18, OR 2.01, 95% CI 0.76 to 5.44). There was a statistically significant increase in fetal recipient mortality in the SFLP arm at 70% (14 of 20) vs the AR arm at 35% (7 of 20) (P = .25, OR 5.31, 95% CI 1.19 to 27.6). This was offset by increased recipient neonatal mortality of 30% (6 of 20) in the AR arm. Echocardiographic abnormality in recipient twin Cardiovascular Profile Score is the most significant predictor of recipient mortality (P = .055, OR 3.025/point) by logistic regression analysis.<h4>Conclusion</h4>The outcome of the trial did not conclusively determine whether AR or SFLP is a superior treatment modality. TTTS cardiomyopathy appears to be an important factor in recipient survival in TTTS.

Breast carcinoma is a rare disease in men. The incidence is 1 per cent of the incidence in women. Relative hyperestrogenemia and environmental factors seem to be important for the development of the disease. In recent years, germline mutations have been observed in male breast carcinoma patients in several genes, BRCA2, the androgene receptor gene and PTEN. Suspected genetic factors include the cell-cycle checkpoint kinase (CHEK)2 protein truncating mutation 1100delC that has been shown to confer a 10-fold increase of breast cancer risk in men. The c.1-34T > C 5' promoter region polymorphism in cytochrome P450c17 (CYP17), a key enzyme in the biosynthesis of estrogen, has been associated with male breast cancer risk, hemochromatosis gene (HFE) mutations, the mismatch repair genes (hMSH2, hMLH1,hPMS1,hPMS2) and PTEN mutations (Cowden syndrome) are associated with male breast cancer. The majority of tumors is seen retromamillarly. Ductal carcinoma in situ comprises 5-10 % of all cancers. In case of invasive growth, 85-90 % are invasive ductal carcinomas (NOS), 2.5 % are papillary tumors; lobular cancers are exceptionally rare. About 3/4 of all cancers express estrogen and progesterone receptor with increasing positivity with increasing patient age. HER-2 / neu overexpression is seen in the same frequency as in female breast cancer. Poor prognostic factors are tumor size > 2 cm, poorly differentiated tumors, receptor negativity, axillary lymph node involvement and more than four affected nodes.

Netrins are secreted molecules with roles in axonal growth and angiogenesis. The Netrin receptor UNC5B is required during embryonic development for vascular patterning, suggesting that it may also contribute to postnatal and pathological angiogenesis. Here we show that unc5b is down-regulated in quiescent adult vasculature, but re-expressed during sprouting angiogenesis in matrigel and tumor implants. Stimulation of UNC5B-expressing neovessels with an agonist (Netrin-1) inhibits sprouting angiogenesis. Genetic loss of function of unc5b reduces Netrin-1-mediated angiogenesis inhibition. Expression of UNC5B full-length receptor also triggers endothelial cell repulsion in response to Netrin-1 in vitro, whereas a truncated UNC5B lacking the intracellular signaling domain fails to induce repulsion. These data show that UNC5B activation inhibits sprouting angiogenesis, thus identifying UNC5B as a potential anti-angiogenic target.

Neural circuits are assembled through the coordinated innervation of pre- and postsynaptic partners. We show that connectivity between two interneurons, AIY and RIA, in Caenorhabditis elegans is orchestrated by a pair of glial cells that express UNC-6 (netrin). In the postsynaptic neuron RIA, the netrin receptor UNC-40 (DCC, deleted in colorectal cancer) plays a conventional guidance role, directing outgrowth of the RIA process ventrally toward the glia. In the presynaptic neuron AIY, UNC-40 (DCC) plays an unexpected and previously uncharacterized role: It cell-autonomously promotes assembly of presynaptic terminals in the immediate vicinity of the glial cell endfeet. These results indicate that netrin can be used both for guidance and local synaptogenesis and suggest that glial cells can function as guideposts during the assembly of neural circuits in vivo.

<h4>Introduction</h4>In spite of the high initial expectancy in preliminary results concerning the genetics of personality, these studies have not provided satisfactory results. The failure could be related to the lack of biological validity of personality concept and the important influence of environmental factors on personality. A possible way to solve this problem is to look at the temperament of preschool children. It is expected that variability in infants' behaviour can be better defined and with less environmental influence.<h4>Development</h4>Firstly, twin and adoption studies of child temperament in comparison with the studies of personality in adults are reviewed. Secondly, the molecular association studies carried out concerning child temperament are analyzed. The serotonin transporter gene (5-HTT), D4 receptor gene (DRD4) and mono amino oxidade-A gene (MAOA) have been considered candidates to explain variability in child temperament because these genes have been related with specific personality dimensions and mental diseases. Finally, the methodological problems and the future direction of research in this field are considered.<h4>Conclusions</h4>Heritability shows higher values in infant temperament than in adult personality. Different gene polymorphisms on 5-HTT, DRD4 and MAOA could explain some individual variability in children's behavior, although replication studies are needed to confirm the role of these genes. Longitudinal studies in large samples that include gene and environmental interactions are one of the best ways to improve our knowledge about the genetics of child temperament.

Improving clinical tests are allowing us to more precisely classify autism spectrum disorders and diagnose them at earlier ages. This raises the possibility of earlier and potentially more effective therapeutic interventions. To fully capitalize on this opportunity, however, will require better understanding of the neurobiological changes underlying this devastating group of developmental disorders. It is becoming clear that the normal trajectory of neurodevelopment is altered in autism, with aberrations in brain growth, neuronal patterning and cortical connectivity. Changes to the structure and function of synapses and dendrites have also been strongly implicated in the pathology of autism by morphological, genetic and animal modeling studies. Finally, environmental factors are likely to interact with the underlying genetic profile, and foster the clinical heterogeneity seen in autism spectrum disorders. In this review we attempt to link the molecular pathways altered in autism to the neurodevelopmental and clinical changes that characterize the disease. We focus on signaling molecules such as neurotrophin, Reelin, PTEN and hepatocyte growth factor, neurotransmitters such as serotonin and glutamate, and synaptic proteins such as neurexin, SHANK and neuroligin. We also discuss evidence implicating oxidative stress, neuroglial activation and neuroimmunity in autism.

<h4>Introduction</h4>Serotonin-1A receptor (5-HT1AR) function appears to be decreased in major depressive disorder (MDD) based on physiological responses to 5-HT1AR agonists in vivo and to 5-HT1AR binding in brain tissues postmortem or antemortem. We have previously assessed 5-HT1AR binding potential (BP) in depression using positron emission tomography (PET) and [carbonyl-(11)C]WAY-100635, and we have demonstrated reduced 5-HT1AR BP in the mesiotemporal cortex (MTC) and raphe in depressives with primary recurrent familial mood disorders (n=12) versus controls (n=8) [Drevets WC, Frank E, Price JC, Kupfer DJ, Holt D, Greer PJ, Huang Y, Gautier C, Mathis C. PET imaging of serotonin 1A receptor binding in depression. Biol Psychiatry 1999;46(10):1375-87]. These findings were replicated by some, but not other, studies performed in depressed samples that were more generally selected using criteria for MDD. In the current study, we attempted to replicate our previous findings in an independent sample of subjects selected according to the criteria for primary recurrent depression applied in our prior study.<h4>Methods</h4>Using PET and [carbonyl-(11)C]WAY-100635, 5-HT1AR BP was assessed in 16 depressed subjects and 8 healthy controls.<h4>Results</h4>Mean 5-HT1AR BP was reduced by 26% in the MTC (P<.005) and by 43% in the raphe (P<.001) in depressives versus controls.<h4>Conclusions</h4>These data replicate our original findings, which showed that BP was reduced by 27% in the MTC (P<.025) and by 42% in the raphe (P<.02) in depression. The magnitudes of these reductions in 5-HT1AR binding were similar to those found postmortem in 5-HT1AR mRNA concentrations in the hippocampus in MDD [López JF, Chalmers DT, Little KY, Watson SJ. Regulation of serotonin 1A, glucocorticoid, and mineralocorticoid receptor in rat and human hippocampus: implications for neurobiology of depression. Biol Psychiatry 1998;43:547-73] and in 5-HT1AR-binding capacity in the raphe in depressed suicide victims [Arango V, Underwood MD, Boldrini M, Tamir H, Kassir SA, Hsiung S, Chen JJ, Mann JJ. Serotonin 1A receptors, serotonin transporter binding and serotonin transporter mRNA expression in the brainstem of depressed suicide victims. Neuropsychopharmacology 2001;25(6):892-903]. There exists disagreement within the literature, however, regarding the presence and direction of 5-HT1AR-binding abnormalities in depression, which may be explained in some cases by differences in anatomical location (e.g., [Stockmeier CA, Shapiro LA, Dilley GE, Kolli TN, Friedman L, Rajkowska G. Increase in serotonin-1A autoreceptors in the midbrain of suicide victims with major depression--postmortem evidence for decreased serotonin activity. J Neurosci 1998;18(18):7394-401]) and in other cases by pathophysiological heterogeneity within MDD (e.g., some depressives hypersecrete cortisol, which would be expected to down-regulate 5-HT1AR expression [López JF, Chalmers DT, Little KY, Watson SJ. Regulation of serotonin 1A, glucocorticoid, and mineralocorticoid receptor in rat and human hippocampus: implications for neurobiology of depression. Biol Psychiatry 1998;43:547-73]). Antidepressant drug treatment does not alter these abnormalities in 5-HT1AR binding [Sargent PA, Kjaer KH, Bench CJ, Rabiner EA, Messa C, Meyer J, Gunn RN, Grasby PM, Cowen PJ. Brain serotonin1A receptor binding measured by positron emission tomography with [11C]WAY-100635: effects of depression and antidepressant treatment. Arch Gen Psychiatry 2000;57(2):174-80; Moses-Kolko EL, Price JC, Thase ME, Meltzer CC, Kupfer DJ, Mathis CA, Bogers WD, Berman SR, Houck PR, Schneider TN, Drevets WC. Measurement of 5-HT1A receptor binding in depressed adults before and after antidepressant drug treatment using positron emission tomography and [11C]WAY-100635. Synapse 2007;61(7):523-30] but may compensate for blunted 5-HT1AR function by increasing post-synaptic 5-HT1AR transmission [Chaput Y, de Montigny C, Blier P. Presynaptic and postsynaptic modifications of the serotonin system by long-term administration of antidepressant treatments. An in vivo electrophysiologic study in the rat. Neuropsychopharmacology 1991;5(4):219-29].

<h4>Background & objective</h4>Tumor suppressor gene PTEN could not only inhibit the proliferation of cancer cells, but also inhibit their metastasis. However, the mechanism is still unclear. This study was to investigate the effects of PTEN gene on the proliferation and metastasis of human breast cancer ZR-75-1 cells, and explore the mechanisms.<h4>Methods</h4>Wild-type PTEN (wt-PTEN) plasmid and phosphatase-defective PTEN (G129R-PTEN) plasmid were transfected into ZR-75-1 cells by liposome, respectively. Cell proliferation was detected by MTT assay. Transfected cells were selected by puromycin. The expression of PTEN protein was detected by Western blot. Cell adhesion and invasion were tested by adhesion test and invasion test.<h4>Results</h4>The proliferation inhibition rate was significantly higher in wt-PTEN-transfected ZR-75-1 cells than in untransfected cells and G129R-PTEN-transfected cells (42.7% vs. 0% and 2.7%, P<0.01); there was no significant difference between untransfected cells and G129R-PTEN-transfected cells(P>0.05). The proliferation inhibition of ZR-75-1 cells was enhanced along with the increase of culture time and concentration of wt-PTEN. wt-PTEN also induced cell apoptosis. PTEN protein was expressed efficiently in the cells transfected with either wt-PTEN or G129R-PTEN. The inhibition rates of adhesion and invasion were significantly higher in wt-PTEN-transfected cells than in G129R-PTEN-transfected cells (65.7% vs. 8.8%, 70.4% vs. 6.9%, P<0.01).<h4>Conclusion</h4>Wild-type PTEN gene with dual-specific phosphatase activity can inhibit the proliferation and metastasis of ZR-75-1 cells.

Huntingtin-interacting protein 1 (HIP1) was identified through its interaction with htt (huntingtin), the Huntington's disease (HD) protein. HIP1 is an endocytic protein that influences transport and function of AMPA and NMDA receptors in the brain. However, little is known about its contribution to neuronal dysfunction in HD. We report that the Caenorhabditis elegans HIP1 homolog hipr-1 modulates presynaptic activity and the abundance of synaptobrevin, a protein involved in synaptic vesicle fusion. Presynaptic function was also altered in hippocampal brain slices of HIP1-/- mice demonstrating delayed recovery from synaptic depression and a reduction in paired-pulse facilitation, a form of presynaptic plasticity. Interestingly, neuronal dysfunction in transgenic nematodes expressing mutant N-terminal huntingtin was specifically enhanced by hipr-1 loss of function. A similar effect was observed with several other mutant proteins that are expressed at the synapse and involved in endocytosis, such as unc-11/AP180, unc-26/synaptojanin, and unc-57/endophilin. Thus, HIP1 is involved in presynaptic nerve terminal activity and modulation of mutant polyglutamine-induced neuronal dysfunction. Moreover, synaptic proteins involved in endocytosis may protect neurons against amino acid homopolymer expansion.

No abstract available.

Huntington's disease is an hereditary dominant neurodegenerative disorder clinically characterised by progressive motor deficits, personality changes, decreased mental capacity and death after about 15-20 years. Most studies are based on the research of intrinsic mechanisms that could be responsible for dysfunction and later degeneration of neuronal subsets. It is only in the last five years that more interest has been focused on another brain cell type : the astrocytes. This review presents evidence that astroglial function is also affected in Huntington's disease. Among the possible mechanisms, Huntington's disease mutation may alter the EGF receptor signaling pathway, that regulates the astrocytic response to neuronal injuries.

We describe the Phase II HapMap, which characterizes over 3.1 million human single nucleotide polymorphisms (SNPs) genotyped in 270 individuals from four geographically diverse populations and includes 25-35% of common SNP variation in the populations surveyed. The map is estimated to capture untyped common variation with an average maximum r2 of between 0.9 and 0.96 depending on population. We demonstrate that the current generation of commercial genome-wide genotyping products captures common Phase II SNPs with an average maximum r2 of up to 0.8 in African and up to 0.95 in non-African populations, and that potential gains in power in association studies can be obtained through imputation. These data also reveal novel aspects of the structure of linkage disequilibrium. We show that 10-30% of pairs of individuals within a population share at least one region of extended genetic identity arising from recent ancestry and that up to 1% of all common variants are untaggable, primarily because they lie within recombination hotspots. We show that recombination rates vary systematically around genes and between genes of different function. Finally, we demonstrate increased differentiation at non-synonymous, compared to synonymous, SNPs, resulting from systematic differences in the strength or efficacy of natural selection between populations.

Many pathways have been proposed as contributing to Huntington's disease (HD) pathogenesis, but generally the in vivo effects of their perturbation have not been compared with reference data from human patients. Here we examine how accurately mechanistically motivated and genetic HD models recapitulate the striatal gene expression phenotype of human HD. The representative genetic model was the R6/2 transgenic mouse, which expresses a fragment of the huntingtin protein containing a long CAG repeat. Pathogenic mechanisms examined include mitochondrial dysfunction; profiled in 3-nitropropionic acid-treated rats, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mice, and PGC-1alpha knock-out mice; and depletion of brain-derived neurotrophic factor (BDNF) using heterozygous and forebrain-specific BDNF-knock-out mice (BDNF(HET), Emx-BDNF(KO)). Based on striatal gene expression, we find the BDNF models, both heterozygous and homozygous knock-outs, to be more like human HD than the other HD models. This implicates reduced trophic support as a major pathway contributing to striatal degeneration in HD. Because the majority of striatal BDNF is synthesized by cortical neurons, the data also imply that cortical dysfunction contributes to HD's hallmark effects on the basal ganglia. Finally, the results suggest that striatal lesions caused by mitochondrial toxins may arise via pathways different from those that drive neurodegeneration in HD. Based on these findings, we present a testable model of HD pathogenesis that, unlike most models, begins to account for regional specificity in human HD and the absence of such specificity in genetic mouse models of HD.

Protein aggregation into amyloid fibrils and protofibrillar aggregates is associated with a number of the most common neurodegenerative diseases. We have established, using a computational approach, that knowledge of the primary sequences of proteins is sufficient to predict their in vitro aggregation propensities. Here we demonstrate, using rational mutagenesis of the Abeta42 peptide based on such computational predictions of aggregation propensity, the existence of a strong correlation between the propensity of Abeta42 to form protofibrils and its effect on neuronal dysfunction and degeneration in a Drosophila model of Alzheimer disease. Our findings provide a quantitative description of the molecular basis for the pathogenicity of Abeta and link directly and systematically the intrinsic properties of biomolecules, predicted in silico and confirmed in vitro, to pathogenic events taking place in a living organism.

<h4>Purpose</h4>The HEIRS Study screened 101,168 primary care participants for iron overload with serum transferrin saturation (TS), serum ferritin (SF), and C282Y and H63D mutations of the HFE gene. The objective of this study was to evaluate the impact of screening on participants' well-being.<h4>Methods</h4>All C282Y homozygotes, participants with an elevated TS and SF concentration, and a control group of phenotype-genotype negative persons, with neither C282Y nor H63D mutations in the HFE gene were recalled for a clinical evaluation. Health-related quality of life was assessed before screening and approximately 1 week after receipt of the results. Health worries were assessed only at follow-up.<h4>Results</h4>Participants (N = 1478) completed both initial and follow-up surveys. After adjusting for model covariates, phenotype and genotype combinations were statistically significant predictors of changes in psychological well-being (P = 0.0001) and general health (P = 0.0014). C282Y homozygotes with transient elevations in TS or SF were significantly more likely to worry about their health compared to study controls. Race, ethnicity, and preferred language subgroups differed on psychological well-being, general health, and health worry.<h4>Conclusion</h4>Iron phenotype and HFE genotype are associated with health-related quality of life. Health worry was greatest among those considered genetically "at risk. " This may have important implications for multi-ethnic population-based screening studies in which genotype and phenotype are communicated.

<h4>Purpose</h4>Diagnostic and predictive testing for Huntington disease requires an accurate measurement of CAG repeats in the HD (IT15) gene. However, precise repeat sizing can be technically challenging, and is complicated by the lack of quality control and reference materials (RM). The aim of this study was to characterize genomic DNA from 14 Huntington cell lines available from the National Institute of General Medical Sciences Human Genetic Cell Repository at the Coriell Cell Repositories for use as reference materials for CAG repeat sizing.<h4>Methods</h4>Fourteen Huntington cell lines were selected for study. The alleles in these materials represent a large range of sizes that include important diagnostic cutoffs and allele combinations. The allele measurement study was conducted by ten volunteer laboratories using a variety of polymerase chain reaction-based in-house developed methods and by DNA sequence analysis.<h4>Results</h4>The Huntington alleles in the 14 genomic DNA samples range in size from 15 to 100 CAG repeats. There was good agreement among the ten laboratories, and thus, the 95% confidence interval was small for each measurement. The allele size determined by DNA sequence analysis agreed with the laboratory developed tests.<h4>Conclusion</h4>These DNA materials, which are available from Coriell Cell Repositories, will facilitate accurate and reliable Huntington genetic testing.

Alzheimer disease (AD) is defined by progressive impairments in memory and cognition and by the presence of extracellular neuritic plaques and intracellular neurofibrillary tangles. However, oxidative stress and impaired mitochondrial function always accompany AD. Mitochondria are a major site of production of free radicals [ie, reactive oxygen species (ROS)] and primary targets of ROS. ROS are cytotoxic, and evidence of ROS-induced damage to cell membranes, proteins, and DNA in AD is overwhelming. Nevertheless, therapies based on antioxidants have been disappointing. Thus, alternative strategies are necessary. ROS also act as signaling molecules including for transcription. Thus, chronic exposure to ROS in AD could activate cascades of genes. Although initially protective, prolonged activation may be damaging. Thus, therapeutic approaches based on modulation of these gene cascades may lead to effective therapies. Genes involved in several pathways including antioxidant defense, detoxification, inflammation, etc, are induced in response to oxidative stress and in AD. However, genes that are associated with energy metabolism, which is necessary for normal brain function, are mostly down-regulated. Redox-sensitive transcription factors such as activator protein-1, nuclear factor-kappaB, specificity protein-1, and hypoxia-inducible factor are important in redox-dependent gene regulation. Peroxisome proliferators-activated receptor-gamma coactivator (PGC-1alpha) is a coactivator of several transcription factors and is a potent stimulator of mitochondrial biogenesis and respiration. Down-regulated expression of PGC-1alpha has been implicated in Huntington disease and in several Huntington disease animal models. PGC-1alpha role in regulation of ROS metabolism makes it a potential candidate player between ROS, mitochondria, and neurodegenerative diseases. This review summarizes the current progress on how oxidative stress regulates the expression of genes that might contribute to AD pathophysiology and the implications of the transcriptional modifications for AD. Finally, potential therapeutic strategies based on the updated understandings of redox state-dependent gene regulation in AD are proposed to overcome the lack of efficacy of antioxidant therapies.

The patient in this study was a 43-year-old woman who had become unconscious after contracting influenza virus type A infection. Brain CT showed severe brain swelling. Brain MRI also showed brain edema with no specific abnormality on T2-weighted images. We diagnosed her as having influenza type A virus-associated encephalopahty and treated her with Oseltamivir, methylprednisolone pulse therapy, and a high dose of intravenous immunoglobulins. In addition, we treated her with hypothermia and a high dose of intravenous ATIII because of the severe brain swelling and possibility of DIC. After the treatments, brain swelling had improved, and she regained consciousness without any sequelae. Adult influenza virus-associated encephalopathy is rare. We were able to successfully treat our patient with primary multidisciplinary treatments without causing sequelae.

Deferiprone is an orally active iron chelator which has emerged from an extensive search for new treatment of iron overload. Comparative studies have shown that at comparable doses deferiprone may be as effective as deferoxamine in removing body iron. Retrospective and prospective studies have shown that deferiprone monotherapy is significantly more effective than deferoxamine in improving myocardial siderosis in thalassemia major. Agranulocytosis is the most serious side effect associated with the use of deferiprone, occurring in about 1% of the patients. More common but less serious side effects are gastrointestinal symptoms, arthralgia, zinc deficiency, and fluctuating transaminases levels. Deferiprone can be used in combination with desferrioxamine. This regimen of chelation is tolerable and attractive for patients unable to comply with standard deferoxamine infusions or with inadequate response to deferiprone alone. Combination therapy has been effectively used in the management of severe cardiac siderosis.

No abstract available.

No abstract available.